Controlling Whole Blood Activation and Resultant Clot ... · Firstly, we synthesised a series of materials composed of acrylic acid (AA), and methyl (MMA), ethyl (EMA) or butyl methacrylates

Controlling Whole Blood Activation and Resultant Clot

Properties on Various Material Surfaces:

A Possible Therapeutic Approach for Enhancing Bone

Healing

Hoi Ting Shiu, BBiomedSc (Hons)

Thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Institute of Health and Biomedical Innovation

Science and Engineering Faculty

Queensland University of Technology

2012

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Keywords

Bone healing, bone graft substitutes, biomaterials, biomaterial-blood interactions,

biocompatibility, blood clot formation, clot lysis, coagulation, complement, fibrin

network, leukocytes, platelets, platelet-derived growth factor, surface chemistry, surface

functional groups.

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Abstract

Injured bone initiates the healing process by forming a blood clot at the

damaged site. However, in severe damage, synthetic bone implants are used to

provide structural integrity and restore the healing process. The implant unavoidably

comes into direct contact with whole blood, leading to a blood clot formation on its

surface. Despite this, most research in bone tissue engineering virtually ignores the

important role of a blood clot in supporting healing.

Surface chemistry of a biomaterial is a crucial property in mediating blood-

biomaterials interactions, and hence the formation of the resultant blood clot. Surfaces

presenting mixtures of functional groups carboxyl (–COOH) and methyl (–CH3) have

been shown to enhance platelet response and coagulation activation, leading to the

formation of fibrin fibres. In addition, it has been shown that varying the compositions of

these functional groups and the length of alkyl groups further modulate the immune

complement response.

In this study, we hypothesised that a biomaterial surface with mixture of –

COOH/–CH3(methyl), –CH2CH3 (ethyl) or –(CH2)3CH3 (butyl) groups at different

ratios would modulate blood coagulation and complement activation, and eventually

tailor the structural and functional properties of the blood clot formed on the surface,

which subsequently impacts new bone formation.

Firstly, we synthesised a series of materials composed of acrylic acid (AA),

and methyl (MMA), ethyl (EMA) or butyl methacrylates (BMA) at different ratios

and coated on the inner surfaces of incubation vials. Our surface analysis showed

that the amount of –COOH groups on the surface coatings was lower than the ratios

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of AA prepared in the materials even though the surface content of –COOH groups

increased with increasing in AA ratios. It was indicated that the surface

hydrophobicity increased with increasing alkyl chain length: –CH3 > –CH2CH3 > –

(CH2)3CH3, and decreased with increasing –COOH groups. No significant

differences in surface hydrophobicity was found on surfaces with –CH3 and –

CH2CH3 groups in the presence of –COOH groups. The material coating was as

smooth as uncoated glass and without any major flaws. The average roughness of

material-coated surface (3.99 ± 0.54 nm) was slightly higher than that of uncoated

glass surface (2.22 ± 0.29 nm). However, no significant differences in surface

average roughness was found among surfaces with the same functionalities at

different –COOH ratios nor among surfaces with different alkyl groups but the same

–COOH ratios. These suggested that the surface functional groups and their

compositions had a combined effect on modulating surface hydrophobicity but not

surface roughness.

The second part of our study was to investigate the effect of surface functional

groups and their compositions on blood cascade activation and structural properties of

the formed clots. It was found that surfaces with –COOH/–(CH2)3CH3 induced a faster

coagulation activation than those with –COOH/–CH3 and –CH2CH3, regardless of the –

COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces

decreased the rate of activation. Moreover, all material-coated surfaces markedly

reduced the complement activation compared to uncoated glass surfaces, and the pattern

of complement activation was entirely similar to that of surface-induced coagulation,

suggesting there is an interaction between two cascades. The clots formed on material-

coated surfaces had thicker fibrin with a tighter network at the exterior when compared

to uncoated glass surfaces. Compared to the clot exteriors, thicker fibrins with a loose

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network were found in clot interiors. Coated surfaces resulted in more rigid clots with a

significantly slower fibrinolysis after 1 h of lysis when compared to uncoated glass

surfaces. Significant differences in fibrinolysis after 1 h of lysis among clots on material-

coated surfaces correlated well with the differences in fibrin thickness and density at clot

exterior. In addition, more growth factors were released during clot formation than

during clot lysis. From an intact clot, there was a correlation between the amount of

PDGF-AB release and fibrin density. Highest amount of PDGF-AB was released from

clots formed on surfaces with 40% –COOH/60% –CH3 (i.e. 65MMA). During clot lysis,

the release of PDGF-AB also correlated with the fibrinolytic rate while the release of

TGF-β1 was influenced by the fibrin thickness. This suggested that different clot

structures led to different release profiles of growth factors in clot intact and degrading

stages.

We further validated whether the clots formed on material-coatings provide

the microenvironment for improved bone healing by using a rabbit femoral defect

model. In this pilot study, the implantation of clots formed on 65MMA coatings

significantly increased new bone formation with enhanced chondrogenesis,

osteoblasts activity and vascularisation, but decreased inflammatory macrophage

number at the defects after 4 weeks when compared to commercial bone grafts

ChronOSTM β-TCP granules. Empty defects were observed when blood clot

formation was inhibited.

In summary, our study demonstrated that surface functional groups and their

relative ratios on material coatings synergistically modulate activation of blood

cascades, resultant fibrin architecture, rigidity, susceptibility to fibrinolysis as well as

growth factor release of the formed clots, which ultimately alter the healing

microenvironment of injured bones.

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Table of Contents

Keywords .......................................................................................................................................... i

Abstract ............................................................................................................................................ ii

Table of Contents.............................................................................................................................. v

List of Figures ................................................................................................................................ vii

List of Tables ................................................................................................................................... ix

List of Abbreviations ........................................................................................................................ x

Statement of Original Authorship .................................................................................................... xii

Acknowledgments ......................................................................................................................... xiii

CHAPTER 1: INTRODUCTION .................................................................................................. 1 1.1 INTRODUCTION ................................................................................................................. 1

1.2 HYPOTHESIS ....................................................................................................................... 5

1.3 SPECIFIC AIMS OF THIS THESIS ...................................................................................... 5

CHAPTER 2: LITERATURE REVIEW ...................................................................................... 7 2.1 BONE NON-UNION IN CRITICAL SIZED DEFECTS ......................................................... 7

2.2 OVERVIEW OF BONE HEALING ....................................................................................... 8 2.2.1 Haemostasis ................................................................................................................ 8 2.2.2 Inflammation............................................................................................................. 18 2.2.3 Proliferation .............................................................................................................. 20 2.2.4 Remodelling .............................................................................................................. 22

2.3 CURRENT THERAPEUTIC APPROACH .......................................................................... 24 2.3.3 Natural Bone Grafts .................................................................................................. 24 2.3.2 Bone Graft Substitutes ............................................................................................... 25 2.3.3 Bone Tissue Engineering ........................................................................................... 26

2.4 PLATELET-RICH PLASMA............................................................................................... 30 2.4.1 Beneficial Role of Platelets & Uses of PRP ................................................................ 30 2.4.2 Role of Thrombin Concentration in Clot Structure ..................................................... 34 2.4.3 Effect of Clot Structure on Fibrinolysis ...................................................................... 35 2.4.4 Effect of Clot Structure on Viscoelastic Properties ..................................................... 37 2.4.5 In Vivo Implications of Altered Clot Structure, Properties and Stability ...................... 37 2.4.6 Differences Between a PRP gel and a Haematoma ..................................................... 41

2.5 IN SITU BLOOD CLOT FORMATION & MODIFICATION - A POSSIBLE TREATMENT FOR BONE DEFECTS ................................................................................................................... 44

2.5.1 Blood and Host Response towards Biomaterial Implants ............................................ 44 2.5.2 Influence of Biomaterial Surface Chemistry on Blood Response ................................ 48

CHAPTER 3: SYNTHESIS AND CHARACTERISATION OF MATERIAL-COATED SURFACES ................................................................................................................................... 56 3.1 INTRODUCTION ............................................................................................................... 56

3.2 MATERIALS ...................................................................................................................... 59

3.3 METHODS .......................................................................................................................... 59 3.3.1 Synthesis of materials ................................................................................................ 59 3.3.2 Preparation of surface coatings .................................................................................. 61 3.3.3 Characterisation of surface ........................................................................................ 61 3.3.4 Statistical analysis ..................................................................................................... 65

3.4 RESULTS ........................................................................................................................... 66

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3.4.1 Surface coating on incubation vials ............................................................................ 66 3.4.2 XPS analysis of material-coated surfaces ................................................................... 67 3.4.3 Surface hydrophobicity.............................................................................................. 72 3.4.4 Surface morphology and roughness ........................................................................... 75

3.5 DISCUSSION ...................................................................................................................... 79

3.6 CONCLUSION.................................................................................................................... 85

CHAPTER 4: THE INFLUENCE OF CARBOXYL AND ALKYL FUNCTIONAL GROUPS AND THEIR RELATIVE COMPOSITIONS ON BLOOD-BIOMATERIAL INTERACTIONS AND CLOT PROPERTIES .......................................................................................................... 86 4.1 INTRODUCTION ............................................................................................................... 86

4.2 MATERIALS & METHODS ............................................................................................... 89 4.2.1 Blood sampling and in vitro incubation ...................................................................... 89 4.2.2 In vitro coagulation activation ................................................................................... 89 4.2.3 In vitro complement activation .................................................................................. 90 4.2.4 Characterisation of clots formed on material-coated surfaces ...................................... 91 4.2.5 Statistical analysis ..................................................................................................... 95

4.3 RESULTS & DISCUSSION ................................................................................................ 96 4.3.1 Surface-initiated coagulation response ....................................................................... 96 4.3.2 Surface-initiated complement response .....................................................................104 4.3.3 SEM analysis of clot morphology and structure ........................................................110 4.3.4 Assessment of clot rigidity by compaction ................................................................121 4.3.5 Clot lysis ..................................................................................................................124 4.3.6 Quantification of PDGF-AB in serum and during clot lysis .......................................134 4.3.7 Quantification of TGF-beta 1 in serum and during clot lysis ......................................138

4.4 CONCLUSION...................................................................................................................146

CHAPTER 5: A PILOT STUDY OF THE OSTEOGENIC PROPERTIES OF EX VIVO BLOOD CLOTS FORMED ON MATERIALS IN A RABBIT FEMORAL DEFECT ............ 148 5.1 INTRODUCTION ..............................................................................................................148

5.2 MATERIALS & METHODS ..............................................................................................150 5.2.1 Preparation of coatings on scaffold surfaces ..............................................................150 5.2.2 Animals ...................................................................................................................151 5.2.3 Ex vivo blood clot formation .....................................................................................151 5.2.4 Surgical procedures in rabbit femur ..........................................................................152 5.2.5 Examination of defects .............................................................................................154 5.2.6 Statistical analysis ....................................................................................................158

5.3 RESULTS ..........................................................................................................................159 5.3.1 Micro-CT analysis for calcification in the defects......................................................159 5.3.2 Histological examination of de novo bone formation with H & E staining .................161 5.3.3 Histological examination of chondrogenesis in the defects ........................................164 5.3.4 Histological examinations of ALP expression ...........................................................167 5.3.5 Vascularisation revealed by vWF ..............................................................................170 5.3.6 Inflammation response revealed by CD68 .................................................................173

5.4 DISCUSSION .....................................................................................................................176

5.5 CONCLUSION...................................................................................................................181

CHAPTER 6: GENERAL DISCUSSION AND FUTURE DIRECTIONS ............................... 182 6.1 GENERAL DISCUSSION ..................................................................................................182

6.2 FUTURE DIRECTIONS .....................................................................................................191

6.3 GENERAL CONCLUSION ................................................................................................192

BIBLIOGRAPHY ....................................................................................................................... 194

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List of Figures

Figure 2-1. Vascular spasm in response to blood vessel damage.. ..................................................... 10

Figure 2-2. Platelet adhesion and aggregation .................................................................................. 12

Figure 2-3. The intrinsic and extrinsic pathways of coagulation system ............................................ 14

Figure 2-4. Conversion of fibrinogen to fibrin is mediated by thrombin ........................................... 16

Figure 2-5. Components of a haemostatic blood clot ........................................................................ 17

Figure 2-6. Haemostasis results in the formation of a haematoma at the injured bone ....................... 17

Figure 2-7. Inflammatory phase in injured bone. .............................................................................. 19

Figure 2-8. Proliferative phase of bone healing ................................................................................ 21

Figure 2-9. Remodelling phase of bone healing. .............................................................................. 22

Figure 2-10. Time sequence of four main phases of bone healing ..................................................... 23 Figure 2-11. The thrombin concentration present at the time of gelation dictates the fibre thickness and

density .................................................................................................................................... 35

Figure 2-12. Plasmin-mediated fibrinolysis ..................................................................................... 36

Figure 2-13. Two distinct patterns of peri-implant endosseous healing ............................................. 39

Figure 2-14. Schematic pictures illustrating differences in cellular components and fibrin scaffold between a) platelet-rich plasma (PRP) gel, and b) a normal haematoma. .................................. 43

Figure 2-15. Activation of complement system ................................................................................ 45

Figure 2-16. A multinucleated foreign body giant cell ..................................................................... 47

Figure 2-17. Self-assembled monolayers (SAMs) ............................................................................ 49

Figure 2-18. Copolymerisation of two monomers M1 and M2 .......................................................... 53

Figure 3-1. Free-radical polymerisation is initiated by benzoyl peroxide .......................................... 60 Figure 3-2. Copolymer surfaces displaying various functional groups .............................................. 63

Figure 3-3. Water contact angle measurement ................................................................................. 64

Figure 3-4. a) Materials formed from free-radical polymerisation. b) An incubation vial treated with material solution resulted in a clear coating. ............................................................................ 66

Figure 3-5. XPS survey spectra of a) uncoated glass, b) PAA, c) PBMA, and d) 45BMA (45% AA/BMA) coated surfaces. ..................................................................................................... 69

Figure 3-6. XPS C1s spectra of a) PAA, b) PMMA, d) PEMA, f) PBMA, and c) 45MMA, e) 45EMA, and g) 45BMA coated surfaces. .............................................................................................. 70

Figure 3-7. Ratio of –COOH groups on the surface coating as a function of mole fraction of –COOH group-containing AA composed with a) MMA, b) EMA or c) BMA........................................ 72

Figure 3-8. Advancing contact angles of different surface coatings. ................................................. 74

Figure 3-9. Representative SEM images of a) uncoated glass, b) PEMA and c) 45 EMA coated surfaces, taken at magnification of 25000 x. ............................................................................ 76

Figure 3-10. AFM images (5 µm x 5 µm areas) of uncoated and coated surfaces. ............................. 77

Figure 4-1. The serum levels of prothrombin F1+2 after 30 min of whole blood incubation with material-coated surfaces relative to the uncoated glass surfaces (%) ........................................ 97

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Figure 4-2. The serum levels of C5a-desArg after 2 h of whole blood incubation with material-coated surfaces relative to the uncoated glass surfaces (%) ................................................................105

Figure 4-3. Cross-talk between complement and coagulation cascades ............................................107

Figure 4-4. Scanning electron microscopy analysis of whole blood clot structures formed on 45BMA, 55BMA, 65BMA and uncoated glass surfaces ........................................................................112

Figure 4-5. Scanning electron microscopy analysis of structure of clots formed on 55MMA, 55EMA, 55BMA and uncoated glass surfaces ......................................................................................114

Figure 4-6. Scanning electron microscopy analysis of structure of clots formed on 65MMA, 65EMA, 65BMA and uncoated glass surfaces ......................................................................................116

Figure 4-7. Compaction studies of clots formed on various material-coated surfaces compared to the uncoated glass surfaces ..........................................................................................................122

Figure 4-8. Release of D-dimer and weight loss over 24 h lysis of clots formed on BMA surfaces compared with uncoated glass surfaces ..................................................................................128

Figure 4-9. Release of D-dimer and weight loss over 24 h lysis of clots formed on 55MMA, 55EMA and 55BMA surfaces compared with 65MMA, 65EMA and 65BMA surfaces ........................132

Figure 4-10. The serum levels of PDGF-AB after 2 h of whole blood incubation with material-coated surfaces compared to the uncoated glass surfaces and the plasma baseline ..............................135

Figure 4-11. In vitro release of PDGF-AB during lysis of clots formed on material-coated surfaces and uncoated glass surfaces ..........................................................................................................137

Figure 4-12. The serum levels of TGF-β1 after 2 h of whole blood incubation with material-coated surfaces compared to the uncoated glass surfaces and plasma baseline....................................139

Figure 4-13. In vitro release of TGF-β1 during lysis of clots formed on material-coated surfaces and uncoated glass surfaces ..........................................................................................................141

Figure 5-1. Stainless steel scaffold coated with material solution ....................................................150

Figure 5-2. Implantation of ex vivo blood clots formed on material-coated scaffolds in rabbit femoral defects...................................................................................................................................153

Figure 5-3. Micro-CT scanning analysis on the femoral defects ......................................................160

Figure 5-4. De novo bone formation in the defects shown by H&E staining ....................................163

Figure 5-5. Chondrogenesis in the defects shown by Alcian Blue staining .......................................166

Figure 5-6. Osteoblasts in the defect shown by ALP staining ..........................................................169

Figure 5-7. Vascularisation of the defects shown by vWF staining ..................................................172

Figure 5-8. Inflammatory response evaluated by the number of macrophages at the defects using CD68 staining .......................................................................................................................175

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List of Tables

Table 2-1. Growth factors of platelets and their functions ................................................................ 31

Table 3-1. Ratio of –COOH groups measured on surface coatings (XCOOH coating) compared to mole fraction of –COOH group-containing AA (XCOOH material) composed with MMA, EMA or BMA ...................................................................................................................................... 71

Table 3-2. Advancing contact angles of surfaces coated with materials composed of varied mole fraction of acrylic acid and alkyl methacrylates ....................................................................... 74

Table 3-3. Average surface roughness measured by AFM ................................................................ 78

Table 5-1. Three treatment groups in the animal study. ...................................................................153 Table 5-2. Primary and secondary antibodies used in immunohistochemistry and immunofluorescence

studies. ..................................................................................................................................158

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List of Abbreviations

AA Acrylic acid ABC Avidin-biotin complex ADP Adenosine-5’ diphosphate AFM Atomic force microscopy ALP Alkaline phosphatase BPO Benzoyl peroxide BMA Butyl methacrylate BMP Bone morphogenic protein Β-TCP Beta tricalcium phosphate CSD Critical sized defect DAB Diaminobenzidine DAPI 4’, 6-diamindino-2-pheylindole EDTA Ethylenediamine tetraacetic acid EGF Epidermal growth factor EMA Ethyl methacrylate ELISA Enzyme-linked immunosorbent assay FBR Foreign body reaction FBGCs Foreign body giant cells FDPs Fibrin degradation products FGF Fibroblast growth factor FII Prothrombin (factor II) FIIa Thrombin (activated factor II) FIXa-FVIIIa Intrinsic tenase complex (Factor IXa–Factor VIIIa) FVII Factor VII FXa-FVa Extrinsic tenase complex (Factor Xa-Factor Va) FXII Factor XII FXIIa Activated factor XII F1+2 Prothrombin fragment 1+2 GP Ib/IX Glycoprotein Ib/IX receptor GP IIb/IIIa Glycoprotein IIb/IIIa receptor HA Hydroxyapaptite H&E Haematoxylin and eosin HMWK High molecular weight kininogen IGF-1 Insulin-like growth factor -1 IL-1 Interleukin-1 L-PRP Leukocyte- and platelet-rich plasma L-PRF Leukocyte- and platelet-rich fibrin MAC Membrane attack complex MMA Methyl methacrylate MP Microparticle PAI Plasminogen activator inhibitor PBS Phosphate buffered saline PDGF Platelet-derived growth factor

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PDEGF Platelet-derived epidermal growth factor PDAF Platelet-derived angiogenesis factor PEG Polyethylene glycol PF-4 Platelet factor 4 PIBMA Poly(isobutyl methacrylate) PL Phospholipid PLGA Poly(lactic-co-glycolic acid) PMMA Poly(methyl methacrylate) P-PPP Pure platelet-rich plasma P-PRF Pure platelet-rich fibrin PRP Platelet-rich plasma RGD Arginine - Glycine - Asparatic acid RSFs Relative sensitivity factors SAMs Self-assembled monolayers SEM Scanning electron microscopy tPA Tissue-type plasminogen activator TAFI Thrombin activatable fibrinolysis inhibitor TCC Terminal complement complex TF Tissue factor TF-FVIIa Extrinsic tenase complex (Tissue factor - Factor VIIa) TFPI Tissue factor pathway inhibitor TGF-β Transforming growth factor- β TNF-α Tumor necrosis factor-α VEGF Vascular endothelial growth factor vWF von Willebrand factor XPS X-ray photoelectron spectroscopy

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Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the best of

my knowledge and belief, the thesis contains no material previously published or written

by another person except where due reference is made.

Signature : _________________________

Hoi Ting Shiu

Date : _________________________ 7th June, 2012

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Acknowledgments

This thesis would not have been possible without the help and encouragement

of many people.

First and foremost, I am indebted to my principal supervisor, Dr Ben Goss, for

providing me with this great opportunity to work on a project in such a dynamic field.

His invaluable suggestions, patience and continuous support throughout my candidature

is greatly appreciated and undoubtedly led to my professional maturation.

My sincere thanks go to my associate supervisor, Dr Cameron Lutton, for his

guidance and enthusiasm which has given me confidence whenever experiments did not

go as planned. His willingness to share his innovative ideas has been truly inspiring and

of great model.

I would like to express my sincere gratitude to my associate supervisor,

Professor Yin Xiao who participated in our team during my PhD journey, for his

motivation, immense knowledge and constructive comments from the initial conception

to the end of this work. Without his technical assistance and scientific criticism, my

project would not have progressed to the stage that it did. Learning and working with

him has been beneficial and enjoyable by his training and his lively attitude.

I also wish to thank my associate supervisor, Professor Ross Crawford for his

stimulating clinical insights as well as persistence in research which have inspired me to

tackle the challenges faced during the period.

A warm thank you goes to all my colleagues, Will, Kunnika, Rinku, Shirly,

Indira, Willa and Navdeep for assisting me with laboratory techniques and for their

friendship which has enriched my PhD years with conversations ranging from food

science to jokes of having “ permanent head damaged (P-h-D)”.

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I wish to thank Dr Sadahiro Sugiyama for his veterinary expertise with animal

surgery. I also thank Dr Barry Wood of UQ for his support with X-ray photoelectron

microscopy and Associate Professor Nunzio Motta for assisting with atomic force

microscopy.

The financial support from IHBI and the Queensland University of Technology

is also gratefully acknowledged.

To my parents goes my deepest gratitude, for their endless love and support

throughout my life. I am grateful to my father for his care and trust, who initially held

opposed opinions on doing research but finally allowed me to explore myself more

through taking honours research project and subsequent this PhD. To my mother, no

words can sufficiently describe my deep gratitude to her, for putting an enormous effort

to provide the best possible environment for me to grow and accepting me as whom I

am. Her accompany during this journey for reminding me to balance my life has been

very important to me. I know no one would be more delighted than her for my

achievement. I wish to dedicate this thesis on her name.

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1Chapter 1: Introduction

1.1 INTRODUCTION

Healing of injured bone begins with haemostasis, leading to the formation of a

blood clot at the injured site (Marieb, 2001c). In severe damage, however, bone healing

is impaired due to a number of reasons, including deficient biological factors, instability

at the site, and uncontrollable inflammation (Doblare et al., 2004, Tsiridis et al., 2006).

Based on these limiting factors, intensive research aims to develop an ideal bone

substitute providing three key elements for bone healing: the scaffolding for

osteoconduction1, growth factors for osteoinduction2, and progenitor cells for

osteogenesis3 (Burg et al., 2000, Parikh, 2002, Crawford and Hatton, 2008, Schliephake,

2009). To date, no engineered material outperforms autograft in bone-forming ability

(Sammarco and Chang, 2002, Avramoglou et al., 2005, Harwood et al., 2010b).

Despite significant progress in biomaterial development and modification, the

use of non-toxic materials is often complicated with immune response and foreign body

reaction, leading to fibrotic encapsulation and implant dysfunction (Anderson et al.,

2004b, Tsai, 2004, Williams, 2008). This indicates that the reduction of immune

response towards the implant and initial formation of a microenvironment at the

1 Capable of supporting the biological process of bone healing such as ingrowth of capillaries and attachment of osteoprogenitor cells by providing a structural scaffold.

2 Capable of inducing bone formation by supplying biological factors.

3 The process of new bone formation by osteoblasts.

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implant/tissue interface are important for bone healing to proceed (Anderson et al.,

2008).

Platelet-rich plasma (PRP) which has been applied in clinical dentistry for over

a decade provides an insight into the microenvironment required. PRP is a fraction of

plasma with a high concentration of platelets and serves as an autologous source of

growth factors (Marx, 2001, Carlson and Roach, 2002, Griffin et al., 2009). Studies have

shown that formation of PRP gels to bone grafts or biomaterials increases the rate of

bone formation and bone density (Marx et al., 1998b, Choi et al., 2004, Wiltfang et al.,

2004, Roussy et al., 2007). Increased concentrations of growth factors derived from

platelets, and adhesive binding of graft particles by the fibrin network are believed to be

responsible for the beneficial effects of PRP gels (Lieberman et al., 2002a, Eppley et al.,

2004, Dolder et al., 2006). However, other studies have shown conflicting results when

PRP gels were prepared with different platelet numbers and thrombin concentration

(Lacoste et al., 2003, Weibrich et al., 2004, Gimeno et al., 2006). Collectively, these

findings imply that the effect of clots on bone healing depends on how the clots are

formed.

Thrombin concentration is known to affect fibrin thickness and density during

clot formation (Carr et al., 2002b, Wolberg, 2007b). Whilst abnormal changes in clot

structure have been shown to influence the viscoelastic properties (Collet et al., 2005,

Liu et al., 2006, Jámbor et al., 2009) and the lysis rate of clots (Gabriel et al., 1992,

Collet et al., 2000), leading to pathological conditions such as cardiovascular thrombosis

and bleeding disorders (Collet et al., 1993a, Mills et al., 2002). Indeed, dental

implantology has long demonstrated that a peri-implant clot plays an important role in

endosseous healing (Davies, 2003a, Carlsson et al., 2004). Chemokines released from

entrapped blood cells and the fibrin network of the peri-implant have been shown to

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support continuous recruitment of osteogenic cells, resulting in a direct bone formation

on the implant surfaces (Davies, 1998, Oprea et al., 2003). These reports indicate that the

properties of a blood clot formed on the implant would influence cellular response and

tissue ingrowth in bone healing.

However, most current approaches in bone tissue engineering focus solely on

the replacement of viable cells, growth factors and functional structure using engineered

scaffolds. It often overlooks the importance of blood clot formation around scaffolds in

controlling bone healing. We hypothesise that the bone-inducing ability of a biomaterial

may be improved by controlling the blood-biomaterial interactions and forming a

desirable peri-implant blood clot with appropriate properties.

Surface chemistry is one of the most crucial parameters in modulating

interactions between blood and biomaterial (Mrksich, 2000, Thevenot et al., 2008,

Tzoneva et al., 2008). Surfaces with mixtures of carboxyl (–COOH) and methyl (–CH3)

groups have been shown to increase platelet adhesion and activation, as well as thrombin

generation leading to the formation of sizeable fibrin fibres on the surfaces, compared to

the surfaces with either groups only. In addition, the extent of complement activation and

leukocyte response were also found to be modulated by the surfaces with –COOH/–CH3

functionalities at different ratios (Sperling et al., 2005a, 2009, Fischer et al., 2010b).

Moreover, the studies of Berglin et al (2004, 2009) indicated that the chain length of

alkyl groups has a regulatory role in surface-mediated coagulation and complement

activation. These findings lead us to consider binary mixtures of –COOH/–CH3(methyl),

–CH2CH3 (ethyl) or –(CH2)3CH3 (butyl) functionalities at different ratios to modulate

blood clot formation and immune response to biomaterials.

To elucidate closely the in vivo blood-biomaterial interactions, several issues

needed to be addressed. Firstly, whole blood must be used. General approaches in

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investigating blood-biomaterial interactions simplifies the whole blood system by

dividing it into plasma, isolated protein solution or single cell types (Eriksson and

Nygren, 2001, Gemmell, 2001, Sperling et al., 2005a, Thor et al., 2007, Fischer et al.,

2010b). Although the simplification permits close examination of protein and cell

reactions involved, which might be otherwise greatly affected by contributions of

interactive events. Secondly, anticoagulation of whole blood is performed in most

studies to achieve sensitivity of the test at manageable incubation periods (Streller et al.,

2003). Heparin is a commonly used anticoagulant (Berry and Chan, 2008). Besides

inactivating coagulation (Fushimi et al., 1998, Klement et al., 2002), heparin also

reduces complement response (Kopp et al., 2002, Lappegård et al., 2004, Sperling et al.,

2006). As such, using an incubation assay of whole blood without anticoagulant would

enable a more accurate evaluation of the coagulation/complement activation, and reflects

closely the structural features of peri-implant clots modified by surface chemistry.

Furthermore, an improvement in in vitro investigations of the surface chemistry would

require a feasible biomaterial, rather than a standardised flat surface model such as self-

assembled monolayer of alkanethiols (SAMs) on gold, which have been employed to

display various functional groups (Faucheux et al., 2004, Tsai et al., 2007, Arima and

Iwata, 2007).

In this study, novel materials composed of acrylic acid and alkyl (methyl, ethyl,

or butyl ) methacrylate at different ratios were formed to present –COOH/–CH3, –

CH2CH3 or –(CH2)3CH3 functionalities. Customised incubation vials coated with the

materials were used to investigate the impact of surface functional groups and their

relative ratios on blood coagulation and complement activation, the formation of a three-

dimensional blood clot, in term of the clot structure, rigidity, susceptibility to lysis and

release of growth factors. To validate the concept that a peri-implant blood clot

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modulated by material coatings could enhance the healing microenvironment, therefore

improving bone regeneration, a pilot study of animal defects treated with an ex vivo

blood clot formed on an optimal material coating was also performed.

1.2 HYPOTHESIS

We hypothesise that the surface functional groups at various compositions on a

biomaterial surface can modulate blood coagulation and complement activation, and

subsequently tailor the architecture and functional properties of the blood clot formed on

the surface, which eventually impacts new bone formation.

1.3 SPECIFIC AIMS OF THIS THESIS

Three specific aims are addressed in this study:

1. The first aim was to synthesise materials of acrylic acid and alkyl

methacrylates and characterise the physiochemical properties of the material

coatings. Materials were prepared by free radical polymerisation of acrylic

acid and alkyl methacrylates ranging from methyl, ethyl or butyl terminal

groups at various ratios. An incubation vial coated with material was

designed to investigate whole blood response to the surface in a three-

dimensional manner. Surface analysis was performed by X-ray photoelectron

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spectroscopy, advancing water contact angle measurement, atomic force

microscopy and scanning electron microscopy.

2. The second aim was to evaluate the influence of surface chemistries of

material coatings on blood coagulation and complement response, and clot

formation after incubation. The extent of coagulation/complement activation

was determined by quantifying cascades related end products using enzyme-

linked immunosorbent assay. The blood clots formed in material-coated vials

were characterised by measuring fibrin thickness and density, clot rigidity,

susceptibility to lysis and the release of growth factors: platelet-derived

growth factors-AB (PDGF-AB) and transforming growth factor-beta 1 (TGF-

β1).

3. The third aim was to perform a pilot animal study to validate the concept that

the blood clots formed on an implant provide the initial microenvironment for

bone healing and assess the in vivo osteogenic potential of a blood clot

formed on material-coated stainless steel scaffolds. The optimal material was

selected based on in vitro analysis from previous aims. A rabbit femoral

defect model was established and autologous rabbit blood was incubated with

the scaffolds to generate an ex vivo blood clot prior to implantation.

Calcification, osteogenesis and chondrogenesis as well as angiogenesis and

prolonged inflammation were analysed using Micro-CT scanning, histology

and immunohistochemistry after 4 weeks.

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2Chapter 2: Literature review

2.1 BONE NON-UNION IN CRITICAL SIZED DEFECTS

Bone healing is a response of injured bone to restore its loss of structure and

function by forming new bone (Marieb, 2001b, Hench, 2005b). However when

extensive bone is lost in large defects such as trauma and tumour resections, the healing

may proceed abnormally slowly or completely attenuate (Caputo, 1999). A critical sized

defect (CSD) is defined as the smallest size of a damaged bone that would not heal

spontaneously during the life time of the animal (e.g. approximately a loss of bone

segment with length exceeding 3 times the diameter of the affected long bone in sheep)

(Hertel et al., 2001, Lindsey et al., 2006, Liu et al., 2008, Reichert et al., 2009). This

leads to a condition termed non-union, as commonly seen in bone fractures (Phillips,

2005, Jahagirdar and Scammell, 2009, Harwood et al., 2010a). Patients with incomplete

healing suffer from prolonged morbidity and dysfunction, resulting in decreased quality

of life (Tsiridis et al., 2006).

More than 500,000 bone graft procedures are performed in the United States

and approximate 2.2 million worldwide every year (Giannoudis et al., 2005, Tosounidis

et al., 2009). Currently, the treatment using autograft is limited by graft availability from

the patients and donor site morbidity (Goulet et al., 1997). In addition, no other implants

are known to perform as well as or better than autograft (Sammarco and Chang, 2002,

Avramoglou et al., 2005). Therefore, a new strategy to improve the capability of

artificial bone grafts in enhancing bone healing is urgently needed. To develop effective

therapeutic interventions, it is important to understand the mechanism of bone healing.

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2.2 OVERVIEW OF BONE HEALING

Bone repairs itself through similar phases of normal wound healing and

some bone tissue-specific processes. Typically, there is an overlap of four major

phases (Caputo, 1999, Marieb, 2001b):

1. Haemostasis

2 Inflammation

3. Proliferation

4. Remodelling

These dynamic processes begin immediately upon injury and include a

series of interactions among various cell types, mediators and extracellular matrix

(Dimitriou et al., 2005, Nurden et al., 2008). Two distinct mechanisms of bone

formation occur in bone repair, namely intramembranous and endochodral

ossification (Marieb, 2001b, Doblare et al., 2004, Harwood et al., 2010a). A bone

fracture where there is a loss of continuity is used herein to illustrate the bone healing

(Tsiridis et al., 2006, Tosounidis et al., 2009).

2.2.1 Haemostasis

As a vascularised tissue, a fracture in bone is associated with the damage of

blood vessels in the periosteum, endosteum and surrounding soft tissue (Harwood et

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al., 2010a). Haemorrhage and oedema at the fracture site disrupt oxygen and nutrient

supply to osteocytes and eventually cause necrosis of broken bone ends (Caputo,

1999, Carano and Filvaroff, 2003, Tosounidis et al., 2009). Haemostasis is a tightly

regulated process that prevents excessive blood loss and maintains blood flow to the

rest of body. It involves vasoconstriction, formation of platelet plugs and coagulation

(Allford and Machin, 2007).

2.2.1.1 Vasoconstriction of damaged vessels

On damage of the endothelium which lines the lumen of blood vessels,

underlying extracellular matrix proteins are exposed to whole blood. Subendothelium

collagen (principally type I and III), von Willebrand factor (vWF) and fibronectin

interact with platelets through various glycoprotein receptors, thereby supporting

platelet adhesion to the damaged site (Hantgan et al., 1990, Sugimoto and Miyata,

2002, Tsai et al., 2002, Ruggeri, 2003, Schmugge et al., 2003, Nurden, 2007, Bennett

et al., 2009). The interactions also stimulate platelets to release contents from their

granules, including serotonin and thromboxane A2 (Watanabe and Kobayashi, 1988,

Gobbi et al., 2003). These vasoactive factors induce contraction of vascular smooth

muscle cells (Golino et al., 1989) and together with cytokine-stimulated endothelial

cells, damaged blood vessels are constricted to reduce extravasation of blood

constituents (Minors, 2004, Anitua et al., 2004) (Figure 2-1).

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Figure 2-1. Vascular spasm in response to blood vessel damage. Exposed subendothelium proteins bind and activate platelets to release vasoactive factors, together with contraction of smooth muscle cells leading to vessel constriction.

2.2.1.2 Platelets and Formation of a Platelet Plug

Platelets (also termed thrombocytes) play a central role in regulating

haemostasis. They are anuclear, disc-shaped cytoplasmic fragments (diameter 3 - 4

µm) derived from megakaryocytes in the bone marrow. They circulate at an average

concentration of 200 million per mL in the blood (Marieb, 2001d, Nurden et al.,

2008). Platelets become active when they are exposed to thrombogenic surfaces (e.g.

injured endothelium and subendothelium collagen) and soluble components such as

adenosine-5’diphosphate (ADP) and thrombin (Brummel et al., 2002, Andrews and

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Berndt, 2004, Allford and Machin, 2007). Activated platelets exhibit five physiologic

responses, including morphological change to spherical with psedudopodia, exposure

of phosphatidylserine and receptors on cell membrane, degranulation, activation of

cytoskeletal contractile apparatus, and formation of platelet microparticles (MP)

(Blockmans et al., 1995, Blair and Flaumenhaft, 2009, Rand et al., 2010, Nurden,

2011). All these responses have been shown to augment platelet adhesion and

activation at the damaged vessels (Andrews and Berndt, 2004, Gibbins, 2004). In

particular, binding of blood protein, fibrinogen, with platelet glycoprotein IIb/IIIa

receptor (GP IIb/IIIa) further facilitates aggregation of adjacent platelets, leading to

the formation of a platelet plug (Bennett, 2001, 2009, Dubois et al., 2004, Sivaraman

and Latour, 2011) (Figure 2-2). This platelet plug not only preserves the integrity of

the vessel, but also supports coagulation activation (Gorbet and Sefton, 2004,

Minors, 2004).

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Figure 2-2. Platelet adhesion and aggregation. a) Platelets adhesion on subendothelium is mediated by the interaction between adsorbed von Willebrand factor (vWF) and surface glycoprotein Ib/IX receptor (GP Ib/IX). Plasma fibrinogen aggregates more platelets onto the subendothelium by binding glycoprotein IIb/IIIa receptors (GP IIb/IIIa) on nearby platelets. b) Scanning electron micrograph of platelet aggregates (pale pink) and red blood cells (pink). Magnification 4000x. Adapted from http://www. inmagine.com/spl012/spl012882-photo.

2.2.1.3 Coagulation and Formation of a Haematoma

The main purpose of coagulation is to produce a stable haemostatic clot by

forming fibrin mesh on the platelet plugs. Coagulation system can be initiated by two

pathways: intrinsic and extrinsic. For both pathways, a series of proteolytic reactions

where an enzyme precursor becomes active will trigger the activation of another

precursor in the downstream cascade. Both pathways converge to a common final

pathway resulting in the formation of thrombin (Davie et al., 1991, Minors, 2004).

Thrombin is an enzyme which mediates the formation of fibrin.

http://www/

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The intrinsic pathway begins when prekallikrein, high molecular weight

kininogen (HMWK) and factor XII (FXII) contact with thrombogenic surfaces

(Yarovaya et al., 2002, Zhuo et al., 2005, Renné and Gailani, 2007). Upon cleavage

on the surfaces, FXII becomes activated FXIIa and subsequently leads to the

formation of an intrinsic tenase complex (FIXa-FVIIIa) (Vogler and Siedlecki, 2009)

(Figure 2-3).

On the other hand, the initiation of extrinsic pathway is dependent on the

presence of tissue factor (TF). TF is highly expressed on or released from various

cells (e.g. endothelial cells, activated platelets and monocytes/macrophages)

following vascular damage and inflammatory stimuli such as endotoxin, tumor

necrosis factor α (TNFα) and interleukin-1α (IL-1α) (Altieri, 1995, Ernofsson et al.,

1997, Bouchard and Tracy, 2001, Esmon, 2004). TF binds and activates factor VII

(FVII) in the plasma, thereby becoming the extrinsic tenase complex (TF-FVIIa).

For each pathway, it has been revealed that activated platelets provide the

phospholipid surfaces for the assembly and function of the complexes, and hence

greatly propagate the coagulation activation (Esmon, 1995, Bouchard and Tracy,

2001, Smyth et al., 2009). Both tenase complexes convert factor X (FX) to FXa via

the common pathway. When FXa binds to an activated factor V (FVa), they form a

prothrombinase complex (FXa-FVa) locally on the platelet membrane. This complex

cleaves prothrombin (Factor II) and produces thrombin (Factor IIa) (Mann et al.,

2003, Minors, 2004, Vogler and Siedlecki, 2009). As a result, thrombin mediates the

conversion of fibrinogen to fibrin, including fibrinogen bound to the platelet plugs,

thereby restricting formation of fibrin mesh to the location of damaged vessel

(Jirousková et al., 1997, Clark, 2001, Di Cera, 2003) (Figure 2-3).

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Figure 2-3. The intrinsic and extrinsic pathways of coagulation system. The intrinsic pathway starting with surface contact activations of prekallikrein, high molecular weight kininogen (HMWK) and factor XII (FXII) is shown as a linear cascade of zymogen activation steps, leading to the formation of intrinsic tenase complex (FIX-FVIIIa). In parallel, the extrinsic pathway is initiated by tissue factor (TF) generated during trauma. TF activates factor VII (FVII) into FVIIa, and form extrinsic tenase complex (TF-FVIIa). Both tenase complexes from respective pathways merge at the common pathway in which factor X (FX) is converted factor Xa (FXa). FXa which in turn binds to activated factor V (FVa) forming the prothrombinase complex (FXa-FVa) that converts prothrombin to thrombin. Thrombin as the end product of coagulation activation subsequently catalyses the formation of fibrin from fibrinogen. Phospholipids (PL) membrane of platelets and calcium ion (Ca2+) serve as cofactors of the process (Minors, 2004).

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In the conversion of fibrinogen to fibrin, thrombin sequentially cleaves

fibrinopeptides A and B from fibrinogen, resulting in the generation of fibrin

monomers (Weisel et al., 1993, Blomback and Bark, 2004, Riedel et al., 2011). The

monomers interact in a half-staggered end-to-end fashion and become double-

stranded protofibrils. Following lateral aggregation of the protofibrils, fibrin fibres

are formed, branched out and eventuated in a three-dimensional network on the

platelet plugs (Brummel et al., 2002, Mosesson, 2005, Wolberg, 2007a). Thrombin-

activated factor XIII forms cross-links between neighbouring fibres in the network.

Hence, the resultant clot is strengthened against flow, mechanical and proteolytic

impacts (Laurens et al., 2006, Rojkjaer and Rojkjaer, 2007, Jámbor et al., 2009)

(Figure 2-4).

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Figure 2-4. Conversion of fibrinogen to fibrin is mediated by thrombin. Fibrinogen is a trinodular protein consisting two sets of three different polypeptide chains: Aα, Bβ, and γ, assembling with their N-termini in a central E domain. The C-termini of Bβ, and γ chains extend to form distal D domains while the C-termini of Aα chains follow to the D domains and extend back to interact intramolecularly with the central E domain (Mosesson, 2005, Weisel, 2005). Thrombin releases the fibrinopeptides A and B from E domain of fibrinogen sequentially, forming the fibrin monomers. Interacting in a half-staggering and end to end manner, fibrin monomers polymerise into protofibrils. By lateral aggregation and branching out of protofibrils, fibrin polymers are formed in a three-dimension. A stable fibrin clot is formed by factor XIIIa-mediated crosslinking between the γ chains in D-domian in the fibrin network.

An array of proteins are incorporated into the clot during fibrin formation.

For example, vWF, fibronectin, collagen, albumin, tissue-type plasminogen activator

(tPA), plasminogen activator inhibitor (PAI), α2-antiplamin and fibroblast growth

factor-2 (FGF-2) (Standeven et al., 2005, Weisel, 2005, Mosesson, 2005). Also,

platelets and other blood cells such as erythrocytes and leukocytes are entrapped in

the growing clot, resulting in a more definitive haemostatic fibrin clot (Laurens et al.,

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2006) (Figure 2-5). A clot, which is formed between the fractured bone ends is called

a haematoma (Figure 2-6) (Street et al., 2000, Marieb, 2001b).

Figure 2-5. Components of a haemostatic blood clot. a) It is composed of platelet aggregates, erythrocytes and b) leukocytes entrapped in the fibrin network. Scanning electron micrograph b) at 5000 x, scale bar 20 µm. a) Adapted from http://www.biocurious.com/new-perspective-on-blood-clot-mechanics.html.

Figure 2-6. Haemostasis results in the formation of a haematoma at the injured bone. Modified from www.uwo.capatholcasesSkeletalfracture.html.

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2.2.2 Inflammation

The second stage of healing is inflammation. In the affected tissue, histamine

and prostaglandins released by local mast cells trigger inflammation (Marieb, 2001a,

Esmon, 2003, Nurden, 2011). These mediators cause vasodilation and increase vascular

permeability, leading to the influx of inflammatory cells, plasma proteins and fluid into

the injured site. The influx produces the cardinal signs of redness, sweating, heat and

pain (Marieb, 2001a, 2001e, Hench, 2005a). In addition, activated platelets in the

haematoma degranulate a series of cytokine and growth factors including platelet factor

4 (PF-4), platelet-derived growth factor (PDGF) and transforming growth factor-beta

(TGF-β) (Hosgood, 1993, Anitua et al., 2004, Frechette et al., 2005, Blair and

Flaumenhaft, 2009, Müller et al., 2009). These factors are revealed to stimulate

chemotaxis of neutrophils, monocytes, fibroblasts and progenitor cells into the damaged

site (Deuel et al., 1981, Pierce et al., 1991, Cross and Mustoe, 2003, Andrae et al., 2008).

Neutrophils are the first type of inflammatory cells that migrate from blood into

the damaged tissue (Marieb, 2001a, Esmon, 2003). Their migration is also mediated by

complement protein C5a, an end product of complement cascade in innate immune

response (Guo and Ward, 2005, Ritis et al., 2006, Nilsson et al., 2007a). Neutrophils

remove necrotic cellular debris, pathogens and foreign materials by phagocytosis. After

24-48 hours, the population of neutrophils diminish due to their short lifespan and are

replaced by blood monocytes (Park and Barbul, 2004, Janeway et al., 2005a, Anderson

et al., 2008). The monocytes differentiate into long-life (up to months) macrophages in

the tissue in response to cytokines, extracellular metabolites and the hypoxic

environment (Hench, 2005a, Anderson et al., 2008). Osteoclasts are also activated to

reabsorb bone debris. In addition to performing phagocytosis, macrophages release

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reactive oxygen species and growth factors. They release fibroblast growth factor (FGF)

and epidermal growth factor (EGF) to mediate the growth of fibroblasts, new blood

vessels and epithelial cells (Harwood et al., 2010a). Fibroblasts and vascular endothelial

cells migrate into the haematoma and contribute to form granulation tissue

approximately 3 to 5 days following the fractures. Undifferentiated mesenchymal cells

which originate from the periosteum of broken bone also migrate into the site (Doblare

et al., 2004, Tsiridis et al., 2006, Tosounidis et al., 2009, Harwood et al., 2010a) (Figure

2-7).

Figure 2-7. Inflammatory phase in injured bone. It involves the influx of inflammatory cells, plasma proteins and fluid to the haematoma, followed by infiltration of fibroblasts which deposit collagen matrix, and endothelial cells which forms new capillaries. Modified from www.uwo.capatholcasesSkeletalfracture.html.

http://www.uwo/

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2.2.3 Proliferation

Granulation tissue is a specialised type of tissue characterised by the

proliferation of new small blood vessels and fibroblasts (Marieb, 2001a). Fibroblasts

synthesise collagen to form a new matrix within the clot while mature endothelial cells

organise into new capillaries from pre-existing vessels in a process known as

angiogenesis (Glowacki, 1998, Carano and Filvaroff, 2003). Angiogenesis is important

in tissue regeneration; as newly established blood flow not only provides oxygen and

nutrient, but also mesenchymal stem cells and signalling molecules (Gerber and Ferrara,

2000, Carano and Filvaroff, 2003, Weiss et al., 2009). During this proliferative phase,

the clot is gradually degraded by plasmin-mediated fibrinolysis while neutrophils and

macrophages clean the remaining by-products of fibrinolysis (Erickson et al., 1985,

Simon et al., 1993, Lijnen and Collen, 1995). Eventually, the haematoma is replaced

with granulation tissue in a controlled pattern. Approximately 12 days after injury a

completely vascularised collagen matrix is formed.

For the majority of severe fractures, there will be a degree of motion during

healing. In this situation, injured bone heals through an indirect (secondary) pathway in

which both intramembranous and endochondral ossification occur (Hench, 2005b,

Phillips, 2005). Endochondral ossification takes place at the centre of the collagen matrix

where mesenchymal stem cells proliferate vigorously and differentiate into chondrocytes

(Figure 2-8). Chondrocytes actively lay down cartilaginous matrix, thus transforming the

matrix to a fibrocartiliaginous callus (Marieb, 2001b). Gradually, the callus is deposited

with osteoid secreted by osteoblasts and calcified with calcium hydroxyapatite. When

mineralization proceeds, the hypertrophic chondrocytes within the callus undergo

apoptosis and a bone callus is formed at the fracture site. Upon union, the callus is

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known as woven bone (Phillips, 2005, Harwood et al., 2010a). Simultaneously,

intramembranous ossification forms a bone callus without a cartilage scaffold at the

periphery of the collagen matrix (Marieb, 2001b, Tsiridis et al., 2006). Mesenchymal

stem cells differentiate to osteoprogenitor cells, which later form the periosteum together

with surrounding fibroblasts. Some osteoprogenitor cells further differentiate into

osteoblasts to synthesise type I collagen of osteoid, resulting in a direct generation of

calcified tissue at the outer surface of fracture (Tosounidis et al., 2009, Harwood et al.,

2010a).

Figure 2-8. Proliferative phase of bone healing. It involves endochondral (at central of matrix through cartilage formation) and intramembranous (at periphery of matrix) ossification leading to woven bone formation from the vascularised granulation tissue. Modified from www.uwo.capatholcasesSkeletalfracture.html.

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2.2.4 Remodelling

The final stage of bone healing is to remodel bone callus into normal lamellar

bone (Hench, 2005b). Collagen fibres are in random orientation in the newly formed

woven bone. This disorganises the calcified components on the fibres, resulting in

reduced mechanical properties of the woven bone (Jahagirdar and Scammell, 2009).

Over a period of months, the collagen fibres are aligned to conform as closely as

possible to the original tissue by dual actions of collagen degradation and synthesis

(Doblare et al., 2004, Phillips, 2005). Together with the coupled actions of osteoblasts

and osteoclasts in bone deposition and resorption respectively, the woven bone is

remodelled into lamellar bone with highly ordered micro-architecture, thereby restoring

the normal mechanical properties (Tsiridis et al., 2006, Jahagirdar and Scammell, 2009)

(Figure 2-9).

Figure 2-9. Remodelling phase of bone healing. It involves collagen alignment and transformation of woven bone into lamella bone as origin. Modified from www.uwo.capatholcasesSkeletalfracture.html.

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To conclude, the bone healing is a highly complex process influenced by

biological and mechanical factors. Despite this, the underlying mechanism is largely

driven by the early microenvironment of which a haematoma forms the critical

component. This suggests that modifying a haematoma might be a primary target for

enhancing bone healing in severe defects. Figure 2-10 summarises the sequence of bone

healing events.

Figure 2-10. Time sequence of four main phases of bone healing: haemostasis (bleeding and blood clotting), inflammation, proliferation and remodelling.

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2.3 CURRENT THERAPEUTIC APPROACH

With an improved understanding of the biology and molecular aspects of bone

healing after injuries, it is generally accepted that the interruption of healing sequences in

non-union is caused by deficiencies of osteogenic cells, growth factors, and a conductive

scaffold at the damaged site (Dimitriou et al., 2005, Tsiridis et al., 2006). Current

therapeutic strategy aims to expedite the natural healing mechanism by providing the

deficient factors to the bone defects. However, treatment options have not been

improved greatly for the past decades.

2.3.3 Natural Bone Grafts

Autograft remains the gold standard for treating CSD. Autograft is harvested

from local bone and implanted in the same patient (Ryzewicz et al., 2009, Cove and

Keenan, 2009). It has been proven clinically efficacious and widely used in

orthopaedics, dentistry, oral and maxillofacial surgeries (Sajjadian et al., 2010). One

important reason is that autograft contains living cells and tissue-inducing substances

with its scaffold, hence imparting the physical structure and supporting osteoconduction,

osteoinduction and osteogenesis (Bauer and Muschler, 2000, Carlsson et al., 2004,

Precheur, 2007). However, the use of autograft is complicated by graft availability,

donor site morbidity and post-operative pain (Goulet et al., 1997, Bimmel and Govaers,

2006, Kolomvos et al., 2010). Despite allograft and xenograft harvested from human

donors or animals respectively overcomes the drawbacks of autograft, their applications

Page 25


are also limited by immune rejection, risk of virus transmission and decreased

biomechanical integrity (Wheeler and Enneking, 2005, Garofalo, 2007).

2.3.2 Bone Graft Substitutes

Due to the problems associated with the uses of natural bone grafts, bone

graft substitutes have been developed. Bone graft substitutes are either proteins

derived from organic bone (e.g. collagen & demineralised bone matrix) or inorganic

(synthetic) materials (Frayssinet, 2004, Giannoudis et al., 2005). Metallic implants

were the first “bone substitutes” to be used in orthopaedic history because of their

high mechanical strength (Precheur, 2007, Kao and Scott, 2007). In particular,

titanium has shown some superior osteointegrating properties (Hong et al., 1999,

2005, Le Guéhennec et al., 2007). Other synthetic materials such as ceramics (e.g.

hydroxyapatite (HA), tricalcium phosphate (TCP)) (Kandaswamy et al., 2000, Hing

et al., 2004), polymers (e.g. poly (lactic-co-glycolic acid)(PLGA)) and composites

are also introduced (Giannoudis et al., 2005, Deb, 2008, Chen et al., 2008). They

could be relatively biodegradable, osteoconductive, and have mechanical strength

similar to bone (McAuliffe, 2003, Ilan and Ladd, 2003).

