DOCTORAL DISSERTATION IN ODONTOLOGY VICTORIA FRöJD ON CA 2+ INCORPORATION AND NANOPOROSITY OF TITANIUM SURFACES AND THE EFFECT ON IMPLANT PERFORMANCE
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VICTORIA FRöjDON CA2+ INCORPORATION AND NANOPOROSITy OF TITANIUm SURFACES AND ThE EFFECT ON ImPLANT PERFORmANCE
isbn 91-7104-315-2
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O N C A 2 + I N C O R P O R A T I O N A N D N A N O P O R O S I T Y O F T I T A N I U M S U R F A C E S A N D T H E E F F E C T O N I M P L A N T P E R F O R M A N C E
Malmo UniversityFaculty of Odontology Doctoral Dissertations 2010
© Victoria Fröjd 2010
ISBN-91-7104-315-2
Holmbergs, Malmö 2010
VICTORIA FRÖJD ON CA2+ INCORPORATION AND NANOPOROSITY OF TITANIUM SURFACES AND THE EFFECT ON IMPLANT PERFORMANCE
Malmo University, 2010Faculty of Odontology
University of GothenburgDepartment of Biomaterials
Publikationen finns även elektroniskt,se www.mah.se/muep
Dedicated to my brilliant brother, my always supporting father, my loving mother,
my darling grandmother, my admirable cousin Camilla, and my dear friend Malin Olsson, who I value deeply.
This thesis represents number 40 in a series of investigations on implants, hard tissue and the locomotor apparatus originating from the department of Biomaterials/Handicap Research, University of Gothenburg and the department of Prosthodontics, Malmö University, Sweden.
1. Anders R Eriksson DDS, 1984. Heat-induced Bone Tissue Injury. An in vivo investigation
of heat tolerance of bone tissue and temperature rise in the drilling of cortical bone. Thesis
defended 21.2.1984. External examiner: Docent K-G. Thorngren.
2. Magnus Jacobsson MD, 1985. On Bone Behaviour after Irradiation. Thesis defended
29.4.1985. External examiner: Docent A. Nathanson.
3. Fredrik Buch MD, 1985. On Electrical Stimulation of Bone Tissue. Thesis defended
28.5.1985. External examiner: Docent T. Ejsing-Jörgensen.
4. Peter Kälebo MD, 1987. On Experimental Bone Regeneration in Titanium Implants.
A quantitative microradiographic and histologic investigation using the Bone Harvest
Chamber. Thesis defended 1.10.1987. External examiner: Docent N. Egund.
5. Lars Carlsson MD, 1989. On the Development of a new Concept for Orthopaedic
Implant Fixation. Thesis defended 2.12.1989. External examiner: Docent L-Å Broström.
6. Tord Röstlund MD, 1990. On the Development of a New Arthroplasty. Thesis defended
19.1.1990. External examiner: Docent Å. Carlsson.
7. Carina Johansson Techn Res, 1991. On Tissue Reactions to Metal Implants. Thesis
defended 12.4.1991. External examiner: Professor K. Nilner.
8. Lars Sennerby DDS, 1991. On the Bone Tissue Response to Titanium Implants. Thesis
defended 24.9.1991. External examiner: Dr J.E. Davis.
9. Per Morberg MD, 1991. On Bone Tissue Reactions to Acrylic Cement. Thesis defended
19.12.1991. External examiner: Docent K. Obrant.
10. Ulla Myhr PT, 1994. On Factors of Importance for Sitting in Children with Cerebral
Palsy. Thesis defended 15.4.1994. External examiner: Docent K. Harms-Ringdahl.
11. Magnus Gottlander MD, 1994. On Hard Tissue Reactions to Hydroxyapatite-Coated
Titanium Implants. Thesis defended 25.11.1994. External examiner: Docent P. Aspenberg.
12. Edward Ebramzadeh MScEng, 1995. On Factors Affecting Long-Term Outcome of
Total Hip Replacements. Thesis defended 6.2.1995. External examiner: Docent L. Linder.
13. Patricia Campbell BA, 1995. On Aseptic Loosening in Total Hip Replacement:
the Role of UHMWPE Wear Particles. Thesis defended 7.2.1995. External examiner:
Professor D. Howie.
14. Ann Wennerberg DDS, 1996. On Surface Roughness and Implant Incorporation.
Thesis defended 19.4.1996. External examiner: Professor P.-O. Glantz.
15. Neil Meredith BDS MSc FDS RCS, 1997. On the Clinical Measurement of Implant
Stability and Osseointegration. Thesis defended 3.6.1997. External examiner: Professor
J. Brunski.
16. Lars Rasmusson DDS, 1998. On Implant Integration in Membrane-Induced and
Grafter Bone. Thesis defended 4.12.1998. External examiner: Professor R. Haanaes.
17. Thay Q Lee MSc, 1999. On the Biomechanics of the Patellofemoral Joint and Patellar
Resurfacing in Total Knee Arthroplasty. Thesis defended 19.4.1999. External examiner:
Docent G. Nemeth.