However, each material has specific disadvantages. For instance fatigue and

stress shielding for non-degradable metals, and deficiency of osteoinductive property

for synthetic biomaterials (Sammarco and Chang, 2002, Hallab et al., 2004, Kao and

Scott, 2007). None of these synthetic materials can perfectly substitute for autograft

in current clinical practice.

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2.3.3 Bone Tissue Engineering

Bone tissue engineering is the latest approach to treat CSD. It combines the

technologies of biomaterial science, cell and molecular biology (Meyer et al., 2004).

The strategy is to confer osteoinductive capabilities on a bone substitute by

incorporating growth factors and/or pre-seeding reparative cells on the biomaterials

(Habibovic and de Groot, 2007, Dawson and Oreffo, 2008, Schliephake, 2009). It is

proposed that a biodegradable material not only serves as an osteoconductive

scaffold for growth, but also as a delivery vehicle of cells and signalling molecules to

accelerate bone healing (Stevens, 2008, Hutmacher et al., 2012).

Growth factor therapy is based on the molecular processes of pro-osteogenic

(e.g. bone morphogenetic protein (BMP)-2, -7, TGF-β) and angiogenic factors (e.g.

PDGF, FGF) in mediating bone formation (Wozney and Rosen, 1998, Lieberman et

al., 2002c, Carano and Filvaroff, 2003, Cross and Mustoe, 2003, Schmidmaier et al.,

2007, Bosetti et al., 2007). However, the degree of bone healing achieved is largely

dependent on the amount of growth factor released by the used scaffold. The

structural properties of the carrier have a dramatic effect on the release profile of

growth factors (Yang et al., 2008). A wide range of materials such as collagen,

hyaluronic acid, polylactic acid, injectable cement and even a biomimetic coating

(e.g. bone-like apatite (carbonated hydroxyapatite) layer) have been investigated to

achieve a controllable and sustained release of growth factor along with their

degradation rate, chemical and physical properties (Seeherman et al., 2006,

Kamitakahara et al., 2007, Liu et al., 2010). So far, design of scaffold architecture

remains a challenging topic in bone tissue engineering (Burg et al., 2000).

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Cell therapy works by grafting normal cells to restore cellular function in

the defects. In bone defects, a number of cell types have been engrafted on

biomaterials in vitro prior to transplantation, including osteoblasts, chondroblasts,

periosteal cells, bone marrow cells, stem cells and vascular cells (Puelacher et al.,

1996, Breitbart et al., 1998, Meyer et al., 2004, Kraus and Kirker-Head, 2006, Nather

et al., 2010). To further enhance the cellular function in producing growth factors,

gene therapy has been combined with this approach. Engrafted cells are transfected

in vitro by viral vectors which contain a gene encoding the expression of desired

growth factor (Kang et al., 2000, Dai et al., 2004, Betz et al., 2010). In addition,

implants might also be coated directly with viral vectors to transduce a patient’s own

cells in vivo (Awad et al., 2007). Recent work by Lin et al. (2010) demonstrated that

the bone marrow stem cells modified to express BMP-2 and vascular endothelial

growth factor (VEGF) accelerated the repair of a CSD in a rabbit femur. Although

these cells execute distinct functions in the bone healing, the cell transplantation has

not been widely applied in clinical practice due to several issues.

Primarily, this approach requires substantial labour and time to isolate and

expand the cells in vitro prior to in vivo application (Avramoglou et al., 2005). While

stem cells derived from periosteum and bone marrow can be expanded readily in

vitro due to their unique self-renewal property, it remains a problem to amplify the

differentiated cells to sufficient populations to rebuild bone mass without losing

viability (Pountos et al., 2007, Shanti et al., 2007). It was also shown that

osteoprogenitor cells only represent approximately 0.001% of the nucleated cells in

healthy adult marrow, making this approach least practical to aged and injured

groups for whom it is most needed. Moreover, concerns of immune rejection, ethical

issues and risk of uncontrolled gene expression also impede the uses of allogeneic

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cells, embryonic stem cells or viral vector transduced cells (Baltzer and Lieberman,

2004).

Together, bone tissue engineering has shown some potential in regenerating

bone. However, there are challenges and limitations in this technique before it can be

routinely translated to clinical situations.

(1) The performance of bioengineered constructs are greatly dependent on

the architecture and property of the biomaterial used (Burg et al., 2000, Blom, 2007,

Stevens, 2008). Slow or incomplete vascularisation of the engineered constructs after

implantation often occurs, causing hypoxia and death of cells seeded deep within the

scaffold and dysfunction of the tissue construct (Avramoglou et al., 2005, Laschke et

al., 2006). This suggests that the scaffold design requires further modification to

achieve rapid and adequate blood vessel ingrowth to include the engraftment of

endothelial cells as well as the release of angiogenic factors. Also, it indicates that

the angiogenic potential of biomaterials with different chemical compounds remain

largely unclear (Burg et al., 2000, Eckhaus et al., 2008, Butler and Sefton, 2007).

Generally, it is suggested that an ideal bone graft substitute for tissue engineering

should have the following characteristics (Giannoudis et al., 2005, Hutmacher and

Williams, 2006, Anderson et al., 2008, Thevenot et al., 2008, Amini et al., 2011):

1. Biocompatible (i.e. defined as the ability of a biomaterial to perform with an

appropriate host response in a specific application without eliciting other

adverse effects) (Williams, 2008).

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2. Three-dimensional and highly interconnected porous network (i.e. for tissue

growth and efficient flow transport of nutrients and metabolic waste).

3. Biodegradable or bioadsorptable at an appropriate rate (i.e. to facilitate the

controlled release of growth factors and match the rate of tissue ingrowth).

4. Possess mechanical properties (i.e. to match those of the tissue at implant

site and support before the regenerated tissue bears an increasing load while

the scaffold is degrading).

5. Suitable surface chemistry for cell adhesion, proliferation and

differentiation.

(2) On the other hand, one limitation of bone tissue engineering is that the

strategy often focuses on replacing a single cell type (differentiated or pluripotent),

or one or more growth factors to enhance bone regeneration, which in reality has a

multi-factorial mechanism. One of the typical examples is that a chondrocyte-seeded

scaffold remains in a cartilaginous stage after implantation into the bone defect, with

no observation of endochondral ossification (Vacanti et al., 1995). This finding

suggests that the healing sequence is important to direct cellular response for bone

formation. It is conceivable that the formation of a haematoma-like clot structure at

the implant site would have a beneficial effect on eliciting bone healing. This idea is

confirmed by studies using platelet-rich plasma.

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2.4 PLATELET-RICH PLASMA

Platelet-rich plasma (PRP) is a fraction of plasma in which platelets are

concentrated in a small volume of plasma (Lozada et al., 2001, Dugrillon et al.,

2002). The rationale behind the use of PRP is to provide autologous platelets, which

secrete their storage pool of growth factors at high concentration to expedite bone

regeneration and soft tissue repair (Anitua et al., 2004, Cenni et al., 2010).

2.4.1 Beneficial Role of Platelets & Uses of PRP

Indeed, activated platelets release a range of osteogenic and angiogenic

growth factors from their alpha (α)-granules, including PDGF, TGF-β, platelet-

derived epidermal growth factor (PDEGF), platelet-derived angiogenesis factor

(PDAF), insulin-like growth factor-I (IGF-I) and platelet factor 4 (PF-4) (Sánchez et

al., 2003, Frechette et al., 2005, Blair and Flaumenhaft, 2009) (Table 2-1). These

growth factors are known to have positive effects on bone healing by stimulating the

proliferation and differentiation of undifferentiated mesenchymal cells and

osteoblasts (Lieberman et al., 2002c, Arpornmaeklong et al., 2003, Bosetti et al.,

2007), angiogenesis as well as chemotaxis for inflammatory cells (Oprea et al., 2003,

Frechette et al., 2005). Primarily, it is believed that the initiation of bone regeneration

begins with the releases of PDGF and TGF-β after platelet aggregation (Kells et al.,

1995, Lieberman et al., 2002b).

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Growth Factor Actions

Platelet-derived growth factor (PDGF)

Chemotaxis and activation: neutrophils, macrophages Chemotaxis, mitogenesis and activation: fibroblasts (collagen synthesis), endothelial cells & smooth muscle cells (angiogenesis) Mitogenesis: mesenchymal and bone marrow stem cells

Transforming growth factor-beta (TGF-β)

Chemotaxis and activation: monocytes, endothelial cells (angiogenesis), preosteoblasts Inhibition: osteoclasts (bone resorption) Stimulation: osteoblasts(osteoid formation, osteogenesis) Regulation of mitogenesis: endothelial cells & fibroblasts (collagen synthesis)

Platelet-derived epidermal growth factor (PDEGF)

Chemotaxis and angiogenesis Mitogenesis: epithelial cells & mesenchymal cells Regulation of collagen synthesis

Insulin-like growth factor-1 (IGF-1)

Stimulation: cartilage & bone matrix formation Mitogenesis: preosteoblasts & osteoblasts Enhancement with PDGF: increases rate & quality of wound healing

Platelet-derived angiogenesis factor (PDAF)

Mitogenesis: endothelial cells Angiogenesis and enhance vascular permeability Upregulated by: IGF-1, TGF-α, -β, PDEGF, FGF & IL-1β

Platelet factor 4 (PF-4)

Chemotaxis: neutrophils, fibroblasts Inhibition: heparin

Table 2-1. Growth factors of platelets and their functions (Sánchez et al., 2003, Frechette et al., 2005).

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Previous studies have shown that growth factor concentrations correlate

directly with platelet number in PRP (Dugrillon et al., 2002, Eppley et al., 2004).

After dual gradient density centrifugation of whole blood to obtain PRP, platelets are

activated by the addition of thrombin and calcium chloride, leading to platelet

degranulation and formation of a PRP gel (Lozada et al., 2001, Grageda, 2004). The

application of PRP offers theoretical advantages over the delivery of a single

recombinant growth factor since PRP releases high concentrations of multiple native

growth factors in their biological ratios. The complex and interdependent nature of

growth factors (i.e. TGF-β, PDAF and IGF-1) suggests that more than one signalling

pathway of bone regeneration could be targeted with the use of PRP (Kells et al.,

1995, Sánchez et al., 2003). Also, when PRP gel is combined with particulate grafts,

better handing characteristics and in vivo stability could be achieved (Roukis et al.,

2006, Mooren et al., 2007).

In vitro studies have shown that PRP supernatants support the viability and

proliferation of human fetal osteoblast-like cells (Slater et al., 1995), alveolar bone

cells (Choi et al., 2005), porcine articular chondrocytes (Akeda et al., 2006) and

human endothelial cells (Roussy et al., 2007, Cenni et al., 2009). Extensive animal

studies have also investigated the effect of PRP gel alone on bone regeneration

(Weibrich et al., 2004, Swennen et al., 2004, Gandhi et al., 2006, Roussy et al.,

2007), or in combination with bone grafts and graft substitutes (Marx et al., 1998a,

Wiltfang et al., 2004, Choi et al., 2004, Mooren et al., 2007, Kasten et al., 2008,

Messora et al., 2008).

However, there is some inconsistency in the literature regarding the benefits

of PRP. While some studies reported significant increases in bone formation and

maturation rates (Marx et al., 1998a, Wiltfang et al., 2004, Gandhi et al., 2006,

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Roussy et al., 2007), others did not observe any improvement or even inhibition of

new bone formation (Choi et al., 2004, Swennen et al., 2004, Weibrich et al., 2004,

Mooren et al., 2007).

One of the possible reasons is the differential use of platelet concentrations

among studies. A typical PRP is defined to have a 5-fold increase platelet

concentration (approximately 1,000,000/µl) over the physiological level (Marx,

2001). Platelet concentration varies greatly due to different baseline values of animal

species and the preparing procedures of PRP (Dolder et al., 2006, Roukis et al.,

2006). In fact, the platelet concentration required for a positive effect on bone

regeneration seems to span a very limited range. Weibrich et al. (2004) reported that

advantageous effects of PRP on peri-implant bone regeneration in rabbits only

occurred when a platelet concentration of approximately 1,000,000/µl was used. At

lower concentration (164,000-373,000/µl), the effect was suboptimal whereas higher

concentration (1,845,000-3,200,000/µl) led to a paradoxically inhibitory effect. This

finding was supported by in vitro work of Choi et al. (2005) and Tomoyasu et al.

(2007) who studied the effect of platelet concentration in PRP alone on human

alveolar cells, and in combination with BMPs on human osteoblasts respectively.

Different protocols for platelet activation may be another reason for the

discrepancy of results (Walkowiak et al., 2000, Lozada et al., 2001). Concentrations

of thrombin and calcium for platelet activation were shown to affect the release of

growth factors, endothelial cell division (Lacoste et al., 2003, Frechette et al., 2005,

Roussy et al., 2007), and the adhesive property of PRP clots on soft tissue (Gimeno

et al., 2006). However, the mechanism of how these activators vary the properties of

the PRP clot is not fully understood. This may be associated with the thrombin

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concentration, which alters platelet activation and fibrin polymerisation, leading to

different kinetics of growth factor release and clot structure (Martineau et al., 2004).

2.4.2 Role of Thrombin Concentration in Clot Structure

The thrombin concentration present at the time of blood clotting has been

shown to profoundly influence the fibrin architecture than fibrinogen concentration, pH

and ionic strength (Nair and Dhall, 1991, Carr et al., 2002b, Wolberg, 2007b). Clots

formed at low thrombin concentration (< 1 nM) are composed of thick fibrin fibres in a

loose configuration. While those formed at high thrombin concentration are composed of

thin fibres in a tight configuration (Figure 2-11) (Wolberg and Campbell, 2008). Using

turbidimetric analysis of plasma, it has been shown that the altered thrombin

concentration contributes to different clot structure through fibrin polymerisation

process. An increase in thrombin concentration leads to a shorter time required for

protofibrils to grow to a sufficient length before they aggregate. It also causes an increase

in maximum rate of turbidity development and a decrease in the maximum final

turbidity, indicating a faster fibrin formation and a decrease in fibrin size (Standeven et

al., 2005).

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Figure 2-11. The thrombin concentration present at the time of gelation dictates the fibre thickness and density. a) Scanning electron micrographs of fibrin clots formed by adding thrombin (0.5-20 nM) to solutions of purified fibrinogen (2 mg/mL). The scale bars indicate 20 μm (top row), and 1 μm (bottom row) Adapted from (Wolberg, 2007a). Laser confocal micrographs of fibrin clots formed by adding thrombin b) 2.5 nM, c) 10 nM to solutions of purified fibrinogen (1 mg/mL). Laser confocal micrographs at 63 x (scale bar 10 µm). Adapted from (Wolberg and Campbell, 2008).

2.4.3 Effect of Clot Structure on Fibrinolysis

Alterations in clot structure have been demonstrated to affect the clot

susceptibility to fibrinolysis. In fibrinolysis, fibrin fibres are digested by plasmin which

is produced from cleavage of inactive plasminogen by tissue type plasminogen activator

Page 36


(tPA) (Figure 2-12) (Medved and Nieuwenhuizen, 2003). Fibrin fibres are shown to be

transverse cut across, rather than by progressive cleavage uniformly around the fibres

(Veklich et al., 1998). A study by Collet et al. (2000) showed that while individual thick

fibres are lysed more slowly than thin fibres, clots with a loose conformation of thick

fibres were lysed more rapidly than those with tightly-packed thin fibres. These findings,

as confirmed by later study of Bhasin et al. (2008) indicate that the network

conformation is a more important determinant for fibrinolysis rate compared with fibre

thickness. The changes in network conformation are believed to regulate the lysis rate by

influencing the fibrin density, tPA bindings on fibrin and transport of fibrinolytic

components throughout the clot (van Gelder et al., 1995, Collet et al., 2000, Medved and

Nieuwenhuizen, 2003, He et al., 2005, Undas et al., 2006).

Figure 2-12. Plasmin-mediated fibrinolysis. Inactive plasminogen is converted to active plasmin by tissue type plasminogen activator (tPA). Plasmin digests fibrin and generates fibrin degradation products (FDPs): D-dimers and E fragments. Antifibrinolysis system which includes proteins such as plasminogen activator inhibitor (PAI) 1 & 2, α2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), inhibits the fibrinolysis at different steps.

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2.4.4 Effect of Clot Structure on Viscoelastic Properties

Individual fibrin fibre has been illustrated to possess extensibility and elasticity

(Liu et al., 2006). The effect of fibre thickness on the properties of individual fibres is

not known yet. Instead, it has been demonstrated that altered fibrin structure may

modulate the clot rigidity as a whole, depending on fibre thickness, length, density,

degree of branching and cross-linking (Standeven et al., 2005, Wolberg and Campbell,

2008). In particular, cross-links that reinforce fibrin contacts within the clot increase the

elasticity of individual fibres and the overall clot elasticity (Collet et al., 2005, Liu et al.,

2006, Jámbor et al., 2009).

2.4.5 In Vivo Implications of Altered Clot Structure, Properties and Stability

A number of studies have shown that an altered clot structure is a causative

mechanism of many thrombotic diseases and bleeding disorders (Collet et al., 1993b,

Mills et al., 2002, Terasawa et al., 2006, Bhasin et al., 2008, Undas et al., 2008, 2009).

Patients with acute ischemic stroke are found to produce in vitro plasma clots that are

denser with thicker fibres and more resistant to fibrinolysis compared to controls (Undas

et al., 2010). In contrast, haemophilia A patients who are deficient of factor VIII, are

shown to produce clots that are much more porous with thicker fibres, and overly more

susceptible to fibrinolysis (Wolberg, 2007a). These findings suggest that the abnormal

clot structure and susceptibility to fibrinolysis are principally related to pathological

conditions.

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In addition to the genetic factors, environmental determinants of fibrin structure

are also of clinical importance (Scott et al., 2004). Changes in thrombin concentration

may indirectly modulate clot architecture by activating factor XIII to cross-link adhesive

proteins to fibrin. Binding of fibronectin to fibrin is essential for cell adhesion and

migration into the clots (Okada et al., 1985, Carr et al., 1987, Weisel, 2005). While

cross-linked collagen also supports the formation of extracellular matrix at the injury

sites, aiding the healing process. Moreover, fibrin-bound actin, myosin and vinculin

together with platelet cytoskeleton have been shown to mediate clot retraction and

subsequent wound narrowing (Asijee et al., 1988, Mosesson, 2005, Weisel, 2005,

Wolberg, 2010). Hence, it possibly explains the changes in growth factor release, cell

division and adhesive properties of PRP clots as previously reported by studies using

thrombin at different concentrations. Moreover, these findings offer an insight into the

pivotal role of a blood clot and its structural properties, which will have a direct effect on

the bone healing by influencing macromolecule transport, cell behaviour and new tissue

ingrowth.

A dental implant inserted in the jaw bone is a typical example of endosseous

(in-bone) implants in which its clinical success is greatly influenced by a blood clot

formed around the implant. During implantation, tissue damage and bleeding are

inevitable. The first tissue that comes into contact with the implant is blood (Anderson,

2001). Subsequently, a blood clot is formed at the gap between host bone and implant, as

a result of coagulation activation. The blood clot not only detains blood flow and anchors

the implant to the endosseous wound site, but most importantly supports two types of

peri-implant endosseous healing: distance and contact osteogenesis (Davies, 2003b).

Distance osteogenesis occurs when new bone is initially formed on the surfaces of

surrounding old bone at a distance from the implant. Conversely, contact osteogenesis

Page 39


takes place when new bone is first formed on the implant surface. Hence, it is clinically

considered as a superior mode of healing in case there is insufficient cortex to provide

early stability (Figure 2-13).

Figure 2-13. Two distinct patterns of peri-implant endosseous healing: a) distance osteogenesis and b) contact osteogenesis where osteoblasts line initially on the host bone or implant surface respectively and lay down matrix. In distance osteogenesis, the bone surfaces provides a population of osteogenic cells which differentiate into osteoblasts and lay down a new matrix that grows slowly towards the implant and results in the approximation of the implant surface shape by the newly formed bone. An intervening layer of non-bone cells is often formed between the bone and implant. Contact osteogenesis occurs with osteoconduction and de novo bone formation, which predominantly require recruitment and migration of osteogenic cells towards the implant surfaces.

Page 40


Clearly, the prerequisite of contact osteogenesis is the continuous

recruitment and migration of osteogenic cells to the implant surface through the

three-dimensional blood clot. The osteoconduction is largely stimulated by activated

platelets and leukocytes entrapped in the compartment. They release a range of

cytokines and growth factors, creating chemoattractant gradients to recruit

undifferentiated or osteogenic cells to the implant site (Oprea et al., 2003, Park and

Barbul, 2004, Dohan Ehrenfest et al., 2006). On the other hand, the three-

dimensional network of fibrin as well as structural proteins of the clot serve as

physical scaffolds to support cell adhesion and migration (Choukroun et al., 2006).

To migrate to the implant, the osteogenic cells impose contractile forces on the fibrin

fibres that attached to the implant surface, where they differentiate into osteoblasts

and directly lay down bone matrix. Following mineralisation, a collagen-free cement

line appears and results in de novo bone formation (Davies, 2003b). Thus, the fibrin

architecture of the clot is important for effective fibrin retention of implant surface

and critically determines the process of contact osteogenesis (Choukroun et al., 2006,

Liu et al., 2006).

Overall, the presence of a blood clot with appropriate clot structural

properties ensures the bone-implant interface environment to support the bone

healing.

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2.4.6 Differences Between a PRP gel and a Haematoma

While PRP gels are mostly formed by adding a fixed and high amount of

thrombin to platelet-concentrated plasma, it is conceivable that a PRP gel would be a

platelet clump tightly networked by thin fibres. These abnormal clot structures and

properties, as well as their impacts on growth factor release, cell proliferation and

physical stability are likely attributed to the negative effect on bone healing as previously

reported. The confusion between the differences in cellular components and structures

between a PRP gel and a haematoma, and how these differences relate to their potentials

in enhancing bone healing may be the source of inconsistent results.

A haematoma contains mostly erythrocytes, approximately 5% of platelets and

less than 1% of leukocytes (Marieb, 2001b, Carlson and Roach, 2002). However, a PRP

gel which contains theoretically no other blood cells but platelets, possessing nearly a

reverse ratio of erythrocytes and platelets compared to a haematoma. In fact, platelets

and circulating blood cells, both their number and interactions play an important role in

clot features. It has long been proposed by Ulevitch and Johnston (1980) that

erythrocytes participate in the intrinsic pathway of coagulation, which is believed to be

associated with the negatively charged phospholipids of the cells (Peyrou et al., 1999,

Iwata et al., 2004). Also, it has been demonstrated that erythrocytes interact with

platelets by promoting platelet aggregation, and inversely activated platelets also

enhance erythrocyte agglomeration (Alkhamis et al., 1988, Valles et al., 1991). In

addition, leukocytes have been suggested to influence coagulation by expressing TF,

activating platelets and factor X (Plescia and Altieri, 1996, Bouchard and Tracy, 2001,

Gorbet and Sefton, 2004, Elalamy et al., 2007), as well as cleavage of tissue factor

pathway inhibitor (TFPI) (Petersen et al., 1992, Sundaram et al., 1996). All these studies

Page 42


are consistent with the findings of Thor et al. (2007), where whole blood induced higher

levels of thrombin generation and platelet activation than PRP on clinically used

titanium, and that the response of PRP could be partially restored in the presence of

erythrocytes (Hong et al., 2001, Horne et al., 2006).

Additionally, it has been shown that cells influence local fibrin structure by

direct interaction between cell surface integrins and fibrin. Platelets have been shown to

organise fibrin into tighter bundles near its cell surface than seen more distally within the

clot (Collet et al., 2002, Campbell et al., 2009). In contrast, the presence of erythrocytes

has been shown to form a more porous fibrin network, facilitating migration of cells into

the area and thus support wound healing (Carr and Hardin, 1987).

Furthermore, cell-associated fibrin is revealed to be more resistant to

fibrinolysis than distally located fibrin (He et al., 2005, Jerome et al., 2005, Campbell et

al., 2008). This might be due to soluble proteins released from the cells which can

regulate the equilibrium between clot formation and dissolution. FXIII and PAI-1,

released from platelets, are known to increase the resistance of the clot to fibrinolysis

(Korbut and Gryglewski, 1995, Handt et al., 1996, Carrieri et al., 2011). On the contrary,

haemoglobin as well as neutrophil elastase and cathepsin G, are shown to enhance

fibrinolysis (Bach-Gansmo et al., 1998, Yoshida et al., 2001). These studies agree that

during in vivo myocardic infarction, normal erythrocytes-rich clots are readily dissolved

by enzymatic lysis whereas platelet-rich clots are more resistant to be degraded (Jang et

al., 1989, Parise and Agnelli, 1991, Gold et al., 1991).

So far, the innovative uses of PRP in bone tissue engineering focus solely on

the biological value of platelet growth factors. There are conflicting results of PRP with

bone healing and its utility remains unsolved. Recently, a second-generation of platelet

concentrates has also been introduced based on leukocyte content and fibrin architecture

Page 43


including: pure platelet-rich plasma (P-PPP), leukocyte- and platelet-rich plasma (L-

PRP), pure platelet-rich fibrin (P-PRF), leukocyte- and platelet-rich fibrin (L-PRF)

(Anitua et al., 2006, Choukroun et al., 2006, Dohan Ehrenfest et al., 2006, Baeyens et al.,

2010, Simonpieri et al., 2012). Little is known about how these two parameters

influences the intrinsic biology of these products (Dohan Ehrenfest et al., 2010, 2012,

Simonpieri et al., 2011), and not to mention other cellular components in the clots are too

often neglected.

Taken together, a PRP gel is different from a natural haematoma in both

cellular components and structure (Figure 2-14). It is likely that these differences

contribute to different molecular and cellular activities as well as mechanical stability at

the injured bone, ultimately dictating the outcome of peri-implant bone healing.

Figure 2-14. Schematic pictures illustrating differences in cellular components and fibrin scaffold between a) platelet-rich plasma (PRP) gel, and b) a normal haematoma.

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2.5 IN SITU BLOOD CLOT FORMATION & MODIFICATION - A

POSSIBLE TREATMENT FOR BONE DEFECTS

Based on in vivo potentials in influencing bone healing, it seems that blood

clots could be classified as “ good” and “ bad”. The fact that the composition of a

blood clot is specific to the circumstances under which it formed opens up a possible

approach to make a “ good ” clot on artificial bone implant as a therapeutic agent for

treating bone defects. A central question is how blood interacts with biomaterials

after implantation.

2.5.1 Blood and Host Response towards Biomaterial Implants

As noted, surgical procedures of implantation induce bleeding which in turn

triggers haemostasis and acute inflammation immediately (Anderson, 2001, Gorbet

and Sefton, 2004). Blood contact with implants leads to rapid adsorption of proteins

on the implant surface. Within seconds, a layer of proteins is formed as a result of

dynamic collision, competitive adsorption and displacement in a process known as

the Vroman effect (Horbett, 1993). This protein deposition on biomaterial surface is

termed as provisional matrix formation (Anderson, 2001). Cells that migrate later

into the site interact with the adsorbed proteins, rather than directly with the material

surface itself. Hence, it is widely believed that the nature of the absorbed proteins

determines subsequent responses of coagulation, platelets, leukocytes, as well as the

immune complement reactions to the implant (Figure 2-15) (Eskin et al., 2004).

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Figure 2-15. Activation of complement system. As part of the innate immune system, complement is a blood cascade for the recognition and clearance of foreign materials. Similar to coagulation system, complement system consists of over 20 proteins and are activated upon cleavage by an upstream enzyme. It can be activated through classical, alternative, and lectin pathways. While classical pathway is initiated by C1q binding on foreign surface, the alternative and lectin pathways are triggered by spontaneous hydrolysis of C3 on foreign surface and mannose-binding lectin, respectively. All pathways converge at the formation of C3 convertase, which cleaves C3 to fragments C3a and C3b. C3a acts as an anaphylatoxin, producing local inflammatory response such as induction of smooth muscle contraction, increase of vascular permeability and chemotaxis. While C3b complexes with C3 convertase and become C5 convertase, which cleaves complement molecule C5 to C5a and C5b. C5a acts as the most potent anaphylatoxin whereas C5b participates in the formation of complement C5b-9 complex (also known as membrane attack complex (MAC) or the terminal complement complex (TCC)). Ultimately, incorporation of C5b-9 into cell membrane of foreign cells achieve defending effects of complement system of destruction of foreign particles by alternating membrane polarization to cause cell rupture, and facilitating recognition and opsonisation by phagocytes.

Page 46


Earlier in vitro studies noted that the adsorption of FXII, HMWK and

prekallikrein on biomaterial surfaces were accompanied with thrombin formation,

suggesting biomaterial-induced coagulation may be initiated by intrinsic pathway

(Mulzer and Brash, 1989, Ziats et al., 1990). Also, the adsorption of fibrinogen,

fibronectin and vWF are known to promote platelet adhesion and activation on

biomaterials (Keuren et al., 2002, Tsai et al., 2002, Kwak et al., 2005). On the other

hand, it is believed that immunglobulin G and complement fragment C3b deposited

on surfaces not only mediate surface-activated complement by classical and

alternative pathways, but also act as opsonins to modulate adhesion and phagocytosis

of neutrophils and macrophages (Gemmell, 1997, Wilson et al., 2005, Sellborn et al.,

2005, Nilsson et al., 2007a).

In acute inflammation, macrophages are beneficial as they replenish

cytokine and growth factors at the implant site, leading to the formation of a

granulation tissue on the implant (Anderson, 2001). However, in some situations,

persistent inflammatory stimuli, chemical and physical properties of the biomaterial

may lead to chronic inflammation that lasts the entire lifetime of the implant (Tsai,

2004, Gorbet and Sefton, 2004, Jones, 2008). Adherent macrophages on implant

surfaces cannot engulf the implant due to size disparity, and therefore experience

“frustrated phagocytosis” (Nilsson et al., 2007b, Anderson et al., 2008). Unlike

normal phagocytosis, they fuse to form multinucleated foreign body giant cells

(FBGCs) surrounding the implant (Figure 2-16). Moreover, the macrophages may

also respond by respiratory burst and protease release, leading to the deterioration of

the implant and injury to peripheral tissue (Janeway et al., 2005b). Thus, the FBGCs

together with granulation tissue in the presence of implant is referred to as foreign

body reaction (FBR) (Tang and Eaton, 1995, Hu et al., 2001, Anderson et al., 2008).

Page 47


Eventually, the host response to implants leads to fibrosis or fibrous

encapsulation. Fibrous capsule and interfacial FBR become a physiochemical barrier

which isolates the implant from the surrounding tissues, severely impairing implant

function and in vivo integration (Anderson et al., 2008, McNally and Anderson,

2011).

In summary, the mechanisms governing the biomaterial-blood interactions,

and the possible role of biomaterial surfaces affecting such responses are crucial for

the design of biocompatible endosseous implants.

Figure 2-16. A multinucleated foreign body giant cell. Adapted from http://www.manana tomy.com/basic-anatomy/reticuloendothelial-system-macrophage-system

http://www/

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2.5.2 Influence of Biomaterial Surface Chemistry on Blood Response

To minimise adverse responses and ensure long term function of implants,

many research efforts have been placed on modifying the surface chemistry of

biomaterials.

The surface chemistry of titanium which offers thrombogenicity to the

endosseous implant has long been suggested to attribute to its superior

osteointegration (Hong et al., 1999). Upon air exposure, a titanium oxide layer is

formed naturally on the surfaces. It displays negative charges that closely resemble

extracellular matrix protein of damaged tissue, therefore resulting in enhanced

attachment and activation of FXII and platelets on the surface. Thrombin generation

was also greatly elevated by titanium when compared to stainless steel, which is

known to have a poorer osteointegration property (Nygren et al., 1997, Broberg et

al., 2002, Hong et al., 2005). Furthermore, using a rat subcutaneous pocket model, it

was demonstrated that the extent of neovascularisation in healing process was

modulated by pre-coating titanium with a thin plasma clot (100 nm) (Jansson et al.,

2001). Hence, these findings suggested that an endosseous implant might generate a

positive osteogenic response through its surface chemistry that modulates blood

cascade activation and the formation of a blood clot.

To determine more specifically what species and density of surface

chemical functionalities affect blood protein adsorption and subsequent cellular

interactions, alkythiols or alkysilanes self-assembled monolayers (SAMs) are widely

used as a flat surface model to presents a single or multiple functional groups (Figure

2-17).

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Figure 2-17. Self-assembled monolayers (SAMs). a) A simple unfunctionalized (methyl-terminated) alkanethiols monolayer on gold substrate. b) ω-functionalized alkanethiols on gold. c) Alkyl silane monolayer on oxidized silicon.

2.5.2.1 Carboxyl and Methyl Functional Groups

Surface chemical functionality has been demonstrated to influence both the

amount and structural changes of protein upon adsorption (Martins et al., 2003,

Ishizaki et al., 2007). SAMs bearing methyl (–CH3) groups were shown to bind more

fibrinogen and cause a greater conformational changes of adsorbed proteins than

carboxyl (–COOH), amino (–NH2) and hydroxyl (–OH) groups (Sit and Marchant,

1999a, Evans-Nguyen and Schoenfisch, 2005b, Roach et al., 2005, Sivaraman et al.,

2009, Sperling et al., 2009, Xu and Siedlecki, 2009, Fischer et al., 2010b).

Such conformational changes of adsorbed fibrinogen on –CH3 SAMs was

also evidenced to associate with stronger platelet adhesion and activation than –

COOH or –OH SAMs (Lin and Chuang, 2000, Sperling et al., 2005a, Rodrigues et

Page 50


al., 2006, Sivaraman and Latour, 2010). In addition, the work of Evans-Nguyen et

al.(2005b) indicated that surface functionalities affect the ability of adsorbed

fibrinogen to interact with thrombin in forming fibrin fibres. Hence, surface

functionality alters the adsorption characteristics of fibrinogen, its ability to support

platelet interaction and fibrin polymerisation on biomaterials.

With regards to surface-associated coagulation, –CH3 SAMs was shown to

induce a stronger overall activation than –COOH SAMs, despite the initiation of

intrinsic pathway being higher on –COOH SAMs. Interestingly, a study which

investigated –COOH and –CH3 mixed SAMs found an addition of –COOH groups

(50-83%) to –CH3 SAMs further elevated coagulation activation compared to –CH3

SAMs alone. Varying compositions of –COOH/–CH3 SAMs was also shown to

modulate platelet adhesion and activation (Sperling et al., 2005a, 2009, Fischer et al.,

2010b). This is consistent with the other studies suggesting that combining –CH3 and

other functional groups may further alter surface-mediated fibrinogen adsorption and

platelet responses (Rodrigues et al., 2006, Tsai et al., 2007).

An opposite trend in immune response was observed on the –COOH and –

CH3 mixed SAMs. Surfaces containing 47% –COOH/53% –CH3 resulted in a lower

level of complement activation followed by –CH3, –COOH and –OH SAMs

(Sperling et al., 2007). The stronger complement response to –OH or –NH2 SAMs is

believed to be mediated through an alternative pathway in which these functional

groups form a direct covalent thioester linkage of complement fragment C3b, leading

to the formations of C3 and C5 convertases. Increasing –OH content on mixed SAMs

was also found to increase complement activation (Tang et al., 1998, Hirata et al.,

2003, Sperling et al., 2005a). Salvador-Morales et al. (2009) who investigated lipid-

Page 51


polymer nanoparticles presenting –COOH/–CH3 groups also demonstrated an

increase in complement activation with increasing –COOH content.

Furthermore, it has been indicated that the complement C3 and

immunoglobulin G deposited on –OH SAMs are associated with the highest number

of leukocyte found on pure –OH3 SAMs than pure –CH3 SAMs which is accounted

for the lowest number of adherent cells in vitro and in vivo, suggesting a relationship

between complement and inflammation (Kalltorp et al., 1999, Barbosa et al., 2003,

2010). Unexpectedly, a noticeably enhanced leukocyte adhesion was seen on 83% –

COOH/17% –CH3 SAMs than either –COOH or –CH3 SAMs alone but no difference

in leukocyte activation was found in vitro (Sperling et al., 2009, Fischer et al., 2010b,

2010a). These studies indicate the surface functionalities modify specific leukocyte

response around implants.

2.5.2.2 Functional Groups from Copolymer Surfaces

Besides using SAMs, an alternative way to generate specific functionalities

on surfaces is by copolymerisation. It is a process of polymerising two monomers,

which contain functional groups. Polymeric materials have been widely generated for

tissue engineering applications.

In synthesis of poly (alkyl methacrylates), Berglin et al. (2004, 2009)

suggested that the chain length of alkyl group (i.e. number of carbons in the alkyl

side chain: 4, 6, 12, 18) had a major influence on the rate of thrombin generation

from coagulation, fibrin deposition and complement activation. A decrease in alkyl

chain length was shown to reduce the rates of thrombin generation and fibrin

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deposition, but increase complement activity. Thus, monomer alkyl methacrylate

with different alkyl chain length: methyl (–CH3), ethyl (–CH2CH3) or butyl (–

(CH2)3CH3)) may modulate activation of coagulation and complement system.

On the other hand, acrylic acid (AA) which contains –COOH group could

be an another desired monomer. A number of studies on graft polymerisation on

various polymers employed acrylic acid to develop functional interfaces for

immobilising biomolecules (Gupta et al., 2001, Alexander et al., 2004, Monien et al.,

2005, Huang and Jang, 2009). Most importantly, AA has been shown to interact with

methyl methacrylate and produce non-toxic copolymers (Yan and Gemeinhart,

2005). Hence, copolymerisation of acrylic acid and alkyl methacrylates may provide

a solid polymeric surface with tailored chemical functionality for a systematic study

in contrast to the model SAMs on gold substrates. However, very little has been

reported on modifying copoly (AA-co-alkyl methacrylate) compositions.

For copolymerisation of two monomers M1 and M2, the reactivity of a

monomer with one another (defined as relative reactivity ratio) and the monomer

concentrations are important. Four propagation reactions are possible with the

assumption that the propagation is dependent on the nature of the monomer at the

growing chain end (Odian, 2004) (Figure 2-18).

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Figure 2-18. Copolymerisation of two monomers M1 and M2. One propagating chain with M1 at the end and the other with M2. The asterisk represents the propagating species such as a radical. k11 and k22 are rate constants of self-propagation; k12 and k21 are rate constants of cross-propagation.

Monomer relative reactivity ratios r1 and r2 are determined as ratios of rate

constants (Ekpenyong, 1985):

The tendency of two monomers to copolymerise is determined by r values

while the type of copolymerisation are classified according to the products r1r2.

When both values of r1 and r2 are zero, it indicates that neither monomer is capable

of adding its own monomer, and propagation results in an alternating copolymer. If

both r1 and r2 values equal to one, it indicates that each monomer shows the same

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preference for one of the monomers, and the sequence of propagating chain is

completely random, resulting in ideal copolymerisation (r1 r2 = 1) and copolymer

composition is the same as the monomer feed (Odian, 2004). When either r values

greatly more than one, homopolymers or block copolymers are obtained.

During copolymerisation, monomers disappear by incorporating into

growing chains. Thus, the rates of disappearance of the two monomers are

synonymous with their rates of entry into the copolymer. The copolymer

composition is given by copolymerisation equation (Ekpenyong, 1985):

where [m1] and [m2] are molar concentrations of monomer M1 and M2 in

copolymer, respectively. [M1] and [M2] are their corresponding concentrations in the

monomer mixture prior to polymerisation.

Hence, different monomer relative ratios and monomer feed concentrations

influence the kinetic of copolymerisation and resultant copolymer composition. This

might result in a complex mixture of random or homopolymers in copolymers,

modulating the bulk and surface properties of the copolymers (Hermitte et al., 2004)

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Taken together, most studies on SAMs target to improve blood

compatibility (i.e. non-thrombogenic and non-immunogenic properties) of blood

contacting devices such as cardiovascular implants, catheters and extracorporeal

circulation. We believe that a blood clot formed on bone implants might constitute a

beneficial microenvironment in bone healing applications, which is currently

overlooked. An understanding of the bioactivities of surface functionalities informs a

rational strategy for developing “prothrombogenic” and non-immunogenic surfaces

on artificial bone implants. The combined use of the copolymer functionalised

surfaces (i.e. –COOH/–CH3, –CH2CH3 or –(CH2)3CH3) at different compositions

may provide a mean of synergistic modulation of the degree of coagulation and

related structure and properties of fibrin clot. In addition, diminishing the immune

response via reduced complement activation may also improve the bone healing

capability of artificial implants.

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3Chapter 3: Synthesis and Characterisation

of Material-Coated Surfaces

3.1 INTRODUCTION

Modification of surface properties is one of the approaches to enhance the

bone healing and in vivo integration of artificial bone substitutes (Ma et al., 2007).

Oral implant with surface roughness of about 1.5 µm Sa value 4 were shown to result

in a stronger bone ingrowth and osteointegration than smoother (< 0.5 µm) and

rougher ( > 2 µm) surfaces (Cooper, 2000, Shalabi et al., 2006, Le Guehennec et al.,

2007, Wennerberg and Albrektsson, 2009). An increase in surface hydrophobicity

was also found to decrease density and spreading of osteoblasts (Lin and Lin-Gibson,

2009, Wei et al., 2009).

Although these studies reported that surface properties affect osteoblast

behaviour and osteointegration, few studies have explored early blood/endosseous

implant interactions and resulting blood clot formation, even if these events occur

prior to bone formation (Hong et al., 2001, Thor et al., 2007).

The effect of surface chemistry on blood-biomaterial interactions has been

intensively studied in developing cardiovascular devices. SAMs displaying 47% –

COOH/53%–CH3 chemical functionalities were shown to increase platelet

interactions and coagulation activation leading to a strong fibrin fibre deposition,

4 Sa value is the arithmetic mean deviation of a surface

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with decreased leukocyte accumulation and complement initiation when compared

with pure –COOH and –CH3 SAMs (Sperling et al., 2005b). In addition, polymer

particles presenting –COOH/–CH3 groups at different ratios, and poly (alkyl

methacrylates) with various alkyl chain lengths were also found to modulate

complement activation, the rates of thrombin generation and fibrin deposition

(Berglin et al., 2004, 2009, Salvador-Morales et al., 2009). These findings highlight

the possible roles of –COOH/–CH3 (methyl), –CH2CH3 (ethyl) or –(CH2)3CH3

(butyl) in the design of a prothrombogenic and immunocompatible polymer surface

for synthetic bone implants.

Methacrylic and acrylic polymers have been long used in medical

applications. Of poly (alkyl methacrylates), poly (methyl methacrylate) (PMMA) is

widely used as a bone cement, dental filling and in intraocular lenses (Lee et al.,

2007). Polymers containing ethyl (EMA) or butyl methacrylate (BMA) have also

been shown to be capable of modulating chondrocyte and osteoblast attachment

(Hutcheon et al., 2001), and induce angiogenesis respectively (Butler and Sefton,

2007). Furthermore, Yan and Gemeinhart (2005) used copolymer microparticles

composed of methyl methacrylate (MMA) and acrylic acid (AA) as a drug delivery

system and confirmed that the copolymer was non-toxic both in vitro and in vivo.

Hence, AA which possess hydrophilic negatively charged –COOH groups provides

an opportunity to present the surface functional groups of our interest together with

hydrophobic non-ionic alkyl methacrylates at different ratios.

In this part of our study, we aim: (1) To synthesise a series of materials by

varying the monomer ratios of AA/MMA, EMA or BMA; (2) To coat materials on

the inner surface of a customised incubation chamber; and (3) To analyse the surface

compositions of functionalities –COOH/–CH3, –CH2CH3 or –(CH2)3CH3,

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hydrophobicity and roughness on coatings. Such knowledge would allow a priori

prediction of physiochemical or even possibly prothrombogenic and non-

immunogenic properties directly from the material formulation.

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3.2 MATERIALS

Acrylic acid (AA), methyl, ethyl and butyl methacrylates were all purchased

from Sigma-Aldrich (New South Wales, Australia) and used as received. Benzoyl

peroxide (BPO; Luperox®, Sigma-Aldrich, Australia), which contained 25% water,

was purified by dissolving in chloroform and recovered by precipitation in excess

methanol. The precipitated BPO was filtered and dried under vacuum before use.

3.3 METHODS

3.3.1 Synthesis of materials

Acrylic acid and alkyl methacrylate were reacted via free-radical

polymerisation using BPO as an initiator. Three types of alkyl methacrylates were

employed: methyl methacrylates (MMA), ethyl methacrylates (EMA) and butyl

methacrylates (BMA). The monomer solutions were added at molar ratios (AA: alkyl

methacrylate; 45, 55 or 65 %) in a glass vial containing 0.5% of BPO. The solutions

were deoxygenated by bubbling argon gas with a syringe through the septum caps on

the vials. The vials were then incubated in an oil bath with increasing temperatures

(45˚C, 55˚C, 65˚C, 75˚C, 85˚C) for 1 h intervals, and at 90 ˚C and 100˚C for 20 min

intervals. Figure 3-1 illustrates the free-radical polymerisation initiated by BPO and

the chemical structures of the comonomers: AA, MMA, EMA and BMA.

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Figure 3-1. Free-radical polymerisation is initiated by benzoyl peroxide, which generates free radicals by heat. Chemical structures of comonomers: acrylic acid, methyl, ethyl and butyl methacrylates were shown with the changes in alkyl chain length highlighted in red boxes.

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3.3.2 Preparation of surface coatings

The impact of the surface functionalities and their relative ratios on blood

responses was studied by incubation vials with surface coatings. The materials

prepared as described above were dissolved in acetone to give a 5 % (w/v) solution.

The solutions were coated on the internal surface of glass vials (4 mL, 15x45 mm;

Waters, Australia) by a solvent-evaporation technique (Fujishita et al., 2009).

Solutions of 1.5 mL were added to the vials and dried in an oven at 50 ˚C until

complete evaporation of acetone. The procedure was repeated three times to ensure

full coverage on the glass surfaces. After drying, the coated vials were capped at

room temperature until used.

For water contact angle measurement, glass coverslips (No. 1, diameter 13

mm, ProSciTech, Australia) were used to provide a flat surface. Coverslips were

placed at the bottom of glass vials before the addition of solution and drying step.

Uncoated glass vials or coverslips were used as controls.

3.3.3 Characterisation of surface

To analyse the surface properties of the coating, the coated vials or

coverslips were cut manually. Macroscopic contamination or dust on the coated

surfaces were removed by purging with argon. Samples were stored in clean sealed

containers until analysis.

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3.3.3.1 X ray photoelectron spectroscopy

The functionalization of coatings was analysed by X-ray photoelectron

spectroscopy (XPS) (Centre for Microscopy and Microanalysis, University of

Queensland). XPS is a surface analysis technique to determine the composition and

chemical state of elements that exist in a material. A spectrometer (Axis ULTRA Kratos

Analytical, Shimadzu, UK) equipped with a monochromatic Al Kα X-rays (1486.6eV)

was operated at 150 W and incident at 45 ⁰ to the sample surface. Photoelectrons emitted

from the surface were collected at a take-off angle of theta 90 ⁰ with a 165 mm

hemispherical electron energy analyser. Elements present in the surface were identified

by survey scans taken at pass energy of 160 eV and at resolution of 1.0 eV. These scans

recorded binding energies of the photoelectrons ranging from 0 - 1200 eV. Multiplex

scans at pass energy of 20 eV and at a higher resolution of 0.05 eV were also performed

to determine the chemical states of carbon atoms. The base pressure in the chamber was

1.0 x 10-8 torr during analysis.

The carbon 1s (C1s) high resolution spectra were processed to determine the

relative oxidation states of carbon atoms. Curve fitting of the spectra was performed

using the Casa XPS software (version 2.3.14) and a linear baseline with Kratos library

Relative Sensitivity Factors (RSFs). The binding energies that are indicated by peaks in

spectra were referenced to the C1s aliphatic carbon peak at 285.0 eV (Takemoto et al.,

2004). This corrected the effect of surface charging during analysis on shifting the peak

positions. Peak areas were normalised and adjusted to obtain a full width at half

maximum between 0.9 and 1.1 eV. By determining the area ratios of carbonyl (C-O)

component derived from methacrylate monomer, and carboxyl (O-C=O) component

derived from both types of comonomers, the compositions of corresponding surface

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functionalities were obtained. Copoly (AA-co-MMA) is shown as an example (Figure 3-

2 a) and the surface ratio of –COOH groups was calculated by using the equation shown

in Figure 3-2 b. Average data were collected from three measurements of each surface.

Figure 3-2. Copolymer surfaces displaying various functional groups. a) Schematic diagram of structure of copoly (AA-co-MMA) illustrates the relative ratios of carbon components C-O (in red box) and O-C=O (in blue circle) which were quantified to determine the compositions of comonomers and corresponding surface functional groups. b) Equation used to calculate the surface ratio of –COOH groups.

3.3.3.2 Water contact angle measurement

Surface hydrophobicity of coatings were assessed by measuring advancing

water contact angle using a FTÅ 200 system (First Ten Ångstroms, Poly-Instruments

Pty. Ltd., Australia) (Figure 3-3 a). Glass coverslips with surface coatings were

employed. After placing the samples on the stage of the goniometer in an environmental

chamber, a volume of degassed deionised water (MiliQ quality) was suspended from the

tip of a microliter syringe. Samples were gently lifted up until the surfaces made contact

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with the droplet. The needle was kept in contact with the droplet and continued

dispensing. Advancing contact angles were achieved when the volume of the droplet

increased to its maximum before spreading across the surfaces. Images were captured

with a horizontal CCD video camera and contact angles were measured from the images

using drop shape analysis software (Fta32 version 2.0) (Figure 3-3 b). Data were the

average of at least six regions of each surface.

Figure 3-3. Water contact angle measurement. a) FTÅ 200 water contact angle goniometer with a horizontal camera. b) The contact angle is the angle at which a liquid/vapour interface meets the solid surface. b) Adapted from http://superhydrophobiccoating.com

3.3.3.3 Scanning electron microscopy

Scanning electron microscopy (SEM) was used to examine the surface

morphology of coatings on glass vials. Specimens were mounted on aluminium stubs,

gold-coated and examined with a Quanta 200 scanning electron microscope (FEI, USA).

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Several fields on each surface were imaged and a representative field was chosen (at a

magnification of 25000 x).

3.3.3.4 Atomic force microscopy

The roughness of surface coating was measured by atomic force microscopy

(AFM) using a Solver P47 Pro scanning probe microscope (NT-MDT Co., Russia).

Sample surfaces were scanned in contact mode (constant force) with a golden silicon

probe (CSG 11 No. 2 rectangular, NT-MDT Co.). Scans size of 5 x 5 μm2 were

recorded under ambient laboratory conditions. Average surface roughness was then

measured from the AFM images using NT-MDT Image Analysis software (version

2.2.0). Data was presented as mean of at least six regions of each surface.

3.3.4 Statistical analysis

Data from the experiments were expressed as the mean values ± standard

derivation. Analysis was performed using SigmaPlot (version 11.0; Systat software

Inc). For multiple comparisons, one-way analysis of variance (ANOVA) was used

with the Holm-Sidak’s test. The significance level was set at p ≤ 0.05.

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3.4 RESULTS

3.4.1 Surface coating on incubation vials

As shown in Figure 3-4, material blocks synthesised from polymerisation were

used to form a translucent coating on the vials. By changing the comonomers and their

molar fractions in the polymerisation, the surface coatings on incubation vials can

display different surface functional groups: –COOH and –CH3, –CH2CH3 or –

(CH2)3CH3. In the present study, the coatings containing homopolymers of acrylic acid,

methyl, ethyl, butyl methacrylates are referred to as PAA, PMMA, PEMA, and PBMA,

respectively, while those containing both acrylic acid and methyl/ethyl/butyl

methacrylates are named according to the mole fractions of acrylic acid (45, 55, 65%),

for example 45% AA/ 55% MMA as 45MMA etc.

Figure 3-4. a) Materials formed from free-radical polymerisation. b) An incubation vial treated with material solution resulted in a clear coating.

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3.4.2 XPS analysis of material-coated surfaces

To confirm the presence of coating on the inner surfaces of glass vials and

to determine the surface compositions of functional groups on the coating, XPS

analysis was performed on uncoated and coated surfaces. A broader range of AA

mole fractions (0.0 to 0.65) were utilised to validate the gradual change in surface

content of –COOH groups.

Figure 3-5 compares XPS survey spectra of the uncoated glass surfaces and

material-coated surfaces, using PAA, PBMA and 45BMA as examples. As expected,

all coated surfaces showed two peaks of element C1s (285.0 eV) and O1s (532.0 eV)

(Figure 3-5 b-d). The oxygen peak was derived from the ester linkage. For PBMA

and 45BMA, the carbon peak increased due to the presence of methyl groups bonded

to the carbon backbone and the terminating –COO((CH2)3CH3) group compared to

that from –COOH group of PAA. This indicated successful surface modification on

the uncoated substrate by these coatings. Detected Si on the spectrum of PAA

reflected trace silicone contaminants.

XPS C1s spectra, which demonstrate different oxidation states of carbon,

were used to investigate the changes in chemical functionalities at surfaces after

coating. Alkyl methacrylates differed from acrylic acid by a prominent peak

corresponding to carbon component C-O (286.8 eV), whereas both comonomers

showed three common components: C-C (285.0 eV), C-COO (285.7 eV), and O-

C=O (289.1 eV) (Figure 3-6 a, b, d, f) (Chuang and Lin, 2007, Minelli et al., 2008).

This sole peak indicated that carbonyl-containing alkyl functional groups of alkyl

methacrylates displayed on the coated surfaces. A minor peak at around that of C-O

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was observed at PAA coated surface which may be due to the contamination. Coated

surfaces showed all four components, as illustrated by spectra of 45MMA, 45EMA

and 45BMA (Figure 3-6 c, e, g). The relative compositions of C-O and O-C=O on

various surface coatings were measured from the peak areas. The surface ratios of –

COOH/–CH3, –CH2CH3 or –(CH2)3CH3 are summarised in Table 3-1.

Overall, the proportion of –COOH group presented on surface coating

(XCOOH coating) was lower than the proportion of AA monomer fed in the material

(XCOOH material). The relationships between XCOOH coating and XCOOH material are

presented in Figure 3-7. As clearly demonstrated by BMA surfaces, the surface

coatings became increasingly richer in –COOH group (i.e. increased XCOOH coating)

with the increase in the AA monomer feed (i.e. increased XCOOH material).