18. Anna Karin Lundgren DDS, 1999. On Factors Influencing Guided Regeneration and
Augmentation of Intramembraneous Bone. Thesis defended 7.5.1999. External examiner:
Professor B. Klinge.
19. Carl-Johan Ivanoff DDS, 1999. On Surgical and Implant Related Factors Influencing
Integration and Function of Titanium Implants. Experimental and Clinical Aspects.
Thesis defended 12.5.1999. External examiner: Professor B. Rosenquist.
20. Bertil Friberg DDS MDS, 1999. On Bone Quality and Implant Stability Measurements.
Thesis defended 12.11.1999. External examiner: Docent P. Åstrand.
21. Åse Allansdotter Johnsson MD, 1999. On Implant Integration in Irradiated Bone.
An Experimental Study of the Effects of Hyberbaric Oxygenation and Delayed Implant
Placement. Thesis defended 8.12.1999. External examiner: Docent K. Arvidsson-Fyrberg.
22. Börje Svensson DDS, 2000. On Costochondral Grafts Replacing Mandibular
Condyles in Juvenile Chronic Arthritis. A Clinical, Histologic and Experimental Study.
Thesis defended 22.5.2000. External examiner: Professor Ch. Lindqvist.
23. Warren Macdonald BEng, MPhil, 2000. On Component Integration in Total Hip
Arthroplasty: Pre-Clinical Evaluations. Thesis defended 1.9.2000. External examiner: Dr
A.J.C. Lee.
24. Magne Røkkum MD, 2001. On Late Complications with HA Coated Hip
Asthroplasties. Thesis defended 12.10.2001. External examiner: Professor P. Benum.
25. Carin Hallgren Höstner DDS, 2001. On the Bone Response to Different Implant
Textures. A 3D analysis of roughness, wavelength and surface pattern of experimental
implants. Thesis defended 9.11.2001. External examiner: Professor S. Lundgren.
26. Young-Taeg Sul DDS, 2002. On the Bone Response to Oxidised Titanium Implants:
The role of microporous structure and chemical composition of the surface oxide in
enhanced osseointegration. Thesis defended 7.6.2002. External examiner: Professor J.-E.
Ellingsen.
27. Victoria Franke Stenport DDS, 2002. On Growth Factors and Titanium Implant
Integration in Bone. Thesis defended 11.6.2002. External examiner: Associate Professor
E. Solheim.
28. Mikael Sundfeldt MD, 2002. On the Aetiology of Aseptic Loosening in Joint
Arthroplasties and Routes to Improved cemented Fixation. Thesis defended 14.6.2002.
External examiner: Professor N Dahlén.
29. Christer Slotte DDS, 2003. On Surgical Techniques to Increase Bone Density and
Volume. Studies in the Rat and the Rabbit. Thesis defended 13.6.2003. External examiner:
Professor C.H.F. Hämmerle.
30. Anna Arvidsson MSc, 2003. On Surface Mediated Interactions Related to Chemo-
mechanical Caries Removal. Effects on surrounding tissues and materials. Thesis defended
28.11.2003. External examiner: Professor P. Tengvall.
31. Pia Bolind DDS, 2004. On 606 retrieved oral and cranio-facial implants. An analysis
of consecutively received human specimens. Thesis defended 17.12. 2004. External
examiner: Professor A. Piattelli.
32. Patricia Miranda Burgos DDS, 2006. On the influence of micro-and macroscopic
surface modifications on bone integration of titanium implants. Thesis defended 1.9.
2006. External examiner: Professor A. Piattelli.
33. Jonas P Becktor DDS, 2006. On factors influencing the outcome of various techniques
using endosseous implants for reconstruction of the atrophic edentulous and partially
dentate maxilla. Thesis defended 17.11.2006. External examiner: Professor K. F. Moos
34. Anna Göransson DDS, 2006. On Possibly Bioactive CP Titanium Surfaces. Thesis
defended 8.12.2006 External examiner: B. Melsen
35. Andreas Thor DDS, 2006. On platelet-rich plasma in reconstructive dental implant
surgery. Thesis defended 8.12.2006. External examiner: Prof E.M. Pinholt.
36. Luiz Meirelles DDS MSc, 2007. On Nano Size Structures For Enhanced Early Bone
Formation. Thesis defended 13.6.2007. External examiner: Professor Lyndon F. Cooper.
37. Pär-Olov Östman DDS, 2007. On various protocols for direct loading of implant-
supported fixed prostheses. Thesis defended 21.12.2007. External examiner: Prof B
Klinge
38. Kerstin Fischer DDS, 2008. On immediate/early loading of implant supported
prostheses in the maxilla. Thesis defended 8.2.2008. External examiner: Professor
Kristina Arvidson Fyrberg
39. Alf Eliasson 2008. On the role of number of fixtures, surgical technique and timing
of loading. Thesis defended 23.5.2008. External examiner: Kristina Arvidson-Fyrberg.
40. Victoria Fröjd DDS, 2010. On Ca2+ incorporation and nanoporosity of titanium
surfaces and the effect on implant performance. Thesis to be defended 26.11.2010.
External examiner: Professor J. E. Ellingsen
List of papersBone tissue (in rabbit): Importance of surface topography as well as anodization and Ca2+ incorporation for osseointegration
Study I Fröjd V, Franke-Stenport V, Meirelles L, Wennerberg A. Increased bone contact to a Ca2+ incorporated oxidized c.p. titanium implant: an in vivo study in rabbit. Int J Oral Maxillofac Surg 2008 37(6): 561-6
Study II Fröjd V, Wennerberg A, Franke-Stenport V. Importance of Ca2+ modifications for osseointegration of smooth and moderately rough anodized titanium implants – a removal torque and histological evaluation in rabbit. Accepted for publication in Clin Impl Dent Relat Res 2010.
Oral mucosa:Impact of nanoporosity for the sealing of oral mucosa
Study III Wennerberg A, Fröjd V, Olsson M, Nannmark U, Emanuelsson L, Johansson P, Yvonne J, Kangasniemi I, Peltola T, Tirri T, Pänkäläinen T, Thomsen P. Nanoporous TiO2 thin film on titanium oral implants for enhanced human soft tissue adhesion - a histological evaluation in three different levels of resolution. Clin Impl Dent Relat Res E published ahead of print 2009.
Biofilm accumulation (in vitro):Influence of surface topography, anodization and Ca2+ incorporation, and nanoporosity on multi-species bacterial adhesion and biofilm formation
Study IV Fröjd V, Chávez de Paz L, Andersson M, Wennerberg A, Davies J, Svensäter G. In situ analysis of biofilm formation on titanium surfaces. Submitted.
Study V Fröjd V, Linderbäck P, Wennerberg A, Chávez de Paz L, Svensäter G, Davies J. Microbial biofilm formation on smooth nanoporous TiO2 coated and anodized Ca2+ modified and titanium surfaces. Submitted.
TAbLE OF CONTENTS
ABstrAct .................................................................. 17
IntrODUctIOn .......................................................... 21Indications for biomedical titanium implants and research within the field ..................................................21Principles for integration of biomaterials ..................................22
Integration into bone tissue................................................22Integration into oral mucosa and soft-tissues ........................26
titanium as a biomaterial ......................................................27Bioactivity ............................................................................29Methods for titanium surface processing ..................................30surface properties of titanium implants ...................................34aimed for bone integration ....................................................34
Macro design ..................................................................34Micro topography ............................................................34nano topography ............................................................35chemistry .......................................................................35
surface properties of titanium implants aimed for soft tissue integration.............................................................44
Macro design ..................................................................44Micro topography ............................................................44nano topography ............................................................45chemistry .......................................................................45
complications with titanium implant treatments .........................48Nature of the complications ..............................................48Extent ...........................................................................48Biofilms and their accumulation on titanium surfaces.............49
AIMs ......................................................................... 55
MAtErIAls AnD MEthODs .......................................... 57surface processing ...............................................................57
Anodic oxidation .............................................................57sol-gel derived nanoporous tiO2 coating ............................58Blasting process ...............................................................58
surface characteristics measurements ......................................59Optical interferometry ......................................................59scanning electron microscopy ...........................................61transmission electron microscopy .......................................62X-ray photoelectron spectroscopy ......................................62Ellipsometry ...................................................................62
In vivo evaluations – rabbit model .........................................62Animals and surgical technique ........................................62Biomechanical evaluation .................................................63Preparation of histological specimen .................................63light microscopy evaluations .............................................63
In vivo evaluations – human model .........................................64Investigation design and patient selection ...........................64X-ray imaging ..................................................................64sample retrieval ...............................................................65Preparation of histological specimen .................................65light microscopy evaluations .............................................65transmission electron microscopy analysis .........................66
In vitro models .....................................................................66Bacterial strains and culture ...............................................6616s rrnA fluorescence in situ hybridization ........................67confocal laser scanning microscopy ..................................68Biofilm biovolume quantifications .......................................69
statistics ..............................................................................69
rEsUlts ..................................................................... 71surface properties ................................................................71
topography .....................................................................71chemistry .......................................................................74
Osseointegration - study I and II .............................................75Interaction with oral mucosa – study III ....................................78
histological investigation ..................................................78Bacterial adhesion and biofilm formation - study IV and V .........79
Biofilm accumulation in presence of saliva ..........................81
DIscUssIOn ............................................................... 83surface processing ...............................................................83surface characteristics measurements ......................................84In vivo evaluations – rabbit models .........................................84In vivo evaluations – human model .........................................86In vitro evaluations ................................................................86surface characteristics ..........................................................87Osseointegration - study I and II .............................................88sealing of oral mucosa – study III ...........................................89Bacterial adhesion and biofilm formation - study IV and V .........89
sUMMAry, FUtUrE PrOsPEctIVEs, AnD DErIVED hyPOthEsIs .............................................................. 93
cOnclUsIOns ........................................................... 95
POPUlärVEtEnskAPlIG sAMMAnFAttnInG .................. 97
AcknOwlEDGEMEnts ............................................... 99
rEFErEncEs ..............................................................103
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AbSTRACT
Introduction: Titanium implants are commonly used as replacements for missing teeth with successful long-term performance. The aim of the research performed in the field is to enable successful osseointegrated implant treatments for compromised as well as healthy bone beds, and to establish a rapid osseointegration to shorten the treatment period for the patients. In some cases bone resorption occurs around oral implants and the surrounding conditions may alter when surfaces aimed at being integrated in the bone are exposed to the extensive oral microbiota. Biofilms are most probably constantly present on exposed intraoral surfaces but may during certain conditions be associated with pathological conditions in the surrounding tissues. Implant treatments depend on a stability through the osseointegration, as well as a sealing of oral mucosa for the defence against extensive biofilm accumulation.