Interestingly, XCOOH material of 0.45 and 0.55 at MMA and EMA surfaces showed

similar XCOOH coating concentrations (i.e. 0.32 – 0.34). Also, XCOOH coating at

MMA, EMA and BMA surfaces were found to be comparable at XCOOH material of

0.55 (0.33, 0.32 &0.34 respectively), and of 0.65 (0.41, 0. 39 & 0.41 respectively)

(Table 3-1).

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Figure 3-5. XPS survey spectra of a) uncoated glass, b) PAA, c) PBMA, and d) 45BMA (45% AA/BMA) coated surfaces.

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Figure 3-6. XPS C1s spectra of a) PAA, b) PMMA, d) PEMA, f) PBMA, and c) 45MMA, e) 45EMA, and g) 45BMA coated surfaces.

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Table 3-1. Ratio of –COOH groups measured on surface coatings (XCOOH coating) compared to mole fraction of –COOH group-containing AA (XCOOH material) composed

with MMA, EMA or BMA. Data were presented as the average value of three

measurements.

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Figure 3-7. Ratio of –COOH groups on the surface coating as a function of mole fraction of –COOH group-containing AA composed with a) MMA, b) EMA or c) BMA.

3.4.3 Surface hydrophobicity

The advancing contact angle of an uncoated glass substrate was 54.1 ± 4.2 º.

With addition of a coating, the values increased to 77.6 ± 3.1 º, 82.9 ± 1.0 º and 91.8

± 4.4 º for PMMA, PEMA and PBMA, respectively (Table 3-2). These results were

in agreement with published data (Takemoto et al., 2004). The contact angle values

of homopolymer-derived coatings increased significantly in the order: –CH3 < –

CH2CH3 < –(CH2)3CH3, suggesting the surface hydrophobicity increased with the

alkyl chain length (p ≤ 0.001).

Similarly, among coated surfaces displaying –COOH/–CH3, –CH2CH3 or –

(CH2)3CH3 functionalities at the same –COOH ratios, a significantly higher contact

angle was observed on surfaces with –(CH2)3CH3 groups compared to those with –

CH3 and –CH2CH3 groups (i.e. At an average 33% –COOH from XCOOH material of

0.55, p=0.005; At an average 40% –COOH from XCOOH material of 0.65, p ≤ 0.001).

This further indicated that surface hydrophobicity was strongly influenced by the

more hydrophobic –(CH2)3CH3 groups. No significant differences in the contact

angles were noted between surfaces with –CH3 and –CH2CH3 groups at both values

of XCOOH material. This suggested that the differences in chain length and

hydrophobic property of –CH3 and –CH2CH3 groups were less likely to affect

surface hydrophobicity in the presence of –COOH groups, unlike on homopolymer-

derived surfaces.

On the other hand, an increase in –COOH ratio (0% to 41%) on –COOH/–

(CH2)3CH3 surfaces led to a decrease in contact angle, suggesting the contact angle

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correlated well with the –COOH ratio (Figure 3-8). This correlation was also

observed on MMA and EMA surfaces in which the relatively similar –COOH ratios

at XCOOH material of 0.45 and 0.55 resulted in similar contact angles. Overall, these

results indicated that the surface functional groups and their compositions had a

combined effect on surface hydrophobicity.

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Table 3-2. Advancing contact angles of surfaces coated with materials composed of varied

mole fraction of acrylic acid and alkyl methacrylates. Measurements were reported as the

average value of contact angles of at least six data points.

Figure 3-8. Advancing contact angles of different surface coatings.

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3.4.4 Surface morphology and roughness

SEM showed that all coated surfaces exhibited as smooth as the uncoated

surfaces (Figure 3-9 a-c).

The smoothness of coatings was further analysed by AFM. The topography

images demonstrated that all coated surfaces had microscopic concave–convex

structures when compared to irregular protrusions on uncoated glass surfaces (Figure

3-10). These surface features were quantitatively verified by analysing the average

roughness (Table 3-3). The average roughness of coated surfaces was 3.99 ± 0.54

nm. No significant differences were found in the average roughness among surfaces

with similar –COOH ratios (i.e. at XCOOH material of 0.55; p = 0.334 or 0.65; p =

0.775) nor among surfaces with the same functionalities but different –COOH ratios

(i.e. –COOH/–(CH2)3CH3 surfaces; p = 0.066). This indicated that the type of

surface functional groups and their relative ratios did not affect the surface

roughness. Indeed, the roughness values of the coated surfaces were higher than that

of uncoated glass surfaces (2.22 ± 0.29 nm), suggesting the application of coatings

did create a small increase in surface roughness.

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Figure 3-9. Representative SEM images of a) uncoated glass, b) PEMA and c) 45 EMA coated surfaces, taken at magnification of 25000 x.

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Figure 3-10. AFM images (5 µm x 5 µm areas) of uncoated and coated surfaces.

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Table 3-3. Average surface roughness measured by AFM. Values are the means of 6 measurements ± SD.

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3.5 DISCUSSION

In this chapter, we developed a series of materials (AA-co-

MMA/EMA/BMA) at varied ratios and coated them on the inner surfaces of

incubation vials. The fabrication of a stable coating onto incubation vials was

achieved by consecutive additions of material solutions (Fujishita et al., 2009). Such

a surface coating technique is a common and efficient way to modify surfaces of

implants having complex geometries without altering the bulk properties for a

specific application (Ma et al., 2007, Werner et al., 2007). By using this model, the

surfaces should exhibit distinct surface chemistry with compositional variation of –

COOH/–CH3, –CH2CH3 or –(CH2)3CH3 functionalities on the coated surfaces.

Chemical composition of surface coatings

Using XPS analysis, it was confirmed that glass substrates were successfully

coated with materials of differing compositions. This was done by identification of the

elements and an additional peak representing C–O binding, appearing on the surfaces

after an alkyl methacrylate was composed with AA. Using this C–O peak contributed

solely from alkyl methacrylates, we quantified the relative compositions of the

comonomers and corresponding functional groups exposed on the surfaces. XPS is a

more sensitive approach in determining surface density of –COOH groups (McArthur,

2006) compared to colorimetric methods using dyes such as Rhodamine 6G (Kang et al.,

1993), toludine blue or thionin acetate (Tzoneva et al., 2008), which are based on an ion

exchange mechanism and the assumption that –COOH groups on surface are able to

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bind equal molar amounts of dye molecules. Since the binding energy differences

between C-O and O-C=O can be resolved clearly by XPS for quantifying the

concentrations of carboxyl and alkyl functionalities, chemical derivatisation in which a

functional group is tagged with an unique element prior to XPS analysis, in this case

trifluoroethanol for –COOH groups, is not necessary (Alexander et al., 2004).

From our XPS results, the surface coating generally had an increase in –COOH

group with increasing AA monomer fed in materials. However, it was found that the

surface –COOH ratio was lower than the expected AA mole fraction. This lower yield of

–COOH groups on the surface is probably due to three factors: the degree of

copolymerisation, polymer chain mobility, and functional group reorientation.

During free-radical copolymerisation, some comonomers may not incorporate

successfully into the propagating polymer chains due to hindered accessibility to the

primary radicals, or undergo homopolymerisation due to the differences in monomer

reactivity (Gupta et al., 2001, Li et al., 2005). It has been shown that in the bulk

copolymerisation of AA and MMA the monomer reactivity ratios are r1= 1.51 and r2 =

0.48, respectively. This indicates that AA tends to form homopolymers subunits in the

resultant copolymers. This homopolymerisation occurs at low conversion at low

temperature (50 ˚C) (Ekpenyong, 1985, Odian, 2004, El-Newehy et al., 2010). As such,

we performed two additional heating steps at 90 ˚C and 100 ˚C to ensure complete

curing of polymerisation to reduce comonomer residues. Hence, this factor is less likely

the primary contributor to a reduced amount of surface –COOH groups.

Similar to our findings, a lower yield of PAA on the surface after grafting AA

onto poly(ethylene terephthalate) films has also been reported by Gupta et al.(2002). It

has been suggested that the PAA chain underwent rearrangement, resulting in chain

distribution not only on the surface but also in the subsurface layer, which was beyond

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the normal sampling depth (<10 nm) of XPS. Such polymer chain flexibility has been

observed on surfaces prepared from PBMA solutions (Berglin et al., 2009, Xu et al.,

2009). In line with these findings, Berglin et al. (2004) identified that the mobility of

poly(alkyl methacrylates) chains was increased systematically with an increase in alkyl

chain length. Moreover, it has been shown that hydrophobic functional groups on

surfaces tend to expose to air whereas hydrophilic ones behave vice versa to minimize

surface energy (Hermitte et al., 2004, Ozcan and Hasirci, 2007, Michiardi et al., 2007).

Therefore, the general lower surface –COOH ratio in regard of the AA mole fraction in

our study is possibly due to the reorientation of hydrophobic alkyl groups to dominate

outmost surface while the hydrophilic carboxyl groups are buried.

Materials composed of BMA contain longer and more hydrophobic side chains

leading to an enhanced polymer chain mobility. This may explain a linear increase in

surface –COOH ratio with increasing AA mole fraction found on these surfaces. In

contrast, the hydrophilic –COOH groups on MMA and EMA surfaces will be less likely

able to orient away from air due to reduction in polymer chain mobility and accessibility

of surface groups reorientation mediated by the shorter and less hydrophobic –CH3 and

–CH2CH3, respectively. This may partly elucidate the higher concentration of –COOH

groups found on MMA and EMA surfaces at a lower AA mole fractions when compared

with BMA surfaces.

Surface hydrophobicity of coatings

Surface hydrophobicity of implant plays an important role in mediating protein

adsorption and cell responses (Arima and Iwata, 2007, Wei et al., 2009, Menzies and

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Jones, 2010). By measuring advancing contact angle, we demonstrated that surface

hydrophobicity of coatings correlates well with the chemical compositions determined

by XPS, a dependence as other papers reported (Tsyganov et al., 2005, Ukiwe et al.,

2005, Lai et al., 2006). Using homopolymeric surfaces with a single functionality, we

found that the contact angle increased with an increase in carbon number in the side

chain: –CH3 < –CH2CH3 < –(CH2)3CH3, indicating a direct correlation of alkyl chain

length and hydrophobicity properties as has been indicated by other studies (Van

Damme et al., 1986, Wulf et al., 2000, Berglin et al., 2004, Hermitte et al., 2004).

Similarly, advancing contact angles on surfaces with two functionalities clearly

demonstrated that surface hydrophobicity was dependent on both the type of functional

groups and their compositions (Tsai et al., 2007). As expected, water contact angle was

higher on surfaces with –(CH2)3CH3 groups than those with –CH3 and –CH2CH3

groups at relatively the same –COOH ratios (at AA mole fraction of 0.55 and 0.65). The

contact angle of the surfaces with –CH3 and –CH2CH3 groups did not differ

significantly. This suggests that the difference in one-carbon length between –CH3 and –

CH2CH3 groups has limited impact on modulating surface hydrophobicity in the

presence of –COOH groups. In contrast, an increase in –COOH ratios on –COOH/–

(CH2)3CH3 surfaces decreased water contact angle. As such, it is reasonable to deduce

that the nature of functionalities and their relative compositions are the key factors in

controlling surface hydrophobicity.

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Surface morphology and roughness of coatings

Coated surfaces were examined by SEM and AFM in an attempt to characterise

surface morphology of coating and determine any relationship between surface chemical

composition or surface hydrophobicity and surface roughness. Our SEM observation on

coated surfaces showed no difference in morphology compared to uncoated surfaces.

However, the surface topography of coated surfaces as examined by AFM underwent

prominent changes as a result of the coating process. Characteristic concave–convex

structures were observed. This may be related to the relative of alkyl methacrylates in

materials with AA, in acetone which determined the evaporation rate of solvent during

coating process. We observed a decrease in material solubility with increasing AA mole

fraction and alkyl chain length. This might be explained by different monomer reactivity

ratios which leads to different copolymer composition and subsequent solubility. Such a

difference in solubility and resultant surface structure has also been reported in mixed

SAM prepared with alkanethiols dissolved in ethanol solution (Chuang and Lin, 2007,

Tsai et al., 2007). It was suggested that the solubility was governed by specific

interactions, such as hydrogen bonds, between terminal functional groups of solute and

solvent, or among the solutes (Xu et al., 2011). In this case, the longer chain length of –

(CH2)3CH3 groups may reduce the evaporation rate of the solvent and produce less

pores on surfaces by strongly interacting with –COOH groups from AA or acetone.

Whereas the shorter length of –CH3 or –CH2CH3 group may give rise to more tiny pores

due to faster evaporation of solvent due to weaker bonds with surrounding components.

To verify these surface structures quantitatively and correlate the surface

topography in relation to surface chemical composition and hydrophobicity, we

measured the average roughness of these coated surfaces. The roughness data showed

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that there was no nano-scale difference in roughness for surfaces with different alkyl

functional groups at the same –COOH ratios (at AA mole fraction of 0.55 and 0.65).

Also, the surface roughness did not differ significantly with an increase in surface –

COOH ratio, as illustrated by BMA surfaces. These results indicate that there is no

evident relationship between surface roughness and surface functional groups and their

compositions. Furthermore, no cracks or other surface imperfections were found.

Consequently, differences in blood response should not be a result of different surface

morphology and roughness between coatings.

The knowledge generated herein would allow a prior prediction of the surface

properties directly from the material formulation. Further studies of whole blood

response on these materials could yield to phenomenological links between the

biocompatibility and formulation.

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3.6 CONCLUSION

In this chapter, we established an incubation model with inner surface

coated with a series of materials consisting of AA and MMA, EMA or BMA at

different ratios. It was demonstrated that the coated surfaces displayed different

contents of carboxyl and alkyl groups and that –COOH ratios were lower than the

AA mole fraction fed in materials. The properties of surface functional groups and

their relative compositions correlated well with the surface hydrophobicity. It

increased with increasing alkyl chain length: –CH3 < –CH2CH3 < –(CH2)3CH3, and

decreased gradually with increasing –COOH groups, suggesting a combined effect of

surface functional groups and their compositions on surface hydrophobicity. No

significant differences in surface hydrophobicity were found on surfaces with –CH3

and –CH2CH3 groups in the presence of –COOH groups. The coating appeared

relative smooth and the surface average roughness was 3.99 ± 0.54 nm, which is

slightly higher than that of uncoated glass surfaces (2.22 ± 0.29 nm). However, we

did not detect significant difference among surfaces with same functionalities at

different –COOH ratios nor among surfaces with different alkyl groups but the same

–COOH ratios, suggesting the surface chemistry did not influence the surface

roughness. Overall, surface functional groups and their relative compositions have a

combined effect on modulating surface chemistry and hydrophobicity on coatings.

The similarity concerning surface roughness may enable a correlation of blood

response and clot formation with the parameters related to the surface chemistry and

hydrophobicity.

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4Chapter 4: The Influence of Carboxyl and

Alkyl Functional Groups and Their Relative

Compositions on Blood-Biomaterial

Interactions and Clot Properties

4.1 INTRODUCTION

Activation of blood coagulation occurs rapidly when whole blood makes

contact with the surface of a synthetic implant. The adsorption of plasma proteins is

believed to initiate platelet reactions and coagulation activation, leading to the

generation of thrombin and fibrin, and ultimately blood clot formation on the implant

surface (Horbett, 1993, Eskin et al., 2004, Gorbet and Sefton, 2004). Although a

peri-implant clot is regarded to be detrimental to the function of cardiovascular

devices (Knetsch, 2008), it may be beneficial for bone repair when the peri-implant

clot has biological and structural properties similar to those of the haematoma

formed on injured bone during normal healing (Tsiridis et al., 2006, Tosounidis et

al., 2009). Moreover, an important concern of the blood-biomaterial interactions is

the activation of the complement pathway and its potential to initiate chronic

inflammation or foreign body reaction (FBR). These adverse reactions may cause the

deterioration of the implanted biomaterial and secondary injury to surrounding tissue

(Tsai, 2004, Anderson et al., 2008).

It is well known that surface chemical functionalities and their relative ratios

on biomaterials affect protein adsorption which in turn affects the activation of

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coagulation and complement cascades (Sperling et al., 2005a, Rodrigues et al., 2006,

Tsai et al., 2007, Barbosa et al., 2010, Fischer et al., 2010b). Using fibrinogen

solution, it was shown that surface functional groups presented on self-assembled

monolayers (SAMs) markedly influenced the extent of thrombin-catalysed fibrin

polymerisation of adsorbed fibrinogen (Evans-Nguyen and Schoenfisch, 2005b,

Evans-Nguyen et al., 2005b). Moreover, other studies investigated the impact of

thrombin concentration on fibrin structure and showed that a lower thrombin

concentration increased the fibre thickness; but decreased the fibre density (Wolberg,

2007a, Wolberg and Campbell, 2008). However, these studies have mainly used

SAMs on flat surfaces and isolated fractions of blood e.g. protein solution, plasma or

single cell culture, which limit the understanding of the complex response of whole

blood in three dimensions. In addition, the influence that the surface functionalities

and their relative ratios have on the ultimate structural properties of a whole blood

clot has not previously been taken into consideration.

In fact, alterations in fibrin structure of blood clots were shown to correlate

with differing clot elasticity and susceptibility to lysis (Collet et al., 2000, 2005, Liu

et al., 2006), and are believed to cause increased risks of bleeding or thrombosis

(Mills et al., 2002, Bhasin et al., 2008, Undas et al., 2009). Theoretically, these

factors could also affect the release of growth factors from the peri-implant clots,

alter the early stage of healing and consequently the extent of new bone formation at

the implant site (Laurens et al., 2006).

To systematically investigate both the surface chemistry-activated cascade

events and the ultimate clot structural properties upon whole blood contact in three-

dimensions, we developed an in vitro incubation vial where the inner surface was

coated with materials of AA/MMA, EMA or BMA at varied ratios. In this chapter,

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we investigated the effect of binary mixture of –COOH/–CH3, –CH2CH3 or –

(CH2)3CH3 surface functionalities and their relative compositions on coagulation

and complement activation as well as alterations in resultant clot structure, elastic

properties, susceptibility to fibrinolysis and the release of PDGF-AB and TGF- β.

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4.2 MATERIALS & METHODS

4.2.1 Blood sampling and in vitro incubation

Whole blood was collected from a healthy volunteer who was not on any

medication for at least 10 days and with no history of coagulation disorders.

Venipuncture was performed by a phlebotomist. Venous blood was drawn into

syringes with a 19-gauge needle and immediately transferred (1.5 mL) to the vials

and incubated at 37 ˚C. After the desired incubation time, all blood contents in the

vials were collected and processed for following experiments. This procedure was

approved by the Human Ethics Committee of the Queensland University of

Technology. Informed consent was also obtained from donor prior to blood

collection.

4.2.2 In vitro coagulation activation

Activation of the coagulation cascade leads to the conversion of

prothrombin into active thrombin, a process accompanied with the production of

prothrombin fragment 1+2 (F1+2). To assess in vitro coagulation activation on the

coated surfaces, prothrombin F1+2 was analysed using an enzyme-linked

immunosorbent assay (ELISA, Enzygnost F1+2; Dade Behring Marburg GmbH,

Germany). Based on our preliminary studies, the coagulation was analysed after 30

min of whole blood incubation as the formation of F1+2 demonstrated well

established differences among surfaces.

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Serum was isolated by transferring samples from incubation vials to

microcentrifuge tubes containing tri-sodium citrate (0.11 mol/L) (one part of tri-

sodium citrate with 9 parts of blood sample) and centrifuged according to the

manufacturer’s protocols. Briefly, the enzyme immunoassay was based on the

sandwich principle in which the monoclonal antibodies specific to F1+2 antigens

were coated on a 96-well plate. Standards and samples were added and any F1+2

antigens present were bound to the immobilised antibodies. After unbound

substances were rinsed away, peroxidise-conjugated antibodies to human

prothrombin were added to bind to F1+2 determinants, producing an antibody-

antigen-antibody “sandwich”. After a second wash, a substrate solution for

peroxidase was added. The enzymatic reaction between hydrogen peroxidase and its

substrate produced a blue colour in direct proportion to the amount of F1+2 present

in the sample. The reaction was terminated by a stop solution as indicated by a colour

change of blue to yellow. The colour intensity was determined by a

spectrophotometer (at 450nm) and quantified from a standard curve. Average data

from 6 replicates of each surface were presented. Plasma levels of F1+2 serve as a

baseline and were obtained by centrifuging blood in Vacuette test tubes containing

sodium citrate (3.5 mL blue capped, Greiner Labortechnik, Austria). All data were

obtained from 6 replicates of each surface.

4.2.3 In vitro complement activation

Initiation of the complement system leads to the formation of a common end

product C5a convertase, which cleaves complement protein C5 to C5a. C5a is

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rapidly transformed to C5a-des Arginine (C5a-desArg) by endogenous

carboxypeptidase N enzyme in plasma or serum (Janeway et al., 2005a). To

determine the extent of complement activation on material-coated surfaces, serum

C5a-desArg was quantified with Human C5a ELISA Kit II (OptEIATM; BD

Bioscience, USA). Serum collected from blood samples after 2 h incubation was

analysed as the formation of C5a-desArg were detectable and more pronounced

based on our preliminary study, which also agrees with earlier findings (Sperling et

al., 2005a). The sandwich ELISA assay was performed as previously described. A

background level of complement activation monitored as plasma level of C5a-

desArg was obtained by centrifuging blood in Vacuette test tubes containing

EDTA (4.0 mL purple capped, Greiner Labortechnik, Austria). All data were

obtained from 6 replicates of each surface.

4.2.4 Characterisation of clots formed on material-coated surfaces

4.2.4.1 Examination of clot structure

To study whether clot architecture was altered by surfaces presenting various

compositions of functional groups, the clots formed in incubation vials were examined

by SEM. After 2 h incubation, the clots were fixed with 4% paraformaldehyde (pH 7.4)

at 4 ˚C overnight. The clots were washed twice with phosphate buffered saline (PBS, pH

7.4) for 30 min, and dehydrated in grades of ethanol (50 %, 70 % and 100 %) for 1 h per

grade. A longitudinal cut was performed on the clots to allow examination of the

ultrastructures at clot/material interface and the centre of the clot. Following dehydration,

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the clots were processed through 100 % amyl acetate twice of 15 min intervals and dried

in a CO2 critical point dryer. The clots were then mounted, gold-coated and examined

with the SEM microscope. Representative images were captured.

4.2.4.2 Fibrin thickness measurement

The effect of material surfaces on the formation of fibrin in the clots was

assessed by measuring the diameter of fibrin strand from SEM images at 5000 x

magnification. The measurement was performed using Image J software (version 1.43)

according to the method of Lai et al. (2010). A transverse line was drawn perpendicular

to long axis of the fibre with clearly defined margins. The pixel value was related to that

obtained for the scale bar on the image. At least forty different fibrin strands were

measured at random fields approximate 50 µm away from the edge of clot and in the

centre of the clot. A minimum of two images were analysed for each sample. The

diameter of individual fibrin of each sample was reported as an average for all fibres

measured.

4.2.4.3 Fibrin density measurement

Quantitative fibrin network analysis was performed using Image J software

(version 1.43) using a modified method of Undas et al. (2008). A 64-field grid was

generated to cover each SEM image and at least twenty fields were selected

randomly at the edge and in the centre of the clots. The density of fibrin fibre was

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determined by counting the number of fibres per field (40 µm2) and the mean value

was presented.

4.2.4.4 Compaction study

To study the viscoelastic properties of the clots formed on material surfaces,

the clot rigidity was assessed by measuring compaction according to Undas et al.

(2009). After 2 h incubation, the clots formed in the incubation vials were transferred

to Eppendorff centrifuge tube (2.0 mL; Hamburg, Germany) and centrifuged at 6000

g for 60 seconds. The volume of fluid expelled from the network by centrifugation

was measured and expressed as a percentage of the initial volume of the clot and was

termed the compaction coefficient. All data were obtained from 6 replicates of each

surface.

4.2.4.5 Clot lysis assay

Clot lysis strongly correlated with fibrin thickness and density. The effect of

material surfaces on overall clot degradability and its relationship to the fibrin

architecture as determined above was studied. Clot lysis was evaluated by the generation

of fibrin degradation product (D-dimer) as the cross-linked clots were digested by

fibrinolytic enzymes in vitro.

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A suspended clot system modified from the protocols of Collet et al. (1993a)

was used in this study. Whole blood was incubated (1.5 mL) in the vials for 2 h at 37 ˚C

to allow complete clot formation and retraction. The clots were removed carefully from

the vials and suspended in 3 mL of PBS containing human plasminogen (Glu-

plasminogen, 5.4 μg/mL final concentration; American Diagnostica Inc., USA)

(Onundarson et al., 1992). Lysis of the clot was induced by adding recombinant tissue-

type plasminogen activator (tPA, 0.25 μg/mL final concentration; American Diagnostica

Inc., USA) at 37 ˚C with gentle agitation. This concentration of tPA was determined as

the lowest one able to induce clot lysis in preliminary study (data not shown). Aliquots

of 300 μL were removed at timed intervals and centrifuged at 1000 g for 3 min. The

supernatants were stored at -70 ˚C before analysis. The same volume of PBS was

supplemented after samplings. The extent of clot lysis was monitored by measuring the

amounts of D-dimer released from the clots using IMUCLONE D-Dimer ELISA

(American Diagnostica Inc., USA). Clots that were suspended in PBS only were used as

control of spontaneous fibrinolysis. Weight loss of clots during lysis was also traced

during the experiments (Prasad et al., 2006, Holland et al., 2008). All data was obtained

from triplicate of clots formed on each surface.

4.2.4.6 Quantification of growth factors

The release of PDGF-AB and TGF-β1 during both clot formation and clot

lysis was assayed by ELISA. After 2 h incubation, supernatant serum above the clots

was collected and the clots were directly subject to clot lysis as described previously.

Supernatant serum and buffer aliquots collected at varied intervals were centrifuged

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for 15 min at 1000 g and assayed according to the manufacturer’s instructions. All

Quantikine ELISA kits were purchased from R&D Systems (Minnesota, USA) and

the sandwich ELISA assay was performed as previously described. To detect the

circulating levels of growth factors, platelet-poor plasma was prepared by

centrifuging blood in Vacuette test tubes containing EDTA for 15 min at 1000 g

and subsequently for 10 min at 10,000g (4.0 mL purple capped, Greiner

Labortechnik, Austria). All data were obtained from triplicates of clots formed on

each surface.


Data from the experiments were expressed as the mean values ± standard

deviation. Analysis was performed using SigmaPlot (version 11.0; Systat software

Inc). The data from control and test surface were compared using the Student’s t-test.

For multiple comparisons, one-way analysis of variance (ANOVA) was used with

the Holm-Sidak’s test. The significance level was set at p ≤ 0.05.

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4.3 RESULTS & DISCUSSION

4.3.1 Surface-initiated coagulation response

To determine the effect of material surfaces on initiating coagulation cascade,

the serum level of prothrombin F1+2 after 30 min incubation was measured by ELISA.

Figure 4-1 shows F1+2 level relative to the uncoated glass surfaces. All material-coated

surfaces had higher levels than the plasma (0.014 ± 0.0007 %, i.e. 0.06 ± 0.003 nmol/L)

but lower levels compared to the uncoated glass surfaces (p ≤ 0.001). All surfaces

resulted in clot formation.

To evaluate the contribution of surface functional groups and their

compositions on activating the coagulation cascade, surfaces with the same

functionalities but different –COOH ratios , and those with different alkyl groups but

relatively similar –COOH ratios were grouped to compare (Figure 4-1). In general, all

BMA surfaces induced significantly higher F1+2 level than MMA and EMA surfaces,

regardless of the –COOH ratios (p ≤ 0.001). Amongst BMA surfaces with various –

COOH ratios, 55BMA had a slightly higher F1+2 level compared to 45BMA and

65BMA but no significant difference was detected (p = 0.305).

Among 55MMA, 55EMA & 55BMA surfaces with approximately 33% –

COOH, 55BMA showed the highest F1+2 level whereas 55EMA showed the lowest

level (p ≤ 0.001). A similar effect of alkyl groups on coagulation activation was also

found on surfaces with a higher content of –COOH (i.e. 40% on 65MMA, 65EMA &

65BMA) as 65BMA and 65EMA showed the higher and lower F1+2 level, respectively

(p ≤ 0.001). In contrast to BMA surfaces, an increase in –COOH ratio on MMA and

EMA surfaces reduced F1+2 level significantly (p ≤ 0.001).

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Figure 4-1. The serum levels of prothrombin F1+2 after 30 min of whole blood incubation with material-coated surfaces relative to the uncoated glass surfaces (%). Plasma levels served as baseline. Data was presented as mean of six replicates of each surface with SD. * p ≤ 0.001

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In all cases, these results demonstrated that the material-coated surfaces, and

not the glass substrate dictated the procoagulant property of the blood-contacting surface.

The assay of prothrombin F1+2 after 30 min incubation indicated that the instant rate of

coagulation initiated by material surfaces within the period. Since the half-life of F1+2 is

approximately 90 min, the level of F1+2 not only represents earlier and more discrete

instances of the prethrombotic state than other markers with shorter half-life (e.g. 3-5

min of fibrinopeptides A), but also is less susceptible to interference from other in vitro

activation, such as venepuncture (Mannucci et al., 1992, Greenberg et al., 1994).

Significant differences found in prothrombin F1+2 level among surfaces after

30 min of incubation indicated that the rates of thrombin generation and coagulation

initiated by the surfaces were different. All material-coated surfaces showed a reduced

rate of coagulation initiation compared to the uncoated glass surfaces. More importantly,

we observed surface functional groups and their compositions strongly influenced the

rate of coagulation activation.

Influence of varying ratios of surface functionalities on coagulation

The rate of coagulation activation was found to be a function of the –COOH/–

CH3 or –CH2CH3 ratio, decreasing as the ratio increases. This finding is surprising since

the initiation of intrinsic pathway on –COOH/–CH3 SAM surfaces has been shown to

increase with increasing –COOH ratio, as measured by bradykinin generation (Sperling

et al., 2005a) or kallikrein/FXIIa activity (Sperling et al., 2009, Fischer et al., 2010b).

Previous studies demonstrated that FXII activation is directly dependent on the amount

of negatively charged functional groups (Sanchez et al., 2002, Zhuo et al., 2006, Chen et

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al., 2007, Tzoneva et al., 2008). Indeed, Sperling et al. (2009) found that –COOH/–CH3

SAM surfaces with less than 50% –COOH did not show FXIIa activity in plasma phase.

Rather, these surfaces had a noticeable effect on activating FXII adsorbed on the

surfaces. Hence, we propose that increasing –COOH from 33% to 40% on our –

COOH/–CH3 surfaces would also increase the activation of absorbed FXII and hence the

rate of coagulation activation within 30 min. However, our results demonstrate a relative

alteration in the rate of coagulation activation on these surfaces. This may be related to

platelet-mediated FXII activation.

As pointed out in a review by van der Meijden and Heemskerk (2010),

activated platelets could trigger FXIIa-mediated intrinsic activation by secreting

polyphosphate, and lead to a high rate of thrombin generation in the presence of

negatively charged surfaces. Bäck et al. (2009, 2010) also provided evidence

suggesting that FXII is activated on the surfaces of platelets in a more physiological

environment, clotting whole blood. While it was showed that almost no platelets

adhered on 100% –COOH surfaces in vitro, however the adhesion increased with

increasing –CH3 content on –COOH/–CH3 surfaces and peaked at 100% –CH3

(Sperling et al., 2009, Fischer et al., 2010b). The platelets exposed to –CH3 surfaces

were also highly active compared to –COOH surfaces, and remained to be

moderately active on surfaces with a range of 50-100% –CH3 (Lin and Chuang,

2000, Sperling et al., 2005a, 2009, Fischer et al., 2010b). These findings support that

neither 100% –COOH surfaces with elevated intrinsic activation nor 100% –CH3

surfaces with strong platelet adhesion alone, was sufficient to boost a strong

coagulation activation (Sperling et al., 2009, Fischer et al., 2010b). It also indicates

that an interplay between FXIIa initiation and activated platelets propagation is

crucial for a substantial coagulation response. This probably explains our finding of

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an alteration of the rate of coagulation activation at 30 min. Increasing –COOH

content from 33% to 40% on –COOH/–CH3 or –CH2CH3 surfaces (which in turn

decreases the alkyl group concentration from 67% to 60%), may lead to a decline of

platelet-dependent amplification of coagulation and therefore decreased rate of

coagulation activation.

Specific activities of surface carboxyl and alkyl groups on coagulation

The surface carboxyl and alkyl groups also showed specific effects on the

rate of coagulation initiation. Regardless of varied –COOH ratios, we found that the

–(CH2)3CH3 groups induced a faster rate of coagulation activation than –CH3 and –

CH2CH3 groups and that varying –COOH ratios in the presence of –(CH2)3CH3

groups were less likely to affect the rate of coagulation. The faster rate of coagulation

response in the presence of more hydrophobic –(CH2)3CH3 groups may be attributed

to the amount and conformation of surface adsorbed fibrinogen, an important ligand

for platelets (Fuss et al., 2001, Tsai et al., 2002, Mosesson, 2005).

It was shown that a higher amount of fibrinogen adsorbed on hydrophobic

materials was associated with stronger platelet adhesion and activation when

compared to hydrophilic materials (Nygren, 1996, Keuren et al., 2002, Tsyganov et

al., 2005, Zha et al., 2009, Li et al., 2009, Faxälv et al., 2010). Similar findings were

also reported when comparing hydrophobic –CH3 to hydrophilic –OH or –COOH

groups on SAMs (Evans-Nguyen and Schoenfisch, 2005a, Rodrigues et al., 2006).

The higher affinity of fibrinogen toward hydrophobic surfaces is proposed to be

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driven by a stronger hydrophobic interaction between D-domain of adsorbed

fibrinogen and the substrate, at the expense of less favourable water-protein

interaction. Conversely, the electrostatic interaction between positively charged αC-

domain of fibrinogen and negatively charged hydrophilic surfaces is weak in nature

(Ta et al., 1998, Krishnan et al., 2006). Moreover, the strong thermodynamic driving

force is believed to cause adsorbed fibrinogen on hydrophobic –CH3 SAMs to

undergo a greater conformational change when compared to hydrophilic –COOH or

–OH SAMs (Sit and Marchant, 1999b, Evans-Nguyen et al., 2005a, Roach et al.,

2005, Xu and Siedlecki, 2009, Koo et al., 2010). A greater extent of unfolding of

adsorbed fibrinogen as indicated by a lower ratio of α-helix to β sheet was shown to

correlate with increased thrombogenicity as two distinctly binding sites for platelet

adhesion and activation were exposed (Koh et al., 2010a, Sivaraman and Latour,

2010).

Hence, these findings explained a polymer surface developed by Sperling et

al. (2007), where –(CH2)3CH3 groups derived from serine-(tert-butyl)-methylester

yielded the highest fibrinogen adsorption, platelet activation and the fastest rate of

coagulation activation when compared to –CH3 and –OH groups after 2 h incubation.

It is also in accordance with our results concerning fastest rate of coagulation

activation on –(CH2)3CH3 bearing surfaces than those with –CH3 or –CH2CH3.

Accordingly, the weaker electrostatic force between negatively charged –COOH

groups and fibrinogen compared to the strong hydrophobic force between

hydrophobic –(CH2)3CH3 and fibrinogen implies that the possible effect of varying

the –COOH ratios in the presence –(CH2)3CH3 groups would be too minor or

limited to influence the overall coagulation activation, unlike that seen on –CH3 and

–CH2CH3 bearing surfaces.

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Interestingly, between two less hydrophobic –CH3 and –CH2CH3 groups,

the –CH3 group displayed a specific activity in enhancing the rate of coagulation

initiation. As the surfaces with these two alkyl groups did not show a significant

difference in water contact angles at the same –COOH ratios, this finding suggests

that the surface hydrophobicity (surface free energy) is not the sole factor that

governs blood coagulation response.

Indeed, Sivaraman et al. (2009) convincingly demonstrated that –COOH

and –OH SAMs with similar levels of surface hydrophilicity induced a significant

difference in the degree of structural change of adsorbed fibrinogen and albumin. A

complex and non-linear relationship between platelet adhesion and water contact

angles has also been reported on –COOH/–NH2 SAM surfaces with graded mole

fractions (Chuang and Lin, 2007) or on a common clinical material oxidized

titanium, on which there is almost no adherent platelets in the range of 20-30 º water

contact angle, but a dramatic increase in platelet number up to 140 % at 80 º, and

then decreased above 110 º (Takemoto et al., 2004). Since surface hydrophobicity is

actually determined by the surface chemical species, the characteristics of the surface

functionalities would play a more significant role in modulating coagulation

activation.

Based on the study of Arima and Iwata (2007), preabsorbed albumin, the

most abundant blood protein, is effectively displaced by cell adhesive proteins on –

COOH/–CH3 surfaces with water contact angle less than 90 º. Surfaces with –

COOH/–CH2CH3 functionalities were not studied. Our data showed that at the same

–COOH ratios all –COOH/–CH3 and –COOH/–CH2CH3 surfaces presented water

contact angle less than 90 º. We postulate that absorbed albumin on –COOH/–CH3

surfaces is more efficiently replaced by fibrinogen than on –COOH/–CH2CH3

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surfaces, leading to increased rate of coagulation activation. However, further studies

such as a detailed time course are required to confirm this.

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4.3.2 Surface-initiated complement response

To assess complement cascade initiated by material surfaces, the serum level of

C5a-desArg after 2 h incubation was measured by ELISA. Figure 4-2 shows C5a-

desArg level relative to the uncoated glass surfaces. All material-coated surfaces had

dramatically reduced level compared with the uncoated glass surfaces, though

expectedly higher than the plasma level (6 ± 1%, i.e. 7 ± 1 ng/mL) (p ≤ 0.001).

Similar to the trends observed for coagulation activation, BMA surfaces

generally had higher C5a-desArg level than MMA and EMA surfaces (p ≤ 0.001).

Among BMA surfaces, 55BMA had a significantly lower C5a-desArg level than

45BMA and 65BMA (p ≤ 0.001). No difference was found between the latter two

surfaces.

For surfaces with approximately 33% –COOH (55MMA, 55EMA & 55BMA),

and 40% –COOH (65MMA, 65EMA & 65BMA), a lower C5a-desArg level was found

on EMA surface for both percentages (p ≤ 0.001). An increase in –COOH ratio on

MMA and EMA surfaces also further reduced C5a-desArg level (p = 0.036; p = 0.04,

respectively). The lowest C5a-desArg level among all coated surfaces was on the

65EMA surface.

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Figure 4-2. The serum levels of C5a-desArg after 2 h of whole blood incubation with material-coated surfaces relative to the uncoated glass surfaces (%). Plasma levels served as baseline. Data was presented as mean of six replicates of each surface with SD.* p ≤ 0.001

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Overall, these results demonstrated that all material-coated surfaces remarkably

reduced complement response compared with uncoated glass surfaces. Moreover, we

observed the effect of surface functional groups and their compositions on complement

activation followed an entirely similar pattern of surface-induced coagulation. The extent

of complement activation was significantly elevated in the presence of –(CH2)3CH3

groups, but reduced in the presence of –CH2CH3 groups. A further reduction in

complement response was observed with increasing –COOH ratio on the surfaces with –

CH3 and –CH2CH3. These data indicate that there is an association between these two

blood cascades.

Cross-talk between complement and coagulation system

In fact, the haematology community has long realised that the complement and

coagulation cascades appear to interact significantly in vivo. Interplay between various

proteins and cells is involved in both cascades (Figure 4-3).

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Figure 4-3. Cross-talk between complement and coagulation cascades. Red arrows illustrate

the amplification of complement activation by coagulation system. Black dash lines show the

procoagulant activities of complement system. i) Binding of C1q, an initiator of classical

pathway of complement is known to activate platelets to express P-selectins (Peerschke et

al., 1993, Peerschke and Ghebrehiwet, 1998). ii) Anaphylatoxin C3a activates platelets,

enhancing their aggregation. iii) Anaphylatoxin C5a initiates the extrinsic pathway of

coagulation by inducing TF expression from endothelial cells (Ikeda et al., 1997, Tedesco et

al., 1997), neutrophils and monocytes (Guo and Ward, 2005, Ritis et al., 2006, Kourtzelis et

al., 2010). iv) It also enhances blood thrombogenicity by upregulating PAI-1 on mast cells

and basophils. v) Furthermore, incorporation of the terminal complement complex (also

known as C5b-9 complex) into platelet membrane activates platelets to expose procoagulant

lipids, TF and release of TF-bearing microparticles (MPs) (Sims and Wiedmer, 1991, 1995).

On the other hand, coagulation contributes significantly to complement response. vi)

Intrinsic coagulation factors FXIIa (Ghebrehiwet et al., 1981, 1983) and kallikrein (Wiggins

et al., 1981, Thoman et al., 1984) are shown to trigger the classical pathway of complement.

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vii) FXIa, FXa and thrombin are also reported to have enzymatic activities for C3 and C5

cleavage in vitro and ex vivo (Amara et al., 2008, 2010, Huber-Lang et al., 2006). Activated

platelets not only viii) trigger classical pathway of complement through reciprocal

interaction of C1q (Peerschke et al., 2006, 2010), xi) stimulate alternative pathway by

expression of MP (Yin et al., 2008), P-selectins (del Conde et al., 2005, Peerschke et al.,

2008, Hamad et al., 2010), release of chondroitin sulphate from its granules (Hamad et al.,

2008), xii) but also amplify complement through phosphorylation of C3b.

The high interdependency between complement and coagulation activation

on material surfaces confirms our whole blood incubation system is able to maintain

free cross-talk between both systems. These data show unambiguously that surface

functional groups and their relative ratios have a synergistic effect on modulating the

activation of both cascades.

We found that complement response on material surfaces was significantly

reduced with the alkyl length in the order: –(CH2)3CH3 > –CH3 > –CH2CH3.

Berglin et al. (2004) suggested that complement activation is reduced with increased

alkyl chain length of poly(alkyl methacrylates) ranged from 4 to 18 carbons.

However, similar to our findings, they also found that PMMA with one carbon in its

alkyl chain (i.e. –CH3) induces slightly less activation than PIBMA (poly (isobutyl

methacrylate) with four carbons in its alkyl chain (i.e. –(CH2)3CH3), of which the

complement activity also did not differ from that of PBMA. On the other hand, we

found that an increase in –COOH contents (from 33% to 40%) on –COOH/–CH3 or

–CH2CH3 surface reduced complement activation upon clot formation, a similar

trend observed in coagulation response. Inconsistently, Salvador-Morales et al.

(2009) demonstrated an increase in –COOH content (0, 25, 50, 75, 100%) on lipid-

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polymer nanoparticles presenting –COOH/–CH3 increases complement activation,

and virtually have no effect on coagulation system after serum incubation. Moreover,

other studies using anticoagulated whole blood also did not find any significant

difference in complement-generated C5a and C3b adsorption among –COOH/–CH3

SAM surfaces with a range of –COOH contents (0, 50, 83, 100%), and reported that

the surface chemistry-initiated coagulation is independent of the complement

response (Fischer et al., 2010a, Sperling et al., 2009). We believe that the difference

in experimental conditions such as uses of heparin or isolated components may

inhibit both systems for cross-talk and thus be attributable to this discrepancy of

results.

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4.3.3 SEM analysis of clot morphology and structure

Despite various rates of coagulation were initiated by the material coatings, all

these surfaces effectively supported clot formation within 30 min. To investigate the

effect of material coating-initiated coagulation on ultimate clot structure, the fibrin

thickness and density at the edge and in the centre of clots were examined by SEM after

2 h.

4.3.3.1 Effect of various ratios of surface carboxyl groups on fibrin structures

As evident from the micrographs shown in Figure 4-4 a-d, the clots formed on

BMA surfaces showed a network of many thicker and highly branched fibrin fibers,

displaying small interstitial pores at the edge. In contrast, the clots formed on uncoated

glass surfaces showed a network of thinner, less branched and small numbers of fibres

displaying large interstitial pores. The fibrin fibres at the edge of clots on glass surfaces

were significantly smaller in diameter (Figure 4-4 i) and lower in density (Figure 4-4 k)

than those of BMA surfaces (p ≤ 0.001).

Among BMA surfaces, the fibrin diameter at the edge of clots was significantly

smaller on 55BMA, while the fibrin density was significantly lower on 45BMA (p ≤

0.001). No difference in fibrin density at the edge was found between 55BMA and

65BMA (p = 0.237).

Fibrin architecture changed dramatically from the edge to the centre of the clot.

The fibrin of all surfaces except 45BMA increased in diameter (Figure 4-4 j), while the

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densities of all surfaces decreased approximately 3 to 5 times (p ≤ 0.001) (Figure 4-4 l).

In the centre of the clots, 45BMA produced significantly thinner fibres (p ≤ 0.05) at

higher density (p ≤ 0.001) than all other surfaces. These results suggested that variation

of the –COOH ratio on –(CH2)3CH3 bearing surfaces led to significant changes in the

fibrin thickness and network density.

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Figure 4-4. Scanning electron microscopy analysis of whole blood clot structures formed on 45BMA, 55BMA, 65BMA and uncoated glass surfaces. Micrographs of the edge of clots (top panel; a-d) and the centre of clots (bottom panel; e–h), scale bar represents 20 µm. Comparison of fibrin thickness (diameter; nm) i) at the edge; j) at the centre of clots. Comparison of fibrin density (fibre number per 40 µm2) k) at the edge; l) at the centre of clots. Data of fibrin thickness was presented as mean of at least 40 fibrin fibres measured at random field while data of fibrin density was presented as mean of fibre numbers quantified in at least 20 random areas of 40 µm2 at the edge and at the centre of the clots of each surface with SD.* p ≤ 0.001

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4.3.3.2 Effect of various surface alkyl groups on fibrin structures

To evaluate the effect of surface alkyl groups –CH3, –CH2CH3 and –

(CH2)3CH3 on clot ultrastructures, the clots formed on surfaces with approximately

33% –COOH (55MMA, 55EMA & 55BMA) and uncoated glass surfaces were

compared.

As shown in Figure 4-5 a-d, the edge of clots formed on 55MMA were a

dense and highly homogenous network with small pores. For 55EMA, the clots were

a dense network similar to that of 55MMA but also had multiple fibres bundled

together resulting in a highly heterogeneous architecture. While the clots of 55BMA

displayed a more porous network relative to the others, a region with tightly packed

fibres was also observed leading to a slightly heterogeneous network. However,

55MMA, 55EMA and 55BMA surfaces did not differ significantly in the fibrin fibre

diameters (p = 0.878) (Figure 4-5 i) and fibrin densities (p = 0.404) (Figure 4-5 k).

Instead, the fibrin fibres at the edge of clots formed on these surfaces were

significantly larger in diameter and higher in density than those of glass surfaces (p ≤

0.001).

Compared to the fibres at the edge, the fibres at the centre of the clots formed

on all surfaces except 55EMA increased in diameter (Figure 4-5 j), and the fibrin

densities of all surfaces decreased approximately 4 to 5 times (p ≤ 0.001) (Figure 4-5 l).

Among 55MMA, 55EMA and 55BMA surfaces, the fibres at the centre of the clots of

55BMA were significantly larger in diameter while those of 55EMA were significantly

higher in density (p ≤ 0.001). Overall, 55MMA, 55EMA and 55BMA surfaces showed a

similar trend of fibrin density at the edge and at the centre of clots, though the difference

at the edge of clots was not significant.

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Figure 4-5. Scanning electron microscopy analysis of structure of clots formed on 55MMA, 55EMA, 55BMA and uncoated glass surfaces. Micrographs of the edge of clot (top panel; a-d) and the centre of clot (bottom panel; e–h), scale bar represents 20 µm. Comparison of fibrin diameter (nm) i) at the edge; j) at the centre of clots. Comparison of fibrin density (fibre number per 40 µm2) k) at the edge; l) at the centre of clots. Data of fibrin thickness was presented as mean of at least 40 fibrin fibres measured at random field while data of fibrin density was presented as mean of fibre numbers quantified in at least 20 random areas of 40 µm2 at the edge and at the centre of the clots of each surface with SD.* p ≤ 0.001

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The clots formed on surfaces with approximately 40% –COOH (65MMA,

65EMA & 65BMA) and uncoated glass surfaces were observed in Figure 4-6 a-d.

Compared to the uncoated glass surfaces, the edge of clots formed on

65MMA, 65EMA and 65BMA showed a highly branched network composed of

numerous, thicker and longer fibres with smaller pores. The fibrin fibres at the edge

of clots formed on 65MMA, 65EMA and 65BMA were significantly larger in

diameter and higher in density than those on glass surfaces (p ≤ 0.001) (Figure 4-6 i,

k). Among 65MMA, 65EMA and 65BMA surfaces, the fibrin diameter at the edge of

the clots of 65BMA was significantly larger than the others (p ≤ 0.001). No

difference was found between those of 65MMA and 65EMA (p = 0.287). Moreover,

the fibrin density was significantly higher on 65MMA while lower on 65EMA (p ≤

0.001).

Compared to the fibres at the edge, the fibres at the centre of the clots on all

surfaces except 65EMA increased in diameter (Figure 4-6 j), and the fibrin densities of

all surfaces decreased approximately 5 times (p ≤ 0.001) (Figure 4-6 l). While the fibrin

diameter of 65EMA was significantly smaller than those of 65MMA and 65BMA (p ≤

0.001), no significant differences were found between the latter (p = 0.105). For the

fibrin density at the centre of the clots, it was significantly higher on 65MMA than all

the others (p ≤ 0.001). Although the mean fibrin densities of 65EMA and 65BMA were

not significant different, a similar trend in fibrin density was observed at the edge and at

the centre of clots formed on 65MMA, 65EMA and 65BMA.

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Figure 4-6. Scanning electron microscopy analysis of structure of clots formed on 65MMA, 65EMA, 65BMA and uncoated glass surfaces. SEM micrographs of the edge of clot (top panel; a-d) and the centre of clot (bottom panel; e–h), scale bar represents 20 µm. Comparison of fibrin diameter (nm) i) at the edge; j) at the centre of clots. Comparison of fibrin density (fibre number per 40 µm2) k) at the edge; l) at the centre of clots. Data of fibrin thickness was presented as mean of at least 40 fibrin fibres measured at random field while data of fibrin density was presented as mean of fibre numbers quantified in at least 20 random areas of 40 µm2 at the edge and at the centre of the clots of each surface with SD.* p ≤ 0.001

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In summary, 65BMA (40% –COOH/60% –(CH2)3CH3) surfaces resulted in

the fibrin fibres at the edge of clots that were significantly larger in diameter than all

other surfaces (p ≤ 0.001). The mean fibrin diameter at the centre of clots on 65BMA

was also larger than all the others, suggesting this composition of –COOH/–(CH2)3CH3

functionalities have an influence throughout the clots. In contrast, the uncoated glass

surfaces produced fibrin with significantly smaller diameter and in lower density at the

edge of clots compared to all other surfaces (p ≤ 0.001). On the other hand, at the centre

of the clots, 65EMA (40% –COOH/60% –CH2CH3) seemed to be more effective in

decreasing both the fibrin diameter and density as the mean values of these two

parameters of this surface were lower than all the others. Furthermore, 65MMA (40% –

COOH/60% –CH3) and 45BMA (25% –COOH/75% –(CH2)3CH3) surfaces led to a

higher fibrin density compared to all other surfaces, at the edge and the centre of the

clots, respectively (p ≤ 0.001).

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Together, our findings indicate that material-coated surfaces modulate the

whole blood clot structure. The changes in alkyl length of –CH3, –CH2CH3 and –

(CH2)3CH3) groups as well as the concentration of –COOH groups on the coated

surfaces showed a combined and direct effect against the fibrin formation, in term of

fibre thickness and density, from the exterior to the interior part of the clots.

The effect of material surfaces on clot fibrin architectures

In direct contact with material-coated surfaces, the clots display a tight

network with thicker fibrin compared to those in contact with uncoated glass

surfaces. Generally, the interior part of clots on all surfaces become an extremely

loose network with thicker fibres. The dramatic changes in fibrin structure at the clot

exterior among various surfaces, and from clot exterior to interior may be due to a

combination of two mechanisms.

(1) The pattern of in situ thrombin generation follows the initiation,

amplification and propagation phases of coagulation. These phases are in turn

profoundly affected by environmental factors (Sauls et al., 2003, Allen et al., 2004,

Scott et al., 2004, Wolberg et al., 2005, Machlus et al., 2009). As such, a dynamic

change in the thrombin concentration (1nM to greater than 500 nM) (Mann et al.,

2003) may lead to significant differences in kinetics of fibrinopeptide release,

protofibril and fibre formation (Blomback, 2000, Carr et al., 2002a, Wolberg and

Campbell, 2008). This is also tied into the fact that normal plasma clots usually

display a bimodal distribution of the fibre diameters (Shah et al., 1982, Collet et al.,

1993b).

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Using turbidity assays of fibrinogen solutions, it has been illustrated that

clots produced in the presence of polymers contains heterogeneous fibrin structure

due to changes in protofibril aggregation rate, the number and size of fibres formed

compared to controls with an addition of a single thrombin concentration (Lai et al.,

2010).

In line with this, it has been demonstrated that the surface functional groups

significantly affect the efficacy of adsorbed fibrinogen to convert to fibrin (Sit and

Marchant, 2001, Wang et al., 2007, Rodriguez Hernandez et al., 2009). With similar

amounts of adsorbed fibrinogen, a denser fibrin network with more branches was

found on –CH3 surface associating with a larger amount of fibrinopeptides released

at a faster rate compared to sparse fibrin observed on –COOH surfaces (Evans-

Nguyen and Schoenfisch, 2005b, Evans-Nguyen et al., 2005b, 2006). The extent of

fibrinopeptides release and fibrin proliferation have been shown to be related to

surface-dependent fibrinopeptide availability. Approximately 2.7 fold more

accessible fibrinopeptide A was found on fibrinogen adsorbed on –CH3 surfaces for

thrombin cleavage than those on –COOH surfaces. Hence, this may explain the

higher efficacy of fibrin proliferation observed on –CH3 surfaces (Geer et al., 2007).

Furthermore, different alkyl chain length of poly (alkyl methacrylates) have also

displayed a major effect on regulating the rate of thrombin generation and fibrin

deposition (Berglin et al., 2004, 2009). This implies that surfaces functionalities –

COOH/–CH3, –CH2CH3 or –(CH2)3CH3) at varied ratios very likely influence the

fibrinopeptides availability, extent and kinetic of fibrinopeptides release leading to

different fibrin architecture as observed in our study.

Indeed, we found that elevated levels of prothrombin F1+2 on uncoated

glass and 55BMA surfaces produced clots with much thinner fibres, compared to

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material-coated surfaces and BMA surfaces, respectively. These findings are

consistent with the work of Wolberg et al. (2003) in which elevated prothrombin

level triggers the formation of thinner fibrin due to increased initial rate, peak and

total amount of thrombin generation.

(2) Highly procoagulant cells (e.g. activated platelets) have been shown to

support the formation of denser fibrin networks that are more resistant to fibrinolysis

and that the density and stability decrease with increasing distance from the cell

surface (Campbell et al., 2009, Wolberg, 2010, Aleman et al., 2011). For instance,

procoagulant human fibroblasts have been shown to produce denser networks in 10-

µm region proximal than distal to (40-50 µm) its surface (Campbell et al., 2008,

2009). Given the differences in procoagulant properties of cells and plasma factors in

surrounding milieu, a thrombin gradient will be formed in space and therefore may

cause the formation of a range of fibre thicknesses and densities across a region of

growing clots (Ovanesov et al., 2005, Panteleev et al., 2006). This likely explains our

observation of spatially heterogeneous clot morphology with fibrin propagation away

from the site of initiation to the interior part of clots.

In addition, we observed a consistent trend on fibrin density at the clot

exterior and interior on surfaces containing same –COOH ratio but different alkyl

groups. This indicates that the surface functionalities and relative ratios have a

specific influence on fibrin density throughout the clots.

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4.3.4 Assessment of clot rigidity by compaction

Our previous results showed that clots formed on material-coated surfaces

had differing fibrin architectures. To determine whether such changes in fibrin

structure influences the mechanical properties of whole blood clot, a compaction

study was performed to investigate the clot rigidity.

The compaction coefficient for clots formed on all material-coated surfaces

was lower than that on uncoated glass surfaces (p ≤ 0.001), suggesting the clot

rigidity was greatly increased by the coated surfaces (Figure 4-7).

For surfaces presenting 33% –COOH (55MMA, 55EMA & 55BMA), and

40% –COOH (65MMA, 65EMA & 65BMA), the clot rigidity was significantly

elevated on MMA surfaces but reduced on BMA surfaces at both percentages (p ≤

0.001) (Figure 4-7 a-b). As evidenced from BMA surfaces, an increase in –COOH

groups resulted in a significant increase in clot rigidity (p ≤ 0.05) (Figure 4-7 c).

Similarly, an increase in –COOH groups on EMA surfaces also resulted in increased

clot rigidity as shown by comparing 55EMA and 65EMA (p ≤ 0.001). However, the

difference between clot rigidity on MMA surfaces due to an increase in –COOH ratio

was not significant (55MMA vs 65MMA; p = 0.921).

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Figure 4-7. Compaction studies of clots formed on various material-coated surfaces compared to the uncoated glass surfaces. a) Among 55MMA, 55EMA and 55BMA surfaces presenting 33% –COOH. b) Among 65MMA, 65EMA and 65BMA surfaces presenting 40% –COOH. c) Among 45BMA, 55BMA and 65BMA surfaces which contains different concentration of –COOH groups. Data was presented as mean of six replicates of each surface with SD.* p ≤ 0.001

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Overall, these findings demonstrated that the stiffness of clots formed on

material-coated surfaces was significantly enhanced by –CH3 groups compared to –

CH2CH3 and –(CH2)3CH3 groups and that the increase in –COOH ratio on surfaces

with –CH2CH3 and –(CH2)3CH3 groups also improved the clot rigidity.

Relationship between fibrin architectures and clot rigidity

With similar clot mass and approximately 3-5 fold less dense fibrin

network in the centre of clots, the densely packed and cross-linked thick fibres at the

clot exterior may contribute mostly to the network strength to resist collapse under

centrifugal force (Nair and Shats, 1997, Collet et al., 2005). Therefore the notable

difference in fibrin structure at the clot exterior between material-coated surfaces and

uncoated glass surfaces may explain the dramatic increase in clot rigidity of coated

surfaces assessed by compaction studies.

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4.3.5 Clot lysis

Having shown the differences in fibrin structural network and overall

rigidity of clots formed on various material-coated surfaces, we further determined

whether such changes in clot structure and elastic properties altered the clot

susceptibly to lysis. This was carried out by degrading the clot with tPA and

plasminogen, and assessing the release of D-dimer during fibrinolysis as a function

of time after an addition of tPA.

4.3.5.1 Effect of various ratios of surface carboxyl groups on fibrinolysis

The levels of D-dimer released from clots formed on BMA and uncoated

glass surfaces over 24 h of lysis were shown in Figure 4-8 b. After 1 h of lysis, a

significant increase in D-dimer concentration was detected from uncoated glass

surfaces compared to BMA surfaces (p ≤ 0.001) (Figure 4-8 a). As the level of D-

dimer is indicative of the rate of fibrinolysis, this finding suggested that the clots of

glass surfaces initially underwent a faster rate of fibrinolysis.

A significant difference was also detected among the BMA surfaces after 1

h of lysis, in which 55BMA led to a faster rate of fibrinolysis when compared to

45BMA and 65BMA (p ≤ 0.001) (Figure 4-8 a). Over the rest of the lysis period, the

mean D-dimer concentration of 55BMA was also higher than the other BMA and

glass surfaces, with a significant difference detected after 8 h of lysis (p ≤ 0.05)

(Figure 4-8 b). No significant differences in D-dimer concentration were found

among 45BMA, 65BMA and glass surfaces except at the early stage of lysis (1 h).

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However, it is worth noting that 45BMA led to a slower rate of fibrinolysis as the

mean D-dimer concentration was lower than all the other surfaces during the

intermediate lysis stage (4 h and 8 h). Whereas during the late stage (18 h and 24 h),

65BMA led to a slower rate of fibrinolysis as the mean value of 65BMA was lower

than other BMA surfaces. Thus, a similar release profile of BMA surfaces was

observed at the early stage (1 h) and late stage (18 h and 24 h) of lysis.

We also measured the weight loss of clots during lysis (Figure 4-8 d-e).

Significant differences in weight loss among BMA surfaces were detected at the late

stage of lysis (18 h and 24 h) with less weight loss from 45BMA than 65BMA (p ≤

0.05). When compared to the clots exposed to tPA and plasminogen in PBS buffer

(Figure 4-8 d-e), the control clots subjected to PBS buffer only showed a little weight

loss over time (Figure 4-8 f-g). A negligible amount of D-dimer was also detected

from control clots of uncoated glass surfaces (0.06 ± 0.005 µg/mL to 2.8 ± 0.287

µg/mL from 1 h to 24 h after lysis) when compared to the plasma level of D-dimer

(0.09 ± 0.008 µg/mL) (Figure 4-8 c). This suggested that the spontaneous fibrinolysis

was not profound under these experimental conditions and that the elevated level of

D-dimer was largely due to the clot lysis by tPA. Moreover, it indicated that our

suspended clot system supplemented with fibrinolytic enzymes was feasible for

assaying clot lysis.

Overall, the initial decrease in the D-dimer concentrations of BMA surfaces

compared to the uncoated glass surfaces indicated a delayed onset of fibrinolysis

during the first hour of lysis. Further, over the rest of lysis period, clots of 55BMA

showed a faster rate of fibrinolysis than other surfaces. Thus, the specific

composition (33% –COOH/ 67% –(CH2)3CH3 ) on 55BMA seemed to contribute to

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a higher clot susceptibility to lysis than lower or higher –COOH ratios presented on

45BMA (25% –COOH) and 65BMA (40% –COOH), respectively.

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Figure 4-8. Release of D-dimer and weight loss over 24 h lysis of clots formed on BMA surfaces compared with uncoated glass surfaces. a) The D-dimer levels of all surfaces after 1 h of lysis. The D-dimer levels over 24 h-lysis period of b) BMA surfaces and c) uncoated glass surfaces and relative control clots. Negligible amount of D-dimer was detected from control clots. The percentage of weight loss over 24 h lysis of clots formed on d) BMA surfaces and e) uncoated glass surfaces, and the control clots relative to f) BMA surfaces and g) uncoated glass surfaces, respectively. Data was presented as mean of three replicates of each surface with SD. * p ≤ 0.001 ** p ≤ 0.05

4.3.5.2 Effect of various surface alkyl groups on fibrinolysis

To evaluate the effect of surface alkyl groups on fibrinolytic potential of

clots, the lysis of clots formed on surfaces exhibiting 33% –COOH (55MMA,

55EMA & 55BMA), and 40% –COOH (65MMA, 65EMA & 65BMA) were

compared respectively. Figure 4-9 shows the D-dimer concentrations and weight loss

of the clots formed on these surfaces over 24 h of lysis. Similarly, after 1 h of lysis, a

faster rate of fibrinolysis was observed from uncoated glass surfaces as indicated by

a significantly higher D-dimer concentration than all other surfaces (p ≤ 0.001)

(Figure 4-9 a-b). Furthermore, 55BMA led to a significantly faster fibrinolysis

compared to 55MMA and 55EMA (Figure 4-9 a), whereas 65EMA also showed a

significantly faster fibrinolysis than 65MMA and 65BMA (p ≤ 0.001) (Figure 4-9 b).

Although no significant differences were detected among 55MMA, 55EMA

and 55BMA for the rest of the lysis period (Figure 4-9 c), a similar pattern was found

between 1 h and 4 h after lysis, in which 55BMA led to a faster rate of lysis while

55EMA led to a lower rate. In addition, the pattern between the late time points 18 h

and 24 h after lysis was also similar, with a faster rate of lysis occurring on 55MMA

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clots compared with a lower rate from 55BMA clots. A transitional pattern of D-

dimer release may be attributed to the difference between early and late time points

at the intermediate stage of lysis (8 h), when 55MMA clots began to display

enhanced lysis. The D-dimer release profile of these surfaces correlated well with the

weight loss measured from 8 h after lysis at which 55MMA showed slightly more

weight loss when compared to 55EMA and 55BMA. At 24 h of lysis, 55MMA

displayed significantly more weight loss compared with 55BMA (p ≤ 0.05) (Figure

4-9 e). The control clots of 55MMA showed more weight loss than that of 55BMA at

24 h of lysis (p ≤ 0.05) (Figure 4-9 g).

Thus, the initial decrease in the D-dimer concentrations compared to the

uncoated glass surfaces indicated that the clots formed on 55MMA, 55EMA and

55BMA surfaces were more resistant to lysis for up to 4 h. The significant

differences in the D-dimer concentration found among 55MMA, 55EMA and

55BMA surfaces after 1 h lysis, indicated that the surface alkyl groups with different

length altered the initial rate of fibrinolysis. It was showed that the clots formed on

33% –COOH/ 67% –(CH2)3CH3 surface were more prone to lysis initially whereas

that formed on 33% –COOH/ 67% –CH2CH3 surface were more resistant to lysis.

For clots formed on 65MMA, 65EMA and 65BMA, no significant

differences were found for the rest of the lysis period except at 1 h of lysis (Figure 4-

9 d). Instead, a similar pattern was found between 4 h and 8 h after lysis with a faster

rate of lysis occurring on 65MMA but a slower rate from 65BMA. In addition, the

pattern between the late time point 18 h and 24 h was also similar with a faster rate

of lysis from 65EMA while a slower rate from 65BMA. No significant differences

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were found in the weight loss of clots formed on these surfaces over time (Figure 4-9

f). In contrast, the weight loss of relative control clots showed a significant increase

from clots on 65MMA than that on 65BMA after 8 h of lysis (p ≤ 0.05) (Figure 4-9

h).

Thus, the initial decrease in the D-dimer concentrations compared to the

glass surfaces indicated that the clots formed on 65MMA, 65EMA and 65BMA

surfaces were also lysed more slowly for up to 4 h. The significant differences in the

D-dimer concentration found among 65MMA, 65EMA and 65BMA after 1 h lysis

not only reconfirmed that the initial rate of fibrinolysis was modulated by surface

alkyl groups but also by the concentration of –COOH groups. The increase in –

COOH ratio on –CH2CH3 bearing surfaces resulted in an increase in lysis

susceptibility (55EMA vs 65EMA; p ≤ 0.001). Whereas intermediate –COOH ratio

(i.e. 33%) on –(CH2)3CH3 bearing surfaces led to a higher susceptibility than low

(25%) or high (40%) ratio (55BMA vs 45BMA, 65BMA respectively; p ≤ 0.001). No

obvious influence of various –COOH ratios was found on –CH3 presenting surfaces

(55MMA vs 65MMA; p = 0.122). This discrepancy in the dependency of –COOH

ratios on surfaces with various alkyl groups suggested that specific functional groups

and their relative compositions had a major effect on the rate of fibrinolysis and

further supported the changes in the clot structure.

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Figure 4-9. Release of D-dimer and weight loss over 24 h lysis of clots formed on 55MMA, 55EMA and 55BMA surfaces compared with 65MMA, 65EMA and 65BMA surfaces. Compared to uncoated glass surfaces, D-dimer levels after 1 h of lysis of a) 55MMA, 55EMA and 55BMA surfaces, and b) 65MMA, 65EMA and 65BMA surfaces. The D-dimer levels over 24 h-lysis period of c) 55MMA, 55EMA and 55BMA surfaces, and d) 65MMA, 65EMA and 65BMA surfaces. The percentage of weight loss over 24 h lysis of clots formed on d) 55MMA, 55EMA and 55BMA surfaces, and e) 65MMA, 65EMA and 65BMA surfaces, and the control clots relative to f) 55MMA, 55EMA and 55BMA surfaces and g) 65MMA, 65EMA and 65BMA surfaces, respectively. Data was presented as mean of three replicates of each surface with SD. * p ≤ 0.001 ** p ≤ 0.05

Relationship between fibrin structures and clot susceptibility to fibrinolysis

Since the fibrin architectures at the clot exterior and interior are different,

we investigated the impact of fibrin structure modification on fibrinolysis using a

suspended clot system. In this case, the exogenous fibrinolytic enzymes would

initially interact with fibrin at the clot exterior and lysis would proceed from the clot

exterior to interior. Our results demonstrate that all material-coated surfaces lead to a

significantly slower fibrinolysis in the first hour of lysis compared to the uncoated

glass surfaces. This slower onset of fibrinolysis is in good agreement with the tight

network and thicker fibrin observed on the clot exterior on coated surfaces, in

accordance with previous studies which indicated that fibrinolysis occurs

predominantly faster on loose network and thinner fibrin (Gabriel et al., 1992, Collet

et al., 2000, Mullin et al., 2001).

Importantly, significant differences in the fibrinolytic rate after 1 h of lysis

in different material surface groups: BMA surfaces with different –COOH ratios,

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surfaces with different alkyl groups combined with 33% –COOH, or with 40% –

COOH, correlated strongly with surface-dependent differences in fibrin thickness

and density at the clot exterior. For instance, a significantly faster fibrinolysis rate on

65EMA clots compared with clots formed on 65BMA and 65MMA surfaces, is

associated with the ascending fibrin densities at the clot exterior. While a faster

fibrinolysis on 55BMA compared to 45BMA and 65BMA is associated with the

significantly thinner fibre observed at the clot exterior of 55BMA. Moreover,

increasing –COOH ratios on –CH2CH3 surfaces with a decrease in fibrin density at

the clot exterior may also account for increasing susceptibility to fibrinolysis. In

contract, an increase in –COOH ratios on –CH3 surfaces with increasing fibrin

density did not result in a significant difference in the initial rate of fibrinolysis.

These results suggest that the composition of the surface, –COOH groups and their

combination with –CH3, –CH2CH3 or –(CH2)3CH3 groups have a specific activity

on regulating initial rate of fibrinolysis through influence on the fibrin thickness and

density at the clot exterior.

After 4-8 h of lysis, we believe that the enzymatic lysis proceeds to the inner

part of the clots. This is illustrated by a slightly more D-dimer released from 45BMA

than other BMA surface during this period, correlating with the significantly thinner

fibres in the clot interior of 45BMA. Also, a shift of lysis pattern observed after 8 h

of lysis in which a slightly more D-dimer released from 55MMA than 55EMA

correlated well with a significantly lower density in the clot interior of 55MMA than

55EMA. Hence, the changes in D-dimer level from these clots during 4-8 h after

lysis may reflect the gross alteration of fibrin architectures from the clot exterior to

the interior.

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4.3.6 Quantification of PDGF-AB in serum and during clot lysis

Given that the clots formed on material-coated surfaces showed differences

in structural network, stability and lysis susceptibility, we next investigated whether

material-coated surfaces influences the biological function of clots for bone repair.

This was assessed by detecting the release of reparative PDGF-AB and TGF-β1 in

supernatant serum after clot formation and in the buffer during clot lysis.

The serum level of PDGF-AB after 2 h whole blood incubation with

surfaces was shown in Figure 4-10. Significantly elevated PDGF-AB level was

found on all surfaces compared to plasma baseline (308 ± 49 pg/mL), confirming the

growth factor is released upon clot formation. The mean values of 65MMA (8017 ±

330 pg/mL) and uncoated glass surfaces (8029 ± 689 pg/mL) was higher than the

other surfaces.

Among 65MMA, 65EMA & 65BMA surfaces, a significantly lower level of

PDGF-AB was released from 65EMA compared to 65MMA (p ≤ 0.05). For surfaces

with 33% –COOH (55MMA, 55EMA & 55BMA), 55MMA resulted in a

significantly lower level of PDGF-AB than 55EMA and 55BMA (p ≤ 0.001), but

there was no differences between the latter (p = 0.88). In addition, no significant

differences were found among the BMA surfaces.

Overall, 65MMA resulted in a significantly higher amount of PDGF-AB

released from intact clots than all other material surfaces.

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Figure 4-10. The serum levels of PDGF-AB after 2 h of whole blood incubation with material-coated surfaces compared to the uncoated glass surfaces and the plasma baseline. Data was presented as mean of triplicates of each surface with SD.* p ≤ 0.001 ** p ≤ 0.05

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The in vitro release of PDGF-AB during clot lysis was illustrated in Figure

4-11. Among the degrading clots of 55MMA, 55EMA and 55BMA surfaces, a

significantly higher amount of PDGF-AB was released from 55MMA throughout the

lysis period compared to the others (p ≤ 0.001) (Figure 4-11 a). A burst release was

observed after 1 h of lysis and peaked at 8 h with approximately 7-fold more than the

others. The level of growth factor remained high up to 24 h. In contrast, both 55EMA

and 55BMA showed similar release curves with no significance difference found.

In contrast, for 65MMA, 65EMA and 65BMA, similar release patterns were

observed with a peak at 8 h of lysis but overall 65MMA led to a higher amount of

PDGF-AB with a significant difference found at 1 h (p ≤ 0.001) and 4h after lysis (p

≤ 0.05) (Figure 4-11 b).

Degrading clots of BMA surfaces exhibited no difference in PDGF-AB

release profile except at the end of lysis period (24 h) 45BMA resulted in a

significantly higher level than the others (p ≤ 0.001) (Figure 4-11 c). The control

clots of glass surfaces showed higher level of PDGF-AB than that underwent

enzymatic lysis after 1 h (p ≤ 0.001) and 24 h of lysis (p ≤ 0.05) (Figure 4-11 d),

implying the presence of tPA and plasminogen may reduce the release of PDGF-AB.

Overall, the release of PDGF-AB during lysis was significantly elevated

from the clots formed on 55MMA than all other surfaces. However, the majority of

PDGF-AB was released during formation of intact clots instead of clot lysis.

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Figure 4-11. In vitro release of PDGF-AB during lysis of clots formed on material-coated surfaces and uncoated glass surfaces. Among clots formed on a) 55MMA, 55EMA and 55BMA surfaces, b) 65MMA, 65EMA and 65BMA surfaces, c) BMA surfaces and d) uncoated glass surfaces and the relative control clots. Data was presented as mean of triplicates of each surface with SD.* p ≤ 0.001 ** p ≤ 0.05

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4.3.7 Quantification of TGF-beta 1 in serum and during clot lysis

The serum level of TGF-β1 after 2 h whole blood incubation with surfaces

was shown in Figure 4-12. The clots formed on all surfaces released a significantly

elevated level of TGF-β1 compared to plasma level (1021 ± 31 pg/mL).

However, no significant differences were found among BMA surfaces

though the mean levels of TGF-β1 increased with increasing –COOH ratios.

Similarly, no difference was found among 65MMA, 65EMA and 65BMA surfaces,

nor among 55MMA, 55EMA and 55BMA surfaces. Also, uncoated glass surfaces

showed no difference to all the other surfaces.

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Figure 4-12. The serum levels of TGF-β1 after 2 h of whole blood incubation with material-coated surfaces compared to the uncoated glass surfaces and plasma baseline. Data was presented as mean of triplicates of each surface with SD.* p ≤ 0.001 ** p ≤ 0.05

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The in vitro release of TGF-β1 from the degrading clots was shown in

Figure 4-13. In general, the level of TGF-β1 increased over time for all surfaces.

Among the degrading clots formed on 55MMA, 55EMA and 55BMA surfaces, a

similar release pattern was observed initially up to 4 h of lysis. At 8 h of lysis,

55MMA showed an approximate two fold increase of TGF-β1 with up to 24 h of

lysis when compared to the others (p ≤ 0.001) (Figure 4-13 a).

The degrading clots of 65MMA, 65EMA and 65BMA also showed a similar

release pattern over the entire lysis period. At 1 h and 4 h of lysis, 65BMA showed a

significantly higher level of TGF-β1 than 65EMA (p ≤ 0.05) (Figure 4-13 b).

Among the BMA surfaces, the degrading clots of 55BMA and 65BMA

showed a very similar pattern of releasing a significantly higher amount of TGF-β1

than 45BMA at 1 h and 4 h of lysis. By 8 h of lysis, the level of TGF-β1 released

from the clot of 65BMA was also significantly higher than that of 55BMA (p ≤

0.001), and remained higher than the others by 24 h (p ≤ 0.05), and no difference was

found then between 55BMA and 45BMA (Figure 4-13 c). Unlike PDGF-AB, the

clots of uncoated glass surfaces subject to enzymatic lysis released a significantly

higher level of TGF-β1 than the control clots over the lysis period (p ≤ 0.001)

(Figure 4-13 d).

In summary, TGF-β1 was released in significantly higher amounts from the

degrading clots of 55MMA than all other surfaces. However, the majority of TGF-β1

was also released during formation of intact clots than during clot lysis.

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Figure 4-13. In vitro release of TGF-β1 during lysis of clots formed on material-coated surfaces and uncoated glass surfaces. Among clots formed on a) 55MMA, 55EMA and 55BMA surfaces, b) 65MMA, 65EMA and 65BMA surfaces, c) BMA surfaces and d) uncoated glass surfaces and the relative control clots. Data was presented as mean of triplicates of each surface with SD.* p ≤ 0.001 ** p ≤ 0.05

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Relationship between growth factor release, clot structure and early healing events

It has been well documented that various growth factors are expressed in

different phases of bone healing (Cross and Mustoe, 2003). In particular, the

initiation of bone regeneration is suggested to begin with the release of PDGF-AB

and TGF-β1 after a clot is formed (Hosgood, 1993, Kells et al., 1995, Lieberman et

al., 2002b). PDGF-AB is most abundant in platelet α-granules and is known to

support chemotaxis and proliferation of fibroblasts, smooth muscle cells as well as

endothelial cells, resulting in collagen synthesis and angiogenesis (Oprea et al., 2003,

Andrae et al., 2008). On the other hand, TGF-β1 is predominant in platelets, bone

and cartilage, and is shown to serve as a mitogen for osteoblasts, fibroblasts and

endothelial cells, as well as an inhibitor of osteoclasts (Zhang et al., 2005, Bosetti et

al., 2007). In addition, both PDGF-AB and TGF-β1 are chemotactic for

inflammatory cells such as neutrophils, monocytes or macrophages, which

establishes a positive feedback loop of growth factors within the injured bone

(Ashcroft, 1999). In view of their function in supporting bone healing, we evaluated

the potentials of modifications in fibrin structure and fibrin structure-dependent

fibrinolysis on affecting the release of these growth factors from the intact and

degrading clots.

Upon clot formation, increased amounts of PDGF-AB and TGF-β1 were

detected in serum compared to platelet-poor plasma. Interestingly, we found that

there was a correlation between the amount of PDGF-AB released in serum and

fibrin density at the clot exterior and interior. This may be related to fibrin-platelet

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interactions and the subsequent platelet-mediated clot retraction that normally occurs

in wound healing (Marieb, 2001b).

It has been shown that a direct interaction between fibrin fibres and platelet

GPIIb/IIIa receptors allows activated platelets to develop contractile forces on fibrin

with the microfilaments in their pseudopodia. These forces condense the fibrin

network, leading to clot retraction to approximately 1/10 of its original volume and

thereby expelling the entrapped the fluid content to the surrounding milieu

(Morgenstern et al., 1984, Katori et al., 2005). Hence, our observations of different

fibrin structures in clots formed on various surfaces may affect clot retraction,

resulting in a different extent of clot content expulsion. This phenomenon is also

supported by Carr and Zekert (1994), which reported an altered fibrin structure

affecting clot retraction.

The higher density of fibrin found throughout the clots of 65MMA when

compared to 65EMA and 65BMA may lead to a stronger retractile force and a higher

extent of clot content expulsion, correlating with the higher level of PDGF-AB

released from the clots of 65MMA. Furthermore, the clots of 65MMA which

released the highest amount of PDGF-AB in serum among all material-coated

surfaces might also be associated with its highest density of fibrin at the clot exterior

which retracts the clot very strongly. Similarly, a significantly lower level of PDGF-

AB released by 55MMA than 55EMA and 55BMA might be due to a lower fibrin

density found in the clots of 55MMA which retracts weakly. These findings suggest

that the surface functionalities and relative ratios influence the release of PDGF-AB

from intact clots through modification of fibrin network density.

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During clot lysis, the amount of PDGF-AB release also appeared to be

dependent on clot retraction. We found that the intact clots of 55MMA which

released the least amount of growth factor showed a burst release of PDGF-AB

during clot lysis, reaffirming a lower fibrin density associated with a weak clot

retraction retains PDGF-AB entrapped. Moreover, the increased release of growth

factor from the clots of 55MMA correlated well with its increased fibrinolytic rate

over time. For other intact clots which previously released a considerable amount of

PDGF-AB, they generally showed a reduced release during clot lysis. Only the intact

clots of 65MMA which released the highest amount of PDGF-AB continued to

release a significantly higher amount compared to 65EMA and 65BMA in early lysis

period, suggesting the clots of 65MMA originally has a higher content of growth

factor.

On the other hand, we found that the release of TGF-β1 from degrading

clots increased gradually over time. The amount of TGF-β1 released during lysis

seems to correlate with fibrin thickness.

We found that the degrading clots of 65BMA released more TGF-β1

compared to 65EMA up to 4 h of lysis. This is associated with the significantly

thicker fibrin of 65BMA than 65EMA at both clot exterior and interior. Moreover,

among BMA surfaces, although the clots of 65BMA showed the most dense and the

thickest fibres at the clot exterior associating with the slowest fibrinolysis at early

lysis, it also released an amount of TGF-β1 as high as that of 55BMA, which

underwent a faster fibrinolysis due to its thin fibres at the exterior. This suggests that

the fibrin thickness has a major influence on the release amount of TGF-β1.

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In fact, fibrin fibres has been characterised to bind a range of growth factors

and adhesive proteins by FXIII-crosslinking, such as FGF-2, VEGF and fibronectin

(Standeven et al., 2005, Weisel, 2005, Mosesson, 2005). Fibronectin has been shown

to bind TGF- β1 with high affinity (Mooradian et al., 1989). Also, TGF- β1 binds

thrombospondin-1 (Murphy-Ullrich et al., 1992, Crawford et al., 1998, Blair and

Flaumenhaft, 2009), which in turn interacts directly with fibronectin,

fibrinogen/fibrin and plasminogen (Wencel-Drake et al., 1985, Panetti et al., 1999).

Hence, these findings imply that TGF- β1 is potentially bound to fibrin fibres. With

the assumption that the amount of TGF- β1 bound to fibrin is directly proportional to

the fibrin thickness, and the fact that fibrin fibres are transversally cleaved rather

than uniformly around during fibrinolysis (Veklich et al., 1998, Collet et al., 2000),

this might explain the correlation between the increased fibrin thickness and the

increased amount of TGF-β1 detected during lysis, and why no significant

differences were seen in the release of TGF-β1 from intact clots.

Taken together, our results showed that more growth factors were released

during clot formation than during clot lysis. From the intact clots, the release of free

growth factor (i.e. PDGF-AB) seems to be strongly influenced by fibrin density,

which alters clot retraction. On the contrary, this influence appeared to be less

significant on the release of fibrin-bound growth factor (i.e. TGF-β1). Instead, during

clot lysis, the release of TGF-β1 is more likely to be associated with the fibrin

thickness while the release of PDGF-AB appeared to be generally associated with the

fibrinolytic rate. Our findings suggest the clots formed on material-coated surfaces

serve as a natural system as a haematoma to provide localised and substantial

releases of growth factors.

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4.4 CONCLUSION

By using an incubation vial where the inner surface was coated with

material, the effect of surface chemical functionalities –COOH/–CH3, –CH2CH3 or –

(CH2)3CH3 and their ratios on whole blood response was studied in a three-

dimensional manner and in the context of clot formation.

The surface functionalities and their ratios showed a modulatory effect on

coagulation response. Surfaces with –COOH/–(CH2)3CH3) induced a faster rate of

coagulation response compared to –COOH/–CH3 and –CH2CH3 regardless of the –

COOH ratios. An increase in –COOH ratio on –COOH/–CH3 and –CH2CH3

surfaces decreased the rate of initiation. However, all material-coated surfaces

resulted in clot formation. All coated surfaces markedly reduced complement

response when compared to uncoated glass surfaces. An entirely similar pattern of

coagulation and complement response was observed on material-coated surfaces. All

coated surfaces resulted in thicker fibrin with a tighter network at the clot exterior

when compared to uncoated glass surfaces. For all surfaces, the interior of clots

showed thicker fibres with loose network when compared to the clot exterior.

Surfaces presenting same –COOH ratio but different alkyl groups showed a

consistent trend in fibrin density throughout the clots. Material-coated surfaces

produced more rigid clots with significantly slower onset of fibrinolysis when

compared to uncoated glass surfaces, which is consistent with the thicker fibres and

tighter network observed on the clot exterior of coated surfaces. Similarly, the

significant differences in fibrinolytic rate of coated surfaces after 1 h of lysis

correlated well with the surface-dependent differences in fibrin thickness and density

at clot exterior. Generally, more PDGF-AB and TGF-β1 were released during clot

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formation than during clot lysis. From an intact clot, 65MMA (40% –COOH/60%–

CH3) released the highest amount of PDGF-AB compared to those of other material

surfaces.

To our best knowledge, this is the first study which provides a more

comprehensive picture of how surface functional groups and their concentrations

considerably modulate blood cascade activation in the context of whole blood clot

formation; subsequent fibrin architecture, clot rigidity, susceptibility to fibrinolysis

and growth factor entrapping/release ability of the modified clots. Further studies are

required to determine whether these changes in fibrin clot structure and function

have any therapeutic relevance to bone regeneration.

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5Chapter 5: A Pilot Study of the Osteogenic

Properties of Ex vivo Blood Clots Formed on

Materials in a Rabbit Femoral Defect

5.1 INTRODUCTION

A blood clot formed at the injured bone is vital in initiating the natural bone

healing response (Tsiridis et al., 2006, Tosounidis et al., 2009). During the

implantation of synthetic bone implants, disruption of bone vasculature and tissue

injury causes blood contact with the implant and ultimately the formation of a peri-

implant clot (Anderson, 2001, Gorbet and Sefton, 2004). Despite its resemblance to

the clot formed normally on injured bone, the structure and properties of peri-implant

clots have not been studied for their effect on the healing capacity of artificial bone

implants. Rather, most work in bone tissue engineering focuses on using synthetic

scaffolds containing osteogenic factors or in combined with PRP gels (Sánchez et al.,

2003, Grageda, 2004, Stevens, 2008, Schliephake, 2009).

Results of the previous chapters have shown that surface functionalities and

their relative compositions on biomaterials can modulate the structural properties of

whole blood clots including fibrin architecture and rigidity, as well as biological

properties such as susceptibility to fibrinolysis and the release of PDGF-AB and

TGF-β1 from clots during intact and degrading stages.

To validate the concept that the blood clots formed on surface coatings

provide the essential microenvironment for bone healing, and that changes in clot

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structure and properties have any clinical implications, a rabbit femoral defect model

was established in this pilot study. In particular, an autologous clot formed on

65MMA surface (40% –COOH/60% –CH3) was employed. From our previous

results, compared to other surface chemistry, this combination of surface

functionalities triggered lower complement activation while also initiating

coagulation response, resulting in the formation of a clot. Moreover, the resultant

clots contained the highest density of thin fibrin fibres, resulting in a high clot

rigidity and increased resistance to fibrinolysis. Moreover, the highest amount of

PDGF-AB was released upon clot formation, and considerable amounts of PDGF-

AB and TGF-β1 were also released during clot lysis.

Ultraporous beta tricalcium phosphate (β-TCP) is a ceramic that has been

widely used as bone fillers in dentistry and in orthopaedics. Its similar chemical

composition to bone precursors, high biocompatibility, mechanical properties and

porous structure are believed to favour bone ingrowth by preferentially promoting

infiltration of cells, vascularisation, mineralisation and resorption for bone recovery

(Johnson et al., 1996, Annaz et al., 2004, Giannoudis et al., 2005, Walsh et al., 2008,

Van Lieshout et al., 2011).

In this chapter, we aimed to confirm the idea that a blood clot generated on

a controlled surface provides a beneficial microenvironment for bone regeneration

when compared to commercial β-TCP bone graft substitute or an empty defect. By

using a rabbit femoral defect model, the in vivo osteogenic potentials of different

treatments were assessed by the extents of calcification, new bone formation,

chondrogenesis as well as vascularisation and inflammation.

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5.2 MATERIALS & METHODS

5.2.1 Preparation of coatings on scaffold surfaces

Stainless steel mesh scaffolds were fashioned with an arm on each side of it

(Figure 5-1). The arms of scaffolds were used to clip around the femur bone for implant

anchorage. The scaffolds were sterilized by soaking in 70% ethanol for 10 min and dried

in a laminar flow hood. The scaffolds were then dip-coated with material solutions and

air-dried. This procedure was repeated three times to achieve multiple coating and

present surface functionalities as on glass surfaces. A visible film was formed across the

centre of the scaffolds. After drying, the film was punched with a hole with a sterile

needle (21G, 0.8x38 mm; TERUMO corporation). This assures clotting over the coated

surface area. The coated scaffolds were kept in tubes in which the internal surfaces were

also coated with the same type of material by solvent evaporation. This was done to

ensure a similar functionalised surface area to blood volume ratio as used in the previous

experiments.

Figure 5-1. Stainless steel scaffold coated with material solution. Arrow pointing to a hole in the middle of film formed across the scaffold. An arm was on each side of the scaffold.

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5.2.2 Animals

Experiments were performed in a total of three New Zealand white rabbits (6-8

month old, 3.9 - 4.6kg) of which the animal ethics were approved by the QUT Animal

Ethics Committee. All animals were housed in the Prince Charles Hospital animal

facility. General anaesthesia was induced after premedication with an intramuscular

injection of atrophine (0.04 mg/kg; AstraZeneca, Australia), by a slow intravenous

injection of ketamine (35mg/kg; Ketalar, Hospira Australia Pty Ltd., Australia) and

midazolam (2.5 mg/kg; Sandoz Pty Ltd., Australia). During the surgery ketamine and

xylazine (5 mg/kg; Troy Laboratories Pty Ltd., Australia) were used in maintenance of

the anaesthesia. After 4 weeks, animals were euthanized by intraperitoneal injection of

1.5 mL sodium pentobarbital. This method of euthanasia was in accordance with

National Health and Medical Research Council of Australia guidelines.

5.2.3 Ex vivo blood clot formation

To prepare ex vivo blood clots on the coated scaffolds, autologous blood was

collected from the marginal ear vein or artery of animals undergoing surgery (Figure 5-2

a). After the rabbits were anaesthetized, fur covering the ear was shaved and the ear was

sterilized with 4 % Chlorhexidine. Whole blood of 4 mL was taken from the tip of the

ear and immediately transferred to the tubes containing the scaffolds (Figure 5-2 b-c).

The tubes were incubated in a water bath at 37˚C for approximate 90-120 min whilst the

defects on the femur were induced.

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5.2.4 Surgical procedures in rabbit femur

An incision was made longitudinally along the midline of the shaved skin

area over the femur. Underlying musculature was dissected to expose the shaft.

Defects of diameter 3.2 mm were made using a bur driven by a drill. Care was taken

during drilling to avoid deep penetration into the marrow cavity. Excessive blood

was removed regularly from the defect sites.

Three defects were made on the femur approximately 1 cm apart and one

femur was operated for each animal (Figure 5-2 d). The defect holes were treated in

the following three ways: ex vivo blood clots formed on scaffolds, ChronOSTM

porous β-TCP granules (60% porosity, pore width 100-500µm, size of granules 0.7-

1.4 mm; Synthes GmbH, Australia) or Surgicel (NU-KNIT, absorbable haemostat,

oxidized regenerated cellulose, sterile; ETHICON, INC., Australia) (Table 5-1). For

the implantation of ex vivo blood clots on scaffolds, the clots were ensured to fill the

defect holes and secured to the position by clipping the arms of scaffolds around the

femur (Figure 5-2 e). Surgicel was served as negative control as it achieved

haemostasis but it prevented formation of complete clots in the defects. After the

implantations, the musculature and skin were closed by suture. Antibiotic therapy

(Alamycin, 200mg/mL; Norbook Laboratories, Australia) and analgesics

(Buprenorphine 324 µg/mL; Reckitt Benckiser Ltd, New Zealand) were

administrated in the immediate postoperative period. The animals were sacrificed

after 4 weeks.

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Figure 5-2. Implantation of ex vivo blood clots formed on material-coated scaffolds in rabbit femoral defects. a) Autologous blood was collected from the marginal ear vein or artery; b) Material-coated scaffold was placed in a tube in which the internal surfaces was coated by the same type of material solution. c) Collected blood was immediately transferred to the tube and incubated at 37˚C. d) Three defects were made on the femur by drilling, and e) clot-embedded scaffold was placed on the defect with clot filling the hole.

Treatment Groups

Ex vivo blood clots on scaffolds (65MMA clots)

ChronOSTM β-TCP granules

Surgicel

Table 5-1. Three treatment groups in the animal study.

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5.2.5 Examination of defects

5.2.5.1 Assessment of calcification by micro-computered tomography

Harvested femurs were fixed with 10% buffered formalin for one week.

Overlying tissue and stainless steel scaffolds were removed after preliminary

examination of the femur with a micro-CT scanner (μCT 40, Scanco Medical,

Switzerland). Cross sectional scans of the femurs were then performed and three

dimensional (3D) images were reconstructed from the scans using the micro-CT system

software package. To evaluate in vivo calcification in the defect areas, the total volume

and the average density against hydroxyapaptite (HA) of calcification within the defect

areas were measured and recorded for statistical analysis. Defect areas were defined by

selecting the area of interest between the lesion edges on each micro-CT slice.

5.2.5.2 Assessment of new bone formation by haematoxylin and eosin staining

After micro-CT analysis, the femurs were segmented according to the defect

sites and decalcified in PBS buffered 10% EDTA (Ethylenediamine tetraacetic acid

Di-sodium salt; AJAX Finechem, Australia). Decalcified samples were washed with

PBS and dehydrated in ethanol as previously described. A midline cut was performed

longitudinally on the defect area on each segment prior to paraffin embedding.

Embedded samples were sectioned at 5 µm on the sagittal plane with a microtome

(Leica Microsystems GmbH, Germany). Sections near the central sagittal plane were

used for all examinations. Paraffin sections were dewaxed three times in xylene for 3

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min intervals, and rehydrated through 100%, 90% and 70% ethanol and distilled

water for 3 min intervals.

For haematoxylin and eosin (H & E) staining, sections were stained with

Mayer’s Haematoxylin (HD Scientific Supplies Pty Ltd., Australia) for 2 min and

excessive stain was rinsed away with running water. After dehydration through 70%,

90% and 100% ethanol the sections were exposed to Eosin stain (HD Scientific

Supplies Pty Ltd., Australia) for 15 s. Excessive stain was removed by dipping in

100% ethanol. Sections were then processed 3 times in xylene for 3 min intervals and

mounted with DePex mounting medium (VWR International Ltd., England). De novo

bone formation in the defect sites was observed under a microscope (Carl Zeiss Inc.,

USA) and the images were captured. Area of new bone tissue per mm2 was measured

in five random fields using AxioVision software.

5.2.5.3 Assessment of chondrogenesis by Alcian Blue staining

Dewaxed and rehydrated sections were first stained with 3% acetic acid for 3

min followed with Alcian blue solution (1% alcian blue in 3 % acetic acid, Fronine

Laboratory Supplies, Australia) for 1 h. After rinsing excessive stain, the sections were

stained with Nuclear fast red (Fronine Laboratory Supplies, Australia) for 5 min and

excessive stain was rinsed. The sections were dehydrated, rinsed with xylene, and

mounted as previously described before observed under the microscopy (Carl Zeiss Inc.,

USA).

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5.2.5.4 Assessment of new bone formation by immunohistochemistry

To identify osteogenic cells in bone newly formed in the defects, an alkaline

phosphatase (ALP) antibody (mouse anti-rat; Santa Cruz Biotechnology Inc., USA) was

used in immunohistochemical staining. Briefly, following dewaxing and rehydration of

the sections, endogenous peroxidise activity of the tissue was quenched by incubating

with 3% H2O2 for 20 min. The sections were blocked with 10% donkey serum (Sigma-

Aldrich) in PBS-Triton at room temperature for 1 h. The blocking solution was aspirated

and ALP primary antibody (1:100) in fresh block solution was added to the section for

overnight at 4˚C. After that, the sections were washed once in PBS-Triton for 3 min and

twice in PBS for 3 min intervals. Biotinylated donkey anti-mouse secondary antibody

(1:200) was then added to the sections for 20 min at room temperature. After washing,

horseradish peroxidase-conjugated avidin-biotin complex (ABC) was added to the

sections for 20 min with excessive complex washed. Antibody bindings were visualised

by the addition of diaminobenzidine (DAB) substrate solution for 3 min. Development

of brown colour was stopped by rinsing the sections with running water. The sections

were counterstained with Mayer’s haematoxylin (HD Scientific Supplies Pty Ltd.,

Australia) for 15 s followed by dehydration, xylene rinsing and mounting. Images were

taken with a microscope (Carl Zeiss Inc., USA).

5.2.5.5 Assessment of vascularisation and inflammation by immunofluorescence

To evaluate the vascularisation and inflammation in the defects, von

Willebrand factor (vWF) antibody (mouse anti-human; Upstate Cell Signaling

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Solution, Australia) and CD68 antibody (mouse anti-rat; Abcam, Australia) were

used to identify the endothelial cells and macrophages, respectively. In brief,

following dewaxing, rehydration and blocking the sections were incubated with vWF

(1:200) or CD68 (1:200) primary antibody at 4˚C overnight. After washing, the

sections were incubated with Alexa Fluor® 488 labelled donkey anti-mouse

secondary antibodies (1:200; Molecular Probes, Australia) overnight at room

temperature. Washed sections were mounted with Vectashield mounting medium

(Vector Laboratories, USA) and observed under Zeiss Z1 ApoTome fluorescence

microscope (Carl Zeiss Inc., USA). Images were captured and the number of vWF+

or CD68+ cells per mm2 were quantified from six random fields using AxioVision

software. Antibodies used in this study were summarized in Table 5-2.

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Primary Antibody Stain For Company Dilution

Mouse anti-rat alkaline phosphatase (ALP)

Osteogenic cells Osteoblasts

Santa Cruz Biotechnology

Inc 1/100

Mouse anti-human von Willebrand factor (vWF) Endothelial cells

Upstate Cell Signaling Solution

1/200

Mouse anti-rat CD68 (CD68) Monocytes/macrophages Abcam 1/200

Secondary Antibody Colour Company Dilution

Biotinylated donkey-anti mouse

Brown Santa Cruz

Biotechnology Inc

1/100

Alexa Fluor ® 488 donkey anti-mouse Green

Molecular Probes 1/200

Table 5-2. Primary and secondary antibodies used in immunohistochemistry and immunofluorescence studies.


Analysis was performed using SigmaPlot (version 11.0; Systat software Inc).

All data were analysed using one-way analysis of variance (ANOVA) for group

differences. The significance level was set at p ≤ 0.05.

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5.3 RESULTS

5.3.1 Micro-CT analysis for calcification in the defects

Segments of defects treated with ex vivo blood clots formed on 65MMA-coated

scaffolds, ChronOSTM granules or Surgicel were shown in Figure 5-3 a-c. Based on

general observation, Surgicel showed very limited healing in the defect compared to

others as demonstrated by red tissue fragments in the defects (Figure 5-3 a-c). Micro-CT

scanning and 3D reconstruction of images were performed to calculate the total volume

and average density of the calcified areas within the defects. In Figure 5-3 d-f, the

defects treated with 65MMA clots or ChronOSTM granules showed significantly higher

volume of calcified areas than that with Surgicel, in which hardly calcification was

found (p ≤ 0.001). However, no significant differences were found between 65MMA

clots and ChronOSTM granules (p=0.0557) (Figure 5-3 g). In addition, no significant

differences were found in the average density of calcified areas among all defects

(p=0.221) (Figure 5-3 h). This was likely due to the radio-opaque nature of ChronOSTM

granules.

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Figure 5-3. Micro-CT scanning analysis on the femoral defects. Representative pictures of segmented femoral defects filled with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel. The 3D reconstruction images of the defect sites corresponding to the treatments a-c) were shown as d-f). g) Measurement of the volume of calcified area in defect sites showed that treatments with 65MMA clots and ChronOSTM porous β-TCP granules resulted in a significantly higher volume of calcified area than with Surgicel, while there was no significant differences in average density among different treatments. Data was presented as the mean ± SD from three measurements. * p ≤ 0.001; Scale =1 mm

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5.3.2 Histological examination of de novo bone formation with H & E staining

The bone regeneration in defects evaluated by H&E staining differed

profoundly. More trabecular bone was formed and bridged the gap in the defects

treated with 65MMA clots (Figure 5-4 a). Much less reparative bone spicules were

identified in the defects treated with ChronOSTM granules. In the space of the

spicules, the myeloid elements of bone marrow were seen as bluish patches. The

positions of ChronOSTM granules that were removed during decalcification were

readily identifiable by the irregular empty gaps around spicules (Figure 5-4 b). The

defects treated with Surgicel remained empty. Bone healing response was restricted

to the defect edges (Figure 5-4 c).

The area of new bone tissues per mm2 in the defects was measured (Figure

5-4 d-f). The defects with 65MMA clots showed a significantly higher density of de

novo bone tissues than other treatments (Figure 5-4 g; p ≤ 0.001). These findings

suggested that the 65MMA clots led to formation of more reparative bones as a result

of faster bone remodelling.

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Figure 5-4. De novo bone formation in the defects shown by H&E staining. Representative images of defects treated with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel (40x) (Scale=500µm). d-f) A higher magnification (100x) views of area seen in the box of a-c) respectively (Scale=200µm). g) Measurement of the de novo bone formation in the defects showed a significantly higher area of new bone per mm2 was formed in the defects treated with 65MMA clots compared to ChronOSTM granules and Surgicel; Data was presented as the average area ± SD from five measurements. * p ≤ 0.001

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5.3.3 Histological examination of chondrogenesis in the defects

The extent of chondrogenesis during new bone formation in the defects was

illustrated by Alcian blue staining (Figure 5-5 a-c). Chondrogenesis was stronger and

more extensive in the defects treated with 65MMA clots than other treatments. Zones of

hypertrophic chondrocytes were found in the top centre of the defects (Figure 5-5 f). The

edges of newly formed trabecular bone and bone spicules in the defects treated with

65MMA clots and ChronOSTM granules also showed some positive blue staining,

indicating the remnants of cartilage. Such chondrogenesic response was not seen in the

defects treated with Surgicel (Figure 5-5 d-g). Overall, these results indicated that the de

novo bone regeneration in defects treated with 65MMA clots and ChronOSTM granules

occurred through cycles of chondrocytes hypertrophy as in natural bone healing.

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Figure 5-5. Chondrogenesis in the defects shown by Alcian Blue staining. Representative images of defects treated with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel (40x) (Scale=500µm). d-g) A higher magnification (100x) views of area seen in the box of a-c) (Scale=200µm). f) Zones of hypertrophic chondrocytes were observed in the defects with 65MMA clots, indicating a stronger response of chondrogenesis than ChronOSTM granules and Surgicel.

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5.3.4 Histological examinations of ALP expression

ALP staining was performed to identify osteoblasts within the defect sites

(Figure 5-6 a-c). In the defects with 65MMA clots, a greater abundance of ALP+

osteoblasts were lining the periphery of trabecular bone and zones of hypertrophic

chondrocytes when compared to those with ChronOSTM granules, in which less ALP+

osteoblasts were around the bone spicules (Figure 5-6 d-f). No ALP+ expression was

observed in the defects with Surgicel (Figure 5-6 g). Consistent with more newly formed

bone shown by H&E staining, these results further confirmed osteogenesis was

remarkably enhanced by 65MMA clots than other treatments.

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Figure 5-6. Osteoblasts in the defect shown by ALP staining. Representative images of defects treated with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel (40x) (Scale=500µm). d-g) A higher magnification (100x) views of area seen in the box of a-c) (Scale=200µm). More ALP+ osteoblasts were shown surrounding the newly formed bone in the defects treated with d) 65MMA clots than e) ChronOSTM granules. f) ALP+ osteoblasts were also found lining the periphery of zones of hypertrophic chondrocytes in the defects with 65MMA clots, confirming an enhanced osteogenesis response. g) No ALP+ expression was detected in the defects with Surgicel.

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5.3.5 Vascularisation revealed by vWF

To determine the vascularisation of the defect sites after treatment, vWF+

endothelial cells and blood vessels were counted using immunofluorescence. vWF+

endothelial cells and blood vessels appeared as circular structure were identified in

the defects with 65MMA clots (Figure 5-7 a, d, g) and ChronOSTM granules (Figure

5-7, b, e, h). Unexpectedly, aggregates of activated platelets which display vWF were

also seen in defects with Surgicel (Figure 5-7 c, f, i). Quantitative analysis showed

that 65MMA clots resulted in a significantly higher density of vWF+ endothelial cells

and blood vessels than ChronOSTM granules (p ≤ 0.001), indicating a higher degree

of vascularisation in the defects. No obvious vascularisation was found in the defects

with Surgicel (Figure 5-7 j).

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Figure 5-7. Vascularisation of the defects shown by vWF staining. Representative immunofluorescence images stained with von Willibrand factor (vWF, green) and nucleus (DAPI, blue) of defects treated with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel (100x) (Scale =200µm). Images at d-f) 200x (scale = 100 µm) and g-i) 400x (scale = 50 µm) magnification for a-c), respectively. vWF+ endothelial cells and blood vessels were seen (pointed by red arrows) in defects treated with g) 65MMA clots and h) ChronOSTM granules. i) No obvious vascularisation was seen in defects treated with Surgicel. Only clusters of activated platelets were positively stained with vWF. j) Measurement of the number of vWF+ endothelial cells and blood vessels per mm2 showed that a significantly higher degree of vascularisation was found in defects treated with 65MMA clots compared to ChronOSTM granules and Surgicel. Data was represented as the mean ± SD from six measurements. * p ≤ 0.001

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5.3.6 Inflammation response revealed by CD68

The extent of inflammation response induced by different implants was

determined by counting macrophages identified by marker CD68. The defects treated

with ChronOSTM granules showed significantly more CD68+ macrophages that were

merged together (red arrows in Figure 5-8 b, e, h) when compared to those with

65MMA clots (Figure 5-8 a, d, g) and Surgicel (Figure 5-8 c, f, i) (p ≤ 0.001). Given

that there was hardly or if any bone formation within the defects treated with

Surgicel, the presence of 65MMA clots resulted in a significantly lower degree of

inflammation response with bone formation compared to ChronOSTM granules

(Figure 5-8 j).

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Figure 5-8. Inflammatory response evaluated by the number of macrophages at the defects using CD68 staining. Representative immunofluorescence images stained with CD68 antibody (green) and nucleus (DAPI, blue) of defects treated with a) an ex vivo blood clot formed on 65MMA-coated scaffold; b) ChronOSTM granules; c) Surgicel (100x) (Scale =200µm). Images at d-f) 200x (scale = 100 µm) and g-i) 400x (scale = 50 µm) magnification for a-c), respectively. j) Measurement of the number of CD68+ macrophages (red arrow) per mm2 showed that a significantly higher density of macrophages was found in defects treated with ChronOSTM granules than with 65MMA clots and Surgicel. Data was represented as the mean ± SD from six measurements. * p ≤ 0.001

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5.4 DISCUSSION

In this proof of concept study, we have demonstrated that ex vivo blood

clots formed on 65MMA-coated scaffolds significantly enhanced bone regeneration

through normal healing process. The 65MMA clots led to increased new bone

formation, stronger chondrogenesis and more osteoblasts activity compared to the

commercial bone substitute: ChronOSTM β-TCP granules. Moreover, we did observe

a beneficial effect of 65MMA clots on increasing vascularisation while reducing

inflammatory response compared to other treatments. When blood clot formation

was disrupted by the presence of Surgicel, the defects remained empty after 4 weeks.

This affirmed that a blood clot provides the vital microenvironment for bone healing.

We observed faster and abundant woven bone ingrowth in defects treated

with 65MMA clots verse less bone formation with bone spicules in defects filled

with ChronOSTM granules. Moreover, it was showed that ChronOSTM granules

resulted in some empty cavities around the bone spicules whereas Micro-CT

scanning showed calcification as high as 65MMA clots. These findings indicate that

ChronOSTM granules (60% porosity) remained present at 4 weeks after implantation.

The superiority of 65MMA clots over ChronOSTM granules in accelerating bone

regeneration is probably related to its fibrin structure and growth factor contents.