Aims: The present thesis has aimed at investigating the impact of Ca2+ incorporation to anodized titanium surfaces for osseointegration, and whether Ca2+ incorporation would compensate for potential shortcomings of a minimal surface roughness. We have further aimed at investigating the adhesion of oral mucosa to nanoporous TiO2 surfaces clinically as well as histologically, and at evaluating the bacterial adhesion and biofilm formation on the test surfaces in vitro.
Methods: The osseointegration of smooth (average height deviation <0.5 µm) and moderately rough (average height deviation 1-2 µm) Ca2+ incorporated anodically oxidized surfaces, minimally (average
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height deviation 0.5-1 µm) and moderately rough anodically oxidized surfaces, and minimally and moderately rough Al2O3 blasted surfaces, was investigated with a rabbit model in two studies: one histological and one combined biomechanical and histological study. Oral mucosa adhesion to sol-gel derived, smooth nanoporous TiO2 coated and turned surfaces with similar microtopography was investigated in an experimental study in humans, where the samples were evaluated clinically and histologically at three different levels of resolution. All histological sections were evaluated both quantitatively and qualitatively. To study bacterial adhesion and biofilm formation on the surfaces as well as the possibility to mechanically remove adhered bacteria with a smooth toothbrush without dentifrice, multi-species bacterial models (with or without the presence of saliva) combining 16S rRNA fluorescence in situ hybridization and confocal laser scanning microscopy were used. Surface topography and chemistry was characterized using optical interferometry, scanning electron microscopy, X-ray photoelectron spectroscopy, and atomic force microscopy.
Results: Smooth Ca2+ incorporated anodically oxidized implants had significantly more bone in contact compared to minimally rough anodically oxidized and blasted implants when placed in rabbit tibia. Moderately rough Ca2+ incorporated anodically oxidized implants had significantly higher removal torque compared to moderately rough anodically oxidized and smooth Ca2+ incorporated anodically oxidized implants, and, at the same time, the removal torque of smooth Ca2+ incorporated anodically oxidized implants did not significantly differ from that of moderately rough blasted or anodically oxidized surfaces when placed in rabbit tibia.
Nanoporous TiO2 coated abutments had significantly more oral mucosa in contact with the surface as well as significantly less marginal bone resorption when only stable implants were evaluated compared to turned control surfaces. The clinical appearance was, furthermore, assumed to be advantageous for the nanoporous surfaces.
Increasing the surface roughness led to larger biofilm biovolumes in vitro. At the same time, Ca2+ incorporation tended to decrease biofilm formation when compared to control surfaces. Nanoporosity
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or Ca2+ incorporation did not seem to effect biofilm formation when compared to turned surfaces. Moderately rough blasted surfaces generally adhered largest biofilm biovolumes and presented the greatest amount of remaining bacteria after mechanical cleaning.
Conclusions: Within the limits of the studies in the present thesis, Ca2+ incorporation may enhance osseointegration and compensate for minimal surface roughness in rabbit tibia. Nanoporosity may hold advantages for oral mucosa adhesion; however, no clear conclusions can be drawn. Increased surface roughness may increase bacterial adhesion and biofilm biovolume in vitro, and moderately rough blasted surfaces were most difficult to clean from adhered bacteria.
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INTRODUCTION
Indications for biomedical titanium implants and research within the fieldSince the discovery of osseointegration of titanium by Brånemark and, almost contemporary Schroeder and Schulte, installation of titanium implants have become an established treatment for replacements of teeth. Today, more than two million oral implants are placed in the United States annually. If implants per capita are considered, South Korea is the leading country closely followed by Italy, Sweden, and Switzerland. Moreover, titanium implants are used for, for example, amputation prostheses and, if rarely, total hip replacements.
Treatment with oral titanium implants may improve the quality of life for edentulous patients11. In general, titanium dental implants have high survival rates of 90-98 percent over twenty years12, 13. However, when approaching compromised patients or patients with unfavourable bone quality there may be a need for specific implant surface designs and/or particular surgical techniques to improve chances for a successful treatment. The complications that do occur in relation to titanium implants, for example bone resorption and biofilm infections, exemplify an area where further research is needed to hopefully control such events. Another aspect is the aesthetics in relation to implants that still could be improved in many cases, and which are of importance for a large group of patients receiving implant treatment.
The field of oral implants is one example of close links between research and industry and findings in the laboratory often becomes clinically applied. Most new designs and surfaces are considered not
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to deviate from existing implants, and the process to reach the clinic is, therefore, rather short. However, a risk with the commercial approach is that the desire to launch “news” may get in conflict with the need for control of possible side-effects with the products, which could result in unnecessary suffering for the patient.