In a rabbit tibial defect, ChronOSTM granules were found to be present up to

26 weeks and its slower resorption was associated with decreased new bone

formation compared to other β-TCP granules with same chemical composition but a

faster resorption profile (Walsh et al., 2008). Reduced porosity and interconnection

of β-TCP granules were demonstrated to correlate with decreased granule

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degradation and subsequent bone formation (Hing et al., 2005, Knabe et al., 2008).

On the contrary, we previously showed that 65MMA clots contain a heterogeneous

fibrin network structure, suggesting the clots would be relatively porous and

interconnected. Moreover, 65MMA clots which are physically rigid and resistant to

early fibrinolysis would therefore provide a stable osteoconductive scaffold for

infiltration of osteoprogenitor cells into the cavity. In addition, the interior of

65MMA clots are less dense in fibrin, which is a fully resorbable biological matrices.

This suggests that 65MMA clots would be readily dissolved in vivo as implicated by

our previous lysis study when compared to ChronOSTM granules. Hence, the

differences in implant porosity and interconnectivity in relation to in vivo resorption

profile likely explains the faster and higher quantity of bone formation with 65MMA

clots whereas ChronOSTM granules appeared to lag behind due to its long-lasting that

potentially limited new bone ingrowth.

On the other hand, ChronOSTM β-TCP granules, like all ceramic scaffolds,

does not have any osteoinductive properties (Hing, 2005, Hak, 2007). In contrast, a

blood clot is well characterised with its inherent chemotactic, angiogenic and

osteogenic factors (Street et al., 2000, Sánchez et al., 2003, Frechette et al., 2005,

Blair and Flaumenhaft, 2009). We have previously revealed that of the clots

generated in vitro, 65MMA clots released the highest level of PDGF-AB and

considerable amount of TGF-β1 upon clot formation than clots generated on other

surfaces. Substantial release of both growth factors were also observed during clot

lysis.

In fact, the ability of PDGF-AB and TGF-β1 to promote bone healing has

been demonstrated in many animal studies (Lind et al., 1996, Geiser et al., 1998,

Vehof et al., 2002, Ehrhart et al., 2005). In the presence of artificial implants, PDGF-

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AB has also been shown to play a crucial role in bone healing. An increased release

of PDGF-AB as a result of enhanced platelet activation on titanium are believed to

be attributed to the enhanced osteogenic properties of titanium (Hong et al., 1999).

As PDGF-AB contributes to half of the mitogenic activity for mesenchymal cells

(Andrae et al., 2008), PDGF-AB released from 65MMA clots may contribute to the

extensive chondrogenic differentiation observed in the defects compared to

ChronOSTM granules by stimulating mitogenesis of bone marrow stem cells and

mesenchymal cells from periosteum of broken bone ends. Moreover, the stronger

chondrogenesic response with 65MMA clots indicates that these clots are capable to

direct normal bone healing sequence similar to a physiological haematoma, to

endochondral ossification through a cartilaginous stage. Furthermore, the higher

number of vWF+ endothelial cells and vessel-like structures present in the defects

with 65MMA clots compared to ChronOSTM granules may be resulted from the

chemotactic and angiogenic properties of PDGF-AB on fibroblasts, smooth muscle

cells and endothelial cells (Lieberman et al., 2002c, Carano and Filvaroff, 2003).

For TGF-β1, its major mechanism on enhancing bone healing is achieved by

mediating chemotaxis, mitogenesis and differentiation of osteoblasts precursor at the

injured sites. This may be attributed to more osteoblasts found in the defects with

65MMA clots than ChronOSTM granules (Hosgood, 1993, Oprea et al., 2003, Celotti

et al., 2006, Bosetti et al., 2007). Moreover, the potential of TGF- β1 in enhancing

implant osteointegration has also been demonstrated on titanium surfaces through

stimulating differentiation and mineralization of human osteoblasts on the surfaces

(Zhang et al., 2005). Given that PDGF-AB acts synergistically with TGF-β1 to

promote osteoblasts growth (Kells et al., 1995, Lieberman et al., 2002b), and

thrombin generated from coagulation activation is able to stimulate osteoblasts

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proliferation (Frost et al., 1999), it is conceivable that the defects filled with entirely

65MMA clots would present more residual thrombin and growth factors for

osteoblast activity than that with ChronOSTM granules. Therefore, our demonstration

of altered structure and growth factor release from 65MMA clots may contribute to

the advanced osteogenesis and angiogenesis in the defects compared to ChronOSTM

granules.

In term of inflammatory response, less CD68+ macrophages were found in

the defects filled with 65MMA clots than with ChronOSTM β-TCP granules. Study of

Hing et al. (2007) indicated the dissolution of β-TCP granules releases degradation

products which subsequently provoked an inflammatory response in rabbit

osteochondral defects. In an analogous investigation by Arvidsson et al. (2011) they

also found that calcium phosphate compound did increase inflammatory response.

Furthermore, using a rat subcutaneous model, Jansson et al. (2002) suggested that

cells at the titanium surface with a plasma clot layer were in a different stage than

those without clot and that these cells were not in a phagocytotic phase as shown by

reduced production of reactive oxygen. It is proposed that the presence of a plasma

clot-coated surface will likely preserve the radical-mediated degradation or killing

capacity for a longer period than those without the clot. Furthermore, Barbosa et al.

(2004, 2005) who used a rat air pouch model to study surface functionalities on in

situ inflammatory response found that –CH3 SAMs recruited similar number of

inflammatory cells as –OH SAMs, but most of the cells were neutrophils and only a

very low density of cells adherent to the surface in contrast to –OH SAMs where

most of the cells were monocytes and a higher density of adherent cells were

detected. These results indicated that the –CH3 groups likely induce neutrophils-

dominated local acute inflammation but is not associated with a significant leukocyte

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adhesion to implant. In line with this, we previously found that 65MMA surfaces

induces a low extent of complement response. This may be accounted for the acute

inflammation which is necessary in normal healing process but not a significant

chronic inflammation up to 4 weeks when compared to ChronOSTM β-TCP granules.

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5.5 CONCLUSION

Using a rabbit femoral defect model, we demonstrated that the ex vivo

65MMA clots provide the essential microenvironment for new bone formation. The

implantation of 65MMA clots led to advances in vascularisation, chondrogenesis,

osteoblasts activity and increased new bone formation compared to ChronOSTM β-

TCP granules. The impact of surface functionalities and their ratios on modulating

ultimate fibrin clot structure and growth factor release were likely contributed to

these differences through mediating infiltrations and activities of reparative cells in

the defect. Moreover, we observed a lower extent of inflammatory response with

65MMA clots compared to ChronOSTM granules, which were probably associated

with lower complement activation in vitro upon clot formation, as well as particulate

products released upon granules degradation. These findings emphasize that 65MMA

serves as a prothrombogenic and immunocompatible surface that alters structural and

biological properties of a consequent clot, which in turn did impact on new bone

regeneration in vivo.

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Chapter 6: General Discussion and Future

Directions

6.1 GENERAL DISCUSSION

It is commonly accepted that the formation of a blood clot at injured bones

is the first step in bone healing process. During bone implantation, blood contacts

with biomaterials and subsequently the formation of a clot around the implant occurs

prior to bone regeneration. Despite both situations being similar in forming a blood

clot at the defect site, there has been no attempt in controlling the biomaterial-blood

interactions and clot formation, such that the result may be beneficial in bone healing

approaches. It is theoretically suggested that applications of PRP gels could improve

de novo bone formation due to its increased contents of growth factors (Roukis et al.,

2006, Griffin et al., 2009, Cenni et al., 2010). While this was borne out in preclinical

experimentation, effective translation to the clinic has been unsuccessful. There has

been limited work addressing the fact that PRP gels contain different cell populations

(i.e. platelets only), and fibrin structure dramatically altered from a normal

haematoma, not to mention any assessment of such differences related to conflicting

effect of PRP gels on bone healing. Indeed, changes in the cellular content and fibrin

structure of a peri-implant clot can greatly affect osteoconduction, which is a vital

phase of peri-implant endosseous healing (Davies, 2003b). The clot architecture

which depends on thrombin concentration plays an important role in various

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thrombotic disorders considering its impact on clot viscoelasticity and degradability

(Collet et al., 2000, 2006, Mills et al., 2002, Wolberg and Campbell, 2008).

In addition to the issues with integration and bone formation, in vivo

function of many implants are often compromised by the blood-based immune

response leading to FBR (Anderson et al., 2008). As such, a control of blood-

biomaterial interactions by modifying biomaterial surface properties has been the

focus in developing an improved biocompatible device. Surface roughness,

hydrophobicity and chemical composition have been shown to influence blood

protein adsorption, cellular interactions, and subsequent immune complement

responses to biomaterials (Albrektsson and Wennerberg, 2004, Anderson et al.,

2004a, Ma et al., 2007, Thevenot et al., 2008, Nilsson et al., 2009). In particular, it

was demonstrated that surface presenting mixtures of –COOH and –CH3 functional

groups at varied ratios modulated profoundly coagulation and complement

activations, as well as extent of fibrin deposition on the surfaces, compared to the

surfaces with only either groups (Sperling et al., 2005a, 2009, Fischer et al., 2010b).

Furthermore, it has been shown that different chain length of alkyl groups provides a

further level to regulate these biomaterial-blood interactions (Berglin et al., 2004,

2009). Theoretically, these factors could modulate healing events and consequently

the extent of new bone formation in injured site. Hence, the rationale of this study

was to create surfaces with –COOH/–CH3, –CH2CH3 or –(CH2)3CH3 functionalities

at an optimal ratio to promote coagulation activation, produce a blood clot with

appropriate cellular content and structure properties while diminish adverse immune

response to the implants, and thus improve the new bone formation in the presence of

synthetic bone grafts.

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To scrutinise this hypothesis, we first fabricated a series of materials

composed of acrylic acid and alkyl methacrylate (MMA, EMA or BMA)) to create a

prothrombogenic and immunocompatible coating of artificial bone implants (Chapter

3). Unlike SAMs on gold substrate that is not feasible clinically, the materials used in

this work have been specifically selected due to their biological relevance. For

instance, PMMA and AA have been widely used as bone cement and protein-

immobilising agent, respectively (Kang et al., 1993, Lu, 2004). The resultant

materials are also shown to be non-toxic while possessing desired functionalities

(Yan and Gemeinhart, 2005). Our results clearly indicated that the inner surface of

incubation vial was modified effectively by material coating. Such coatings based on

materials can be adapted to most of the current biomedical materials without

affecting bulk properties. Our strategy of varying the types and mole fractions of

comonomers in forming materials was ascribed to different surface functional

groups: –COOH/–CH3, –CH2CH3 or –(CH2)3CH3) at different compositions. An

increase in AA proportion generally increased the surface content of –COOH groups

but the content of –COOH groups was lower than the expected AA fraction. Similar

observations were also seen in other studies and might be attributed to different

degrees of copolymerisation, polymer chain mobility and functional group

reorientation (Gupta et al., 2002, Berglin et al., 2009, Xu et al., 2009, Hermitte et al.,

2004, Ozcan and Hasirci, 2007).

Our results demonstrated that surface hydrophobicity of coated surfaces

correlates well with the chemical compositions, as in accordance to the literature

(Tsyganov et al., 2005, Ukiwe et al., 2005, Lai et al., 2006). At relatively the same –

COOH ratios, surfaces presenting –(CH2)3CH3) groups exhibited a higher

hydrophobicity than –CH3 and –CH2CH3 groups whereas the latter two did not differ

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significantly. This suggests that both the surface functional groups and their relative

compositions synergistically influence surface hydrophobicity (Tsai et al., 2007).

Moderate hydrophobicity of our surfaces is consistent with what is commonly

suggested to be the optimal than extremely hydrophilic or hydrophobic surfaces as

they support protein adsorption with preserved conformation (Hermitte et al., 2004).

Moreover, we observed that the material coatings were as smooth as

uncoated surfaces and without apparent cracks. The average surface roughness was

in nano-scale in which material coating (3.99 ± 0.54 nm) was slightly higher than

uncoated glass surfaces (2.22 ± 0.29 nm). Recently, in vivo studies have shown that

nano-scale roughness enhanced early stage of healing but micro-scale roughness

improved overall bone-implant contact and bone density of peri-implant endosseous

healing (Telleman et al., 2010, de Barros et al., 2011). Microstructure has also been

demonstrated to greatly increase fibrinogen adsorption, subsequent conformation

changes and platelet responses compared to sub-micron structure (Xie et al., 2009,

Koh et al., 2010a, 2010b). Another studies of Ferraz et al. (2008, 2010) demonstrated

that complement activation was stronger on surface with 200 nm pore sizes rather

than 20 nm whereas within this nano-range platelet response was influenced

differently over time. So far, it remains largely unknown whether nano-scale

roughness as low as 2-4 nm would have any impact on protein adsorption or cellular

interaction. Our results showed no significant differences in average roughness

among surfaces with same functionalities at different –COOH ratios nor among

surfaces with different alkyl groups but the same –COOH ratios. This suggests the

surface functional groups and their relative ratios do not influence the surface

roughness. Hence, these results imply that any difference in blood response is less

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likely a result of different surface roughness but surface functionalities and relative

compositions.

To link the surface chemical formulation of the material coating and blood

response, we carried out a comprehensive investigation of whole blood interaction on

the customised incubation vials (Chapter 4). The uses of the incubation vials and

whole blood were designed to mimic closely the formation of three-dimensional peri-

implant clots during surgery, allow blood cascades to cross-talk and avoid the impact

of anticoagulant in interrupting the measurement of blood response.

The prothrombogenic properties of surfaces was assessed by the rate of

coagulation initiation. Surfaces presenting –COOH/–(CH2)3CH3) groups showed a

faster rate of coagulation activation compared to those with –COOH/–CH3 and –

CH2CH3 groups, regardless of the –COOH ratios. Specifically, increasing –COOH

ratios on surfaces with –COOH/–CH3 and –CH2CH3 decrease the rate of the

activation. Analysis of complement activation revealed that all material-coated

surfaces induced a weaker response compared to uncoated surfaces, clearly

indicating these surfaces had a reduced immunogenic property. In addition, the

complement response followed an entirely similar pattern of surface-activated

coagulation, suggesting our in vitro incubation system allows an interaction between

these two cascades to take place as it is found in vivo (Markiewski et al., 2007,

Peerschke et al., 2008, Amara et al., 2010). This also reflects the acute inflammation

caused by the implanted biomaterials will occur naturally following thrombotic

events.

Examination of resultant blood clots showed that the material-coated

surfaces modulated the fibrin architecture resulting in a thicker fibre at tighter

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configuration at the clot exterior when compared to uncoated glass surfaces. In

addition, our observation of the clot interior revealed that fibrin architectures were

also different from the exterior for all surfaces in which fibres were thicker and with

very loose network. Differences in fibre thickness and density was also detected on

material-coated surfaces, reaffirming the specific effects of surface functionalities

and their relative ratios on controlling the kinetic of coagulation initiation and

subsequent fibrin structure throughout the clots. The heterogeneous morphology of

fibrin structures was also noted in other studies and was attributed to surface

functionalities-dependent differences in pattern of thrombin generation leading to

different kinetics of fibrinopeptides cleavage and fibrin polymerization (Geer et al.,

2007, Wolberg and Campbell, 2008), as well as different procoagulant potential of

entrapped cells in the clots in organising the fibrin bundles (Gugutkov et al., 2011).

To verify in vivo stability of the altered clot structure which is important for

physical support at the injured sites and subsequent new bone ingrowth, we measured

the clot rigidity and the rate of clot lysis. Material-coated surfaces were shown to

change the clot mechanical properties leading to more rigid clots than those formed

on uncoated glass surfaces. Also, it was shown that all coated surfaces resulted in a

slower initial fibrinolysis (i.e. first hour after lysis) when compared to uncoated glass

surfaces. These findings were consistent with the tighter network and thicker fibrin

observed on the clot exterior on material-coated surfaces. Similarly, surface-

dependent differences in fibrin thickness and density at the clot exterior were also in

good agreement with the difference in initial fibrinolytic rates observed among

coated surfaces. These results suggests that the material-coated surface modulates the

clot susceptibility to fibrinolysis by changing fibrin architectures in the clots.

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As an assessment of biological function of the altered clots for enhancing

bone regeneration, we measured the content of PDGF-AB and TGF-1 released upon

clot formation and lysis. Both growth factors were released in higher levels during

clot formation than during clot lysis. Our results revealed that from an intact clot the

release of PDGF-AB correlated with the fibrin density at the clot exterior and

interior, resulting in the highest level of PDGF-AB being released from clots formed

on 65MMA (40% –COOH/60% –CH3). The correlation of PDGF-AB released upon

clot formation with the surface-dependent difference in fibrin density is likely due to

fibrin-mediated clot retraction which expels the content entrapped within the clots

(Morgenstern et al., 1984, Carr and Zekert, 1994). During lysis, however, the release

of PDGF-AB seemed to correlate with fibrinolytic rate, whereas the release of TGF-

β1 was likely influenced by the surface-dependent fibrin thickness, implicating TGF-

β1 was fibrin-bound and PDGF-AB was soluble within the clots (Mooradian et al.,

1989, Murphy-Ullrich et al., 1992). Overall, these results suggested that the growth

factor release of the clots was modulated by surface-dependent fibrin architecture

and fibrinolysis. Next, we questioned whether such modification of resultant clots

have any functional impact in bone healing. This would implicate whether the clots

generated in vitro on material-coated surfaces can form the basis of truly therapeutic

agents.

To answer the question above, we implanted ex vivo 65MMA clots into the

rabbit femoral defect model and analysed after 4 weeks (Chapter 5). Implanting

65MMA clots into the defects led to significantly stronger chondrogenesis, increased

new bone formation, more osteoblasts activity as well as increased vascularisation

when compared to ChronOSTM porous β-TCP granules. Unlike β-TCP granules

which is known to be devoid of osteoinductive properties, 65MMA clots which was

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previously shown to release a high amount of growth factors such as PDGF-AB and

TGF-β1 were likely to be responsible for the enhanced osteogenesis and

angiogenesis. In contrast, negative control with Surgicel which inhibits blood clot

formation resulted in a empty defect, indicating the clots formed on material-coated

surfaces provide the essential healing microenvironment as normal haematoma does.

Previous studies indicated that the slow in vivo resolution profile associated with

ChronOSTM granules resulted in significantly lower amount of new bone formation

(Walsh et al., 2008, Hing, 2005, Knabe et al., 2008). This implies that the advance in

bone formation with 65MMA clots are possibly attributed to a better fibrin porous

structure and faster degradation profile compared to ChronOSTM granules. In

particular, 65MMA clots resulted in less macrophages in the defects compared to

ChronOSTM β-TCP granules over the time course of study. This affirms our previous

demonstration of 65MMA surfaces which had less immunogenic effect as it induced

a low extent of complement activation in vitro and did not show a potential in

promoting chronic inflammation nor FBR in vivo. In contrast to normal implant

coating which would be broken down by proteolytic enzymes from neutrophils in

initial inflammation and expose underlying synthetic materials that would evoke

FBR, our strategy of pre-formed blood clots around material-coated scaffolds could

completely hide the materials, mimic ECM for cell recognition and be relatively

resistant to premature enzymatic degradation.

Altogether, this project opens the new scope of blood clots generated on

various surface functionalities for treating severe bone injuries. Our studies on the

effect of surface chemistry on blood clots were initiated based on the notion that the

normal mechanism of bone healing could be useful for enhancing the healing

microenvironment in the presence of synthetic bone implants. As far as we can

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ascertain, this is the first study that demonstrates systematically surface functional

groups and their relative ratios on material coatings modulate initiation of blood

cascades in the context of whole blood clot formation; and subsequent fibrin

architecture, clot rigidity, susceptibility to lysis and growth factor entrapping/release.

Importantly, these clots generated on material-coated surfaces are in many ways

comparable to the natural haematoma, in both structural and functional aspects. The

provision of such pre-formed blood clots may recreate the healing microenvironment

and serve as therapeutic agents for improved bone regeneration.

Page 191


6.2 FUTURE DIRECTIONS

The present study raises many questions of phenomenological differences

observed and an extension of this study is to unravel the underlying mechanisms of

differences in clot structure on various compositions of implant surfaces. First question

is how different combinations of surface functional groups at varied ratios affects the

early mechanism of blood coagulation resulting in different fibrin thickness and density

as detected. Hence, future studies will apply circular dichroism spectropolarimetry to

investigate the dynamic interactions between surfaces and adsorbed protein in term of

surface coverage and conformational change. Furthermore, the blood transition of flow

to stasis will be characterised by using the technique of thromboelastography (TEG),

which records the overall coagulation profile, the time of initial fibrin formation, overall

clot strength and the dynamic properties between fibrin and platelet bonding via GP

IIb/IIIa (Lai et al., 2010).

In addition, the clinical implications of our material-coated surfaces on

supporting bone healing, apart from 65MMA, remain to be further defined. A larger

scale of animal study with critical sized defects is required to clarify any therapeutic

differences in the clots generated on various material surfaces, in order to establish the

corresponding surface chemical formulation for the development of a truly

prothrombogenic and immunocompatible synthetic bone grafts.

Page 192


6.3 GENERAL CONCLUSION

In summary, this thesis has demonstrated that materials composed of AA

and MMA, EMA or BMA at varied mole fraction provided solid coatings displaying

–COOH/–CH3, –CH2CH3 or –(CH2)3CH3 functionalities at varied ratios. Although

the material-coated surfaces yielded a lower content of –COOH groups compared to

the AA fraction fed in the material, the measured surface hydrophobicity correlated

well with the corresponding chemical composition. Surface functionalities and their

relative ratios on material coatings had no effect on surface average roughness. The

roughness of material-coated surfaces (3.99 ± 0.54 nm) was higher than uncoated

glass surfaces (2.22 ± 0.29 nm). Upon whole blood contact with coated surfaces on

incubation vials, the COOH/–(CH2)3CH3 functionalities significantly increased the

rate of coagulation initiation than other functionalities at all ratios. All material-

coated surfaces significantly reduced the complement activation than uncoated glass

surfaces. The similar pattern of material surface-mediated complement and

coagulation activation suggests that there is interaction of the two cascades.

Moreover, all material-coated surfaces produced clots with thicker fibrins and tighter

network at the exterior than uncoated glass surfaces, while the clot interior of all

surfaces contained thicker fibrins with very loose network than the clot exterior. This

affirms coated surfaces control the kinetics of coagulation initiation and alter fibrin

architectures in the clots. Material-coated surfaces resulted in more rigid clots with a

significantly slower onset of fibrinolysis than that of uncoated glass surfaces,

indicating the material coatings change clot mechanical property and stability.

Significant difference in fibrinolytic rate after 1 h of lysis among coated surfaces

were consistent to the surface-dependent differences in fibrin thickness and density at

Page 193


clot exterior, suggesting the surface chemistry influences clot susceptibility to lysis

by modifying the clot fibrin architectures. PDGF-AB and TGF-β1 were released in

greater amounts during clot formation than during clot lysis. In addition, the amount

of PDGF-AB released from intact clots correlated with the fibrin density at both

exterior and interior of the clots. During clot lysis, the release of PDGF-AB was

influenced by fibrinolytic rate whereas that of TGF-β1 was associated with the

surface-dependent fibrin thickness. Furthermore, in a rabbit femoral defect, the

implantation of ex vivo clots generated on 65MMA surfaces (40% –COOH/60% –

CH3) significantly increased vascularisation, chondrogenesis, osteoblast activity and

new bone formation but reduced chronic inflammatory response when compared to

ChronOSTM β-TCP granules. The empty cavities in defects treated with Surgicel

which inhibits proper clot formation versus abundant new bone ingrowth in defects

filled with 65MMA clots indicated that the blood clots provide the vital healing

microenvironment for bone regeneration. Overall, this study has emphasized the

important role of surface functionalities and their relative ratios on controlling the

initiations of blood cascades, the structural properties of resultant clots and the

ultimate effect of the clots on bone healing. These results explore the future potential

of applying blood clot regulation by various material coatings to improve the

efficacy of synthetic bone grafts.

Bibliography Page 194


Bibliography

AKEDA, K., AN, H. S., OKUMA, M., ATTAWIA, M., MIYAMOTO, K., THONAR, E. J.-M. A., LENZ, M. E., SAH, R. L. & MASUDA, K. 2006. Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis Osteoarthritis and Cartilage, 14, 1272-1280.

ALBREKTSSON, T. & WENNERBERG, A. 2004. Oral implant surfaces: Part 1--review focusing on topographic and chemical properties of different surfaces and in vivo responses to them. Int J Prosthodont, 17, 536-43.

ALEMAN, M. M., GARDINER, C., HARRISON, P. & WOLBERG, A. S. 2011. Differential contributions of monocyte- and platelet-derived microparticles towards thrombin generation and fibrin formation and stability. J Thromb Haemost.

ALEXANDER, M. R., WHITTLE, J. D., BARTON, D. & SHORT, R. D. 2004. Plasma polymer chemical gradients for evaluation of surface reactivity: epoxide reaction with carboxylic acid surface groups. Journal of Materials Chemistry, 14, 408-412.

ALKHAMIS, T. M., BEISSINGER, R. L. & CHEDIAK, J. R. 1988. Red blood cell effect on platelet adhesion and aggregation in low-stress shear flow: myth or fact? ASAIO Journal, 34, 868-873.

ALLEN, G. A., WOLBERG, A. S., OLIVER, J. A., HOFFMAN, M., ROBERTS, H. R. & MONROE, D. M. 2004. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost, 2, 402-13.

ALLFORD, S. L. & MACHIN, S. J. 2007. Haemostasis. Surgery, 25, 241-244. ALTIERI, D. C. 1995. Inflammatory cell participation in coagulation. Seminars in Cell Biology,

6, 269-274. AMARA, U., FLIERL, M. A., RITTIRSCH, D., KLOS, A., CHEN, H. C., ACKER, B.,

BRÜCKNER, U. B., NILSSON, B., GEBHARD, F., LAMBRIS, J. D. & HUBER-LANG, M. 2010. Molecular intercommunication between the complement and coagulation systems. The Journal of Immunology, 185, 5628-5636.

AMARA, U., RITTIRSCH, D., FLIERL, M. A., BRUCKNER, U., KLOS, A., GEBHARD, F., LAMBRIS, J. D. & HUBER-LANG, M. 2008. Interaction between the coagulation and complement system. Advances in experimental medicine and biology, 632, 71-79.

AMINI, A. R., WALLACE, J. S. & NUKAVARAPU, S. P. 2011. Short-term and long-term effects of orthopedic biodegradable implants. Journal of Long-Term Effects of Medical Implants, 21, 93-122.

ANDERSON, A. B., DALLMIER, A. W., CHUDZIK, S. J., DURAN, L. W., GUIRE, P. E., HERGENROTHER, R. W., LODHI, M. A., NOVAK, A. E., OFSTEAD, R. F. & WORMUTH, K. 2004a. Technologies for the surface modification of biomaterials. In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthropedics. New York: Marcel Dekker, Inc.

ANDERSON, J. M. 2001. Biological responses to materials. Annual Reviews of Materials Resources, 31, 81-110.

ANDERSON, J. M., COOK, G., COSTERTON, B., HANSON, S. R., PETTERSEN, A. H., JACOBSEN, N., JOHNSON, R. J., MITCHELL, R. N., PASMORE, M., SCHOEN, F. J., SHIRTLIFF, M. & STOODLEY, P. 2004b. Host reactions to biomaterial and their evaluation. In: RATNER, B. D., HOFFMAN, A. S. & SCHOEN, F. S. (eds.) Biomaterial science: an introduction to materials in medicine. 2nd ed. New York: Elsevier Academic Press.



ANDERSON, J. M., RODRIGUEZ, A. & CHANG, D. T. 2008. Foreign body reaction to biomaterials. Seminars in Immunology, 20, 86-100.

ANDRAE, J., GALLINI, R. & BETSHOLTZ, C. 2008. Role of platelet-derived growth factors in physiology and medicine. Genes & Development, 22, 1276-1312.

ANDREWS, R. K. & BERNDT, M. C. 2004. Platelet physiology and thrombosis. Thromb Res, 114, 447-53.

ANITUA, E., ANDIA, I., ARDANZA, B., NURDEN, P. & NURDEN, A. T. 2004. Autologous platelets as a source of proteins for healing and tissue regeneration. Thrombosis and Haemostasis, 91, 4-15.

ANITUA, E., SÁNCHEZ, M., NURDEN, A. T., NURDEN, P., ORIVE, G. & ANDÍA, I. 2006. New insights into and novel applications for platelet-rich fibrin therapies. Trends in Biotechnology, 24, 227-234.

ANNAZ, B., HING, K. A., KAYSER, M., BUCKLAND, T. & DI SILVIO, L. 2004. Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study. J Microsc, 215, 100-10.

ARIMA, Y. & IWATA, H. 2007. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials, 28, 3074-3082.

ARPORNMAEKLONG, P., KOCHEL, M., DEPPRICH, R., KÜBLER, N. R. & WÜRZLER, K. K. 2003. Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. International Journal of Oral and Maxillofacial Surgery, 33, 60-70.

ARVIDSSON, A., MALMBERG, P., KJELLIN, P., CURRIE, F., ARVIDSSON, M. & FRANKE STENPORT, V. 2011. Early interactions between leukocytes and three different potentially bioactive titanium surface modifications. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 97B, 364-372.

ASHCROFT, G. S. 1999. Bidirectional regulation of macrophage function by TGF-[beta]. Microbes and Infection, 1, 1275-1282.

ASIJEE, G. M., MUSZBEK, L., KAPPELMAYER, J., POLGAR, J., HORVATH, A. & STURK, A. 1988. Platelet vinculin: a substrate of activated factor XIII. Biochim Biophys Acta, 954, 303-8.

AVRAMOGLOU, D. L., ANAGNOSTOU, F., BIZIOS, R. & PETITE, H. 2005. Engineering bone: challenges and obstacles. Journal of Cell and Molecular Medicine, 9, 72-84.

AWAD, H. A., ZHANG, X., REYNOLDS, D. G., GULDBERG, R. E., O'KEEFE, R. J. & SCHWARZ, E. M. 2007. Recent advances in gene delivery for structural bone allografts. Tissue Eng, 13, 1973-85.

BACH-GANSMO, E. T., GODAL, H. C. & SKJONSBERG, O. H. 1998. Degradation of fibrinogen and cross-linked fibrin by human neutrophil elastase generates D-like fragments detected by ELISA but not latex D-dimer test. Thromb Res, 92, 125-34.

BÄCK, J., HUBER LANG, M., ELGUE, G., KALBITZ, M., SANCHEZ, J., NILSSON EKDAHL, K. & NILSSON, B. 2009. Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces. Biomaterials, 30, 6573-6580.

BÄCK, J., SANCHEZ, J., ELGUE, G., EKDAHL, K. N. & NILSSON, B. 2010. Activated human platelets induce factor XIIa-mediated contact activation. Biochemical and Biophysical Research Communications, 391, 11-17.

BAEYENS, W., GLINEUR, R. & EVRARD, L. 2010. The use of platelet concentrates: platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in bone reconstruction prior to dental implant surgery. Rev Med Brux, 31, 521-527.

BALTZER, A. W. A. & LIEBERMAN, J. R. 2004. Regional gene therapy to enhance bone repair. Gene Therapy, 11, 344-350.

BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2003. Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces. J Biomed Mater Res A, 65, 429-34.



BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2004. Inflammatory responses and cell adhesion to self-assembled monolayers of alkanethiolates on gold. Biomaterials, 25, 2557-63.

BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2005. Inflammatory cell recruitment and adhesion to methyl-terminated self-assembled monolayers: effect of implantation time. Microsc Res Tech, 66, 37-42.

BARBOSA, J. N., MARTINS, M. C., FREITAS, S. C., GONCALVES, I. C., AGUAS, A. P. & BARBOSA, M. A. 2010. Adhesion of human leukocytes on mixtures of hydroxyl- and methyl-terminated self-assembled monolayers: effect of blood protein adsorption. J Biomed Mater Res A, 93, 12-9.

BAUER, T. W. & MUSCHLER, G. F. 2000. Bone graft materials. An overview of the basic science. Clin Orthop Relat Res, 10-27.

BENNETT, J. S. 2001. Platelet-fibrinogen interactions. Ann N Y Acad Sci, 936, 340-54. BENNETT, J. S., BERGER, B. W. & BILLINGS, P. C. 2009. The structure and function of

platelet integrins. J Thromb Haemost, 7 Suppl 1, 200-5. BERGLIN, M., ANDERSSON, M., SELLBORN, A. & ELWING, H. 2004. The effect of

substrate molecular mobility on surface induced immune complement activation and blood plasma coagulation. Biomaterials, 25, 4581-4590.

BERGLIN, M., PINORI, E., SELLBORN, A., ANDERSSON, M., HULANDER, M. & ELWING, H. 2009. Fibrinogen adsorption and conformational change on model polymers: Novel aspects of mutual molecular rearrangement. Langmuir, 25, 5602-5608.

BERRY, L. R. & CHAN, A. K. C. 2008. Improving blood compatibility of biomaterials using a novel antithrombin-heparin covalent complex. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

BETZ, O. B., BETZ, V. M., ABDULAZIM, A., PENZKOFER, R., SCHMITT, B., SCHRODER, C., MAYER-WAGNER, S., AUGAT, P., JANSSON, V. & MULLER, P. E. 2010. The repair of critical-sized bone defects using expedited, autologous BMP-2 gene-activated fat implants. Tissue Eng Part A, 16, 1093-101.

BHASIN, N., ARIENS, R. A., WEST, R. M., PARRY, D. J., GRANT, P. J. & SCOTT, D. J. 2008. Altered fibrin clot structure and function in the healthy first-degree relatives of subjects with intermittent claudication. J Vasc Surg, 48, 1497-503, 1503 e1.

BIMMEL, R. & GOVAERS, K. 2006. Does harvesting of iliac bone grafts with an acetabular reamer reduce complication rate? Acta Orthop Belg, 72, 598-602.

BLAIR, P. & FLAUMENHAFT, R. 2009. Platelet [alpha]-granules: basic biology and clinical correlates. Blood Reviews, 23, 177-189.

BLOCKMANS, D., DECKMYN, H. & VERMYLEN, J. 1995. Platelet activation. Blood Reviews, 9, 143-156.

BLOM, A. 2007. (V) Which scaffold for which application? Current Orthopaedics, 21, 280-287. BLOMBACK, B. 2000. Fibrin formation in whole blood. Thromb Res, 99, 307-10. BLOMBACK, B. & BARK, N. 2004. Fibrinopeptides and fibrin gel structure. Biophys Chem,

112, 147-51. BOSETTI, M., BOCCAFOSCHI, F., LEIGHEB, M. & CANNAS, M. F. 2007. Effect of

different growth factors on human osteoblasts activities: a possible application in bone regeneration for tissue engineering. Biomolecular Engineering, 24, 613-618.

BOUCHARD, B. A. & TRACY, P. B. 2001. Platelets, leukocytes, and coagulation. Current Opinion in Hematology, 8, 263-269.

BREITBART, A. S., GRANDE, D. A., KESSLER, R., RYABY, J. T., FITZSIMMONS, R. J. & GRANT, R. T. 1998. Tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg, 101, 567-74; discussion 575-6.

BROBERG, M., ERIKSSON, C. & NYGREN, H. 2002. GPIIb/IIIa is the main receptor for initial platelet adhesion to glass and titanium surfaces in contact with whole blood. Journal of Laboratory and Clinical Medicine, 139, 163-172.

BRUMMEL, K. E., PARADIS, S. G., BUTENAS, S. & MANN, K. G. 2002. Thrombin functions during tissue factor-induced blood coagulation Blood, 100, 148-152.



BURG, K. J. L., PORTER, S. & KELLAM, J. F. 2000. Biomaterial developments for bone tissue engineering. Biomaterials, 21, 2347-2359.

BUTLER, M. J. & SEFTON, M. V. 2007. Poly(butyl methacrylate-co-methacrylic acid) tissue engineering scaffold with pro-angiogenic potential in vivo. J Biomed Mater Res A, 82, 265-73.

CAMPBELL, R. A., OVERMYER, K. A., BAGNELL, C. R. & WOLBERG, A. S. 2008. Cellular procoagulant activity dictates clot structure and stability as a function of distance from the cell surface. Arterioscler Thromb Vasc Biol, 28, 2247-54.

CAMPBELL, R. A., OVERMYER, K. A., SELZMAN, C. H., SHERIDAN, B. C. & WOLBERG, A. S. 2009. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood, 114, 4886-96.

CAPUTO, A. E. 1999. Healing of bone and connective tissues. In: BRONNER, F. & WORRELL, R. V. (eds.) Structure and function of bone tissues. New York: CRC Press LLC.

CARANO, R. A. D. & FILVAROFF, E. H. 2003. Angiogenesis and bone repair. Drug Discovery Today, 8, 980-989.

CARLSON, N. E. & ROACH, R. B., JR. 2002. Platelet-rich plasma: clinical applications in dentistry. J Am Dent Assoc, 133, 1383-6.

CARLSSON, L. V., MACDONALD, W., JACOBSSON, C. M. & ALBREKTSSON, T. 2004. Osseointegration principles in orthopedics: basic research and clinical applications. In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthopedics. New York: Marcel Dekker, Inc.

CARR, M. E. & HARDIN, C. L. 1987. Fibrin has larger pores when formed in the presence of erythrocytes. American Journal of Physiology - Heart and Circulatory Physiology, 253, H1069-H1073.

CARR, M. E., JR., GABRIEL, D. A. & MCDONAGH, J. 1987. Influence of factor XIII and fibronectin on fibre size and density in thrombin-induced fibrin gels. J Lab Clin Med, 110, 747-52.

CARR, M. E., JR., MARTIN, E. J. & CARR, S. L. 2002a. Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation. Blood Coagul Fibrinolysis, 13, 193-7.

CARR, M. E., JR., MARTIN, E. J. & CARR, S. L. 2002b. Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation. Blood Coagulation & Fibrinolysis, 13, 193-197.

CARR, M. E., JR. & ZEKERT, S. L. 1994. Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma. Am J Physiol, 266, H1195-201.

CARRIERI, C., GALASSO, R., SEMERARO, F., AMMOLLO, C. T., SEMERARO, N. & COLUCCI, M. 2011. The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet-mediated fibrinolysis resistance: a thromboelastographic study in whole blood. Journal of Thrombosis and Haemostasis, 9, 154-162.

CELOTTI, F., COLCIAGO, A., NEGRI-CESI, P., PRAVETTONI, A., ZANINETTI, R. & SACCHI, M. C. 2006. Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-β. Wound Repair and Regeneration, 14, 195-202.

CENNI, E., CIAPETTI, G., GRANCHI, D., FOTIA, C., PERUT, F., GIUNTI, A. & BALDINI, N. 2009. Endothelial cells incubated with platelet-rich plasma express PDGF-B and ICAM-1 and induce bone marrow stromal cell migration. J Orthop Res, 27, 1493-8.

CENNI, E., SAVARINO, L., PERUT, F., FOTIA, C., AVNET, S. & SABBIONI, G. 2010. Background and rationale of platelet gel in orthopaedic surgery. Musculoskelet Surg, 94, 1-8.

CHEN, Q. Z., BRETCANU, O. & BOCCACCINI, A. R. 2008. Inorganic and composite bioactive scaffolds for bone tissue engineering. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.



CHEN, X., WANG, J., PASZTI, Z., WANG, F., SCHRAUBEN, J., TARABARA, V., SCHMAIER, A. & CHEN, Z. 2007. Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy. Analytical and Bioanalytical Chemistry, 388, 65-72.

CHOI, B.-H., IM, C.-J., HUH, J.-Y., SUH, J.-J. & LEE, S.-H. 2004. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. International Journal of Oral and Maxillofacial Surgery, 33, 56-59.

CHOI, B.-H., ZHU, S.-J., B.-Y., K., HUH, J.-Y., LEE, S.-H. & JUNG, J.-H. 2005. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. International Journal of Oral and Maxillofacial Surgery, 34, 420-424.

CHOUKROUN, J., DISS, A., SIMONPIERI, A., GIRARD, M. O., SCHOEFFLER, C., DOHAN, S. L., DOHAN, A. J. J., MOUHYI, J. & DOHAN EHRENFEST, D. M. 2006. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 101, e56-e60.

CHUANG, W. H. & LIN, J. C. 2007. Surface characterization and platelet adhesion studies for the mixed self-assembled monolayers with amine and carboxylic acid terminated functionalities. Journal of Biomedical Materials Research Part A, 82A, 820-830.

CLARK, R. A. 2001. Fibrin and wound healing. Ann N Y Acad Sci, 936, 355-67. COLLET, J. P., ALLALI, Y., LESTY, C., TANGUY, M. L., SILVAIN, J., ANKRI, A.,

BLANCHET, B., DUMAINE, R., GIANETTI, J., PAYOT, L., WEISEL, J. W. & MONTALESCOT, G. 2006. Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol, 26, 2567-73.

COLLET, J. P., MONTALESCOT, G., LESTY, C. & WEISEL, J. W. 2002. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circ Res, 90, 428-34.

COLLET, J. P., PARK, D., LESTY, C., SORIA, J., SORIA, C., MONTALESCOT, G. & WEISEL, J. W. 2000. Influence of fibrin network conformation and fibrin fibre diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol, 20, 1354-61.

COLLET, J. P., SHUMAN, H., LEDGER, R. E., LEE, S. & WEISEL, J. W. 2005. The elasticity of an individual fibrin fibre in a clot. Proc Natl Acad Sci U S A, 102, 9133-9137.

COLLET, J. P., SORIA, J., MIRSHAHI, M., HIRSCH, M., DAGONNET, F. B., CAEN, J. & SORIA, C. 1993a. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood, 82, 2462-9.

COLLET, J. P., SORIA, J., MIRSHAHI, M., HIRSCH, M., DAGONNET, F. B., CAEN, J. & SORIA, C. 1993b. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood, 82, 2462-2469.

COOPER, L. F. 2000. A role for surface topography in creating and maintaining bone at titanium endosseous implants. J Prosthet Dent, 84, 522-34.

COVE, R. & KEENAN, J. 2009. Tibial bone grafting for lateral tibial plateau fractures. Ann R Coll Surg Engl, 91, 268-9.

CRAWFORD, A. & HATTON, P. V. 2008. Cell-biomaterial interactions in skeletal tissue engineering. Biomedicine & Pharmacotherapy, 62, 493-494.

CRAWFORD, S. E., STELLMACH, V., MURPHY-ULLRICH, J. E., RIBEIRO, S. M. F., LAWLER, J., HYNES, R. O., BOIVIN, G. P. & BOUCK, N. 1998. Thrombospondin-1 is a major activator of TGF-b1 in vivo. Cell, 93, 1159-1170.

CROSS, K. J. & MUSTOE, T. A. 2003. Growth factors in wound healing. The Surgical clinics of North America, 83, 531-545.

DAI, J., RABIE, A. B., HAGG, U. & XU, R. 2004. Alternative gene therapy strategies for the repair of craniofacial bone defects. Curr Gene Ther, 4, 469-85.



DAVIE, E. W., FUJIKAWA, K. & KISIEL, W. 1991. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry, 30, 10363-10370.

DAVIES, J. E. 1998. Mechanisms of endosseous integration. The international journal of prosthodontics, 11, 391.

DAVIES, J. E. 2003a. Understanding peri-implant endosseous healing. Journal of dental education, 67, 932-949.

DAVIES, J. E. 2003b. Understanding peri-implant endosseous healing. Journal of dental education, 67, 932.

DAWSON, J. I. & OREFFO, R. O. 2008. Bridging the regeneration gap: stem cells, biomaterials and clinical translation in bone tissue engineering. Arch Biochem Biophys, 473, 124-31.

DE BARROS, R. R., NOVAES, A. B., JR., QUEIROZ, A. & DE ALMEIDA, A. L. 2011. Early peri-implant endosseous healing of two implant surfaces placed in surgically created circumferential defects. A histomorphometric and fluorescence study in dogs. Clin Oral Implants Res.

DEB, S. 2008. Synthetic and natural degradable polymeric biomaterials. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication amd processing handbook. New York: Taylor & Francis Group, LLC.

DEL CONDE, I., CRÚZ, M. A., ZHANG, H., LÓPEZ, J. A. & AFSHAR-KHARGHAN, V. 2005. Platelet activation leads to activation and propagation of the complement system. The Journal of Experimental Medicine, 201, 871-879.

DEUEL, T. F., SENIOR, R. M., CHANG, D., GRIFFIN, G. L., HEINRIKSON, R. L. & KAISER, E. T. 1981. Platelet factor 4 is chemotactic for neutrophils and monocytes. PNAS, 78, 4584-4587.

DI CERA, E. 2003. Thrombin interactions. Chest, 124, 11S-17S. DIMITRIOU, R., TSIRIDIS, E. & GIANNOUDIS, P. V. 2005. Current concepts of molecular

aspects of bone healing. Injury, 36, 1392-404. DOBLARE, M., GARCIA, J. M. & GOMEZ, M. J. 2004. Modelling bone tissue fracture and

healing: a review. Engineering Fracure Mechanics, 71, 1809-1840. DOHAN EHRENFEST, D. M., CHOUKROUN, J., DISS, A., DOHAN, S. L., DOHAN, A. J. J.,

MOUHYI, J. & GOGLY, B. 2006. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: leucocyte activation: a new feature for platelet concentrates? Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 101, e51-e55.

DOHAN EHRENFEST, D. M., DEL CORSO, M., INCHINGOLO, F., SAMMARTINO, G. & CHARRIER, J. B. 2010. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in human cell cultures: growth factor release and contradictory results. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 110, 418-21; author reply 421-2.

DOLDER, J. V. D., MOOREN, R., VLOON, A. P. G., STOELINGA, P. J. W. & JANSEN, J. A. 2006. Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells. Tissue Engineering, 12, 3067-3073.

DUBOIS, C., REIGNER, S. C., STEINER, B. & RIEDERER, M. A. 2004. Thrombin binding to GPIbalpha induces integrin alphaIIbbeta3 dependent platelet adhesion to fibrin in ex vivo flowing whole blood. Thromb Haemost, 91, 233-7.

DUGRILLON, A., EICHLER, H., KERN, S. & KLÜTER, H. 2002. Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration. The International Journal of Oral and Maxillofacial Surgery, 31, 615-619.

ECKHAUS, A. A., FISH, J. S., SKARJA, G., SEMPLE, J. L. & SEFTON, M. V. 2008. A preliminary study of the effect of poly(methacrylic acid-co-methyl methacrylate) beads on angiogenesis in rodent skin grafts and the quality of the panniculus carnosus. Plast Reconstr Surg, 122, 1361-70.

EHRHART, N. P., HONG, L., MORGAN, A. L., EURELL, J. A. & JAMISON, R. D. 2005. Effect of transforming growth factor-beta1 on bone regeneration in critical-sized bone defects after irradiation of host tissues. Am J Vet Res, 66, 1039-45.

ELALAMY, I., CHAKROUN, T., GEROTZIAFAS, G. T., PETROPOULOU, A., ROBERT, F., KARROUM, A., ELGRABLY, F., SAMAMA, M. M. & HATMI, M. 2007. Circulating



platelet-leukocyte aggregates: A marker of microvascular injury in diabetic patients. Thrombosis Research, In Press, Corrected Proof.

EPPLEY, B. L., WOODELL, J. E. & HIGGINS, J. 2004. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plastic and Reconstructive Surgery, 114, 1502-1508.

ERICKSON, L. A., R., S. R., NY, T. & LOSKUTOFF, D. J. 1985. The fibrinolytic system of the vascular wall. Clinics in Haematology, 14, 513-530.

ERIKSSON, C. & NYGREN, H. 2001. Polymorphonuclear leukocytes in coagulating whole blood recognize hydrophilic and hydrophobic titanium surfaces by different adhesion receptors and show different patterns of receptor expression. Journal of Laboratory and Clinical Medicine, 137, 296-302.

ERNOFSSON, M., THELIN, S. & SIEGBAHN, A. 1997. Monocyte tissue factor expression, cell activation and thrombin formation during cardiopulmonary bypass: a clinical study. The Journal of Thoracic and Cardiovascular Surgery, 113, 576-584.

ESKIN, S. G., HORBETT, T. A., MCINTIRE, L. V., MITCHELL, R. N., RATNER, B. D., SCHOEN, F. J. & YEE, A. 2004. Some background concepts. In: RATNER, B. D., HOFFMAN, A. S. & SCHOEN, F. S. (eds.) Biomaterial science: an introduction to materials in medicine. New York: Elsevier Academic Press.

ESMON, C. T. 1995. Introduction: cellular regulation of blood coagulation. Seminars in Cell Biology, 6, 257-258.

ESMON, C. T. 2003. Inflammation and thrombosis. Journal of Thromosis and Haemostasis, 1, 1343-1348.

ESMON, C. T. 2004. The impact of the inflammatory response on coagulation. Thrombosis Research, 114, 321-327.

EVANS-NGUYEN, K. M., FUIERER, R. R., FITCHETT, B. D., TOLLES, L. R., CONBOY, J. C. & SCHOENFISCH, M. H. 2006. Changes in adsorbed fibrinogen upon conversion to fibrin. Langmuir, 22, 5115-21.

EVANS-NGUYEN, K. M. & SCHOENFISCH, M. H. 2005a. Fibrin proliferation at model surfaces: influence of surface properties. Langmuir, 21, 1691-4.

EVANS-NGUYEN, K. M. & SCHOENFISCH, M. H. 2005b. Fibrin proliferation at model surfaces: influence of surface properties. Langmuir, 21, 1691-1694.

EVANS-NGUYEN, K. M., TOLLES, L. R., GORKUN, O. V., LORD, S. T. & SCHOENFISCH, M. H. 2005a. Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers. Biochemistry, 44, 15561-8.

EVANS-NGUYEN, K. M., TOLLES, L. R., GORKUN, O. V., LORD, S. T. & SCHOENFISCH, M. H. 2005b. Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers. Biochemistry, 44, 15561–15568.

FAUCHEUX, N., SCHWEISS, R., LÜTZOW, K., WERNER, C. & GROTH, T. 2004. Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies. Biomaterials, 25, 2721-2730.

FAXÄLV, L., EKBLAD, T., LIEDBERG, B. & LINDAHL, T. L. 2010. Blood compatibility of photografted hydrogel coatings. Acta Biomaterialia, 6, 2599-2608.

FERRAZ, N., NILSSON, B., HONG, J. & KARLSSON OTT, M. 2008. Nanoporesize affects complement activation. J Biomed Mater Res A, 87, 575-81.

FERRAZ, N., OTT, M. K. & HONG, J. 2010. Time sequence of blood activation by nanoporous alumina: studies on platelets and complement system. Microscopy Research and Technique, 73, 1101-1109.

FISCHER, M., SPERLING, C., TENGVALL, P. & WERNER, C. 2010a. The ability of surface characteristics of materials to trigger leukocyte tissue factor expression. Biomaterials, 31, 2498-2507.

FISCHER, M., SPERLING, C. & WERNER, C. 2010b. Synergistic effect of hydrophobic and anionic surface groups triggers blood coagulation in vitro. Journal of Materials Science: Materials in Medicine, 21, 931-937.



FRAYSSINET, P. 2004. Biocompatible materials: calcium phosphates in orthopedic surgery. In: POITOUT, D. G. (ed.) Biomechanics and biomaterials in orthopedics. Singapore: Springer-Verlag London Limited.

FRECHETTE, J.-P., MARTINEAU, I. & GAGNON, G. 2005. Platelet-rich plasmas: Growth factor content and roles in wound healing. Journal of Dental Research, 84, 434-439.

FROST, A., JONSSON, K. B., RIDEFELT, P., NILSSON, O., LJUNGHALL, S. & LJUNGGREN, O. 1999. Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation. Acta Orthop Scand, 70, 497-503.

FUJISHITA, S., INABA, C., ISHISAKA, T., GEMMEI-IDE, M. & KITANO, H. 2009. Effect of end groups of poly(n-butyl methacrylate) on its biocompatibility. Colloids and Surfaces B: Biointerfaces, 74, 45-50.

FUSHIMI, F., NAKAYAMA, M., NISHIMURA, K. & HIYOSHI, T. 1998. Platelet adhesion, contact phase coagulation activation, and C5a generation of polyethylene glycol acid-grafted high flux cellulosic membrane with varieties of grafting amounts. Artif Organs, 22, 821-6.

FUSS, C., PALMAZ, J. C. & SPRAGUE, E. A. 2001. Fibrinogen: structure, function, and surface interactions. Journal of Vascular and Interventional Radiology, 12, 677-682.

GABRIEL, D. A., MUGA, K. & BOOTHROYD, E. M. 1992. The effect of fibrin structure on fibrinolysis. Journal of Biological Chemistry, 267, 24259-24263.

GANDHI, A., DOUMAS, C., O'CONNOR, J. P., PARSONS, J. R. & LIN, S. S. 2006. The effects of local platelet rich plasma delivery on diabetic fracture healing. Bone, 38, 540-546.

GAROFALO, G. S. 2007. Autogenous, allogenetic and xenogenetic grafts for maxillary sinus elevation: literature review, current status and prospects. Minerva Stomatol, 56, 373-92.

GEER, C. B., RUS, I. A., LORD, S. T. & SCHOENFISCH, M. H. 2007. Surface-dependent fibrinopeptide A accessibility to thrombin. Acta Biomaterialia, 3, 663-668.

GEISER, A. G., ZENG, Q. Q., SATO, M., HELVERING, L. M., HIRANO, T. & TURNER, C. H. 1998. Decreased bone mass and bone elasticity in mice lacking the transforming growth factor-beta 1 gene. Bone, 23, 87-93.

GEMMELL, C. H. 1997. A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces. Journal of biomedical materials research, 37, 474-480.

GEMMELL, C. H. 2001. Activation of platelets by in vitro whole blood contact with materials: Increases in microparticle, procoagulant activity, and soluble P-selectin blood levels Journal of Biomaterials Science, Polymer Edition,, 12, 933-943.

GERBER, H. & FERRARA, N. 2000. Angiogenesis and bone growth. Trends in Cardiovascular Medicine, 10, 223-228.

GHEBREHIWET, B., RANDAZZO, B. P., DUNN, J. T., SILVERBERG, M. & KAPLAN, A. P. 1983. Mechanisms of activation of the classical pathway of complement by Hageman factor fragment. J Clin Invest, 71, 1450-6.