In conclusion, implants need to perform in three biological arenas: in relation to bone tissue, soft tissue, and microbial biofilms, and all these aspects should be considered when developing new implant surfaces. This thesis has aimed at initiating investigations of surfaces in relation to more than one of these “arenas”.
Principles for integration of biomaterialsSurface characteristics of implants seem to effect the inflammatory response in the surrounding tissues 14. An inflammatory response to installed titanium implants, added to the one caused by the surgical trauma have been noted15, 16. One aspect of the modulation of the body response to biomaterials is complement activation (mainly C3 derivates and C5a mediates inflammation)17, where there are differences between various surfaces18-20. However, numerous studies and clinical experience have proven titanium to be a most proper material to use for replacements of lost teeth. Furthermore, the inflammation process may be of importance for the tissue healing around implants14 and, therefore, a controlled activation of a transient inflammatory response may even be a positive reaction. A literature review reveals that implant surface characteristics may influence the biological response.
Integration into bone tissueBrånemark et al. coined the term osseointegration in 1977 and defined it as: “re- and new-formed bone tissues enclose the implant with perfect congruency to the implant form and surface irregularities thus establishing a true osseointegration of the implant without any interpositioned connective tissue”21. Studies report an unmineralized zone of some hundred nanometres between titanium implants and bone22, 23. This layer is suggested to mainly consist of proteoglycans 23, 24. However, studies using transmission electron microscopy of the interface between bone and titanium show an intimate contact and presence of hydroxyapatite at the immediate implant surface 25, 26.
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The integration of biomaterials into bone, or any tissue, relies on healing mechanisms involving the stages of haemostasis, inflammation, regeneration, and remodelling. Chemotaxis and recruitment of cells are of crucial importance. However, with new techniques and knowledge, trials to modify the mechanisms after implant installation, to speed up the process as well as having an enhanced healing and integration, are performed with many implant surfaces. The terms distance and contact osteogenesis was first coined by Osborn and Newsely in 198027, reflecting on whether bone formation is initiated at the border of the old bone or at the implant surface. Suggestively, contact osteogenesis or a combination of contact and distance osteogenesis may occur with certain surface characteristics of bioactive nature2. Possible ways to affect the osseointegration is either via the living compartments of the bone, i.e. the cells, or by physico-chemistry with mechanical interlocking28 or attraction forces. In order to modulate the response, an understanding of the naturally occurring events is of great importance.
Today, research starts to focus on the gene-regulation and molecular mechanisms of cells involved in osteogenesis around implant29. In a thesis by Omar (2010), the molecular mechanisms of osseointegration, the importance of the mesenchymal progenies and the hematopoietic derived cells (for example platelets, neutrophils, endothelial cells, monocytes, lymphocytes, and osteoclasts), and growth factors that initiate other cellular events have been discussed. Polymorphonuclear leukocytes, macrophages, and osteoclasts will phagocytise injured tissues to allow for regeneration of newly formed tissue. The effect of cytokines or other transcription factors varies with the cell type and the specific cell surface receptor they bind to; consequently, one substance may activate various intracellular cascades30. The multi-function of several molecules involved in the healing cascades reveal the complexity of the biological system as well as the fact that we do not have complete knowledge of the mechanisms. There is, furthermore, a close relation between all cells involved, from the mesenchymal stem cells to the osteoclasts; for example osteoblasts release factors that activates osteoclasts31.
Cells may interact with surfaces via receptors/adhesion molecules, such as immunoglobulin’s, cadherins, and integrins. Integrins
24
may form complexes with RGD (Arginine-Glycine-Aspartic acid) sequences and may, thereby, be attached to certain RGD containing proteins (e.g. fibronectin) or other with cell receptors coating a surface 32.
When bone formation occurs, osteoblasts first lay down a seam of osteoid, an unorganized non-mineralized matrix of collagen. Mineral/hydroxyapatite crystals are, thereafter, being deposited in gaps within the arranged collagen α-triple helix33. Examples of key factors in bone healing that enable osseointegration are presented in Figure 1.
Summary, discussion, and specific relevance to the present thesis:Osseointegration of titanium implants have been extensively investigated, however, we do not possess complete understanding of the mechanisms behind osteogenesis and bone healing around implants. Studies on biomolecular and cellular effects of titanium surface characteristics are being more common and will probably improve the possibility to more accurately modify an “optimal” surface. Whether this “optimal surface” would possess properties that have desired effects not only regarding bone-tissue, but in addition with respect to soft-tissue and microbial biofilms, is to be discussed in this thesis.