GHEBREHIWET, B., SILVERBERG, M. & KAPLAN, A. P. 1981. Activation of the classical pathway of complement by Hageman factor fragment. J Exp Med, 153, 665-76.

GIANNOUDIS, P. V., DINOPOULOS, H. & TSIRIDIS, E. 2005. Bone substitutes: an update. Injury, 36, S20-27.

GIBBINS, J. M. 2004. Platelet adhesion signalling and the regulation of thrombus formation. Journal of Cell Science, 117, 3415-3425.

GIMENO, F. L., GATTO, S., FERRO, J., CROXATTO, J. O. & GOLLA, J. E. 2006. Preparation of platelet-rich plasma as a tissue adhesive for experimental transplantation in rabbits. Thrombosis Journal, 4, 18-24.

GLOWACKI, J. 1998. Angiogenesis in fracture repair. Clinical Orthopaedics and Related Research, 355, S82.

GOBBI, G., MIRANDOLA, P., TAZZARI, P. L., RICCI, F., CAIMI, L., CACCHIOLI, A., PAPA, S., CONTE, R. & VITALE, M. 2003. Flow cytometry detection of serotonin content and release in resting and activated platelets. Br J Haematol, 121, 892-6.



GOLD, H. K., YASUDA, T., JANG, I. K., GUERRERO, J. L., FALLON, J. T., LEINBACH, R. C. & COLLEN, D. 1991. Animal models for arterial thrombolysis and prevention of reocclusion. Erythrocyte-rich versus platelet-rich thrombus. Circulation, 83, IV26-40.

GOLINO, P., ASHTON, J. H., BUJA, L. M., ROSOLOWSKY, M., TAYLOR, A. L., MCNATT, J., CAMPBELL, W. B. & WILLERSON, J. T. 1989. Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators. Circulation, 79, 154-66.

GORBET, M. B. & SEFTON, M. V. 2004. Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelet and leukocytes. Biomaterials, 25, 5681-5703.

GOULET, J. A., SENUNAS, L. E., DESILVA, G. L. & GREENFIELD, M. L. V. H. 1997. Autogenous iliac crest bone graft: complications and functional assessment. Clinical Orthopaedic & Related Research, 339, 76-81.

GRAGEDA, E. 2004. Platelet-rich plasma and bone graft materials: a review and a standardized research protocol. Implant Dentistry, 13, 301-309.

GREENBERG, C. S., HURSTING, M. J., MACIK, B. G., ORTEL, T. L., KANE, W. H. & MOORE, B. M. 1994. Evaluation of preanalytical variables associated with measurement of prothrombin fragment 1.2. Clin Chem, 40, 1962-9.

GRIFFIN, X. L., SMITH, C. M. & COSTA, M. L. 2009. The clinical use of platelet-rich plasma in the promotion of bone healing: a systematic review. Injury, 40, 158-162.

GUGUTKOV, D., GONZÁLEZ-GARCÍA, C., ALTANKOV, G. & SALMERÓN-SÁNCHEZ, M. 2011. Fibrinogen organization at the cell-material interface directs endothelial cell behavior. Journal of Bioactive and Compatible Polymers, 26, 375-387.

GUO, R. F. & WARD, P. A. 2005. Role of C5a in inflammatory responses. Annu. Rev. Immunol., 23, 821-852.

GUPTA, B., HILBORN, J. G., BISSON, I. & FREY, P. 2001. Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films. Journal of Applied Polymer Science, 81, 2993-3001.

GUPTA, B., PLUMMER, C., BISSON, I., FREY, P. & HILBORN, J. 2002. Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films: characterization and human smooth muscle cell growth on grafted films. Biomaterials, 23, 863-871.

HABIBOVIC, P. & DE GROOT, K. 2007. Osteoinductive biomaterials--properties and relevance in bone repair. J Tissue Eng Regen Med, 1, 25-32.

HAK, D. J. 2007. The use of osteoconductive bone graft substitutes in orthopaedic trauma. J Am Acad Orthop Surg, 15, 525-36.

HALLAB, J. N., URBAN, R. M. & JACOBS, J. J. 2004. Corrosion and biocompatibility of orthopedic implants In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthopedics. New York: Marcel Dekker, Inc.

HAMAD, O. A., EKDAHL, K. N., NILSSON, P. H., ANDERSSON, J., MAGOTTI, P., LAMBRIS, J. D. & NILSSON, B. 2008. Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets. J Thromb Haemost, 6, 1413-1421.

HAMAD, O. A., NILSSON, P. H., WOUTERS, D., LAMBRIS, J. D., EKDAHL, K. N. & NILSSON, B. 2010. Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1. The Journal of Immunology, 184, 2686-2692.

HANDT, S., JEROME, W. G., TIETZE, L. & HANTGAN, R. R. 1996. Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance. Blood, 87, 4204-13.

HANTGAN, R. R., HINDRIKS, G., TAYLOR, R. G., SIXMA, J. J. & DE GROOT, P. G. 1990. Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood. Blood, 76, 345-353.



HARWOOD, P. J., NEWMAN, J. B. & MICHAEL, A. L. R. 2010a. (ii) An update on fracture healing and non-union. 24, 9-23.

HARWOOD, P. J., NEWMAN, J. B. & MICHAEL, A. L. R. 2010b. (ii) An update on fracture healing and non-union. Orthopaedics and Trauma, 24, 9-23.

HE, S., CAO, H., ANTOVIC, A. & BLOMBACK, M. 2005. Modifications of flow measurement to determine fibrin gel permeability and the preliminary use in research and clinical materials. Blood Coagulation & Fibrinolysis, 16, 61-67.

HENCH, J. W. 2005a. Inflammation and wound healing. In: HENCH, L. L. & JONES, J. R. (eds.) Biomaterials, artificial organs and tissue engineering. New York: Woodhead Publishing Limited.

HENCH, L. L. 2005b. Repair of skeletal tissue. In: HENCH, L. L. & JONES, J. R. (eds.) Biomaterials, artifical organs and tissue engineering. New York: Woodhead Publishing Limited.

HERMITTE, L., THOMAS, F., BOUGARAN, R. & MARTELET, C. 2004. Contribution of the comonomers to the bulk and surface properties of methacrylate copolymers. Journal of Colloid and Interface Science, 272, 82-89.

HERTEL, R., EIJER, H., MEISSER, A., HAUKE, C. & PERREN, S. M. 2001. Biomechanical and biological considerations relating to the clinical use of the Point Contact-Fixator--evaluation of the device handling test in the treatment of diaphyseal fractures of the radius and/or ulna. Injury, 32 Suppl 2, B10-4.

HING, K. A. 2005. Bioceramic bone graft substitutes: influence of porosity and chemistry. International Journal of Applied Ceramic Technology, 2, 184-199.

HING, K. A., ANNAZ, B., SAEED, S., REVELL, P. A. & BUCKLAND, T. 2005. Microporosity enhances bioactivity of synthetic bone graft substitutes. J Mater Sci Mater Med, 16, 467-75.

HING, K. A., BEST, S. M., TANNER, K. E., BONFIELD, W. & REVELL, P. A. 2004. Mediation of bone ingrowth in porous hydroxyapaptite bone graft substitutes. Journal of Biomedical Materials Research. Part A., 68, 187-200.

HING, K. A., WILSON, L. F. & BUCKLAND, T. 2007. Comparative performance of three ceramic bone graft substitutes. Spine J, 7, 475-90.

HIRATA, I., HIOKI, Y., TODA, M., KITAZAWA, T., MURAKAMI, Y., KITANO, E., KITAMURA, H., IKADA, Y. & IWATA, H. 2003. Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance. J Biomed Mater Res A, 66, 669-76.

HOLLAND, C. K., VAIDYA, S. S., DATTA, S., COUSSIOS, C. C. & SHAW, G. J. 2008. Ultrasound-enhanced tissue plasminogen activator thrombolysis in an in vitro porcine clot model. Thromb Res, 121, 663-73.

HONG, J., ANDERSSON, J., EKDAHL, K. N., ELGUE, G., AXEN, N., LARSSON, R. & NILSSON, B. 1999. Titanium is a highly thrombogenic biomaterial: possible implications for osetogenesis. Thrombosis and Haemostasis, 82, 58-64.

HONG, J., AZENS, A., EKDAHL, K. N., GRANQVIST, C. G. & NILSSON, B. 2005. Material-specific thrombin generation following contact between metal surfaces and whole blood. Biomaterials, 26, 1397-1403.

HONG, J., LARSSON, A., EKDAHL, K. N., ELGUE, G., LARSSON, R. & NILSSON, B. 2001. Contact between a polymer and whole blood: sequence of events leading to thrombin generation. Journal of Laboratory and Clinical Medicine, 138, 139-145.

HORBETT, T. A. 1993. Chapter 13: Principles underlying the role of adsorbed plasma proteins in blood interactions with foreign materials. Cardiovascular Pathology, 2, 137-148.

HORNE, M. K., CULLINANE, A. M., MERRYMAN, P. K. & HODDESON, E. K. 2006. The effect of red blood cells on thrombin generation. British Journal of Haematology, 133, 403-408.

HOSGOOD, G. 1993. Wound healing: the role of platelet-derived growth factor and transforming growth factor beta. Veterinary Surgery, 22, 490-495.

HU, W. J., EATON, J. W., UGAROVA, T. P. & TANG, L. P. 2001. Molecular basis of biomaterial-mediated foreign body reactions. Blood, 98, 1231-1238.



HUBER-LANG, M., SARMA, J. V., ZETOUNE, F. S., RITTIRSCH, D., NEFF, T. A., MCGUIRE, S. R., LAMBRIS, J. D., WARNER, R. L., FLIERL, M. A., HOESEL, L. M., GEBHARD, F., YOUNGER, J. G., DROUIN, S. M., WETSEL, R. A. & WARD, P. A. 2006. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med, 12, 682-7.

HUTCHEON, G. A., MESSIOU, C., WYRE, R. M., DAVIES, M. C. & DOWNES, S. 2001. Water absorption and surface properties of novel poly(ethylmethacrylate) polymer systems for use in bone and cartilage repair. Biomaterials, 22, 667-676.

HUTMACHER, D. W. & WILLIAMS, D. F. 2006. Scaffolds in tissue engineering bone and cartilage. The Biomaterials: Silver Jubilee Compendium. Oxford: Elsevier Science.

IKEDA, K., NAGASAWA, K., HORIUCHI, T., TSURU, T., NISHIZAKA, H. & NIHO, Y. 1997. C5a induces tissue factor activity on endothelial cells. Thromb Haemost, 77, 394-8.

ILAN, D. I. & LADD, A. L. 2003. Bone graft substitutes. Operative Techniques in Plastics and Reconstructive Surgery, 9, 151-160.

ISHIZAKI, T., SAITO, N., SATO, Y. & TAKAI, O. 2007. Probing into adsorption behavior of human plasma fibrinogen on self-assembled monolayers with different chemical properties by scanning probe microscopy. Surface Science, 601, 3861-3865.

IWATA, H., KAIBARA, M., DOHMAE, N., TAKIO, K., HIMENO, R. & KAWAKAMI, S. 2004. Purification, identification, and characterization of elastase on erythrocyte membrane as factor IX-activating enzyme. Biochemical and Biophysical Research Communications, 316, 65-70.

JAHAGIRDAR, R. & SCAMMELL, B. E. 2009. Principles of fracture healing and disorders of bone union. Surgery (Oxford), 27, 63-69.

JÁMBOR, C., REUL, V., SCHNIDER, T. W., DEGIACOMI, P., METZNER, H. & KORTE, W. C. 2009. In vitro inhibition of Factor XIII retards clot formation, reduces clot firmness, and increases fibrinolytic effects in whole blood. Anesthesia & Analgesia, 109, 1023-1028.

JANEWAY, C. A., TRAVERS, P., WALPORT, M. & SHLOMCHIK, M. J. 2005a. Innate immunity. Immunobiology: the immune system in health and disease. 6th ed. Oxford: Garland Science Publishing.

JANEWAY, C. A., TRAVERS, P., WALPORT, M. & SHLOMCHIK, M. L. 2005b. Chapter 2: Innate Immunity. Immunology: the immune system in health and disease 6th ed. New York: Garland Science Publishing.

JANG, I. K., GOLD, H. K., ZISKIND, A. A., FALLON, J. T., HOLT, R. E., LEINBACH, R. C., MAY, J. W. & COLLEN, D. 1989. Differential sensitivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator. A possible explanation for resistance to coronary thrombolysis. Circulation, 79, 920-928.

JANSSON, E., KALLTORP, M., JOHANSSON, A., TENGVALL, P. & THOMSEN, P. 2001. On the formation of fibrous capsule and fluid space around machined and porous blood plasma clot coated titanium. J Mater Sci Mater Med, 12, 1019-24.

JANSSON, E., KÄLLTORP, M., THOMSEN, P. & TENGVALL, P. 2002. Ex vivo PMA-induced respiratory burst and TNF- alpha secretion elicited from inflammatory cells on machined and porous blood plasma clot-coated titanium. Biomaterials, 23, 2803-2815.

JEROME, W. G., HANDT, S. & HANTGAN, R. R. 2005. Endothelial cells organize fibrin clots into structures that are more resistant to lysis. Microsc Microanal, 11, 268-77.

JIROUSKOVÁ, M., DYR, J. E., SUTTNAR, J., HOLADA, K. & TRNKOVÁ, B. 1997. Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation. Thromb Haemost., 78, 1125-1131.

JOHNSON, K. D., FRIERSON, K. E., KELLER, T. S., COOK, C., SCHEINBERG, R., ZERWEKH, J., MEYERS, L. & SCIADINI, M. F. 1996. Porous ceramics as bone graft substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. Journal of Orthopaedic Research, 14, 351-369.



JONES, K. S. 2008. Effects of biomaterial-induced inflammation on fibrosis and rejection. Seminars in Immunology, 20, 130-136.

KALLTORP, M., OBLOGINA, S., JACOBSSON, S., KARLSSON, A., TENGVALL, P. & THOMSEN, P. 1999. In vivo cell recruitment, cytokine release and chemiluminescence response at gold, and thiol functionalized surfaces. Biomaterials, 20, 2123-37.

KAMITAKAHARA, M., OHTSUKI, C. & MIYAZAKI, T. 2007. Coating of bone-like apatite for development of bioactive materials for bone reconstruction. Biomed Mater, 2, R17-23.

KANDASWAMY, D., RAMACHANDRAN, G., MAHESHWARI, S. & MOHAN, B. 2000. Bone regenration using hydroxyapatite crystals for periapical lesions. Endodontology, 12, 51-54.

KANG, I. K., KWON, B. K., LEE, J. H. & LEE, H. B. 1993. Immobilization of proteins on poly(methyl methacrylate) films. Biomaterials, 14, 787-792.

KANG, R., GHIVIZZANI, S. C., MUZZONIGRO, T. S., HERNDON, J. H., ROBBINS, P. D. & EVANS, C. H. 2000. Orthopaedic applications of gene therapy: from concept to clinic. Clinical Orthopaedic & Related Research, 375, 324-337.

KAO, S. T. & SCOTT, D. D. 2007. A review of bone substitutes. Oral Maxillofac Surg Clin North Am, 19, 513-21, vi.

KASTEN, P., VOGEL, J., GEIGER, F., NIEMEYER, P., LUGINBÜHL, R. & SZALAY, K. 2008. The effect of platelet-rich plasma on healing in critical-size long-bone defects. Biomaterials, 29, 3983-3992.

KATORI, N., TANAKA, K. A., SZLAM, F. & LEVY, J. H. 2005. The effects of platelet count on clot retraction and tissue plasminogen activator-induced fibrinolysis on thrombelastography. Anesth Analg, 100, 1781-5.

KELLS, A. F., COATS, S. R., SCHWARTZ, H. S. & HOOVER, R. L. 1995. TGF-beta and PDGF act synergistically in affecting the growth of human osteoblast-enriched cultures. Connective Tissue Research, 31, 117-124.

KEUREN, J. F., WIELDERS, S. J., WILLEMS, G. M., MORRA, M. & LINDHOUT, T. 2002. Fibrinogen adsorption, platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood. Thromb Haemost, 87, 742-747.

KLEMENT, P., DU, Y. J., BERRY, L., ANDREW, M. & CHAN, A. K. C. 2002. Blood-compatible biomaterials by surface coating with a novel antithrombin-heparin covalent complex. Biomaterials, 23, 527-535.

KNABE, C., KOCH, C., RACK, A. & STILLER, M. 2008. Effect of β-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans. Biomaterials, 29, 2249-2258.

KNETSCH, M. L. W. 2008. Blood-contacting surfaces. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

KOH, L. B., RODRIGUEZ, I. & VENKATRAMAN, S. S. 2010a. Conformational behavior of fibrinogen on topographically modified polymer surfaces. Physical Chemistry Chemical Physics 12, 10301-10308.

KOH, L. B., RODRIGUEZ, I. & VENKATRAMAN, S. S. 2010b. The effect of topography of polymer surfaces on platelet adhesion. Biomaterials, 31, 1533-45.

KOLOMVOS, N., IATROU, I., THEOLOGIE-LYGIDAKIS, N., TZERBOS, F. & SCHOINOHORITI, O. 2010. Iliac crest morbidity following maxillofacial bone grafting in children: a clinical and radiographic prospective study. J Craniomaxillofac Surg, 38, 293-302.

KOO, J., RAFAILOVICH, M. H., MEDVED, L., TSURUPA, G., KUDRYK, B. J., LIU, Y. & GALANAKIS, D. K. 2010. Evaluation of fibrinogen self-assembly: role of its αC region. Journal of Thrombosis and Haemostasis, 8, 2727-2735.

KOPP, R., MOTTAGHY, K. & KIRSCHFINK, M. 2002. Mechanism of complement activation during extracorporeal blood-biomaterial interaction: effects of heparin coated and uncoated surfaces. ASAIO J, 48, 598-605.



KORBUT, R. & GRYGLEWSKI, R. J. 1995. Platelets in fibrinolytic system. J Physiol Pharmacol, 46, 409-18.

KOURTZELIS, I., MARKIEWSKI, M. M., DOUMAS, M., RAFAIL, S., KAMBAS, K., MITROULIS, I., PANAGOUTSOS, S., PASSADAKIS, P., VARGEMEZIS, V., MAGOTTI, P., QU, H., MOLLNES, T. E., RITIS, K. & LAMBRIS, J. D. 2010. Complement anaphylatoxin C5a contributes to hemodialysis-associated thrombosis. Blood, 116, 631-9.

KRAUS, K. H. & KIRKER-HEAD, C. 2006. Mesenchymal stem cells and bone regeneration. Vet Surg, 35, 232-42.

KRISHNAN, A., CHA, P., LIU, Y. H., ALLARA, D. & VOGLER, E. A. 2006. Interfacial energetics of blood plasma and serum adsorption to a hydrophobic self-assembled monolayer surface. Biomaterials, 27, 3187-3194.

KWAK, D., WU, Y. & HORBETT, T. A. 2005. Fibrinogen and von Willebrand's factor adsorption are both required for platelet adhesion from sheared suspensions to polyethylene preadsorbed with blood plasma. Journal of Biomedical Materials Research. Part A., 74, 69-83.

LACOSTE, E., MARTINEAU, I. & GAGNON, G. 2003. Platelet concentrates: effect of calcium and thrombin on endothelial cell proliferation and growth factor release. Journal of Periodontology, 74, 1498-1507.

LAI, B. F. L., ZOU, Y., BROOKS, D. E. & KIZHAKKEDATHU, J. N. 2010. The influence of poly-N-(2,2-dimethyl-1,3-dioxolane) methyl]acrylamide on fibrin polymerization, cross-linking and clot structure. Biomaterials, 31, 5749-5758.

LAI, J., SUNDERLAND, B., XUE, J., YAN, S., ZHAO, W., FOLKARD, M., MICHAEL, B. D. & WANG, Y. 2006. Study on hydrophilicity of polymer surfaces improved by plasma treatment. Applied Surface Science, 252, 3375-3379.

LAPPEGÅRD, K. T., FUNG, M., BERGSETH, G., RIESENFELD, J., LAMBRIS, J. D., VIDEM, V. & MOLLNES, T. E. 2004. Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation. The Annals of Thoracic Surgery, 77, 932-941.

LASCHKE, M. W., HARDER, Y., AMON, M., MARTIN, I., FARHADI, J., RING, A., TORIO-PADRON, N., SCHRAMM, R., RUCKER, M., JUNKER, D., HAUFEL, J. M., CARVALHO, C., HEBERER, M., GERMANN, G., VOLLMAR, B. & MENGER, M. D. 2006. Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng, 12, 2093-104.

LAURENS, N., KOOLWIJK, P. & DE MAAT, M. P. 2006. Fibrin structure and wound healing. Journal of thrombosis and haemostasis, 4, 932-939.

LE GUEHENNEC, L., SOUEIDAN, A., LAYROLLE, P. & AMOURIQ, Y. 2007. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater, 23, 844-54.

LE GUÉHENNEC, L., SOUEIDAN, A., LAYROLLE, P. & AMOURIQ, Y. 2007. Surface treatments of titanium dental implants for rapid osseointegration. Dental Materials, 23, 844-854.

LEE, H. B., KHANG, G. & LEE, J. H. 2007. Polymeric Biomaterials. Biomaterials, 3-1 - 3-21. LI, B., MA, Y., WANG, S. & MORAN, P. M. 2005. Influence of carboxyl group density on

neuron cell attachment and differentiation behavior: Gradient-guided neurite outgrowth. Biomaterials, 26, 4956-4963.

LI, D., CHEN, H. C., GLENN MCCLUNG, W. & BRASH, J. L. 2009. Lysine-PEG-modified polyurethane as a fibrinolytic surface: Effect of PEG chain length on protein interactions, platelet interactions and clot lysis. Acta Biomaterialia, 5, 1864-1871.

LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002a. The role of growth factors in the repair of bone : biology and clinical applications. J Bone Joint Surg Am, 84, 1032-1044.

LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002b. The role of growth factors in the repair of bone: biology and clinical applications. The Journal of Bone and Joint Surgery, 84A, 1032-1044.



LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002c. The role of growth factors in the repair of bone: biology and clinical applications. J Bone Joint Surg Am, 84, 1032-1044.

LIJNEN, H. R. & COLLEN, D. 1995. Mechanisms of physiological fibrinolysis. Baillieres Clin Haematol., 8, 277-290.

LIN, C. Y., CHANG, Y. H., LIN, K. J., YEN, T. C., TAI, C. L., CHEN, C. Y., LO, W. H., HSIAO, I. T. & HU, Y. C. 2010. The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells. Biomaterials, 31, 3222-30.

LIN, J. C. & CHUANG, W. H. 2000. Synthesis, surface characterization, and platelet reactivity evaluation for the self-assembled monolayer of alkanethiol with sulfonic acid functionality. Journal of Biomedical Materials Research, 51, 413-423.

LIN, N. J. & LIN-GIBSON, S. 2009. Osteoblast response to dimethacrylate composites varying in composition, conversion and roughness using a combinatorial approach. Biomaterials, 30, 4480-4487.

LIND, M., OVERGAARD, S., ONGPIPATTANAKUL, B., NGUYEN, T., BUNGER, C. & SOBALLE, K. 1996. Transforming growth factor-beta 1 stimulates bone ongrowth to weight-loaded tricalcium phosphate coated implants: an experimental study in dogs. J Bone Joint Surg Br, 78, 377-82.

LIU, G., ZHAO, L., ZHANG, W., CUI, L., LIU, W. & CAO, Y. 2008. Repair of goat tibial defects with bone marrow stromal cells and beta-tricalcium phosphate. J Mater Sci Mater Med, 19, 2367-76.

LIU, W., JAWERTH, L. M., SPARKS, E. A., FALVO, M. R., HANTGAN, R. R., SUPERFINE, R., LORD, S. T. & GUTHOLD, M. 2006. Fibrin fibres have extraordinary extensibility and elasticity. Science, 313, 634.

LIU, Y., WU, G. & DE GROOT, K. 2010. Biomimetic coatings for bone tissue engineering of critical-sized defects. J R Soc Interface, 7 Suppl 5, S631-47.

LOZADA, J. L., CAPLANIS, N., PROUSSAEFS, P., WILLARDSEN, J. & KAMMEYER, G. 2001. Platelet-rich plasma application in sinus graft surgery: Part I--Background and processing techniques. J Oral Implantol, 27, 38-42.

LU, J. 2004. Biocompatible materials: orthopedic bone cements. In: POITOUT, D. G. (ed.) Biomechanics and biomaterials in orthopedics. Singapore: Springer-Verlag London Limited.

MA, Z., MAO, Z. & GAO, C. 2007. Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids and Surfaces B: Biointerfaces, 60, 137-157.

MACHLUS, K. R., COLBY, E. A., WU, J. R., KOCH, G. G., KEY, N. S. & WOLBERG, A. S. 2009. Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram. Thromb Haemost, 102, 936-44.

MANN, K. G., BRUMMEL, K. E. & BUTENAS, S. 2003. What is all that thrombin for? Journal of Thromosis and Haemostasis, 1, 1504-1514.

MANNUCCI, P. M., GIANGRANDE, P. L. F. & BONOMI, A. B. 1992. Detection of the prethrombotic state due to procoagulant imbalance. European Journal of Haematology, 48, 65-69.

MARIEB, E. N. 2001a. Chapter 4: Tissue: the living fabric. Human anatomy & physiology. 5th ed. New York: Benjamin Cummings.

MARIEB, E. N. 2001b. Chapter 6: Bone and skeletal tissues. Human anatomy & physiology. 5th ed. New York: Benjamin Cummings.

MARIEB, E. N. 2001c. Chapter 6: Bone and skeletal tissues. Human anatomy & physiology, 172-197.

MARIEB, E. N. 2001d. Chapter 18: Blood. Human anatomy & physiology. New York: Benjamin Cummings.

MARIEB, E. N. 2001e. Chapter 22: The immune system: innate and adaptive body defenses. Human anatomy & physiology. New York: Benjamin Cummings.



MARKIEWSKI, M. M., NILSSON, B., NILSSON EKDAHL, K., MOLLNES, T. E. & LAMBRIS, J. D. 2007. Complement and coagulation: strangers or partners in crime? Trends in Immunology, 28, 184-192.

MARTINEAU, I., LACOSTE, E. & GAGNON, G. 2004. Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials, 25, 4489-502.

MARTINS, M. C., RATNER, B. D. & BARBOSA, M. A. 2003. Protein adsorption on mixtures of hydroxyl- and methyl-terminated alkanethiols self-assembled monolayers. J Biomed Mater Res A, 67, 158-71.

MARX, R. E. 2001. Platelet-rich plasma (PRP): What is PRP and what is not PRP? Implant Dentistry, 10, 225-228.

MARX, R. E., CARLSON, E. R., EICHSTAEDT, R. M., SCHIMMELE, S. R., STRAUSS, J. E. & GEORGEFF, K. R. 1998a. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, 85, 638-646.

MARX, R. E., CARLSON, E. R., EICHSTAEDT, R. M., SCHIMMELE, S. R., STRAUSS, J. E. & GEORGEFF, K. R. 1998b. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, 85, 638-646.

MCARTHUR, S. L. 2006. Applications of XPS in bioengineering. Surface and Interface Analysis, 38, 1380-1385.

MCAULIFFE, J. A. 2003. Bone graft substitutes. J Hand Ther, 16, 180-7. MCNALLY, A. K. & ANDERSON, J. M. 2011. Macrophage fusion and multinucleated giant

cells of inflammation, New York, NY, ETATS-UNIS, Springer Science+Business Media.

MEDVED, L. & NIEUWENHUIZEN, W. 2003. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thrombosis and Haemostasis, 89, 409-419.

MENZIES, K. L. & JONES, L. 2010. The impact of contact angle on the biocompatibility of biomaterials. Optometry & Vision Science, 87, 387-399.

MESSORA, M. R., NAGATA, M. J. H., MARIANO, R. C., DORNELLES, R. C. M., BOMFIM, S. R. M., FUCINI, S. E., GARCIA, V. G. & BOSCO, A. F. 2008. Bone healing in critical-size defects treated with platelet-rich plasma: a histologic and histometric study in rat calvaria Journal of Periodontal Research, 43, 217-223.

MEYER, U., JOOS, U. & WIESMANN, H. P. 2004. Biological and biophysical principles in extracorporal bone tissue engineering: Part I. International Journal of Oral and Maxillofacial Surgery, 33, 325-332.

MICHIARDI, A., APARICIO, C., RATNER, B. D., PLANELL, J. A. & GIL, J. 2007. The influence of surface energy on competitive protein adsorption on oxidized NiTi surfaces. Biomaterials, 28, 586-594.

MILLS, J. D., ARIENS, R. A. S., MANSFIELD, M. W. & GRANT, P. J. 2002. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation, 106, 1938-1942.

MINELLI, C., KIKUTA, A., TSUD, N., BALL, M. D. & YAMAMOTO, A. 2008. A micro-fluidic study of whole blood behaviour on PMMA topographical nanostructures. Journal of nanobiotechnology, 6, 3-13.

MINORS, D. S. 2004. Haemostasis, blood platelets and coagulation. Anaesthesia and intensive care medicine, 5, 189-191.

MOORADIAN, D. L., LUCAS, R. C., WEATHERBEE, J. A. & FURCHT, L. T. 1989. Transforming growth factor-β1 binds to immobilized fibronectin. Journal of Cellular Biochemistry, 41, 189-200.

MOOREN, R. E. C. M., MERKX, M. A. W., BRONKHORST, E. M., JANSEN, J. A. & STOELINGA, P. J. W. 2007. The effect of platelet-rich plasma on early and late bone healing: an experimental study in goats. International Journal of Oral and Maxillofacial Surgery, 36, 626-631.



MORGENSTERN, E., KORELL, U. & RICHTER, J. 1984. Platelets and fibrin strands during clot retraction. Thromb Res, 33, 617-23.

MOSESSON, M. W. 2005. Fibrinogen and fibrin structure and functions. Journal of Thrombosis and Haemostasis, 3, 1894-1904.

MRKSICH, M. 2000. A surface chemistry approach to studying cell adhesion. Chemical Society Reviews, 29, 267-273.

MÜLLER, F., MUTCH, N. J., SCHENK, W. A., SMITH, S. A., ESTERL, L., SPRONK, H. M., SCHMIDBAUER, S., GAHL, W. A., MORRISSEY, J. H. & RENNÉ, T. 2009. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell, 139, 1143-1156.

MULLIN, J. L., NORFOLK, S. E., WEISEL, J. E. & LORD, S. T. 2001. Clot lysis of variant recombinant fibrinogens confirms that fibre diameter is a major determinant of lysis rate. Annals of the New York Academy of Sciences, 936, 331-334.

MULZER, S. R. & BRASH, J. L. 1989. Identification of plasma protein adsorbed to hemodialyzers during clinical use. Journal of Biomedical Materials Research, 23, 1483-1504.

MURPHY-ULLRICH, J. E., SCHULTZ-CHERRY, S. & HOOK, M. 1992. Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell, 3, 181-8.

NAIR, C. H. & DHALL, D. P. 1991. Studies on fibrin network structure: the effect of some plasma proteins. Thrombosis Research, 61, 315-325.

NAIR, C. H. & SHATS, E. A. 1997. Compaction as a method to characterise fibrin network structure: kinetic studies and relationship to crosslinking. Thrombosis Research, 88, 381-387.

NATHER, A., DAVID, V., TENG, J. W., LEE, C. W. & PEREIRA, B. P. 2010. Effect of autologous mesenchymal stem cells on biological healing of allografts in critical-sized tibial defects simulated in adult rabbits. Ann Acad Med Singapore, 39, 599-606.

NILSSON, B., EKDAHL, K. N., MOLLNES, T. E. & LAMBRIS, J. D. 2007a. The role of complement in biomaterial-induced inflammation. Molecular Immunology, 44, 82-94.

NILSSON, B., EKDAHL, K. N., MOLLNES, T. E. & LAMBRIS, J. D. 2007b. The role of complement in biomaterial-induced inflammation. Molecular Immunology, 44, 82-94.

NILSSON, B., KORSGREN, O., LAMBRIS, J. D. & EKDAHL, K. N. 2009. Can cells and biomaterials in therapeutic medicine be shielded from innate immune recognition? Trends in Immunology, 31, 32-38.

NURDEN, A. T. 2007. Platelet membrane glycoproteins: a look back into the past and a view to the future. Thromb Haemost, 98, 49-54.

NURDEN, A. T. 2011. Platelets, inflammation and tissue regeneration. Thromb Haemost, 105 Suppl 1, S13-33.

NURDEN, A. T., NURDEN, P., SANCHEZ, M., ANDIA, I. & ANITUA, E. 2008. Platelets and wound healing. Front Biosci, 13, 3532-48.

NYGREN, H. 1996. Initial reactions of whole blood with hydrophilic and hydrophobic titanium surfaces. Colloids and Surfaces B: Biointerfaces, 6, 329-333.

NYGREN, H., TENGVALL, P. & LUNDSTROM, I. 1997. The initial reactions of TiO2 with blood. J Biomed Mater Res, 34, 487-92.

OKADA, M., BLOMBACK, B., CHANG, M. D. & HOROWITZ, B. 1985. Fibronectin and fibrin gel structure. J Biol Chem, 260, 1811-20.

ONUNDARSON, P. T., FRANCIS, C. W. & MARDER, V. J. 1992. Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential. J Lab Clin Med, 120, 120-128.

OPREA, W. E., KARP, J. M., HOSSEINI, M. M. & DAVIES, J. E. 2003. Effect of platelet releasate on bone cell migration and recruitment in vitro. Journal of Craniofacial Surgery, 14, 292-300.

OVANESOV, M. V., ANANYEVA, N. M., PANTELEEV, M. A., ATAULLAKHANOV, F. I. & SAENKO, E. L. 2005. Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost, 3, 321-31.



OZCAN, C. & HASIRCI, N. 2007. Plasma modification of PMMA films: surface free energy and cell-attachment studies. J. Biomater. Sci. Polymer Edn, 18, 759-773.

PANETTI, T. S., KUDRYK, B. J. & MOSHER, D. F. 1999. Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin. Journal of Biological Chemistry, 274, 430-437.

PANTELEEV, M. A., OVANESOV, M. V., KIREEV, D. A., SHIBEKO, A. M., SINAURIDZE, E. I., ANANYEVA, N. M., BUTYLIN, A. A., SAENKO, E. L. & ATAULLAKHANOV, F. I. 2006. Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively. Biophys J, 90, 1489-500.

PARHI, P., GOLAS, A. & VOGLER, E. A. 2010. Role of proteins and water in the initial attachment of mammalian cells to biomedical surfaces: A review. Journal of Adhesion Science and Technology, 24, 853-888.

PARIKH, S. N. 2002. Bone graft substitutes: past, present, future. Journal of Postgraduate Medicine, 48, 142-148.

PARISE, P. & AGNELLI, G. 1991. Thrombus resistance to lysis and reocclusion after thrombolysis: the role of platelets. Blood Coagul Fibrinolysis, 2, 749-58.

PARK, J. E. & BARBUL, A. 2004. Understanding the role of immune regulation in wound healing. The American Journal of Surgery, 187, S11-S16.

PEERSCHKE, E. I., YIN, W., GRIGG, S. E. & GHEBREHIWET, B. 2006. Blood platelets activate the classical pathway of human complement. J Thromb Haemost, 4, 2035-42.

PEERSCHKE, E. I. B. & GHEBREHIWET, B. 1998. Platelet receptors for the complement component C1q: implication for hemostasis and thrombosis. Immunobiology, 199, 239-249.

PEERSCHKE, E. I. B., REID, K. B. & GHEBREHIWET, B. 1993. Platelet activation by C1q results in the induction of alpha IIb/beta 3 integrins (GPIIb-IIIa) and the expression of P-selectin and procoagulant activity. The Journal of Experimental Medicine, 178, 579-587.

PEERSCHKE, E. I. B., YIN, W. & GHEBREHIWET, B. 2008. Platelet mediated complement activation. Adv Exp Med Biol., 632, 81-91.

PEERSCHKE, E. I. B., YIN, W. & GHEBREHIWET, B. 2010. Complement activation on platelets: implications for vascular inflammation and thrombosis. Molecular Immunology, 47, 2170-2175.

PETERSEN, L. C., BJORN, S. E. & NORDFANG, O. 1992. Effect of leukocyte proteinase on tissue factor pathway inhibitor. Thromb Haemost, 67, 537-541.

PEYROU, V., LORMEAU, J. C., HERAULT, J. P., GAICH, C., PFLIEGGER, A. M. & HERBERT, J. M. 1999. Contribution of erythrocytes to thrombin generation in whole blood, Stuttgart, ALLEMAGNE, Schattauer.

PHILLIPS, A. M. 2005. Overview of the fracture healing cascade. Injury, 36 Suppl 3, S5-7. PIERCE, G. F., MUSTOE, T. A., ALTROCK, B. W., DEUEL, T. F. & THOMASON, A. 1991.

Role of platelet-derived growth factor in wound healing. Journal of Cellular Biochemistry, 45, 319-326.

PLESCIA, J. & ALTIERI, D. C. 1996. Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation. Biochem J, 319, 873–879.

POUNTOS, I., CORSCADDEN, D., EMERY, P. & GIANNOUDIS, P. V. 2007. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury, 38 Suppl 4, S23-33.

PRASAD, S., KASHYAP, R. S., DEOPUJARI, J. Y., PUROHIT, H. J., TAORI, G. M. & DAGINAWALA, H. F. 2006. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J, 4, 14.

PRECHEUR, H. V. 2007. Bone graft materials. Dent Clin North Am, 51, 729-46, viii. PUELACHER, W. C., VACANTI, J. P., FERRARO, N. F., SCHLOO, B. & VACANTI, C. A.

1996. Femoral shaft reconstruction using tissue-engineered growth of bone. Int J Oral Maxillofac Surg, 25, 223-8.



RAND, M. L., WANG, H., BANG, K. W., TEITEL, J. M., BLANCHETTE, V. S., FREEDMAN, J. & NURDEN, A. T. 2010. Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets. Am J Hematol, 85, 584-92.

REICHERT, J. C., SAIFZADEH, S., WULLSCHLEGER, M. E., EPARI, D. R., SCHUTZ, M. A., DUDA, G. N., SCHELL, H., VAN GRIENSVEN, M., REDL, H. & HUTMACHER, D. W. 2009. The challenge of establishing preclinical models for segmental bone defect research. Biomaterials, 30, 2149-63.

RENNÉ, T. & GAILANI, D. 2007. Role of Factor XII in hemostasis and thrombosis: clinical implications. Expect Review of Cardiovascular Therapy, 5, 733-741.

RIEDEL, T., SUTTNAR, J., BRYNDA, E., HOUSKA, M., MEDVED, L. & DYR, J. E. 2011. Fibrinopeptides A and B release in the process of surface fibrin formation. Blood, 117, 1700-1705.

RITIS, K., DOUMAS, M., MASTELLOS, D., MICHELI, A., GIAGLIS, S., MAGOTTI, P., RAFAIL, S., KARTALIS, G., SIDERAS, P. & LAMBRIS, J. D. 2006. A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways. J Immunol, 177, 4794-802.

ROACH, P., FARRAR, D. & PERRY, C. C. 2005. Interpretation of protein adsorption: surface-induced conformational changes. J Am Chem Soc, 127, 8168-73.

RODRIGUES, S. N., GONCALVES, I. C., MARTINS, M. C. L., BARBOSA, M. A. & RATNER, B. D. 2006. Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methy-terminated self-assembled monolayers. Biomaterials, 27, 5357-5367.

RODRIGUEZ HERNANDEZ, J. C., RICO, P., MORATAL, D., MONLEON PRADAS, M. & SALMERON-SANCHEZ, M. 2009. Fibrinogen patterns and activity on substrates with tailored hydroxy density. Macromol Biosci, 9, 766-75.

ROJKJAER, L. P. & ROJKJAER, R. 2007. Clot stabilization for the prevention of bleeding. Hematol Oncol Clin North Am, 21, 25-32.

ROUKIS, T. S., ZGONIS, T. & TIERNAN, B. 2006. Autologous platelet-rich plasma for wound and osseous healing: a review of literature and commercial available products. Advances in therapy, 23, 128-137.

ROUSSY, Y., BERTRAND DUCHESNE, M.-P. & GAGNON, G. 2007. Activation of human platelet-rich plasmas: effect on growth factor release, cell division and in vivo bone formation. Clinical oral implants research, 18, 639-648.

RUGGERI, Z. M. 2003. Von Willebrand factor, platelets and endothelial cell interactions. Journal of Thromosis and Haemostasis, 1, 1335-1342.

RYZEWICZ, M., MORGAN, S. J., LINFORD, E., THWING, J. I., DE RESENDE, G. V. & SMITH, W. R. 2009. Central bone grafting for nonunion of fractures of the tibia: a retrospective series. J Bone Joint Surg Br, 91, 522-9.

SAJJADIAN, A., RUBINSTEIN, R. & NAGHSHINEH, N. 2010. Current status of grafts and implants in rhinoplasty: part I. Autologous grafts. Plast Reconstr Surg, 125, 40e-49e.

SALVADOR-MORALES, C., ZHANG, L., LANGER, R. & FAROKHZAD, O. C. 2009. Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials, 30, 2231-40.

SAMMARCO, V. J. & CHANG, L. 2002. Modern issues in bone graft substitutes and advances in bone tissue technology. Foot And Ankle Clinics, 7, 19-41.

SÁNCHEZ, A. R., SHERIDAN, P. J. & KUPP, L. I. 2003. Is platelet-rich plasma the perfect enhancement factor? A current review. Int J Oral Maxillofac Implants, 18, 93-103.

SANCHEZ, J., LUNDQUIST, P. B., ELGUE, G., LARSSON, R. & OLSSON, P. 2002. Measuring the degree of plasma contact activation induced by artificial materials. Thromb Res, 105, 407-12.

SAULS, D. L., WOLBERG, A. S. & HOFFMAN, M. 2003. Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: implications for the mechanism of thrombosis in hyperhomocysteinemia. J Thromb Haemost, 1, 300-6.

SCHLIEPHAKE, H. 2009. Growth factors and cell based devices in bone tissue engineering. International Journal of Oral and Maxillofacial Surgery, 38, 413-413.



SCHMIDMAIER, G., SCHWABE, P., WILDEMANN, B. & HAAS, N. P. 2007. Use of bone morphogenetic proteins for treatment of non-unions and future perspectives. Injury, 38 Suppl 4, S35-41.

SCHMUGGE, M., RAND, M. L. & FREEDMAN, J. 2003. Platelets and von Willebrand factor. Transfus Apher Sci, 28, 269-77.

SCOTT, E. M., ARIENS, R. A. & GRANT, P. J. 2004. Genetic and environmental determinants of fibrin structure and function: relevance to clinical disease. Arterioscler Thromb Vasc Biol, 24, 1558-66.

SEEHERMAN, H. J., AZARI, K., BIDIC, S., ROGERS, L., LI, X. J., HOLLINGER, J. O. & WOZNEY, J. M. 2006. rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radii. J Bone Joint Surg Am, 88, 1553-65.

SELLBORN, A., ANDERSSON, M., HEDLUND, J., ANDERSSON, J., BERGLIN, M. & ELWING, H. 2005. Immune complement activation on polystyrene and silicon dioxide surfaces. Impact of reversible IgG adsorption. Mol Immunol, 42, 569-74.

SHAH, G. A., FERGUSON, I. A., DHALL, T. Z. & DHALL, D. P. 1982. Polydispersion in the diameter of fibres in fibrin networks: Consequences on the measurement of mass-length ratio by permeability and turbidity. Biopolymers, 21, 1037-1047.

SHALABI, M. M., GORTEMAKER, A., VAN'T HOF, M. A., JANSEN, J. A. & CREUGERS, N. H. 2006. Implant surface roughness and bone healing: a systematic review. J Dent Res, 85, 496-500.

SHANTI, R. M., LI, W. J., NESTI, L. J., WANG, X. & TUAN, R. S. 2007. Adult mesenchymal stem cells: biological properties, characteristics, and applications in maxillofacial surgery. J Oral Maxillofac Surg, 65, 1640-7.

SIMON, D. I., EZRATTY, A. M., FRANCIS, S. A., RENNKE, H. & LOSCALZO, J. 1993. Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway. Blood, 82, 2414-22.

SIMONPIERI, A., DEL CORSO, M., VERVELLE, A., JIMBO, R., INCHINGOLO, F., SAMMARTINO, G. & DOHAN EHRENFEST, D. M. 2011. Current knowledge and perspectives for the use of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial surgery. Part 2: bone graft, implant and reconstructive surgery. Curr Pharm Biotechnol.

SIMS, P. J. & WIEDMER, T. 1991. The response of human platelets to activated components of the complement system. Immunol Today, 12, 338-42.

SIMS, P. J. & WIEDMER, T. 1995. Induction of cellular procoagulant activity by the membrane attack complex of complement. Seminars in Cell Biology, 6, 275-282.

SIT, P. S. & MARCHANT, R. E. 1999a. Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions. Thromb Haemost, 82, 1053-60.

SIT, P. S. & MARCHANT, R. E. 1999b. Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions. Thrombosis and haemostasis, 82, 1053-1060.

SIT, P. S. & MARCHANT, R. E. 2001. Surface-dependent differences in fibrin assembly visualized by atomic force microscopy. Surface Science, 491, 421-432.

SIVARAMAN, B., FEARS, K. P. & LATOUR, R. A. 2009. Investigation of the effects of surface chemistry and solution concentration on the conformation of adsorbed proteins using an improved circular dichroism method. Langmuir, 25, 3050-6.

SIVARAMAN, B. & LATOUR, R. A. 2010. The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen. Biomaterials, 31, 832-839.

SIVARAMAN, B. & LATOUR, R. A. 2011. Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin. Biomaterials, 32, 5365-70.

SLATER, M., PATAVA, J., KINGHAM, K. & MASON, R. S. 1995. Involvement of platelets in stimulating osteogenic activity. Journal of Orthopaedic Research, 13, 655-663.



SMYTH, S. S., MCEVER, R. P., WEYRICH, A. S., MORRELL, C. N., HOFFMAN, M. R., AREPALLY, G. M., FRENCH, P. A., DAUERMAN, H. L. & BECKER, R. C. 2009. Platelet functions beyond hemostasis. Journal of Thrombosis and Haemostasis, 7, 1759-1766.

SPERLING, C., FISCHER, M., MAITZ, M. F. & WERNER, C. 2009. Blood coagulation on biomaterials requires the combination of distinct activation processes. Biomaterials, 30, 4447-4456.

SPERLING, C., HOUSKA, M., BRYNDA, E., STRELLER, U. & WERNER, C. 2006. In vitro hemocompatibility of albumin–heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique. Journal of Biomedical Materials Research Part A, 76A, 681-689.

SPERLING, C., MATIZ, M. F., TALKENBERGER, S., GOUZY, M. F. & WERNER, C. 2007. In vitro blood reactivity of hydroxylated and non-hydroxylated polymer surfaces. Biomaterials, 28, 3617-3625.

SPERLING, C., SCHWEISS, R. B., STRELLER, U. & WERNER, C. 2005a. In vitro hemocompatibility of self-assembled monolayers displaying various functional groups. Biomaterials, 25, 6547-6557.

SPERLING, C., SCHWEISS, R. B., STRELLER, U. & WERNER, C. 2005b. In vitro hemocompatibility of self-assembled monolayers displaying various functional groups. Biomaterials, 25, 6547-6557.

STANDEVEN, K. F., ARIËNS, R. A. S. & GRANT, P. J. 2005. The molecular physiology and pathology of fibrin structure/function. Blood Reviews, 19, 275-288.

STEVENS, M. M. 2008. Biomaterials for bone tissue engineering. Materials Today, 11, 18-25. STREET, J., WINTER, D., WANG, J. H., WAKAI, A., MCGUINNESS, A. & REDMOND, H.

P. 2000. Is human fracture hematoma inherently angiogenic? Clinical Orthopaedics and Related Research, 378, 224-237.

STRELLER, U., SPERLING, C., HUBNER, J., HANKE, R. & WERNER, C. 2003. Design and evaluation of novel blood incubation systems for in vitro hemocompatibility assessment of planar solid surfaces. J Biomed Mater Res B Appl Biomater, 66, 379-90.

SUGIMOTO, M. & MIYATA, S. 2002. Functional property of von Willebrand factor under flowing blood. Int J Hematol, 75, 19-24.

SUNDARAM, S., LIM, F., COOPER, S. L. & COLMAN, R. W. 1996. Role of leucocytes in coagulation induced by artificial surfaces: investigation of expression of Mac-1, granulocyte elastase release and leucocyte adhesion on modified polyurethanes. Biomaterials, 17, 1041-1047.

SWENNEN, G. R. J., SCHUTYSER, F., MUELLER, M. C., KRAMER, F.-J., C., E. & SCHLIEPHAKE, H. 2004. Effect of platelet-rich plasma on cranial distraction osteogenesis in sheep: preliminary clinical and radiographic results. International Journal of Oral and Maxillofacial Surgery, 34, 294-304.

TA, T. C., SYKES, M. T. & MCDERMOTT, M. T. 1998. Real-time observation of plasma protein film formation on well-defined surfaces with scanning force microscopy. Langmuir, 14, 2435-2443.

TAKEMOTO, S., YAMAMOTO, T., TSURU, K., HAYAKAWA, S., OSAKA, A. & TAKASHIMA, S. 2004. Platelet adhesion on titanium oxide gels: effect of surface oxidation. Biomaterials, 25, 3485-3492.

TANG, L. & EATON, J. W. 1995. Inflammatory responses to biomaterials. American journal of clinicalpathology 103, 466-471.

TANG, L., LIU, L. & ELWING, H. B. 1998. Complement activation and inflammation triggered by model biomaterial surfaces. Journal of biomedical materials research, 41, 333-340.

TEDESCO, F., PAUSA, M., NARDON, E., INTRONA, M., MANTOVANI, A. & DOBRINA, A. 1997. The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity. J Exp Med, 185, 1619-27.

TELLEMAN, G., ALBREKTSSON, T., HOFFMAN, M., JOHANSSON, C. B., VISSINK, A., MEIJER, H. J. & RAGHOEBAR, G. M. 2010. Peri-implant endosseous healing



properties of dual acid-etched mini-implants with a nanometer-sized deposition of CaP: a histological and histomorphometric human study. Clin Implant Dent Relat Res, 12, 153-60.

TERASAWA, F., KANI, S., HONGO, M. & OKUMURA, N. 2006. In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from gammaAsn319, Asp320 deletion dysfibrinogen, Otsu I. Thromb Res, 118, 651-61.

THEVENOT, P., HU, W. & TANG, L. 2008. Surface chemistry influences implant biocompatibility. Current Topics in Medicinal Chemistry, 8, 270-280.

THOMAN, M. L., MEUTH, J. L., MORGAN, E. L., WEIGLE, W. O. & HUGLI, T. E. 1984. C3d-K, a kallikrein cleavage fragment of iC3b is a potent inhibitor of cellular proliferation. J Immunol, 133, 2629-33.

THOR, A., RASMUSSON, L., WENNERBERG, A., THOMSEN, P., HIRSCH, J. M., NILSSON, B. & HONG, J. 2007. The role of whole blood in thrombin generation in contact with various titanium surfaces. Biomaterials, 28, 966-974.

TOMOYASU, A., HIGASHIO, K., KANOMATA, K., GOTO, M., KODAIRA, K., SERIZAWA, H., SUDA, T., NAKAMURA, A., NOJIMA, J., FUKUDA, T. & KATAGIRI, T. 2007. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs. Biochemical and Biophysical Research Communications, 36, 62-67.

TOSOUNIDIS, T., KONTAKIS, G., NIKOLAOU, V. S., PAPATHANASSOPOULOS, A. & GIANNOUDIS, P. V. 2009. Facture healing and bone repair: an update. Trauma, 11, 145-156.

TSAI, A. T. 2004. Wound healing and the foreign body reaction [Online]. University of Washington Engineered Biomaterials. Available: http://www.uweb.engr.washington. edu/research/tutorials/woundhealing.html [Accessed 1st August 2007 2007].

TSAI, M. Y., SUN, Y. T. & LIN, J. C. 2007. Surface characterization and platelet compatibility evaluation of the binary mixed self-assembled monolayers. Journal of Colloid and Interface Science, 308, 474-484.

TSAI, W. B., GRUNKEMEIER, J. M., MCFARLAN, C. D. & HORBETT, T. A. 2002. Platelet adhesion to polystyrene-based surfaces preadsorbed with plasma selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor. Journal of biomedical materials research, 60, 348-359.

TSIRIDIS, E., KAKAR, S. & EINHORN, T. A. 2006. Fracture healing and bone grafting. In: ELSTROM, J. A., VIRKUS, W. W. & PANKOVICH, A. M. (eds.) Handbook of fractures. Third ed. New York: The McGraw-Hill Companies, Inc.

TSYGANOV, I., MAITZ, M. F., WIESER, E., RICHTER, E. & REUTHER, H. 2005. Correlation between blood compatibility and physical surface properties of titanium-based coatings. Surface and Coatings Technology, 200, 1041-1044.

TZONEVA, R., SEIFERT, B., ALBRECHT, W., RICHAU, K., GROTH, T. & LENDLEIN, A. 2008. Hemocompatibility of poly(ether imide) membranes functionalized with carboxylic groups. Journal of Materials Science: Materials in Medicine, 19, 3203-3210.

UKIWE, C., MANSOURI, A. & KWOK, D. Y. 2005. The dynamics of impacting water droplets on alkanethiol self-assembled monolayers with co-adsorbed CH3 and CO2H terminal groups. Journal of Colloid and Interface Science, 285, 760-768.

ULEVITCH, R. J. & JOHNSTON, A. R. 1980. In vitro studies of prekallikrein activation in rabbit blood: the participation of red blood cells. J Immunol, 124, 2027-33.

UNDAS, A., CELINSKA-LÖWENHOFF, M., LÖWENHOFF, T. & SZCZEKLIK, A. 2006. Statins, fenofibrate, and quinapril increase clot permeability and enhance fibrinolysis in patients with coronary artery disease. Journal of Thrombosis and Haemostasis, 4, 1029-1036.

UNDAS, A., SLOWIK, A., WOLKOW, P., SZCZUDLIK, A. & TRACZ, W. 2010. Fibrin clot properties in acute ischemic stroke: relation to neurological deficit. Thromb Res, 125, 357-61.

UNDAS, A., SZULDRZYNSKI, K., STEPIEN, E., ZALEWSKI, J., GODLEWSKI, J., TRACZ, W., PASOWICZ, M. & ZMUDKA, K. 2008. Reduced clot permeability and



susceptibility to lysis in patients with acute coronary syndrome: effects of inflammation and oxidative stress. Atherosclerosis, 196, 551-557.

UNDAS, A., ZAWILSKA, K., CIESLA-DUL, M., LEHMANN-KOPYDLOWSKA, A., SKUBISZAK, A., CIEPLUCH, K. & TRACZ, W. 2009. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood, 114, 4272-4278.

VACANTI, C. A., KIM, W., UPTON, J., MOONEY, D. & VACANTI, J. P. 1995. The efficacy of periosteal cells compared to chondrocytes in the tissue engineered repair of bone defects. Tissue Eng, 1, 301-8.

VALLES, J., SANTOS, M., AZNAR, J., MARCUS, A., MARTINEZ-SALES, V., PORTOLES, M., BROEKMAN, M. & SAFIER, L. 1991. Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment. Blood, 78, 154-162.

VAN DAMME, H. S., HOGT, A. H. & FEIJEN, J. 1986. Surface mobility and structural transitions of poly(n-alkyl methacrylates) probed by dynamic contact angle measurements. Journal of Colloid and Interface Science, 114, 167-172.

VAN DER MEIJDEN, P. E. & HEEMSKERK, J. W. M. 2010. Polyphosphates: a link between platelet activation, intrinsic coagulation and inflammation? Expert Review of Hematology, 3, 269-272.

VAN GELDER, J. M., NAIR, C. H. & DHALL, D. P. 1995. The permeability of fibrin network developed in human plasma. Blood Coagulation & Fibrinolysis, 6, 293-301.

VAN LIESHOUT, E. M. M., VAN KRALINGEN, G. H., EL-MASSOUDI, Y., WEINANS, H. & PATKA, P. 2011. Microstructure and biomechanical characteristics of bone substitutes for trauma and orthopaedic surgery. BMC Musculoskeletal Disorders, 12, 1-14.

VEHOF, J. W., HAUS, M. T., DE RUIJTER, A. E., SPAUWEN, P. H. & JANSEN, J. A. 2002. Bone formation in transforming growth factor beta-1-loaded titanium fibre mesh implants. Clin Oral Implants Res, 13, 94-102.

VEKLICH, Y., FRANCIS, C. W., WHITE, J. & WEISEL, J. W. 1998. Structural studies of fibrinolysis by electron microscopy. Blood, 92, 4721-9.

VOGLER, E. A. & SIEDLECKI, C. A. 2009. Contact activation of blood-plasma coagulation. Biomaterials, In Press, Corrected Proof.

WALKOWIAK, B., KRALISZ, U., MICHALEC, L., MAJEWSKA, E., KOZIOLKIEWICZ, W., LIGOCKA, A. & CIERNIEWSKI, C. S. 2000. Comparison of platelet aggregability and p-selectin surface expression on platelets isolated by different methods. Thrombosis Research, 99, 495-502.

WALSH, W. R., VIZESI, F., MICHAEL, D., AULD, J., LANGDOWN, A., OLIVER, R., YU, Y., IRIE, H. & BRUCE, W. 2008. [beta]-TCP bone graft substitutes in a bilateral rabbit tibial defect model. Biomaterials, 29, 266-271.

WANG, H., RATNER, B. D., SAGE, E. H. & JIANG, S. 2007. Stepwise assembly of fibrin bilayers on self-assembled monolayers of alkanethiolates: Influence of surface chemistry. The Journal of Physical Chemistry C, 111, 8504-8508.

WATANABE, Y. & KOBAYASHI, B. 1988. Differential release of calcium, magnesium and serotonin by rabbit and human platelets. J Pharmacobiodyn, 11, 268-76.

WEI, J., IGARASHI, T., OKUMORI, N., IGARASHI, T., MAETANI, T., LIU, B. & YOSHINARI, M. 2009. Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts. Biomedical Materials, 4, 045002.

WEIBRICH, G., HANSEN, T., KLEIS, W., BUCH, R. & HITZLER, W. E. 2004. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone, 34, 665-671.

WEISEL, J. W. 2005. Fibrinogen and fibrin. WEISEL, J. W., VEKLICH, Y. & GORKUN, O. 1993. The sequence of cleavage of

fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots. Journal of Molecular Biology, 232, 285-297.



WEISS, S., ZIMMERMANN, G., PUFE, T., VAROGA, D. & HENLE, P. 2009. The systemic angiogenic response during bone healing Archives of Orthopaedic and Trauma Surgery, 129, 989-997.

WENCEL-DRAKE, J. D., PAINTER, R. G., ZIMMERMAN, T. S. & GINSBERG, M. H. 1985. Ultrastructural localisation of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section. Blood, 65, 929-938.

WENNERBERG, A. & ALBREKTSSON, T. 2009. Effects of titanium surface topography on bone integration: a systematic review. Clin Oral Implants Res, 20 Suppl 4, 172-84.

WERNER, C., MAITZ, M. F. & SPERLING, C. 2007. Current strategies towards hemocompatible coatings. Journal of Materials Chemistry, 17, 3376-3384.

WHEELER, D. L. & ENNEKING, W. F. 2005. Allograft bone decreases in strength in vivo over time. Clinical Orthopaedic & Related Research, 435, 36-42.

WIGGINS, R. C., GICLAS, P. C. & HENSON, P. M. 1981. Chemotactic activity generated from the fifth component of complement by plasma kallikrein of the rabbit. Journal of Experimental Medicine, 153, 1391-1404.

WILLIAMS, D. F. 2008. On the mechanisms of biocompatibility. Biomaterials, 29, 2941-2953. WILSON, C. J., CLEGG, R. E., LEAVESLEY, D. I. & PEARCY, M. J. 2005. Mediation of

biomaterial-cell interactions by adsorbed proteins: a review. Tissue Engineering, 11, 1-18.

WILTFANG, J., KLOSS, F. R., KESSLER, P., NKENKE, E., SCHULTZE-MOSGAU, S., ZIMMERMANN, R. & SCHLEGEL, K. A. 2004. Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clinical oral implants research, 15, 187-193.

WOLBERG, A. S. 2007a. Thrombin generation and fibrin clot structure. Blood Rev, 21, 131-42. WOLBERG, A. S. 2007b. Thrombin generation and fibrin clot structure. Blood Reviews, 21,

131-142. WOLBERG, A. S. 2010. Plasma and cellular contributions to fibrin network formation, structure

and stability. Haemophilia, 16 Suppl 3, 7-12. WOLBERG, A. S., ALLEN, G. A., MONROE, D. M., HEDNER, U., ROBERTS, H. R. &

HOFFMAN, M. 2005. High dose factor VIIa improves clot structure and stability in a model of haemophilia B. Br J Haematol, 131, 645-55.

WOLBERG, A. S. & CAMPBELL, R. A. 2008. Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci, 38, 15-23.

WOLBERG, A. S., MONROE, D. M., ROBERTS, H. R. & HOFFMAN, M. 2003. Elevated prothrombin results in clots with an altered fibre structure: a possible mechanism of the increased thrombotic risk. Blood, 101, 3008-13.

WOZNEY, J. M. & ROSEN, V. 1998. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clinical Orthopaedic & Related Research, 346, 26-37.

WULF, M., GRUNDKE, K., KWOK, D. Y. & NEUMANN, A. W. 2000. Influence of different alkyl side chains on solid surface tension of polymethacrylates. Journal of Applied Polymer Science, 77, 2493-2504.

XIE, H. G., ZHENG, J. N., LI, X. X., LIU, X. D., ZHU, J., WANG, F. X., XIE, W. Y. & MA, X. J. 2009. Effect of surface morphology and charge on the amount and conformation of fibrinogen adsorbed onto alginate/chitosan microcapsules. Langmuir, 26, 5587–5594.

XU, L. C. & SIEDLECKI, C. A. 2009. Atomic force microscopy studies of the initial interactions between fibrinogen and surfaces. Langmuir, 25, 3675-3681.

XU, Y., TAKAI, M. & ISHIHARA, K. 2009. Protein adsorption and cell adhesion on cationic, neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces. Biomaterials, 30, 4930-4938.

XU, Z., SONG, K., YUAN, S. L. & LIU, C. B. 2011. Microscopic wetting of self-assembled monolayers with different surfaces: a combined molecular dynamics and quantum mechanics study. Langmuir, 27, 8611-20.

YAN, X. & GEMEINHART, R. A. 2005. Cisplatin delivery from poly(acrylic acid-co-methyl methacrylate) microparticles. J Control Release, 106, 198-208.



YANG, Y., CUNHA-REIS, C., BAGNANINCHI, P. O. & AYDIN, H. M. 2008. Control and monitoring of scaffold architecture for tissue engineering. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

YAROVAYA, G. A., BLOKHINA, T. B. & NESHKOVA, E. A. 2002. Contact system. New concepts on activation mechanisms and bioregulatory functions. Biochemistry (Mosc), 67, 13-24.

YIN, W., GHEBREHIWET, B. & PEERSCHKE, E. I. B. 2008. Expression of complement components and inhibitors on platelet microparticles. Platelets, 19, 225-233.

YOSHIDA, E., OHMURA, S., SUGIKI, M., ANAI, K. & MARUYAMA, M. 2001. A novel function of extraerythrocytic hemoglobin: identification of globin as a stimulant of plasminogen activator biosynthesis in human fibroblasts. Thromb Haemost, 86, 1521-7.

ZHA, Z., MA, Y., YUE, X. L., LIU, M. & DAI, Z. 2009. Self-assembled hemocompatible coating on poly (vinyl chloride) surface. Applied Surface Science, 256, 805-814.

ZHANG, H., ARONOW, M. S. & GRONOWICZ, G. A. 2005. Transforming growth factor-beta 1 (TGF-beta1) prevents the age-dependent decrease in bone formation in human osteoblast/implant cultures. Journal of Biomedical Materials Research Part A, 75A, 98-105.

ZHUO, R., MILLER, R., BUSSARD, K. M., SIEDLECKI, C. A. & VOGLER, E. A. 2005. Procoagulant stimulus processing by the intrinsic pathway of blood plasma coagulation. Biomaterials, 26, 2965-2973.

ZHUO, R., SIEDLECKI, C. A. & VOGLER, E. A. 2006. Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces. Biomaterials, 27, 4325-4332.

ZIATS, N. P., PANKOWSKY, D. A., TIERNEY, B. P., RATNOFF, O. D. & ANDERSON, J.M. 1990. Adsorption of Hageman Factor (Factor XII) and other human plasma proteins to biomaterial polymers. Journal of Laboratory and Clinical Medicine, 116, 687-696.

AKEDA, K., AN, H. S., OKUMA, M., ATTAWIA, M., MIYAMOTO, K., THONAR, E. J.-M. A., LENZ, M. E., SAH, R. L. & MASUDA, K. 2006. Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis Osteoarthritis and Cartilage, 14, 1272-1280.

ALBREKTSSON, T. & WENNERBERG, A. 2004. Oral implant surfaces: Part 1--review focusing on topographic and chemical properties of different surfaces and in vivo responses to them. Int J Prosthodont, 17, 536-43.

ALEMAN, M. M., GARDINER, C., HARRISON, P. & WOLBERG, A. S. 2011. Differential contributions of monocyte- and platelet-derived microparticles towards thrombin generation and fibrin formation and stability. J Thromb Haemost.

ALEXANDER, M. R., WHITTLE, J. D., BARTON, D. & SHORT, R. D. 2004. Plasma polymer chemical gradients for evaluation of surface reactivity: epoxide reaction with carboxylic acid surface groups. Journal of Materials Chemistry, 14, 408-412.

ALKHAMIS, T. M., BEISSINGER, R. L. & CHEDIAK, J. R. 1988. Red blood cell effect on platelet adhesion and aggregation in low-stress shear flow: myth or fact? ASAIO Journal, 34, 868-873.

ALLEN, G. A., WOLBERG, A. S., OLIVER, J. A., HOFFMAN, M., ROBERTS, H. R. & MONROE, D. M. 2004. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost, 2, 402-13.

ALLFORD, S. L. & MACHIN, S. J. 2007. Haemostasis. Surgery, 25, 241-244. ALTIERI, D. C. 1995. Inflammatory cell participation in coagulation. Seminars in Cell Biology,

6, 269-274.



AMARA, U., FLIERL, M. A., RITTIRSCH, D., KLOS, A., CHEN, H. C., ACKER, B., BRÜCKNER, U. B., NILSSON, B., GEBHARD, F., LAMBRIS, J. D. & HUBER-LANG, M. 2010. Molecular intercommunication between the complement and coagulation systems. The Journal of Immunology, 185, 5628-5636.

AMARA, U., RITTIRSCH, D., FLIERL, M. A., BRUCKNER, U., KLOS, A., GEBHARD, F., LAMBRIS, J. D. & HUBER-LANG, M. 2008. Interaction between the coagulation and complement system. Advances in experimental medicine and biology, 632, 71-79.

AMINI, A. R., WALLACE, J. S. & NUKAVARAPU, S. P. 2011. Short-term and long-term effects of orthopedic biodegradable implants. Journal of Long-Term Effects of Medical Implants, 21, 93-122.

ANDERSON, A. B., DALLMIER, A. W., CHUDZIK, S. J., DURAN, L. W., GUIRE, P. E., HERGENROTHER, R. W., LODHI, M. A., NOVAK, A. E., OFSTEAD, R. F. & WORMUTH, K. 2004a. Technologies for the surface modification of biomaterials. In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthropedics. New York: Marcel Dekker, Inc.

ANDERSON, J. M. 2001. Biological responses to materials. Annual Reviews of Materials Resources, 31, 81-110.

ANDERSON, J. M., COOK, G., COSTERTON, B., HANSON, S. R., PETTERSEN, A. H., JACOBSEN, N., JOHNSON, R. J., MITCHELL, R. N., PASMORE, M., SCHOEN, F. J., SHIRTLIFF, M. & STOODLEY, P. 2004b. Host reactions to biomaterial and their evaluation. In: RATNER, B. D., HOFFMAN, A. S. & SCHOEN, F. S. (eds.) Biomaterial science: an introduction to materials in medicine. 2nd ed. New York: Elsevier Academic Press.

ANDERSON, J. M., RODRIGUEZ, A. & CHANG, D. T. 2008. Foreign body reaction to biomaterials. Seminars in Immunology, 20, 86-100.

ANDRAE, J., GALLINI, R. & BETSHOLTZ, C. 2008. Role of platelet-derived growth factors in physiology and medicine. Genes & Development, 22, 1276-1312.

ANDREWS, R. K. & BERNDT, M. C. 2004. Platelet physiology and thrombosis. Thromb Res, 114, 447-53.

ANITUA, E., ANDIA, I., ARDANZA, B., NURDEN, P. & NURDEN, A. T. 2004. Autologous platelets as a source of proteins for healing and tissue regeneration. Thrombosis and Haemostasis, 91, 4-15.

ANITUA, E., SÁNCHEZ, M., NURDEN, A. T., NURDEN, P., ORIVE, G. & ANDÍA, I. 2006. New insights into and novel applications for platelet-rich fibrin therapies. Trends in Biotechnology, 24, 227-234.

ANNAZ, B., HING, K. A., KAYSER, M., BUCKLAND, T. & DI SILVIO, L. 2004. Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study. J Microsc, 215, 100-10.

ARIMA, Y. & IWATA, H. 2007. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials, 28, 3074-3082.

ARPORNMAEKLONG, P., KOCHEL, M., DEPPRICH, R., KÜBLER, N. R. & WÜRZLER, K. K. 2003. Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. International Journal of Oral and Maxillofacial Surgery, 33, 60-70.

ARVIDSSON, A., MALMBERG, P., KJELLIN, P., CURRIE, F., ARVIDSSON, M. & FRANKE STENPORT, V. 2011. Early interactions between leukocytes and three different potentially bioactive titanium surface modifications. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 97B, 364-372.

ASHCROFT, G. S. 1999. Bidirectional regulation of macrophage function by TGF-[beta]. Microbes and Infection, 1, 1275-1282.

ASIJEE, G. M., MUSZBEK, L., KAPPELMAYER, J., POLGAR, J., HORVATH, A. & STURK, A. 1988. Platelet vinculin: a substrate of activated factor XIII. Biochim Biophys Acta, 954, 303-8.



AVRAMOGLOU, D. L., ANAGNOSTOU, F., BIZIOS, R. & PETITE, H. 2005. Engineering bone: challenges and obstacles. Journal of Cell and Molecular Medicine, 9, 72-84.

AWAD, H. A., ZHANG, X., REYNOLDS, D. G., GULDBERG, R. E., O'KEEFE, R. J. & SCHWARZ, E. M. 2007. Recent advances in gene delivery for structural bone allografts. Tissue Eng, 13, 1973-85.

BACH-GANSMO, E. T., GODAL, H. C. & SKJONSBERG, O. H. 1998. Degradation of fibrinogen and cross-linked fibrin by human neutrophil elastase generates D-like fragments detected by ELISA but not latex D-dimer test. Thromb Res, 92, 125-34.

BÄCK, J., HUBER LANG, M., ELGUE, G., KALBITZ, M., SANCHEZ, J., NILSSON EKDAHL, K. & NILSSON, B. 2009. Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces. Biomaterials, 30, 6573-6580.

BÄCK, J., SANCHEZ, J., ELGUE, G., EKDAHL, K. N. & NILSSON, B. 2010. Activated human platelets induce factor XIIa-mediated contact activation. Biochemical and Biophysical Research Communications, 391, 11-17.

BAEYENS, W., GLINEUR, R. & EVRARD, L. 2010. The use of platelet concentrates: platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in bone reconstruction prior to dental implant surgery. Rev Med Brux, 31, 521-527.

BALTZER, A. W. A. & LIEBERMAN, J. R. 2004. Regional gene therapy to enhance bone repair. Gene Therapy, 11, 344-350.

BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2003. Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces. J Biomed Mater Res A, 65, 429-34.

BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2004. Inflammatory responses and cell adhesion to self-assembled monolayers of alkanethiolates on gold. Biomaterials, 25, 2557-63.

BARBOSA, J. N., BARBOSA, M. A. & AGUAS, A. P. 2005. Inflammatory cell recruitment and adhesion to methyl-terminated self-assembled monolayers: effect of implantation time. Microsc Res Tech, 66, 37-42.

BARBOSA, J. N., MARTINS, M. C., FREITAS, S. C., GONCALVES, I. C., AGUAS, A. P. & BARBOSA, M. A. 2010. Adhesion of human leukocytes on mixtures of hydroxyl- and methyl-terminated self-assembled monolayers: effect of blood protein adsorption. J Biomed Mater Res A, 93, 12-9.

BAUER, T. W. & MUSCHLER, G. F. 2000. Bone graft materials. An overview of the basic science. Clin Orthop Relat Res, 10-27.

BENNETT, J. S. 2001. Platelet-fibrinogen interactions. Ann N Y Acad Sci, 936, 340-54. BENNETT, J. S., BERGER, B. W. & BILLINGS, P. C. 2009. The structure and function of

platelet integrins. J Thromb Haemost, 7 Suppl 1, 200-5. BERGLIN, M., ANDERSSON, M., SELLBORN, A. & ELWING, H. 2004. The effect of

substrate molecular mobility on surface induced immune complement activation and blood plasma coagulation. Biomaterials, 25, 4581-4590.

BERGLIN, M., PINORI, E., SELLBORN, A., ANDERSSON, M., HULANDER, M. & ELWING, H. 2009. Fibrinogen adsorption and conformational change on model polymers: novel aspects of mutual molecular rearrangement. Langmuir, 25, 5602-5608.

BERRY, L. R. & CHAN, A. K. C. 2008. Improving blood compatibility of biomaterials using a novel antithrombin-heparin covalent complex. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

BETZ, O. B., BETZ, V. M., ABDULAZIM, A., PENZKOFER, R., SCHMITT, B., SCHRODER, C., MAYER-WAGNER, S., AUGAT, P., JANSSON, V. & MULLER, P. E. 2010. The repair of critical-sized bone defects using expedited, autologous BMP-2 gene-activated fat implants. Tissue Eng Part A, 16, 1093-101.

BHASIN, N., ARIENS, R. A., WEST, R. M., PARRY, D. J., GRANT, P. J. & SCOTT, D. J. 2008. Altered fibrin clot structure and function in the healthy first-degree relatives of subjects with intermittent claudication. J Vasc Surg, 48, 1497-503, 1503 e1.



BIMMEL, R. & GOVAERS, K. 2006. Does harvesting of iliac bone grafts with an acetabular reamer reduce complication rate? Acta Orthop Belg, 72, 598-602.

BLAIR, P. & FLAUMENHAFT, R. 2009. Platelet [alpha]-granules: basic biology and clinical correlates. Blood Reviews, 23, 177-189.

BLOCKMANS, D., DECKMYN, H. & VERMYLEN, J. 1995. Platelet activation. Blood Reviews, 9, 143-156.

BLOM, A. 2007. (V) Which scaffold for which application? Current Orthopaedics, 21, 280-287. BLOMBACK, B. 2000. Fibrin formation in whole blood. Thromb Res, 99, 307-10. BLOMBACK, B. & BARK, N. 2004. Fibrinopeptides and fibrin gel structure. Biophys Chem,

112, 147-51. BOSETTI, M., BOCCAFOSCHI, F., LEIGHEB, M. & CANNAS, M. F. 2007. Effect of

different growth factors on human osteoblasts activities: a possible application in bone regeneration for tissue engineering. Biomolecular Engineering, 24, 613-618.

BOUCHARD, B. A. & TRACY, P. B. 2001. Platelets, leukocytes, and coagulation. Current Opinion in Hematology, 8, 263-269.

BREITBART, A. S., GRANDE, D. A., KESSLER, R., RYABY, J. T., FITZSIMMONS, R. J. & GRANT, R. T. 1998. Tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg, 101, 567-74; discussion 575-6.

BROBERG, M., ERIKSSON, C. & NYGREN, H. 2002. GPIIb/IIIa is the main receptor for initial platelet adhesion to glass and titanium surfaces in contact with whole blood. Journal of Laboratory and Clinical Medicine, 139, 163-172.

BRUMMEL, K. E., PARADIS, S. G., BUTENAS, S. & MANN, K. G. 2002. Thrombin functions during tissue factor-induced blood coagulation Blood, 100, 148-152.

BURG, K. J. L., PORTER, S. & KELLAM, J. F. 2000. Biomaterial developments for bone tissue engineering. Biomaterials, 21, 2347-2359.

BUTLER, M. J. & SEFTON, M. V. 2007. Poly(butyl methacrylate-co-methacrylic acid) tissue engineering scaffold with pro-angiogenic potential in vivo. J Biomed Mater Res A, 82, 265-73.

CAMPBELL, R. A., OVERMYER, K. A., BAGNELL, C. R. & WOLBERG, A. S. 2008. Cellular procoagulant activity dictates clot structure and stability as a function of distance from the cell surface. Arterioscler Thromb Vasc Biol, 28, 2247-54.

CAMPBELL, R. A., OVERMYER, K. A., SELZMAN, C. H., SHERIDAN, B. C. & WOLBERG, A. S. 2009. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood, 114, 4886-96.

CAPUTO, A. E. 1999. Healing of bone and connective tissues. In: BRONNER, F. & WORRELL, R. V. (eds.) Structure and function of bone tissues. New York: CRC Press LLC.

CARANO, R. A. D. & FILVAROFF, E. H. 2003. Angiogenesis and bone repair. Drug Discovery Today, 8, 980-989.

CARLSON, N. E. & ROACH, R. B., JR. 2002. Platelet-rich plasma: clinical applications in dentistry. J Am Dent Assoc, 133, 1383-6.

CARLSSON, L. V., MACDONALD, W., JACOBSSON, C. M. & ALBREKTSSON, T. 2004. Osseointegration principles in orthopedics: basic research and clinical applications. In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthopedics. New York: Marcel Dekker, Inc.

CARR, M. E. & HARDIN, C. L. 1987. Fibrin has larger pores when formed in the presence of erythrocytes. American Journal of Physiology - Heart and Circulatory Physiology, 253, H1069-H1073.

CARR, M. E., JR., GABRIEL, D. A. & MCDONAGH, J. 1987. Influence of factor XIII and fibronectin on fiber size and density in thrombin-induced fibrin gels. J Lab Clin Med, 110, 747-52.

CARR, M. E., JR., MARTIN, E. J. & CARR, S. L. 2002a. Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation. Blood Coagul Fibrinolysis, 13, 193-7.



CARR, M. E., JR., MARTIN, E. J. & CARR, S. L. 2002b. Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation. Blood Coagulation & Fibrinolysis, 13, 193-197.

CARR, M. E., JR. & ZEKERT, S. L. 1994. Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma. Am J Physiol, 266, H1195-201.

CARRIERI, C., GALASSO, R., SEMERARO, F., AMMOLLO, C. T., SEMERARO, N. & COLUCCI, M. 2011. The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet-mediated fibrinolysis resistance: a thromboelastographic study in whole blood. Journal of Thrombosis and Haemostasis, 9, 154-162.

CELOTTI, F., COLCIAGO, A., NEGRI-CESI, P., PRAVETTONI, A., ZANINETTI, R. & SACCHI, M. C. 2006. Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-β. Wound Repair and Regeneration, 14, 195-202.

CENNI, E., CIAPETTI, G., GRANCHI, D., FOTIA, C., PERUT, F., GIUNTI, A. & BALDINI, N. 2009. Endothelial cells incubated with platelet-rich plasma express PDGF-B and ICAM-1 and induce bone marrow stromal cell migration. J Orthop Res, 27, 1493-8.

CENNI, E., SAVARINO, L., PERUT, F., FOTIA, C., AVNET, S. & SABBIONI, G. 2010. Background and rationale of platelet gel in orthopaedic surgery. Musculoskelet Surg, 94, 1-8.

CHEN, Q. Z., BRETCANU, O. & BOCCACCINI, A. R. 2008. Inorganic and composite bioactive scaffolds for bone tissue engineering. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

CHEN, X., WANG, J., PASZTI, Z., WANG, F., SCHRAUBEN, J., TARABARA, V., SCHMAIER, A. & CHEN, Z. 2007. Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy. Analytical and Bioanalytical Chemistry, 388, 65-72.

CHOI, B.-H., IM, C.-J., HUH, J.-Y., SUH, J.-J. & LEE, S.-H. 2004. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. International Journal of Oral and Maxillofacial Surgery, 33, 56-59.

CHOI, B.-H., ZHU, S.-J., B.-Y., K., HUH, J.-Y., LEE, S.-H. & JUNG, J.-H. 2005. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. International Journal of Oral and Maxillofacial Surgery, 34, 420-424.

CHOUKROUN, J., DISS, A., SIMONPIERI, A., GIRARD, M. O., SCHOEFFLER, C., DOHAN, S. L., DOHAN, A. J. J., MOUHYI, J. & DOHAN EHRENFEST, D. M. 2006. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 101, e56-e60.

CHUANG, W. H. & LIN, J. C. 2007. Surface characterization and platelet adhesion studies for the mixed self-assembled monolayers with amine and carboxylic acid terminated functionalities. Journal of Biomedical Materials Research Part A, 82A, 820-830.

CLARK, R. A. 2001. Fibrin and wound healing. Ann N Y Acad Sci, 936, 355-67. COLLET, J. P., ALLALI, Y., LESTY, C., TANGUY, M. L., SILVAIN, J., ANKRI, A.,

BLANCHET, B., DUMAINE, R., GIANETTI, J., PAYOT, L., WEISEL, J. W. & MONTALESCOT, G. 2006. Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol, 26, 2567-73.

COLLET, J. P., MONTALESCOT, G., LESTY, C. & WEISEL, J. W. 2002. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circ Res, 90, 428-34.

COLLET, J. P., PARK, D., LESTY, C., SORIA, J., SORIA, C., MONTALESCOT, G. & WEISEL, J. W. 2000. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol, 20, 1354-61.



COLLET, J. P., SHUMAN, H., LEDGER, R. E., LEE, S. & WEISEL, J. W. 2005. The elasticity of an individual fibrin fiber in a clot. Proc Natl Acad Sci U S A, 102, 9133-9137.

COLLET, J. P., SORIA, J., MIRSHAHI, M., HIRSCH, M., DAGONNET, F. B., CAEN, J. & SORIA, C. 1993a. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood, 82, 2462-2469.

COLLET, J. P., SORIA, J., MIRSHAHI, M., HIRSCH, M., DAGONNET, F. B., CAEN, J. & SORIA, C. 1993b. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood, 82, 2462-9.

COOPER, L. F. 2000. A role for surface topography in creating and maintaining bone at titanium endosseous implants. J Prosthet Dent, 84, 522-34.

COVE, R. & KEENAN, J. 2009. Tibial bone grafting for lateral tibial plateau fractures. Ann R Coll Surg Engl, 91, 268-9.

CRAWFORD, A. & HATTON, P. V. 2008. Cell-biomaterial interactions in skeletal tissue engineering. Biomedicine & Pharmacotherapy, 62, 493-494.

CRAWFORD, S. E., STELLMACH, V., MURPHY-ULLRICH, J. E., RIBEIRO, S. M. F., LAWLER, J., HYNES, R. O., BOIVIN, G. P. & BOUCK, N. 1998. Thrombospondin-1 is a major activator of TGF-b1 in vivo. Cell, 93, 1159-1170.

CROSS, K. J. & MUSTOE, T. A. 2003. Growth factors in wound healing. The Surgical clinics of North America, 83, 531-545.

DAI, J., RABIE, A. B., HAGG, U. & XU, R. 2004. Alternative gene therapy strategies for the repair of craniofacial bone defects. Curr Gene Ther, 4, 469-85.

DAVIE, E. W., FUJIKAWA, K. & KISIEL, W. 1991. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry, 30, 10363-10370.

DAVIES, J. E. 1998. Mechanisms of endosseous integration. The international journal of prosthodontics, 11, 391.

DAVIES, J. E. 2003a. Understanding peri-implant endosseous healing. Journal of dental education, 67, 932.

DAVIES, J. E. 2003b. Understanding peri-implant endosseous healing. Journal of dental education, 67, 932-949.

DAWSON, J. I. & OREFFO, R. O. 2008. Bridging the regeneration gap: stem cells, biomaterials and clinical translation in bone tissue engineering. Arch Biochem Biophys, 473, 124-31.

DE BARROS, R. R., NOVAES, A. B., JR., QUEIROZ, A. & DE ALMEIDA, A. L. 2011. Early peri-implant endosseous healing of two implant surfaces placed in surgically created circumferential defects. A histomorphometric and fluorescence study in dogs. Clin Oral Implants Res.

DEB, S. 2008. Synthetic and natural degradable polymeric biomaterials. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication amd processing handbook. New York: Taylor & Francis Group, LLC.

DEL CONDE, I., CRÚZ, M. A., ZHANG, H., LÓPEZ, J. A. & AFSHAR-KHARGHAN, V. 2005. Platelet activation leads to activation and propagation of the complement system. The Journal of Experimental Medicine, 201, 871-879.

DEUEL, T. F., SENIOR, R. M., CHANG, D., GRIFFIN, G. L., HEINRIKSON, R. L. & KAISER, E. T. 1981. Platelet factor 4 is chemotactic for neutrophils and monocytes. PNAS, 78, 4584-4587.

DI CERA, E. 2003. Thrombin interactions. Chest, 124, 11S-17S. DIMITRIOU, R., TSIRIDIS, E. & GIANNOUDIS, P. V. 2005. Current concepts of molecular

aspects of bone healing. Injury, 36, 1392-404. DOBLARE, M., GARCIA, J. M. & GOMEZ, M. J. 2004. Modelling bone tissue fracture and

healing: a review. Engineering Fracure Mechanics, 71, 1809-1840. DOHAN EHRENFEST, D. M., BIELECKI, T., JIMBO, R., BARBE, G., DEL CORSO, M.,

INCHINGOLO, F. & SAMMARTINO, G. 2012. Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An



evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol, 13, 1145-52.

DOHAN EHRENFEST, D. M., CHOUKROUN, J., DISS, A., DOHAN, S. L., DOHAN, A. J. J., MOUHYI, J. & GOGLY, B. 2006. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: leucocyte activation: a new feature for platelet concentrates? Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 101, e51-e55.

DOHAN EHRENFEST, D. M., DEL CORSO, M., INCHINGOLO, F., SAMMARTINO, G. & CHARRIER, J. B. 2010. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in human cell cultures: growth factor release and contradictory results. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 110, 418-21; author reply 421-2.

DOLDER, J. V. D., MOOREN, R., VLOON, A. P. G., STOELINGA, P. J. W. & JANSEN, J. A. 2006. Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells. Tissue Engineering, 12, 3067-3073.

DUBOIS, C., REIGNER, S. C., STEINER, B. & RIEDERER, M. A. 2004. Thrombin binding to GPIbalpha induces integrin alphaIIbbeta3 dependent platelet adhesion to fibrin in ex vivo flowing whole blood. Thromb Haemost, 91, 233-7.

DUGRILLON, A., EICHLER, H., KERN, S. & KLÜTER, H. 2002. Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration. The International Journal of Oral and Maxillofacial Surgery, 31, 615-619.

ECKHAUS, A. A., FISH, J. S., SKARJA, G., SEMPLE, J. L. & SEFTON, M. V. 2008. A preliminary study of the effect of poly(methacrylic acid-co-methyl methacrylate) beads on angiogenesis in rodent skin grafts and the quality of the panniculus carnosus. Plast Reconstr Surg, 122, 1361-70.

EHRHART, N. P., HONG, L., MORGAN, A. L., EURELL, J. A. & JAMISON, R. D. 2005. Effect of transforming growth factor-beta1 on bone regeneration in critical-sized bone defects after irradiation of host tissues. Am J Vet Res, 66, 1039-45.

EKPENYONG, K. I. 1985. Monomer reactivity ratios: acrylic acid-methylmethacrylate copolymerization in dimethylsulfoxide. Journal of Chemical Education, 62, 173-174.

EL-NEWEHY, M. H., AL-DEYAB, S. S. & AL-HAZMI, A. M. A. 2010. Reactivity ratios for organotin copolymer systems. Molecules, 15, 2749-2758.

ELALAMY, I., CHAKROUN, T., GEROTZIAFAS, G. T., PETROPOULOU, A., ROBERT, F., KARROUM, A., ELGRABLY, F., SAMAMA, M. M. & HATMI, M. 2007. Circulating platelet-leukocyte aggregates: A marker of microvascular injury in diabetic patients. Thrombosis Research, In Press, Corrected Proof.

EPPLEY, B. L., WOODELL, J. E. & HIGGINS, J. 2004. Platelet quantification and growth factor analysis from platelet-rich plasma: Implications for wound healing. Plastic and Reconstructive Surgery, 114, 1502-1508.

ERICKSON, L. A., R., S. R., NY, T. & LOSKUTOFF, D. J. 1985. The fibrinolytic system of the vascular wall. Clinics in Haematology, 14, 513-530.

ERIKSSON, C. & NYGREN, H. 2001. Polymorphonuclear leukocytes in coagulating whole blood recognize hydrophilic and hydrophobic titanium surfaces by different adhesion receptors and show different patterns of receptor expression. Journal of Laboratory and Clinical Medicine, 137, 296-302.

ERNOFSSON, M., THELIN, S. & SIEGBAHN, A. 1997. Monocyte tissue factor expression, cell activation and thrombin formation during cardiopulmonary bypass: a clinical study. The Journal of Thoracic and Cardiovascular Surgery, 113, 576-584.

ESKIN, S. G., HORBETT, T. A., MCINTIRE, L. V., MITCHELL, R. N., RATNER, B. D., SCHOEN, F. J. & YEE, A. 2004. Some background concepts. In: RATNER, B. D., HOFFMAN, A. S. & SCHOEN, F. S. (eds.) Biomaterial science: an introduction to materials in medicine. New York: Elsevier Academic Press.

ESMON, C. T. 1995. Introduction: cellular regulation of blood coagulation. Seminars in Cell Biology, 6, 257-258.

ESMON, C. T. 2003. Inflammation and thrombosis. Journal of Thromosis and Haemostasis, 1, 1343-1348.



ESMON, C. T. 2004. The impact of the inflammatory response on coagulation. Thrombosis Research, 114, 321-327.

EVANS-NGUYEN, K. M., FUIERER, R. R., FITCHETT, B. D., TOLLES, L. R., CONBOY, J. C. & SCHOENFISCH, M. H. 2006. Changes in adsorbed fibrinogen upon conversion to fibrin. Langmuir, 22, 5115-21.

EVANS-NGUYEN, K. M. & SCHOENFISCH, M. H. 2005a. Fibrin proliferation at model surfaces: influence of surface properties. Langmuir, 21, 1691-1694.

EVANS-NGUYEN, K. M. & SCHOENFISCH, M. H. 2005b. Fibrin proliferation at model surfaces: influence of surface properties. Langmuir, 21, 1691-4.

EVANS-NGUYEN, K. M., TOLLES, L. R., GORKUN, O. V., LORD, S. T. & SCHOENFISCH, M. H. 2005a. Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers. Biochemistry, 44, 15561–15568.

EVANS-NGUYEN, K. M., TOLLES, L. R., GORKUN, O. V., LORD, S. T. & SCHOENFISCH, M. H. 2005b. Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers. Biochemistry, 44, 15561-8.

FAUCHEUX, N., SCHWEISS, R., LÜTZOW, K., WERNER, C. & GROTH, T. 2004. Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies. Biomaterials, 25, 2721-2730.

FAXÄLV, L., EKBLAD, T., LIEDBERG, B. & LINDAHL, T. L. 2010. Blood compatibility of photografted hydrogel coatings. Acta Biomaterialia, 6, 2599-2608.

FERRAZ, N., NILSSON, B., HONG, J. & KARLSSON OTT, M. 2008. Nanoporesize affects complement activation. J Biomed Mater Res A, 87, 575-81.

FERRAZ, N., OTT, M. K. & HONG, J. 2010. Time sequence of blood activation by nanoporous alumina: studies on platelets and complement system. Microscopy Research and Technique, 73, 1101-1109.

FISCHER, M., SPERLING, C., TENGVALL, P. & WERNER, C. 2010a. The ability of surface characteristics of materials to trigger leukocyte tissue factor expression. Biomaterials, 31, 2498-2507.

FISCHER, M., SPERLING, C. & WERNER, C. 2010b. Synergistic effect of hydrophobic and anionic surface groups triggers blood coagulation in vitro. Journal of Materials Science: Materials in Medicine, 21, 931-937.

FRAYSSINET, P. 2004. Biocompatible materials: calcium phosphates in orthopedic surgery. In: POITOUT, D. G. (ed.) Biomechanics and biomaterials in orthopedics. Singapore: Springer-Verlag London Limited.

FRECHETTE, J.-P., MARTINEAU, I. & GAGNON, G. 2005. Platelet-rich plasmas: growth factor content and roles in wound healing. Journal of Dental Research, 84, 434-439.

FROST, A., JONSSON, K. B., RIDEFELT, P., NILSSON, O., LJUNGHALL, S. & LJUNGGREN, O. 1999. Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation. Acta Orthop Scand, 70, 497-503.

FUJISHITA, S., INABA, C., ISHISAKA, T., GEMMEI-IDE, M. & KITANO, H. 2009. Effect of end groups of poly(n-butyl methacrylate) on its biocompatibility. Colloids and Surfaces B: Biointerfaces, 74, 45-50.

FUSHIMI, F., NAKAYAMA, M., NISHIMURA, K. & HIYOSHI, T. 1998. Platelet adhesion, contact phase coagulation activation, and C5a generation of polyethylene glycol acid-grafted high flux cellulosic membrane with varieties of grafting amounts. Artif Organs, 22, 821-6.

FUSS, C., PALMAZ, J. C. & SPRAGUE, E. A. 2001. Fibrinogen: structure, function, and surface interactions. Journal of Vascular and Interventional Radiology, 12, 677-682.

GABRIEL, D. A., MUGA, K. & BOOTHROYD, E. M. 1992. The effect of fibrin structure on fibrinolysis. Journal of Biological Chemistry, 267, 24259-24263.



GANDHI, A., DOUMAS, C., O'CONNOR, J. P., PARSONS, J. R. & LIN, S. S. 2006. The effects of local platelet rich plasma delivery on diabetic fracture healing. Bone, 38, 540-546.

GAROFALO, G. S. 2007. Autogenous, allogenetic and xenogenetic grafts for maxillary sinus elevation: literature review, current status and prospects. Minerva Stomatol, 56, 373-92.

GEER, C. B., RUS, I. A., LORD, S. T. & SCHOENFISCH, M. H. 2007. Surface-dependent fibrinopeptide A accessibility to thrombin. Acta Biomaterialia, 3, 663-668.

GEISER, A. G., ZENG, Q. Q., SATO, M., HELVERING, L. M., HIRANO, T. & TURNER, C. H. 1998. Decreased bone mass and bone elasticity in mice lacking the transforming growth factor-beta 1 gene. Bone, 23, 87-93.

GEMMELL, C. H. 1997. A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces. Journal of biomedical materials research, 37, 474-480.

GEMMELL, C. H. 2001. Activation of platelets by in vitro whole blood contact with materials: Increases in microparticle, procoagulant activity, and soluble P-selectin blood levels Journal of Biomaterials Science, Polymer Edition,, 12, 933-943.

GERBER, H. & FERRARA, N. 2000. Angiogenesis and bone growth. Trends in Cardiovascular Medicine, 10, 223-228.

GHEBREHIWET, B., RANDAZZO, B. P., DUNN, J. T., SILVERBERG, M. & KAPLAN, A. P. 1983. Mechanisms of activation of the classical pathway of complement by Hageman factor fragment. J Clin Invest, 71, 1450-6.

GHEBREHIWET, B., SILVERBERG, M. & KAPLAN, A. P. 1981. Activation of the classical pathway of complement by Hageman factor fragment. J Exp Med, 153, 665-76.

GIANNOUDIS, P. V., DINOPOULOS, H. & TSIRIDIS, E. 2005. Bone substitutes: an update. Injury, 36, S20-27.

GIBBINS, J. M. 2004. Platelet adhesion signalling and the regulation of thrombus formation. Journal of Cell Science, 117, 3415-3425.

GIMENO, F. L., GATTO, S., FERRO, J., CROXATTO, J. O. & GOLLA, J. E. 2006. Preparation of platelet-rich plasma as a tissue adhesive for experimental transplantation in rabbits. Thrombosis Journal, 4, 18-24.

GLOWACKI, J. 1998. Angiogenesis in fracture repair. Clinical Orthopaedics and Related Research, 355, S82.

GOBBI, G., MIRANDOLA, P., TAZZARI, P. L., RICCI, F., CAIMI, L., CACCHIOLI, A., PAPA, S., CONTE, R. & VITALE, M. 2003. Flow cytometry detection of serotonin content and release in resting and activated platelets. Br J Haematol, 121, 892-6.

GOLD, H. K., YASUDA, T., JANG, I. K., GUERRERO, J. L., FALLON, J. T., LEINBACH, R. C. & COLLEN, D. 1991. Animal models for arterial thrombolysis and prevention of reocclusion. Erythrocyte-rich versus platelet-rich thrombus. Circulation, 83, IV26-40.

GOLINO, P., ASHTON, J. H., BUJA, L. M., ROSOLOWSKY, M., TAYLOR, A. L., MCNATT, J., CAMPBELL, W. B. & WILLERSON, J. T. 1989. Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators. Circulation, 79, 154-66.

GORBET, M. B. & SEFTON, M. V. 2004. Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelet and leukocytes. Biomaterials, 25, 5681-5703.

GOULET, J. A., SENUNAS, L. E., DESILVA, G. L. & GREENFIELD, M. L. V. H. 1997. Autogenous iliac crest bone graft: complications and functional assessment. Clinical Orthopaedic & Related Research, 339, 76-81.

GRAGEDA, E. 2004. Platelet-rich plasma and bone graft materials: a review and a standardized research protocol. Implant Dentistry, 13, 301-309.

GREENBERG, C. S., HURSTING, M. J., MACIK, B. G., ORTEL, T. L., KANE, W. H. & MOORE, B. M. 1994. Evaluation of preanalytical variables associated with measurement of prothrombin fragment 1.2. Clin Chem, 40, 1962-9.

GRIFFIN, X. L., SMITH, C. M. & COSTA, M. L. 2009. The clinical use of platelet-rich plasma in the promotion of bone healing: a systematic review. Injury, 40, 158-162.



GUGUTKOV, D., GONZÁLEZ-GARCÍA, C., ALTANKOV, G. & SALMERÓN-SÁNCHEZ, M. 2011. Fibrinogen organization at the cell-material interface directs endothelial cell behavior. Journal of Bioactive and Compatible Polymers, 26, 375-387.

GUO, R. F. & WARD, P. A. 2005. Role of C5a in inflammatory responses. Annu. Rev. Immunol., 23, 821-852.

GUPTA, B., HILBORN, J. G., BISSON, I. & FREY, P. 2001. Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films. Journal of Applied Polymer Science, 81, 2993-3001.

GUPTA, B., PLUMMER, C., BISSON, I., FREY, P. & HILBORN, J. 2002. Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films: characterization and human smooth muscle cell growth on grafted films. Biomaterials, 23, 863-871.

HABIBOVIC, P. & DE GROOT, K. 2007. Osteoinductive biomaterials--properties and relevance in bone repair. J Tissue Eng Regen Med, 1, 25-32.

HAK, D. J. 2007. The use of osteoconductive bone graft substitutes in orthopaedic trauma. J Am Acad Orthop Surg, 15, 525-36.

HALLAB, J. N., URBAN, R. M. & JACOBS, J. J. 2004. Corrosion and biocompatibility of orthopedic implants In: YASZEMSKI, M. J., TRANTOLO, D. J., LEWANDROWSKI, K. U., HASIRCI, V., ALTOBELLI, D. E. & WISE, D. L. (eds.) Biomaterials in orthopedics. New York: Marcel Dekker, Inc.

HAMAD, O. A., EKDAHL, K. N., NILSSON, P. H., ANDERSSON, J., MAGOTTI, P., LAMBRIS, J. D. & NILSSON, B. 2008. Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets. J Thromb Haemost, 6, 1413-1421.

HAMAD, O. A., NILSSON, P. H., WOUTERS, D., LAMBRIS, J. D., EKDAHL, K. N. & NILSSON, B. 2010. Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1. The Journal of Immunology, 184, 2686-2692.

HANDT, S., JEROME, W. G., TIETZE, L. & HANTGAN, R. R. 1996. Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance. Blood, 87, 4204-13.

HANTGAN, R. R., HINDRIKS, G., TAYLOR, R. G., SIXMA, J. J. & DE GROOT, P. G. 1990. Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood. Blood, 76, 345-353.

HARWOOD, P. J., NEWMAN, J. B. & MICHAEL, A. L. R. 2010a. (ii) An update on fracture healing and non-union. Orthopaedics and Trauma, 24, 9-23.

HARWOOD, P. J., NEWMAN, J. B. & MICHAEL, A. L. R. 2010b. (ii) An update on fracture healing and non-union. 24, 9-23.

HE, S., CAO, H., ANTOVIC, A. & BLOMBACK, M. 2005. Modifications of flow measurement to determine fibrin gel permeability and the preliminary use in research and clinical materials. Blood Coagulation & Fibrinolysis, 16, 61-67.

HENCH, J. W. 2005a. Inflammation and wound healing. In: HENCH, L. L. & JONES, J. R. (eds.) Biomaterials, artificial organs and tissue engineering. New York: Woodhead Publishing Limited.

HENCH, L. L. 2005b. Repair of skeletal tissue. In: HENCH, L. L. & JONES, J. R. (eds.) Biomaterials, artifical organs and tissue engineering. New York: Woodhead Publishing Limited.

HERMITTE, L., THOMAS, F., BOUGARAN, R. & MARTELET, C. 2004. Contribution of the comonomers to the bulk and surface properties of methacrylate copolymers. Journal of Colloid and Interface Science, 272, 82-89.

HERTEL, R., EIJER, H., MEISSER, A., HAUKE, C. & PERREN, S. M. 2001. Biomechanical and biological considerations relating to the clinical use of the Point Contact-Fixator--evaluation of the device handling test in the treatment of diaphyseal fractures of the radius and/or ulna. Injury, 32 Suppl 2, B10-4.



HING, K. A. 2005. Bioceramic bone graft substitutes: influence of porosity and chemistry. International Journal of Applied Ceramic Technology, 2, 184-199.

HING, K. A., ANNAZ, B., SAEED, S., REVELL, P. A. & BUCKLAND, T. 2005. Microporosity enhances bioactivity of synthetic bone graft substitutes. J Mater Sci Mater Med, 16, 467-75.

HING, K. A., BEST, S. M., TANNER, K. E., BONFIELD, W. & REVELL, P. A. 2004. Mediation of bone ingrowth in porous hydroxyapaptite bone graft substitutes. Journal of Biomedical Materials Research. Part A., 68, 187-200.

HING, K. A., WILSON, L. F. & BUCKLAND, T. 2007. Comparative performance of three ceramic bone graft substitutes. Spine J, 7, 475-90.

HIRATA, I., HIOKI, Y., TODA, M., KITAZAWA, T., MURAKAMI, Y., KITANO, E., KITAMURA, H., IKADA, Y. & IWATA, H. 2003. Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance. J Biomed Mater Res A, 66, 669-76.

HOLLAND, C. K., VAIDYA, S. S., DATTA, S., COUSSIOS, C. C. & SHAW, G. J. 2008. Ultrasound-enhanced tissue plasminogen activator thrombolysis in an in vitro porcine clot model. Thromb Res, 121, 663-73.

HONG, J., ANDERSSON, J., EKDAHL, K. N., ELGUE, G., AXEN, N., LARSSON, R. & NILSSON, B. 1999. Titanium is a highly thrombogenic biomaterial: possible implications for osetogenesis. Thrombosis and Haemostasis, 82, 58-64.

HONG, J., AZENS, A., EKDAHL, K. N., GRANQVIST, C. G. & NILSSON, B. 2005. Material-specific thrombin generation following contact between metal surfaces and whole blood. Biomaterials, 26, 1397-1403.

HONG, J., LARSSON, A., EKDAHL, K. N., ELGUE, G., LARSSON, R. & NILSSON, B. 2001. Contact between a polymer and whole blood: sequence of events leading to thrombin generation. Journal of Laboratory and Clinical Medicine, 138, 139-145.

HORBETT, T. A. 1993. Chapter 13: Principles underlying the role of adsorbed plasma proteins in blood interactions with foreign materials. Cardiovascular Pathology, 2, 137-148.

HORNE, M. K., CULLINANE, A. M., MERRYMAN, P. K. & HODDESON, E. K. 2006. The effect of red blood cells on thrombin generation. British Journal of Haematology, 133, 403-408.

HOSGOOD, G. 1993. Wound healing: the role of platelet-derived growth factor and transforming growth factor beta. Veterinary Surgery, 22, 490-495.

HU, W. J., EATON, J. W., UGAROVA, T. P. & TANG, L. P. 2001. Molecular basis of biomaterial-mediated foreign body reactions. Blood, 98, 1231-1238.

HUANG, W. W. & JANG, J. H. 2009. Hydrophilic modification of PET fabric via continuous photografting of acrylic acid (AA) and hydroxyethyl methacrylate (HEMA). Fibers and Polymers, 10, 27-33.

HUBER-LANG, M., SARMA, J. V., ZETOUNE, F. S., RITTIRSCH, D., NEFF, T. A., MCGUIRE, S. R., LAMBRIS, J. D., WARNER, R. L., FLIERL, M. A., HOESEL, L. M., GEBHARD, F., YOUNGER, J. G., DROUIN, S. M., WETSEL, R. A. & WARD, P. A. 2006. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med, 12, 682-7.

HUTCHEON, G. A., MESSIOU, C., WYRE, R. M., DAVIES, M. C. & DOWNES, S. 2001. Water absorption and surface properties of novel poly(ethylmethacrylate) polymer systems for use in bone and cartilage repair. Biomaterials, 22, 667-676.

HUTMACHER, D. W., DUDA, G. & GULDBERG, R. E. 2012. Endogenous musculoskeletal tissue regeneration. Cell Tissue Res, 347, 485-8.

HUTMACHER, D. W. & WILLIAMS, D. F. 2006. Scaffolds in tissue engineering bone and cartilage. The Biomaterials: Silver Jubilee Compendium. Oxford: Elsevier Science.

IKEDA, K., NAGASAWA, K., HORIUCHI, T., TSURU, T., NISHIZAKA, H. & NIHO, Y. 1997. C5a induces tissue factor activity on endothelial cells. Thromb Haemost, 77, 394-8.

ILAN, D. I. & LADD, A. L. 2003. Bone graft substitutes. Operative Techniques in Plastics and Reconstructive Surgery, 9, 151-160.



ISHIZAKI, T., SAITO, N., SATO, Y. & TAKAI, O. 2007. Probing into adsorption behavior of human plasma fibrinogen on self-assembled monolayers with different chemical properties by scanning probe microscopy. Surface Science, 601, 3861-3865.

IWATA, H., KAIBARA, M., DOHMAE, N., TAKIO, K., HIMENO, R. & KAWAKAMI, S. 2004. Purification, identification, and characterization of elastase on erythrocyte membrane as factor IX-activating enzyme. Biochemical and Biophysical Research Communications, 316, 65-70.

JAHAGIRDAR, R. & SCAMMELL, B. E. 2009. Principles of fracture healing and disorders of bone union. Surgery (Oxford), 27, 63-69.

JÁMBOR, C., REUL, V., SCHNIDER, T. W., DEGIACOMI, P., METZNER, H. & KORTE, W. C. 2009. In vitro inhibition of Factor XIII retards clot formation, reduces clot firmness, and increases fibrinolytic effects in whole blood. Anesthesia & Analgesia, 109, 1023-1028.

JANEWAY, C. A., TRAVERS, P., WALPORT, M. & SHLOMCHIK, M. J. 2005a. Innate immunity. Immunobiology: the immune system in health and disease. 6th ed. Oxford: Garland Science Publishing.