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Integration into oral mucosa and soft-tissuesThe oral mucosa surrounding implants has been suggested to have both similarities and differences to the gingiva around teeth. From the marginal bone level to the marginal gingiva about 1-1.5 mm of connective tissue are suggestively in close contact with the implant, followed by about 2 mm of junctional epithelium, and a keratinized oral epithelium34. Hemidesmosomes as well as a basal lamina have been found at the interface between titanium and epithelial cells35. This relation does seem to be re-established if disrupted followed by healing36. If only disrupted by clinical probing, the epithelial attachment is suggested to heal in five days37. One difference between the peri-implant mucosa and the gingiva surrounding a tooth is the arrangement of the subepithelial connective tissue. Collagen fibres project from the root cement into the connective tissue proper, while a commercially pure titanium implant is surrounded by dense, connective tissue with collagen fibres and fibroblasts extended mainly in parallel with the implant surface in man38-40. There are, however, findings of random 41, circular42 , or even perpendicular fibre orientations43, around specific surfaces. A tight sealing between the oral mucosa and the implants is believed to be of importance for the defence against extensive biofilms44 and the protective mechanisms seem to correspond to the gingiva surrounding a tooth42. However, an in vivo study in dogs suggested that peri-implant tissues may have decreased defence capacity when it is poorly vascularised45.
In an in vitro study, multiple integrin subunits in human gingival fibroblasts were found grown in contact with titanium implant surfaces and, furthermore, titanium surface roughness altered cellular morphology but appeared to have limited effects on the integrin expression46. However, connective tissue cells are greatly influenced by an extra-cellular matrix, and in vitro results may only vaguely reflect the physiological situation47.
Summary, discussion, and specific relevance to the present thesis:For oral implants, the relation to soft-tissue has not been explored in as great extent as regarding bone-tissue. However, soft-tissue attachment is of importance for the infection resistance and the aesthetics of implant treatments. There are surface modifications suggested to improve soft-tissue adhesion and one such has been investigated in this thesis.
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Titanium as a biomaterialA major reason behind titanium being a gold standard for osseointegration is the native titanium oxide layer (about 2-7 nm thick) established instantly when in contact with oxygen. The oxide layer offers a stable and corrosion resistant outer layer of the bulk material, giving the material outward characteristics more of a ceramic and makes the metal biocompatible. The native oxide of a titanium surface is depending on the bulk material but mainly consists of titanium oxide and has an amorphous structure. If titanium is oxidized further (thermally or electrochemically), TiO2 may assume three crystalline phases: anatase, rutile, and, if not as common, brookite48.
Titanium may exist in two crystallographic forms: the hexagonal close-packed crystal structure, named the alpha structure, and a body-centred cubic structure, named the beta structure. In few words, the two phases, or a combination of them, gives the metal somewhat different properties. The beta phase of titanium appears when titanium is heated above 883°C and solidified rapidly and stabilized using stabilizing elements. 49 Commercially pure titanium (ASTM F67, grade 1 to 4) is composed of 98.9-99.5 weight percent pure titanium; all has alpha crystallinity, and the differences between the grades are the content of impurities (carbon, nitrogen, iron, hydrogen, and oxygen). To further increase the mechanical properties, for example, when used as total hip replacements or amputation prosthesis carrying substantial load, alloys of titanium are used for biomedical applications and have been shown reasonably biocompatible. Titanium alloys are considerably harder materials compared to commercially pure titanium. Ti-6Al-4V is the most commonly used titanium alloy in the biomedical field and also called titanium grade 5; it has a mixture of alpha and beta phased crystals, as have many of the other titanium alloys.49
Some studies indicate similar response to titanium alloys as to commercially pure titanium50, whereas others have indicated somewhat weaker bone responses to the alloy compared to commercially pure titanium51. Originally, the Brånemark concept strictly advocated titanium grade 1 for osseointegrated implants, but today most commercial titanium oral implants are made from commercially pure grade 4, and some from titanium alloy (grade
28
5). What needs to be kept in mind is that the resultant surface characteristics of a certain surface process may differ between different grades of titanium when these possess different mechanical as well as chemical properties. Focus will be on commercially pure titanium in the following parts of the thesis, where all studies were performed with titanium grade 4.
The primary interaction between an implant and the host is by adsorption of water molecules and ions followed by proteins from the blood plasma. Titanium and titanium alloys have the ability to spontaneously allow calcium phosphate nucleation on the surface in a solution; the calcium phosphate formed on commercially pure titanium most resembles apatite52. Furthermore, ions generally modulate the adhesion of proteins and extracellular Ca2+ has been suggested to link proteins to TiO2
53, 54. The part of the implants positioned in the bone mainly interacts with plasma proteins, for example, fibrinogen, albumin, immunoglobulin G53, 55. Albumin has been suggested the main salivary protein adsorbed on titanium surfaces56.
Proteins generally adsorb to a surface, possibly unfold, and thereafter bind firmly or are exchanged or covered by other proteins. The protein adsorption may, in addition, be altered by surface characteristics, such as the topography55 and the physico-chemical properties57. Possibly, a protein with an RGD sequence could be sought to express its RGD sequence to the surroundings after having adsorbed to a surface in order to promote cell adhesion. It has also been suggested that surface chemistry increases the biological activity of, e.g. the integrin-binding protein fibronectin, resulting in enhancement of cell adhesion58.
Summary, discussion, and specific relevance to the present thesis:For many years it has been known that titanium as a bulk material possesses qualities appropriate for biomedical implants, nevertheless, it is the native or modified titanium oxide layer that enables the tissue integration. The properties of the surface impact the protein adsorption, and, furthermore, the proteins are of importance for the biological response (by bone cells, soft-tissue cells, as well as bacteria). However, the protein adsorption to the titanium surfaces used has not been investigated within the frame of this thesis.