JANEWAY, C. A., TRAVERS, P., WALPORT, M. & SHLOMCHIK, M. L. 2005b. Chapter 2: Innate Immunity. Immunology: the immune system in health and disease 6th ed. New York: Garland Science Publishing.

JANG, I. K., GOLD, H. K., ZISKIND, A. A., FALLON, J. T., HOLT, R. E., LEINBACH, R. C., MAY, J. W. & COLLEN, D. 1989. Differential sensitivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator. A possible explanation for resistance to coronary thrombolysis. Circulation, 79, 920-928.

JANSSON, E., KALLTORP, M., JOHANSSON, A., TENGVALL, P. & THOMSEN, P. 2001. On the formation of fibrous capsule and fluid space around machined and porous blood plasma clot coated titanium. J Mater Sci Mater Med, 12, 1019-24.

JANSSON, E., KÄLLTORP, M., THOMSEN, P. & TENGVALL, P. 2002. Ex vivo PMA-induced respiratory burst and TNF- alpha secretion elicited from inflammatory cells on machined and porous blood plasma clot-coated titanium. Biomaterials, 23, 2803-2815.

JEROME, W. G., HANDT, S. & HANTGAN, R. R. 2005. Endothelial cells organize fibrin clots into structures that are more resistant to lysis. Microsc Microanal, 11, 268-77.

JIROUSKOVÁ, M., DYR, J. E., SUTTNAR, J., HOLADA, K. & TRNKOVÁ, B. 1997. Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation. Thromb Haemost., 78, 1125-1131.

JOHNSON, K. D., FRIERSON, K. E., KELLER, T. S., COOK, C., SCHEINBERG, R., ZERWEKH, J., MEYERS, L. & SCIADINI, M. F. 1996. Porous ceramics as bone graft substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. Journal of Orthopaedic Research, 14, 351-369.

JONES, K. S. 2008. Effects of biomaterial-induced inflammation on fibrosis and rejection. Seminars in Immunology, 20, 130-136.

KALLTORP, M., OBLOGINA, S., JACOBSSON, S., KARLSSON, A., TENGVALL, P. & THOMSEN, P. 1999. In vivo cell recruitment, cytokine release and chemiluminescence response at gold, and thiol functionalized surfaces. Biomaterials, 20, 2123-37.

KAMITAKAHARA, M., OHTSUKI, C. & MIYAZAKI, T. 2007. Coating of bone-like apatite for development of bioactive materials for bone reconstruction. Biomed Mater, 2, R17-23.

KANDASWAMY, D., RAMACHANDRAN, G., MAHESHWARI, S. & MOHAN, B. 2000. Bone regenration using hydroxyapatite crystals for periapical lesions. Endodontology, 12, 51-54.

KANG, I. K., KWON, B. K., LEE, J. H. & LEE, H. B. 1993. Immobilization of proteins on poly(methyl methacrylate) films. Biomaterials, 14, 787-792.



KANG, R., GHIVIZZANI, S. C., MUZZONIGRO, T. S., HERNDON, J. H., ROBBINS, P. D. & EVANS, C. H. 2000. Orthopaedic applications of gene therapy: from concept to clinic. Clinical Orthopaedic & Related Research, 375, 324-337.

KAO, S. T. & SCOTT, D. D. 2007. A review of bone substitutes. Oral Maxillofac Surg Clin North Am, 19, 513-21, vi.

KASTEN, P., VOGEL, J., GEIGER, F., NIEMEYER, P., LUGINBÜHL, R. & SZALAY, K. 2008. The effect of platelet-rich plasma on healing in critical-size long-bone defects. Biomaterials, 29, 3983-3992.

KATORI, N., TANAKA, K. A., SZLAM, F. & LEVY, J. H. 2005. The effects of platelet count on clot retraction and tissue plasminogen activator-induced fibrinolysis on thrombelastography. Anesth Analg, 100, 1781-5.

KELLS, A. F., COATS, S. R., SCHWARTZ, H. S. & HOOVER, R. L. 1995. TGF-beta and PDGF act synergistically in affecting the growth of human osteoblast-enriched cultures. Connective Tissue Research, 31, 117-124.

KEUREN, J. F., WIELDERS, S. J., WILLEMS, G. M., MORRA, M. & LINDHOUT, T. 2002. Fibrinogen adsorption, platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood. Thromb Haemost, 87, 742-747.

KLEMENT, P., DU, Y. J., BERRY, L., ANDREW, M. & CHAN, A. K. C. 2002. Blood-compatible biomaterials by surface coating with a novel antithrombin-heparin covalent complex. Biomaterials, 23, 527-535.

KNABE, C., KOCH, C., RACK, A. & STILLER, M. 2008. Effect of β-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans. Biomaterials, 29, 2249-2258.

KNETSCH, M. L. W. 2008. Blood-contacting surfaces. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

KOH, L. B., RODRIGUEZ, I. & VENKATRAMAN, S. S. 2010a. Conformational behavior of fibrinogen on topographically modified polymer surfaces. Physical Chemistry Chemical Physics 12, 10301-10308.

KOH, L. B., RODRIGUEZ, I. & VENKATRAMAN, S. S. 2010b. The effect of topography of polymer surfaces on platelet adhesion. Biomaterials, 31, 1533-45.

KOLOMVOS, N., IATROU, I., THEOLOGIE-LYGIDAKIS, N., TZERBOS, F. & SCHOINOHORITI, O. 2010. Iliac crest morbidity following maxillofacial bone grafting in children: a clinical and radiographic prospective study. J Craniomaxillofac Surg, 38, 293-302.

KOO, J., RAFAILOVICH, M. H., MEDVED, L., TSURUPA, G., KUDRYK, B. J., LIU, Y. & GALANAKIS, D. K. 2010. Evaluation of fibrinogen self-assembly: role of its αC region. Journal of Thrombosis and Haemostasis, 8, 2727-2735.

KOPP, R., MOTTAGHY, K. & KIRSCHFINK, M. 2002. Mechanism of complement activation during extracorporeal blood-biomaterial interaction: effects of heparin coated and uncoated surfaces. ASAIO J, 48, 598-605.

KORBUT, R. & GRYGLEWSKI, R. J. 1995. Platelets in fibrinolytic system. J Physiol Pharmacol, 46, 409-18.

KOURTZELIS, I., MARKIEWSKI, M. M., DOUMAS, M., RAFAIL, S., KAMBAS, K., MITROULIS, I., PANAGOUTSOS, S., PASSADAKIS, P., VARGEMEZIS, V., MAGOTTI, P., QU, H., MOLLNES, T. E., RITIS, K. & LAMBRIS, J. D. 2010. Complement anaphylatoxin C5a contributes to hemodialysis-associated thrombosis. Blood, 116, 631-9.

KRAUS, K. H. & KIRKER-HEAD, C. 2006. Mesenchymal stem cells and bone regeneration. Vet Surg, 35, 232-42.

KRISHNAN, A., CHA, P., LIU, Y. H., ALLARA, D. & VOGLER, E. A. 2006. Interfacial energetics of blood plasma and serum adsorption to a hydrophobic self-assembled monolayer surface. Biomaterials, 27, 3187-3194.

KWAK, D., WU, Y. & HORBETT, T. A. 2005. Fibrinogen and von Willebrand's factor adsorption are both required for platelet adhesion from sheared suspensions to



polyethylene preadsorbed with blood plasma. Journal of Biomedical Materials Research. Part A., 74, 69-83.

LACOSTE, E., MARTINEAU, I. & GAGNON, G. 2003. Platelet concentrates: effect of calcium and thrombin on endothelial cell proliferation and growth factor release. Journal of Periodontology, 74, 1498-1507.

LAI, B. F. L., ZOU, Y., BROOKS, D. E. & KIZHAKKEDATHU, J. N. 2010. The influence of poly-N-(2,2-dimethyl-1,3-dioxolane) methyl]acrylamide on fibrin polymerization, cross-linking and clot structure. Biomaterials, 31, 5749-5758.

LAI, J., SUNDERLAND, B., XUE, J., YAN, S., ZHAO, W., FOLKARD, M., MICHAEL, B. D. & WANG, Y. 2006. Study on hydrophilicity of polymer surfaces improved by plasma treatment. Applied Surface Science, 252, 3375-3379.

LAPPEGÅRD, K. T., FUNG, M., BERGSETH, G., RIESENFELD, J., LAMBRIS, J. D., VIDEM, V. & MOLLNES, T. E. 2004. Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation. The Annals of Thoracic Surgery, 77, 932-941.

LASCHKE, M. W., HARDER, Y., AMON, M., MARTIN, I., FARHADI, J., RING, A., TORIO-PADRON, N., SCHRAMM, R., RUCKER, M., JUNKER, D., HAUFEL, J. M., CARVALHO, C., HEBERER, M., GERMANN, G., VOLLMAR, B. & MENGER, M. D. 2006. Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng, 12, 2093-104.

LAURENS, N., KOOLWIJK, P. & DE MAAT, M. P. 2006. Fibrin structure and wound healing. Journal of thrombosis and haemostasis, 4, 932-939.

LE GUEHENNEC, L., SOUEIDAN, A., LAYROLLE, P. & AMOURIQ, Y. 2007. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater, 23, 844-54.

LE GUÉHENNEC, L., SOUEIDAN, A., LAYROLLE, P. & AMOURIQ, Y. 2007. Surface treatments of titanium dental implants for rapid osseointegration. Dental Materials, 23, 844-854.

LEE, H. B., KHANG, G. & LEE, J. H. 2007. Polymeric Biomaterials. Biomaterials, 3-1 - 3-21. LI, B., MA, Y., WANG, S. & MORAN, P. M. 2005. Influence of carboxyl group density on

neuron cell attachment and differentiation behavior: Gradient-guided neurite outgrowth. Biomaterials, 26, 4956-4963.

LI, D., CHEN, H. C., GLENN MCCLUNG, W. & BRASH, J. L. 2009. Lysine-PEG-modified polyurethane as a fibrinolytic surface: Effect of PEG chain length on protein interactions, platelet interactions and clot lysis. Acta Biomaterialia, 5, 1864-1871.

LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002a. The role of growth factors in the repair of bone : biology and clinical applications. J Bone Joint Surg Am, 84, 1032-1044.

LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002b. The role of growth factors in the repair of bone: biology and clinical applications. The Journal of Bone and Joint Surgery, 84A, 1032-1044.

LIEBERMAN, J. R., DALUISKI, A. & EINHORN, T. A. 2002c. The role of growth factors in the repair of bone: biology and clinical applications. J Bone Joint Surg Am, 84, 1032-1044.

LIJNEN, H. R. & COLLEN, D. 1995. Mechanisms of physiological fibrinolysis. Baillieres Clin Haematol., 8, 277-290.

LIN, C. Y., CHANG, Y. H., LIN, K. J., YEN, T. C., TAI, C. L., CHEN, C. Y., LO, W. H., HSIAO, I. T. & HU, Y. C. 2010. The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells. Biomaterials, 31, 3222-30.

LIN, J. C. & CHUANG, W. H. 2000. Synthesis, surface characterization, and platelet reactivity evaluation for the self-assembled monolayer of alkanethiol with sulfonic acid functionality. Journal of Biomedical Materials Research, 51, 413-423.



LIN, N. J. & LIN-GIBSON, S. 2009. Osteoblast response to dimethacrylate composites varying in composition, conversion and roughness using a combinatorial approach. Biomaterials, 30, 4480-4487.

LIND, M., OVERGAARD, S., ONGPIPATTANAKUL, B., NGUYEN, T., BUNGER, C. & SOBALLE, K. 1996. Transforming growth factor-beta 1 stimulates bone ongrowth to weight-loaded tricalcium phosphate coated implants: an experimental study in dogs. J Bone Joint Surg Br, 78, 377-82.

LINDSEY, R. W., GUGALA, Z., MILNE, E., SUN, M., GANNON, F. H. & LATTA, L. L. 2006. The efficacy of cylindrical titanium mesh cage for the reconstruction of a critical-size canine segmental femoral diaphyseal defect. Journal of Orthopaedic Research, 24, 1438-1453.

LIU, G., ZHAO, L., ZHANG, W., CUI, L., LIU, W. & CAO, Y. 2008. Repair of goat tibial defects with bone marrow stromal cells and beta-tricalcium phosphate. J Mater Sci Mater Med, 19, 2367-76.

LIU, W., JAWERTH, L. M., SPARKS, E. A., FALVO, M. R., HANTGAN, R. R., SUPERFINE, R., LORD, S. T. & GUTHOLD, M. 2006. Fibrin fibers have extraordinary extensibility and elasticity. Science, 313, 634.

LIU, Y., WU, G. & DE GROOT, K. 2010. Biomimetic coatings for bone tissue engineering of critical-sized defects. J R Soc Interface, 7 Suppl 5, S631-47.

LOZADA, J. L., CAPLANIS, N., PROUSSAEFS, P., WILLARDSEN, J. & KAMMEYER, G. 2001. Platelet-rich plasma application in sinus graft surgery: Part I--Background and processing techniques. J Oral Implantol, 27, 38-42.

LU, J. 2004. Biocompatible materials: orthopedic bone cements. In: POITOUT, D. G. (ed.) Biomechanics and biomaterials in orthopedics. Singapore: Springer-Verlag London Limited.

MA, Z., MAO, Z. & GAO, C. 2007. Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids and Surfaces B: Biointerfaces, 60, 137-157.

MACHLUS, K. R., COLBY, E. A., WU, J. R., KOCH, G. G., KEY, N. S. & WOLBERG, A. S. 2009. Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram. Thromb Haemost, 102, 936-44.

MANN, K. G., BRUMMEL, K. E. & BUTENAS, S. 2003. What is all that thrombin for? Journal of Thromosis and Haemostasis, 1, 1504-1514.

MANNUCCI, P. M., GIANGRANDE, P. L. F. & BONOMI, A. B. 1992. Detection of the prethrombotic state due to procoagulant imbalance. European Journal of Haematology, 48, 65-69.

MARIEB, E. N. 2001a. Chapter 4: Tissue: the living fabric. Human anatomy & physiology. 5th ed. New York: Benjamin Cummings.

MARIEB, E. N. 2001b. Chapter 6: Bone and skeletal tissues. Human anatomy & physiology, 172-197.

MARIEB, E. N. 2001c. Chapter 6: Bone and skeletal tissues. Human anatomy & physiology. 5th ed. New York: Benjamin Cummings.

MARIEB, E. N. 2001d. Chapter 18: Blood. Human anatomy & physiology. New York: Benjamin Cummings.

MARIEB, E. N. 2001e. Chapter 22: The immune system: innate and adaptive body defenses. Human anatomy & physiology. New York: Benjamin Cummings.

MARKIEWSKI, M. M., NILSSON, B., NILSSON EKDAHL, K., MOLLNES, T. E. & LAMBRIS, J. D. 2007. Complement and coagulation: strangers or partners in crime? Trends in Immunology, 28, 184-192.

MARTINEAU, I., LACOSTE, E. & GAGNON, G. 2004. Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials, 25, 4489-502.



MARTINS, M. C., RATNER, B. D. & BARBOSA, M. A. 2003. Protein adsorption on mixtures of hydroxyl- and methyl-terminated alkanethiols self-assembled monolayers. J Biomed Mater Res A, 67, 158-71.

MARX, R. E. 2001. Platelet-rich plasma (PRP): What is PRP and what is not PRP? Implant Dentistry, 10, 225-228.

MARX, R. E., CARLSON, E. R., EICHSTAEDT, R. M., SCHIMMELE, S. R., STRAUSS, J. E. & GEORGEFF, K. R. 1998a. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, 85, 638-646.

MARX, R. E., CARLSON, E. R., EICHSTAEDT, R. M., SCHIMMELE, S. R., STRAUSS, J. E. & GEORGEFF, K. R. 1998b. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, 85, 638-646.

MCARTHUR, S. L. 2006. Applications of XPS in bioengineering. Surface and Interface Analysis, 38, 1380-1385.

MCAULIFFE, J. A. 2003. Bone graft substitutes. J Hand Ther, 16, 180-7. MCNALLY, A. K. & ANDERSON, J. M. 2011. Macrophage fusion and multinucleated giant

cells of inflammation, New York, NY, ETATS-UNIS, Springer Science+Business Media.

MEDVED, L. & NIEUWENHUIZEN, W. 2003. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thrombosis and Haemostasis, 89, 409-419.

MENZIES, K. L. & JONES, L. 2010. The impact of contact angle on the biocompatibility of biomaterials. Optometry & Vision Science, 87, 387-399.

MESSORA, M. R., NAGATA, M. J. H., MARIANO, R. C., DORNELLES, R. C. M., BOMFIM, S. R. M., FUCINI, S. E., GARCIA, V. G. & BOSCO, A. F. 2008. Bone healing in critical-size defects treated with platelet-rich plasma: a histologic and histometric study in rat calvaria Journal of Periodontal Research, 43, 217-223.

MEYER, U., JOOS, U. & WIESMANN, H. P. 2004. Biological and biophysical principles in extracorporal bone tissue engineering: Part I. International Journal of Oral and Maxillofacial Surgery, 33, 325-332.

MICHIARDI, A., APARICIO, C., RATNER, B. D., PLANELL, J. A. & GIL, J. 2007. The influence of surface energy on competitive protein adsorption on oxidized NiTi surfaces. Biomaterials, 28, 586-594.

MILLS, J. D., ARIENS, R. A. S., MANSFIELD, M. W. & GRANT, P. J. 2002. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation, 106, 1938-1942.

MINELLI, C., KIKUTA, A., TSUD, N., BALL, M. D. & YAMAMOTO, A. 2008. A micro-fluidic study of whole blood behaviour on PMMA topographical nanostructures. Journal of nanobiotechnology, 6, 3-13.

MINORS, D. S. 2004. Haemostasis, blood platelets and coagulation. Anaesthesia and intensive care medicine, 5, 189-191.

MONIEN, B. H., CHEANG, K. I. & DESAI, U. R. 2005. Mechanism of poly(acrylic acid) acceleration of antithrombin inhibition of thrombin: Implications for the design of novel heparin mimics. Journal of Medicinal Chemistry, 48, 5360-5368.

MOORADIAN, D. L., LUCAS, R. C., WEATHERBEE, J. A. & FURCHT, L. T. 1989. Transforming growth factor-β1 binds to immobilized fibronectin. Journal of Cellular Biochemistry, 41, 189-200.

MOOREN, R. E. C. M., MERKX, M. A. W., BRONKHORST, E. M., JANSEN, J. A. & STOELINGA, P. J. W. 2007. The effect of platelet-rich plasma on early and late bone healing: an experimental study in goats. International Journal of Oral and Maxillofacial Surgery, 36, 626-631.

MORGENSTERN, E., KORELL, U. & RICHTER, J. 1984. Platelets and fibrin strands during clot retraction. Thromb Res, 33, 617-23.

MOSESSON, M. W. 2005. Fibrinogen and fibrin structure and functions. Journal of Thrombosis and Haemostasis, 3, 1894-1904.



MRKSICH, M. 2000. A surface chemistry approach to studying cell adhesion. Chemical Society Reviews, 29, 267-273.

MÜLLER, F., MUTCH, N. J., SCHENK, W. A., SMITH, S. A., ESTERL, L., SPRONK, H. M., SCHMIDBAUER, S., GAHL, W. A., MORRISSEY, J. H. & RENNÉ, T. 2009. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell, 139, 1143-1156.

MULLIN, J. L., NORFOLK, S. E., WEISEL, J. E. & LORD, S. T. 2001. Clot lysis of variant recombinant fibrinogens confirms that fiber diameter is a major determinant of lysis rate. Annals of the New York Academy of Sciences, 936, 331-334.

MULZER, S. R. & BRASH, J. L. 1989. Identification of plasma protein adsorbed to hemodialyzers during clinical use. Journal of Biomedical Materials Research, 23, 1483-1504.

MURPHY-ULLRICH, J. E., SCHULTZ-CHERRY, S. & HOOK, M. 1992. Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell, 3, 181-8.

NAIR, C. H. & DHALL, D. P. 1991. Studies on fibrin network structure: the effect of some plasma proteins. Thrombosis Research, 61, 315-325.

NAIR, C. H. & SHATS, E. A. 1997. Compaction as a method to characterise fibrin network structure: kinetic studies and relationship to crosslinking. Thrombosis Research, 88, 381-387.

NATHER, A., DAVID, V., TENG, J. W., LEE, C. W. & PEREIRA, B. P. 2010. Effect of autologous mesenchymal stem cells on biological healing of allografts in critical-sized tibial defects simulated in adult rabbits. Ann Acad Med Singapore, 39, 599-606.

NILSSON, B., EKDAHL, K. N., MOLLNES, T. E. & LAMBRIS, J. D. 2007a. The role of complement in biomaterial-induced inflammation. Molecular Immunology, 44, 82-94.

NILSSON, B., EKDAHL, K. N., MOLLNES, T. E. & LAMBRIS, J. D. 2007b. The role of complement in biomaterial-induced inflammation. Molecular Immunology, 44, 82-94.

NILSSON, B., KORSGREN, O., LAMBRIS, J. D. & EKDAHL, K. N. 2009. Can cells and biomaterials in therapeutic medicine be shielded from innate immune recognition? Trends in Immunology, 31, 32-38.

NURDEN, A. T. 2007. Platelet membrane glycoproteins: a look back into the past and a view to the future. Thromb Haemost, 98, 49-54.

NURDEN, A. T. 2011. Platelets, inflammation and tissue regeneration. Thromb Haemost, 105 Suppl 1, S13-33.

NURDEN, A. T., NURDEN, P., SANCHEZ, M., ANDIA, I. & ANITUA, E. 2008. Platelets and wound healing. Front Biosci, 13, 3532-48.

NYGREN, H. 1996. Initial reactions of whole blood with hydrophilic and hydrophobic titanium surfaces. Colloids and Surfaces B: Biointerfaces, 6, 329-333.

NYGREN, H., TENGVALL, P. & LUNDSTROM, I. 1997. The initial reactions of TiO2 with blood. J Biomed Mater Res, 34, 487-92.

ODIAN, G. 2004. Chain polymerization. Principles of polymerization. Fourth ed. New York: John Wiley & Sons, Inc.

OKADA, M., BLOMBACK, B., CHANG, M. D. & HOROWITZ, B. 1985. Fibronectin and fibrin gel structure. J Biol Chem, 260, 1811-20.

ONUNDARSON, P. T., FRANCIS, C. W. & MARDER, V. J. 1992. Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential. J Lab Clin Med, 120, 120-128.

OPREA, W. E., KARP, J. M., HOSSEINI, M. M. & DAVIES, J. E. 2003. Effect of platelet releasate on bone cell migration and recruitment in vitro. Journal of Craniofacial Surgery, 14, 292-300.

OVANESOV, M. V., ANANYEVA, N. M., PANTELEEV, M. A., ATAULLAKHANOV, F. I. & SAENKO, E. L. 2005. Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost, 3, 321-31.

OZCAN, C. & HASIRCI, N. 2007. Plasma modification of PMMA films: surface free energy and cell-attachment studies. J. Biomater. Sci. Polymer Edn, 18, 759-773.



PANETTI, T. S., KUDRYK, B. J. & MOSHER, D. F. 1999. Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin. Journal of Biological Chemistry, 274, 430-437.

PANTELEEV, M. A., OVANESOV, M. V., KIREEV, D. A., SHIBEKO, A. M., SINAURIDZE, E. I., ANANYEVA, N. M., BUTYLIN, A. A., SAENKO, E. L. & ATAULLAKHANOV, F. I. 2006. Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively. Biophys J, 90, 1489-500.

PARIKH, S. N. 2002. Bone graft substitutes: past, present, future. Journal of Postgraduate Medicine, 48, 142-148.

PARISE, P. & AGNELLI, G. 1991. Thrombus resistance to lysis and reocclusion after thrombolysis: the role of platelets. Blood Coagul Fibrinolysis, 2, 749-58.

PARK, J. E. & BARBUL, A. 2004. Understanding the role of immune regulation in wound healing. The American Journal of Surgery, 187, S11-S16.

PEERSCHKE, E. I., YIN, W., GRIGG, S. E. & GHEBREHIWET, B. 2006. Blood platelets activate the classical pathway of human complement. J Thromb Haemost, 4, 2035-42.

PEERSCHKE, E. I. B. & GHEBREHIWET, B. 1998. Platelet receptors for the complement component C1q: implication for hemostasis and thrombosis. Immunobiology, 199, 239-249.

PEERSCHKE, E. I. B., REID, K. B. & GHEBREHIWET, B. 1993. Platelet activation by C1q results in the induction of alpha IIb/beta 3 integrins (GPIIb-IIIa) and the expression of P-selectin and procoagulant activity. The Journal of Experimental Medicine, 178, 579-587.

PEERSCHKE, E. I. B., YIN, W. & GHEBREHIWET, B. 2008. Platelet mediated complement activation. Adv Exp Med Biol., 632, 81-91.

PEERSCHKE, E. I. B., YIN, W. & GHEBREHIWET, B. 2010. Complement activation on platelets: implications for vascular inflammation and thrombosis. Molecular Immunology, 47, 2170-2175.

PETERSEN, L. C., BJORN, S. E. & NORDFANG, O. 1992. Effect of leukocyte proteinase on tissue factor pathway inhibitor. Thromb Haemost, 67, 537-541.

PEYROU, V., LORMEAU, J. C., HERAULT, J. P., GAICH, C., PFLIEGGER, A. M. & HERBERT, J. M. 1999. Contribution of erythrocytes to thrombin generation in whole blood, Stuttgart, ALLEMAGNE, Schattauer.

PHILLIPS, A. M. 2005. Overview of the fracture healing cascade. Injury, 36 Suppl 3, S5-7. PIERCE, G. F., MUSTOE, T. A., ALTROCK, B. W., DEUEL, T. F. & THOMASON, A. 1991.

Role of platelet-derived growth factor in wound healing. Journal of Cellular Biochemistry, 45, 319-326.

PLESCIA, J. & ALTIERI, D. C. 1996. Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation. Biochem J, 319, 873–879.

POUNTOS, I., CORSCADDEN, D., EMERY, P. & GIANNOUDIS, P. V. 2007. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury, 38 Suppl 4, S23-33.

PRASAD, S., KASHYAP, R. S., DEOPUJARI, J. Y., PUROHIT, H. J., TAORI, G. M. & DAGINAWALA, H. F. 2006. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J, 4, 14.

PRECHEUR, H. V. 2007. Bone graft materials. Dent Clin North Am, 51, 729-46, viii. PUELACHER, W. C., VACANTI, J. P., FERRARO, N. F., SCHLOO, B. & VACANTI, C. A.

1996. Femoral shaft reconstruction using tissue-engineered growth of bone. Int J Oral Maxillofac Surg, 25, 223-8.

RAND, M. L., WANG, H., BANG, K. W., TEITEL, J. M., BLANCHETTE, V. S., FREEDMAN, J. & NURDEN, A. T. 2010. Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets. Am J Hematol, 85, 584-92.

REICHERT, J. C., SAIFZADEH, S., WULLSCHLEGER, M. E., EPARI, D. R., SCHUTZ, M. A., DUDA, G. N., SCHELL, H., VAN GRIENSVEN, M., REDL, H. &



HUTMACHER, D. W. 2009. The challenge of establishing preclinical models for segmental bone defect research. Biomaterials, 30, 2149-63.

RENNÉ, T. & GAILANI, D. 2007. Role of Factor XII in hemostasis and thrombosis: clinical implications. Expect Review of Cardiovascular Therapy, 5, 733-741.

RIEDEL, T., SUTTNAR, J., BRYNDA, E., HOUSKA, M., MEDVED, L. & DYR, J. E. 2011. Fibrinopeptides A and B release in the process of surface fibrin formation. Blood, 117, 1700-1705.

RITIS, K., DOUMAS, M., MASTELLOS, D., MICHELI, A., GIAGLIS, S., MAGOTTI, P., RAFAIL, S., KARTALIS, G., SIDERAS, P. & LAMBRIS, J. D. 2006. A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways. J Immunol, 177, 4794-802.

ROACH, P., FARRAR, D. & PERRY, C. C. 2005. Interpretation of protein adsorption: surface-induced conformational changes. J Am Chem Soc, 127, 8168-73.

RODRIGUES, S. N., GONCALVES, I. C., MARTINS, M. C. L., BARBOSA, M. A. & RATNER, B. D. 2006. Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methy-terminated self-assembled monolayers. Biomaterials, 27, 5357-5367.

RODRIGUEZ HERNANDEZ, J. C., RICO, P., MORATAL, D., MONLEON PRADAS, M. & SALMERON-SANCHEZ, M. 2009. Fibrinogen patterns and activity on substrates with tailored hydroxy density. Macromol Biosci, 9, 766-75.

ROJKJAER, L. P. & ROJKJAER, R. 2007. Clot stabilization for the prevention of bleeding. Hematol Oncol Clin North Am, 21, 25-32.

ROUKIS, T. S., ZGONIS, T. & TIERNAN, B. 2006. Autologous platelet-rich plasma for wound and osseous healing: a review of literature and commercial available products. Advances in therapy, 23, 128-137.

ROUSSY, Y., BERTRAND DUCHESNE, M.-P. & GAGNON, G. 2007. Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation. Clinical oral implants research, 18, 639-648.

RUGGERI, Z. M. 2003. Von Willebrand factor, platelets and endothelial cell interactions. Journal of Thromosis and Haemostasis, 1, 1335-1342.

RYZEWICZ, M., MORGAN, S. J., LINFORD, E., THWING, J. I., DE RESENDE, G. V. & SMITH, W. R. 2009. Central bone grafting for nonunion of fractures of the tibia: a retrospective series. J Bone Joint Surg Br, 91, 522-9.

SAJJADIAN, A., RUBINSTEIN, R. & NAGHSHINEH, N. 2010. Current status of grafts and implants in rhinoplasty: part I. Autologous grafts. Plast Reconstr Surg, 125, 40e-49e.

SALVADOR-MORALES, C., ZHANG, L., LANGER, R. & FAROKHZAD, O. C. 2009. Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials, 30, 2231-40.

SAMMARCO, V. J. & CHANG, L. 2002. Modern issues in bone graft substitutes and advances in bone tissue technology. Foot And Ankle Clinics, 7, 19-41.

SÁNCHEZ, A. R., SHERIDAN, P. J. & KUPP, L. I. 2003. Is platelet-rich plasma the perfect enhancement factor? A current review. Int J Oral Maxillofac Implants, 18, 93-103.

SANCHEZ, J., LUNDQUIST, P. B., ELGUE, G., LARSSON, R. & OLSSON, P. 2002. Measuring the degree of plasma contact activation induced by artificial materials. Thromb Res, 105, 407-12.

SAULS, D. L., WOLBERG, A. S. & HOFFMAN, M. 2003. Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: implications for the mechanism of thrombosis in hyperhomocysteinemia. J Thromb Haemost, 1, 300-6.

SCHLIEPHAKE, H. 2009. Growth factors and cell based devices in bone tissue engineering. International Journal of Oral and Maxillofacial Surgery, 38, 413-413.

SCHMIDMAIER, G., SCHWABE, P., WILDEMANN, B. & HAAS, N. P. 2007. Use of bone morphogenetic proteins for treatment of non-unions and future perspectives. Injury, 38 Suppl 4, S35-41.

SCHMUGGE, M., RAND, M. L. & FREEDMAN, J. 2003. Platelets and von Willebrand factor. Transfus Apher Sci, 28, 269-77.



SCOTT, E. M., ARIENS, R. A. & GRANT, P. J. 2004. Genetic and environmental determinants of fibrin structure and function: relevance to clinical disease. Arterioscler Thromb Vasc Biol, 24, 1558-66.

SEEHERMAN, H. J., AZARI, K., BIDIC, S., ROGERS, L., LI, X. J., HOLLINGER, J. O. & WOZNEY, J. M. 2006. rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radii. J Bone Joint Surg Am, 88, 1553-65.

SELLBORN, A., ANDERSSON, M., HEDLUND, J., ANDERSSON, J., BERGLIN, M. & ELWING, H. 2005. Immune complement activation on polystyrene and silicon dioxide surfaces. Impact of reversible IgG adsorption. Mol Immunol, 42, 569-74.

SHAH, G. A., FERGUSON, I. A., DHALL, T. Z. & DHALL, D. P. 1982. Polydispersion in the diameter of fibers in fibrin networks: Consequences on the measurement of mass-length ratio by permeability and turbidity. Biopolymers, 21, 1037-1047.

SHALABI, M. M., GORTEMAKER, A., VAN'T HOF, M. A., JANSEN, J. A. & CREUGERS, N. H. 2006. Implant surface roughness and bone healing: a systematic review. J Dent Res, 85, 496-500.

SHANTI, R. M., LI, W. J., NESTI, L. J., WANG, X. & TUAN, R. S. 2007. Adult mesenchymal stem cells: biological properties, characteristics, and applications in maxillofacial surgery. J Oral Maxillofac Surg, 65, 1640-7.

SIMON, D. I., EZRATTY, A. M., FRANCIS, S. A., RENNKE, H. & LOSCALZO, J. 1993. Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway. Blood, 82, 2414-22.

SIMONPIERI, A., DEL CORSO, M., VERVELLE, A., JIMBO, R., INCHINGOLO, F., SAMMARTINO, G. & DOHAN EHRENFEST, D. M. 2011. Current knowledge and perspectives for the use of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial surgery. Part 2: bone graft, implant and reconstructive surgery. Curr Pharm Biotechnol.

SIMONPIERI, A., DEL CORSO, M., VERVELLE, A., JIMBO, R., INCHINGOLO, F., SAMMARTINO, G. & DOHAN EHRENFEST, D. M. 2012. Current knowledge and perspectives for the use of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial surgery part 2: Bone graft, implant and reconstructive surgery. Curr Pharm Biotechnol, 13, 1231-56.

SIMS, P. J. & WIEDMER, T. 1991. The response of human platelets to activated components of the complement system. Immunol Today, 12, 338-42.

SIMS, P. J. & WIEDMER, T. 1995. Induction of cellular procoagulant activity by the membrane attack complex of complement. Seminars in Cell Biology, 6, 275-282.

SIT, P. S. & MARCHANT, R. E. 1999a. Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions. Thromb Haemost, 82, 1053-60.

SIT, P. S. & MARCHANT, R. E. 1999b. Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions. Thrombosis and haemostasis, 82, 1053-1060.

SIT, P. S. & MARCHANT, R. E. 2001. Surface-dependent differences in fibrin assembly visualized by atomic force microscopy. Surface Science, 491, 421-432.

SIVARAMAN, B., FEARS, K. P. & LATOUR, R. A. 2009. Investigation of the effects of surface chemistry and solution concentration on the conformation of adsorbed proteins using an improved circular dichroism method. Langmuir, 25, 3050-6.

SIVARAMAN, B. & LATOUR, R. A. 2010. The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen. Biomaterials, 31, 832-839.

SIVARAMAN, B. & LATOUR, R. A. 2011. Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin. Biomaterials, 32, 5365-70.

SLATER, M., PATAVA, J., KINGHAM, K. & MASON, R. S. 1995. Involvement of platelets in stimulating osteogenic activity. Journal of Orthopaedic Research, 13, 655-663.



SMYTH, S. S., MCEVER, R. P., WEYRICH, A. S., MORRELL, C. N., HOFFMAN, M. R., AREPALLY, G. M., FRENCH, P. A., DAUERMAN, H. L. & BECKER, R. C. 2009. Platelet functions beyond hemostasis. Journal of Thrombosis and Haemostasis, 7, 1759-1766.

SPERLING, C., FISCHER, M., MAITZ, M. F. & WERNER, C. 2009. Blood coagulation on biomaterials requires the combination of distinct activation processes. Biomaterials, 30, 4447-4456.

SPERLING, C., HOUSKA, M., BRYNDA, E., STRELLER, U. & WERNER, C. 2006. In vitro hemocompatibility of albumin–heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique. Journal of Biomedical Materials Research Part A, 76A, 681-689.

SPERLING, C., MATIZ, M. F., TALKENBERGER, S., GOUZY, M. F. & WERNER, C. 2007. In vitro blood reactivity of hydroxylated and non-hydroxylated polymer surfaces. Biomaterials, 28, 3617-3625.

SPERLING, C., SCHWEISS, R. B., STRELLER, U. & WERNER, C. 2005a. In vitro hemocompatibility of self-assembled monolayers displaying various functional groups. Biomaterials, 25, 6547-6557.

SPERLING, C., SCHWEISS, R. B., STRELLER, U. & WERNER, C. 2005b. In vitro hemocompatibility of self-assembled monolayers displaying various functional groups. Biomaterials, 25, 6547-6557.

STANDEVEN, K. F., ARIËNS, R. A. S. & GRANT, P. J. 2005. The molecular physiology and pathology of fibrin structure/function. Blood Reviews, 19, 275-288.

STEVENS, M. M. 2008. Biomaterials for bone tissue engineering. Materials Today, 11, 18-25. STREET, J., WINTER, D., WANG, J. H., WAKAI, A., MCGUINNESS, A. & REDMOND, H.

P. 2000. Is human fracture hematoma inherently angiogenic? Clinical Orthopaedics and Related Research, 378, 224-237.

STRELLER, U., SPERLING, C., HUBNER, J., HANKE, R. & WERNER, C. 2003. Design and evaluation of novel blood incubation systems for in vitro hemocompatibility assessment of planar solid surfaces. J Biomed Mater Res B Appl Biomater, 66, 379-90.

SUGIMOTO, M. & MIYATA, S. 2002. Functional property of von Willebrand factor under flowing blood. Int J Hematol, 75, 19-24.

SUNDARAM, S., LIM, F., COOPER, S. L. & COLMAN, R. W. 1996. Role of leucocytes in coagulation induced by artificial surfaces: investigation of expression of Mac-1, granulocyte elastase release and leucocyte adhesion on modified polyurethanes. Biomaterials, 17, 1041-1047.

SWENNEN, G. R. J., SCHUTYSER, F., MUELLER, M. C., KRAMER, F.-J., C., E. & SCHLIEPHAKE, H. 2004. Effect of platelet-rich plasma on cranial distraction osteogenesis in sheep: preliminary clinical and radiographic results. International Journal of Oral and Maxillofacial Surgery, 34, 294-304.

TA, T. C., SYKES, M. T. & MCDERMOTT, M. T. 1998. Real-time observation of plasma protein film formation on well-defined surfaces with scanning force microscopy. Langmuir, 14, 2435-2443.

TAKEMOTO, S., YAMAMOTO, T., TSURU, K., HAYAKAWA, S., OSAKA, A. & TAKASHIMA, S. 2004. Platelet adhesion on titanium oxide gels: effect of surface oxidation. Biomaterials, 25, 3485-3492.

TANG, L. & EATON, J. W. 1995. Inflammatory responses to biomaterials. American journal of clinicalpathology 103, 466-471.

TANG, L., LIU, L. & ELWING, H. B. 1998. Complement activation and inflammation triggered by model biomaterial surfaces. Journal of biomedical materials research, 41, 333-340.

TEDESCO, F., PAUSA, M., NARDON, E., INTRONA, M., MANTOVANI, A. & DOBRINA, A. 1997. The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity. J Exp Med, 185, 1619-27.

TELLEMAN, G., ALBREKTSSON, T., HOFFMAN, M., JOHANSSON, C. B., VISSINK, A., MEIJER, H. J. & RAGHOEBAR, G. M. 2010. Peri-implant endosseous healing



properties of dual acid-etched mini-implants with a nanometer-sized deposition of CaP: a histological and histomorphometric human study. Clin Implant Dent Relat Res, 12, 153-60.

TERASAWA, F., KANI, S., HONGO, M. & OKUMURA, N. 2006. In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from gammaAsn319, Asp320 deletion dysfibrinogen, Otsu I. Thromb Res, 118, 651-61.

THEVENOT, P., HU, W. & TANG, L. 2008. Surface chemistry influences implant biocompatibility. Current Topics in Medicinal Chemistry, 8, 270-280.

THOMAN, M. L., MEUTH, J. L., MORGAN, E. L., WEIGLE, W. O. & HUGLI, T. E. 1984. C3d-K, a kallikrein cleavage fragment of iC3b is a potent inhibitor of cellular proliferation. J Immunol, 133, 2629-33.

THOR, A., RASMUSSON, L., WENNERBERG, A., THOMSEN, P., HIRSCH, J. M., NILSSON, B. & HONG, J. 2007. The role of whole blood in thrombin generation in contact with various titanium surfaces. Biomaterials, 28, 966-974.

TOMOYASU, A., HIGASHIO, K., KANOMATA, K., GOTO, M., KODAIRA, K., SERIZAWA, H., SUDA, T., NAKAMURA, A., NOJIMA, J., FUKUDA, T. & KATAGIRI, T. 2007. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs. Biochemical and Biophysical Research Communications, 36, 62-67.

TOSOUNIDIS, T., KONTAKIS, G., NIKOLAOU, V. S., PAPATHANASSOPOULOS, A. & GIANNOUDIS, P. V. 2009. Facture healing and bone repair: an update. Trauma, 11, 145-156.

TSAI, A. T. 2004. Wound healing and the foreign body reaction [Online]. University of Washington Engineered Biomaterials. Available: http://www.uweb.engr.washington.edu/research/tutorials/woundhealing.html [Accessed 1st August 2007 2007].

TSAI, M. Y., SUN, Y. T. & LIN, J. C. 2007. Surface characterization and platelet compatibility evaluation of the binary mixed self-assembled monolayers. Journal of Colloid and Interface Science, 308, 474-484.

TSAI, W. B., GRUNKEMEIER, J. M., MCFARLAN, C. D. & HORBETT, T. A. 2002. Platelet adhesion to polystyrene-based surfaces preadsorbed with plasma selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor. Journal of biomedical materials research, 60, 348-359.

TSIRIDIS, E., KAKAR, S. & EINHORN, T. A. 2006. Fracture healing and bone grafting. In: ELSTROM, J. A., VIRKUS, W. W. & PANKOVICH, A. M. (eds.) Handbook of fractures. Third ed. New York: The McGraw-Hill Companies, Inc.

TSYGANOV, I., MAITZ, M. F., WIESER, E., RICHTER, E. & REUTHER, H. 2005. Correlation between blood compatibility and physical surface properties of titanium-based coatings. Surface and Coatings Technology, 200, 1041-1044.

TZONEVA, R., SEIFERT, B., ALBRECHT, W., RICHAU, K., GROTH, T. & LENDLEIN, A. 2008. Hemocompatibility of poly(ether imide) membranes functionalized with carboxylic groups. Journal of Materials Science: Materials in Medicine, 19, 3203-3210.

UKIWE, C., MANSOURI, A. & KWOK, D. Y. 2005. The dynamics of impacting water droplets on alkanethiol self-assembled monolayers with co-adsorbed CH3 and CO2H terminal groups. Journal of Colloid and Interface Science, 285, 760-768.

ULEVITCH, R. J. & JOHNSTON, A. R. 1980. In vitro studies of prekallikrein activation in rabbit blood: the participation of red blood cells. J Immunol, 124, 2027-33.

UNDAS, A., CELINSKA-LÖWENHOFF, M., LÖWENHOFF, T. & SZCZEKLIK, A. 2006. Statins, fenofibrate, and quinapril increase clot permeability and enhance fibrinolysis in patients with coronary artery disease. Journal of Thrombosis and Haemostasis, 4, 1029-1036.

UNDAS, A., SLOWIK, A., WOLKOW, P., SZCZUDLIK, A. & TRACZ, W. 2010. Fibrin clot properties in acute ischemic stroke: relation to neurological deficit. Thromb Res, 125, 357-61.

UNDAS, A., SZULDRZYNSKI, K., STEPIEN, E., ZALEWSKI, J., GODLEWSKI, J., TRACZ, W., PASOWICZ, M. & ZMUDKA, K. 2008. Reduced clot permeability and

http://www.uweb.engr.washington.edu/research/tutorials/woundhealing.html



susceptibility to lysis in patients with acute coronary syndrome: effects of inflammation and oxidative stress. Atherosclerosis, 196, 551-557.

UNDAS, A., ZAWILSKA, K., CIESLA-DUL, M., LEHMANN-KOPYDLOWSKA, A., SKUBISZAK, A., CIEPLUCH, K. & TRACZ, W. 2009. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood, 114, 4272-4278.

VACANTI, C. A., KIM, W., UPTON, J., MOONEY, D. & VACANTI, J. P. 1995. The efficacy of periosteal cells compared to chondrocytes in the tissue engineered repair of bone defects. Tissue Eng, 1, 301-8.

VALLES, J., SANTOS, M., AZNAR, J., MARCUS, A., MARTINEZ-SALES, V., PORTOLES, M., BROEKMAN, M. & SAFIER, L. 1991. Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment. Blood, 78, 154-162.

VAN DAMME, H. S., HOGT, A. H. & FEIJEN, J. 1986. Surface mobility and structural transitions of poly(n-alkyl methacrylates) probed by dynamic contact angle measurements. Journal of Colloid and Interface Science, 114, 167-172.

VAN DER MEIJDEN, P. E. & HEEMSKERK, J. W. M. 2010. Polyphosphates: a link between platelet activation, intrinsic coagulation and inflammation? Expert Review of Hematology, 3, 269-272.

VAN GELDER, J. M., NAIR, C. H. & DHALL, D. P. 1995. The permeability of fibrin network developed in human plasma. Blood Coagulation & Fibrinolysis, 6, 293-301.

VAN LIESHOUT, E. M. M., VAN KRALINGEN, G. H., EL-MASSOUDI, Y., WEINANS, H. & PATKA, P. 2011. Microstructure and biomechanical characteristics of bone substitutes for trauma and orthopaedic surgery. BMC Musculoskeletal Disorders, 12, 1-14.

VEHOF, J. W., HAUS, M. T., DE RUIJTER, A. E., SPAUWEN, P. H. & JANSEN, J. A. 2002. Bone formation in transforming growth factor beta-1-loaded titanium fiber mesh implants. Clin Oral Implants Res, 13, 94-102.

VEKLICH, Y., FRANCIS, C. W., WHITE, J. & WEISEL, J. W. 1998. Structural studies of fibrinolysis by electron microscopy. Blood, 92, 4721-9.

VOGLER, E. A. & SIEDLECKI, C. A. 2009. Contact activation of blood-plasma coagulation. Biomaterials, In Press, Corrected Proof.

WALKOWIAK, B., KRALISZ, U., MICHALEC, L., MAJEWSKA, E., KOZIOLKIEWICZ, W., LIGOCKA, A. & CIERNIEWSKI, C. S. 2000. Comparison of platelet aggregability and p-selectin surface expression on platelets isolated by different methods. Thrombosis Research, 99, 495-502.

WALSH, W. R., VIZESI, F., MICHAEL, D., AULD, J., LANGDOWN, A., OLIVER, R., YU, Y., IRIE, H. & BRUCE, W. 2008. [beta]-TCP bone graft substitutes in a bilateral rabbit tibial defect model. Biomaterials, 29, 266-271.

WANG, H., RATNER, B. D., SAGE, E. H. & JIANG, S. 2007. Stepwise assembly of fibrin bilayers on self-assembled monolayers of alkanethiolates: Influence of surface chemistry. The Journal of Physical Chemistry C, 111, 8504-8508.

WATANABE, Y. & KOBAYASHI, B. 1988. Differential release of calcium, magnesium and serotonin by rabbit and human platelets. J Pharmacobiodyn, 11, 268-76.

WEI, J., IGARASHI, T., OKUMORI, N., IGARASHI, T., MAETANI, T., LIU, B. & YOSHINARI, M. 2009. Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts. Biomedical Materials, 4, 045002.

WEIBRICH, G., HANSEN, T., KLEIS, W., BUCH, R. & HITZLER, W. E. 2004. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone, 34, 665-671.

WEISEL, J. W. 2005. Fibrinogen and fibrin. WEISEL, J. W., VEKLICH, Y. & GORKUN, O. 1993. The sequence of cleavage of

fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots. Journal of Molecular Biology, 232, 285-297.



WEISS, S., ZIMMERMANN, G., PUFE, T., VAROGA, D. & HENLE, P. 2009. The systemic angiogenic response during bone healing Archives of Orthopaedic and Trauma Surgery, 129, 989-997.

WENCEL-DRAKE, J. D., PAINTER, R. G., ZIMMERMAN, T. S. & GINSBERG, M. H. 1985. Ultrastructural localisation of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section. Blood, 65, 929-938.

WENNERBERG, A. & ALBREKTSSON, T. 2009. Effects of titanium surface topography on bone integration: a systematic review. Clin Oral Implants Res, 20 Suppl 4, 172-84.

WERNER, C., MAITZ, M. F. & SPERLING, C. 2007. Current strategies towards hemocompatible coatings. Journal of Materials Chemistry, 17, 3376-3384.

WHEELER, D. L. & ENNEKING, W. F. 2005. Allograft bone decreases in strength in vivo over time. Clinical Orthopaedic & Related Research, 435, 36-42.

WIGGINS, R. C., GICLAS, P. C. & HENSON, P. M. 1981. Chemotactic activity generated from the fifth component of complement by plasma kallikrein of the rabbit. Journal of Experimental Medicine, 153, 1391-1404.

WILLIAMS, D. F. 2008. On the mechanisms of biocompatibility. Biomaterials, 29, 2941-2953. WILSON, C. J., CLEGG, R. E., LEAVESLEY, D. I. & PEARCY, M. J. 2005. Mediation of

biomaterial-cell interactions by adsorbed proteins: a review. Tissue Engineering, 11, 1-18.

WILTFANG, J., KLOSS, F. R., KESSLER, P., NKENKE, E., SCHULTZE-MOSGAU, S., ZIMMERMANN, R. & SCHLEGEL, K. A. 2004. Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clinical oral implants research, 15, 187-193.

WOLBERG, A. S. 2007a. Thrombin generation and fibrin clot structure. Blood Rev, 21, 131-42. WOLBERG, A. S. 2007b. Thrombin generation and fibrin clot structure. Blood Reviews, 21,

131-142. WOLBERG, A. S. 2010. Plasma and cellular contributions to fibrin network formation, structure

and stability. Haemophilia, 16 Suppl 3, 7-12. WOLBERG, A. S., ALLEN, G. A., MONROE, D. M., HEDNER, U., ROBERTS, H. R. &

HOFFMAN, M. 2005. High dose factor VIIa improves clot structure and stability in a model of haemophilia B. Br J Haematol, 131, 645-55.

WOLBERG, A. S. & CAMPBELL, R. A. 2008. Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci, 38, 15-23.

WOLBERG, A. S., MONROE, D. M., ROBERTS, H. R. & HOFFMAN, M. 2003. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk. Blood, 101, 3008-13.

WOZNEY, J. M. & ROSEN, V. 1998. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clinical Orthopaedic & Related Research, 346, 26-37.

WULF, M., GRUNDKE, K., KWOK, D. Y. & NEUMANN, A. W. 2000. Influence of different alkyl side chains on solid surface tension of polymethacrylates. Journal of Applied Polymer Science, 77, 2493-2504.

XIE, H. G., ZHENG, J. N., LI, X. X., LIU, X. D., ZHU, J., WANG, F. X., XIE, W. Y. & MA, X. J. 2009. Effect of surface morphology and charge on the amount and conformation of fibrinogen adsorbed onto alginate/chitosan microcapsules. Langmuir, 26, 5587–5594.

XU, L. C. & SIEDLECKI, C. A. 2009. Atomic force microscopy studies of the initial interactions between fibrinogen and surfaces. Langmuir, 25, 3675-3681.

XU, Y., TAKAI, M. & ISHIHARA, K. 2009. Protein adsorption and cell adhesion on cationic, neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces. Biomaterials, 30, 4930-4938.

XU, Z., SONG, K., YUAN, S. L. & LIU, C. B. 2011. Microscopic wetting of self-assembled monolayers with different surfaces: a combined molecular dynamics and quantum mechanics study. Langmuir, 27, 8611-20.

YAN, X. & GEMEINHART, R. A. 2005. Cisplatin delivery from poly(acrylic acid-co-methyl methacrylate) microparticles. J Control Release, 106, 198-208.



YANG, Y., CUNHA-REIS, C., BAGNANINCHI, P. O. & AYDIN, H. M. 2008. Control and monitoring of scaffold architecture for tissue engineering. In: CHU, P. K. & LIU, X. (eds.) Biomaterials fabrication and processing handbook. New York: Taylor & Francis Group, LLC.

YAROVAYA, G. A., BLOKHINA, T. B. & NESHKOVA, E. A. 2002. Contact system. New concepts on activation mechanisms and bioregulatory functions. Biochemistry (Mosc), 67, 13-24.

YIN, W., GHEBREHIWET, B. & PEERSCHKE, E. I. B. 2008. Expression of complement components and inhibitors on platelet microparticles. Platelets, 19, 225-233.

YOSHIDA, E., OHMURA, S., SUGIKI, M., ANAI, K. & MARUYAMA, M. 2001. A novel function of extraerythrocytic hemoglobin: identification of globin as a stimulant of plasminogen activator biosynthesis in human fibroblasts. Thromb Haemost, 86, 1521-7.

ZHA, Z., MA, Y., YUE, X. L., LIU, M. & DAI, Z. 2009. Self-assembled hemocompatible coating on poly (vinyl chloride) surface. Applied Surface Science, 256, 805-814.

ZHANG, H., ARONOW, M. S. & GRONOWICZ, G. A. 2005. Transforming growth factor-beta 1 (TGF-beta1) prevents the age-dependent decrease in bone formation in human osteoblast/implant cultures. Journal of Biomedical Materials Research Part A, 75A, 98-105.

ZHUO, R., MILLER, R., BUSSARD, K. M., SIEDLECKI, C. A. & VOGLER, E. A. 2005. Procoagulant stimulus processing by the intrinsic pathway of blood plasma coagulation. Biomaterials, 26, 2965-2973.

ZHUO, R., SIEDLECKI, C. A. & VOGLER, E. A. 2006. Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces. Biomaterials, 27, 4325-4332.

ZIATS, N. P., PANKOWSKY, D. A., TIERNEY, B. P., RATNOFF, O. D. & ANDERSON, J. M. 1990. Adsorption of Hageman Factor (Factor XII) and other human plasma proteins to biomaterial polymers. Journal of Laboratory and Clinical Medicine, 116, 687-696.

Controlling Whole Blood Activation and Resultant Clot ... · Firstly, we synthesised a series of materials composed of acrylic acid (AA), and methyl (MMA), ethyl (EMA) or butyl methacrylates

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