29
bioactivityThe term bioactivity indicates that something interacts or stimulates an effect in biological structures. Suggestively, the term “bioactivity” should be addressing a phenomenon and not a specific surface or material. Bioactivity may be defined as involving biochemical bonding, or that a surface attracts certain proteins and/or stimulates bone cells, having a catalytic effect on other processes within the cell. Hench, who has mainly been working with bioglass or bioglass ceramics, defined bioactivity, referring to the biochemical bonding, as: “Bioactivity is the characteristic of an implant material which allows it to form a bond with living tissues” 59. Williams defined bioactivity, aiming at the stimulating effect rather than the bonding, as: “Phenomenon by which a biomaterial elicits or modulates biological activity” 60. One example of bioactive processes is discussed by Davies, with the importance of platelet activation and osteoblasts and pre-osteoblasts migrating through the fibrin network to attach to a surface and deposit osteoid; resulting in de novo bone formation 2, which may also be described as bone induction.
Regardless of the definition, the aim of a surface with bioactive characteristics would be more rapid integration, resulting in shorter healing periods, and, possibly, stronger anchorage of the implant. In the thesis by Göransson (2006) the following surface modifications result in surfaces with properties of possibly bioactive character: fluoride etching, alkali heat treatment, anodization with e.g. ion incorporation into the oxide layer, hydroxyapatite coating, and covalent immobilization of proteins61.
Summary, discussion, and specific relevance to the present thesis:Bioactivity is a widely used term. The concept should suggestively be used to describe the nature of surface characteristics, instead of being considered a particular surface character. Suggested bioactive surface characteristics have indicated advantages for osseointegration, and one surface with allegedly bioactive characteristics (i.e. anodically oxidized and calcium ion incorporated) have been investigated in this thesis.
30
Methods for titanium surface processingThere are a number of processes used to modify titanium implant surfaces. The selected process is depending on the mechanical properties required, as well as which parameters that are aimed to be modified. However, it is difficult to alter the chemistry without altering the topography and vice versa. It is common that methods are combined to achieve the preferred surface characteristics. The resulting surface can be controlled to various degrees with different methods. Some examples will follow.
Physical treatments:Turning processThe turning process of dental implants is often used to gain the macro design of the implant that may, thereafter, be modified. Turned surfaces have mainly been found smooth (average height deviation <0.5 µm) or minimally rough (average height deviation of 0.5-1 µm) and anisotropic due to the turning process. The original Brånemark implant has a turned, minimally rough surface. Today, studies of more than 20 years follow-up times for functional implants are reported with the original Brånemark system implants 13. M ost abutment surfaces have smooth turned or polished surfaces 62.
Grit/abrasive blastingThe processing can produce isotropic surfaces with various roughnesses and chemistries depending on the blasting particle (TiO2 and Al2O3 are commonly used) and its size, as well as the pressure and the distance of the blasting instrument. There are commercial implants with a blasted surface alone (TiOblast™ from Astra Tech AB, Gothenburg, Sweden) or in combination with other surface processes (for example OsseoSpeed™ from Astra Tech AB, and SLA®/SLActive® from Institute Straumann AG, Basel, Switzerland). Most blasted surfaces achieve a minimally to moderately rough (average height deviation of 1-2 µm) surface.
Ultraviolet irradiation of TiO2 crystalline surfacesBy treating a crystalline surface with ultraviolet irradiation, decomposition of organic compounds occur and an extremely clean surface is achieved 63. Furthermore, surface oxygen vacancies appear,
31
which interact with water molecules and forms hydroxyl-groups with hydrophilic domains on the outermost layer 64. A surface need to be crystalline in order to respond to the treatment in the wanted manner, and cannot have an amorphous outer titania layer. After the surface processing the surface, although being highly hydrophilic, becomes amphiphilic 63 and may, thereby, attract different proteins at different regions. Specific surface chemistry may, in addition, be applied to the surfaces and may, further, influence the photocatalytic effect 65. The surface topography depends on the original surface, but is in general isotropic, and, mostly, minimally 65 and moderately rough surfaces have been investigated in the literature 66, 67.
Electrochemical treatments:Micro-arch oxidation/anodic oxidationBy using electrochemical oxidation surface topography as well as chemistry may be altered 68. Anodized surfaces acquire an isotropic, porous appearance with pore size and distribution depending on the electrolyte as well as the voltage/current of the oxidizing process. The process can be modified to achieve a relatively high control of the resulting surface, for example, nanotubular structures. The TiUnite® surface of Nobel Biocare™ is anodized and has a moderately rough surface, with porous structures of a diameter 0.5-3 µm in general; it presents phosphor ions in the oxide layer69, which is about 2-8 µm thick and has both anatase crystallinity and an amorphous phase70,
71. The Ospol AB, Malmö, Sweden, surface is a smooth anodized surface with calcium ions within its oxide layer. The Ca (study I) or OxCa (study II, IV, and V) is processed according to the same protocol as the Ospol surface and similar to that presented by Sul et al. (2002)72.
Chemical treatments:Acid etchingEtching of a surface, mainly via a thermal process using acids that resolves the outermost layer of a material, in general creates an isotropic surface with a negative skewness. Commercial dual acid-etched implants are OSSEOTITE® (BIOMET 3i™), which are etched with HCL/H2SO4
73 and has a minimally rough surface74. The SLA®/SLActive® surfaces from Institute Straumann AG
32
(Waldenburg, Switzerland) is firstly blasted, secondly acid etched, resulting in a moderately rough surface. The OsseoSpeed™ surface from Astra Tech AB is the TiOblast™ surface with further etching of hydrofluoric acid, resulting in a chemically modified, moderately rough surface with nanofeatures; the oxide thickness is up to 1 µm and consisting of an inner amorphous layer followed by an outer crystalline layer (anatase and rutile)71.
Alkali heat treatmentBy treatment of NaOH followed by heating, an amorphous sodium titanate surface is achieved. The surface have been found to initiate apatite formation in simulated body fluid; the scenario has been suggested to begin with ion exchange between the material (sodium) and the solution (hydronium and, thereafter, calcium)75. When calcium titanate constitutes the outermost surface, adsorption of phosphate and calcium ions occurs. The processing can be performed on various initial surfaces. The surface orientation and roughness depend on the original surface; however, the process tends to create an isotropic surface.
Depositional treatments:Ion implantationIon implantation involves an ion source, an accelerator, and a chamber where the surface is positioned. When ions are sputtered towards a surface using lower energies the process is defined as ”ion beam deposition”. Various ions can be implanted to various depths and in controlled concentrations. Ion implantation can be executed on any initial surface, but with somewhat different outcome (depending on the hardness etc.). The surface topography and orientation is nearly solely depending on the original surface. For a review, see Rautray et al. (2010).76
Sol-gel coatingsSol-gel technology allows preparation of materials with a wide range of topographical, physical, and chemical properties. Most achieve an isotropic appearance. The sol-gel technique, furthermore, provides a surface coating that is relatively easy to control in great quantities and for larger implant sizes. A solution (“sol”) is prepared with a
33
specific composition and often matured to retain a solid and a liquid phase. The specimen is, thereafter, dipped at a specific number of times, with drying in between, and finally heat-treated to solidify and sinter the coating. It has been shown that sol-gel matrices can be modified with organic functional groups77, that may incorporate proteins and release them at a controlled rate78. Sol-gel techniques are used to achieve nanotopographical features. An example of sol-gel coating used commercially is the dental implant NanoTite® (BIOMET 3i™), which has a surface treated with discrete crystalline deposition of calcium phosphate that is sol-gel derived.
Summary, discussion, and specific relevance to the present thesis:There are a number of available processes to modify titanium surfaces. Many of them allow for great control of the effect on the surface, which is of importance in the production of commercial implants as well as for research studies. However, it is still difficult to alter only one specific characteristic, since they often affect one another. The surfaces used in the studies of this thesis have frequently been aimed to posses either similar topographic or chemical features. The methods used have been: turning process, blasting with Al
2O3, anodic oxidation and incorporation of calcium ions, sol-gel deposition of TiO2, or combinations thereof.
34
Surface properties of titanium implants aimed for bone integrationMacro designThe threaded, screw shaped implant is today the dominant design, although, the ideal thread type remains to be described. Screw shaped implants are advantageous to cylindrical implants since they, for example, have improved load distribution and a larger area with close fit to the bone 79. Companies offer both straight walled or tapered implants, and the shape and depth of the threads also varies. Hansson et al.80 suggested enhanced load distribution with microthreads resulting in significantly less bone resorption compared to implants without microthreads81.
Modern implants today presents high survival rates, even if short implants (<10 mm) are assessed82, 83.
Micro topographyWennerberg proposed in her thesis (1996) an optimal topography for bone integration of titanium implants to be a surface with an average height deviation of 1.5 µm, an average wavelength of about 11.1 µm, and a developed area ratio of 50 percent. The following section will mainly focus on average height deviation or roughness in height, the Sa parameter.
Albrektsson and Wennerberg84 suggest titanium implant surfaces to be divided into smooth (<0.5 µm), minimally rough (0.5-1.0 µm), moderately rough (1.0-1.5 µm), or rough (>2.0 µm) according to their average roughness in height. As can be understood from earlier presented findings, moderately rough surfaces may be considered to stimulate the strongest bone integration. Although the importance of surface parameters has been questioned85, most commonly used commercial implants today have minimally or moderately rough surfaces86. Whereas these modern surfaces have been backed up by published clinical studies with survival rates of about 97 percent for five to ten years follow-up times87-89, possible risks with moderately rough surfaces may be increased risks in cases with peri-implantitis and, to a minor extent, ion leakage. These will further be discussed under the section “Complications with implant treatments”.
35
nano topographyNano features of titanium implants have been suggested of importance for bone integration mostly by affecting the wettability, ion and protein adsorption, as well as the cells90. The bone response in a rabbit model was enhanced by a nanofeatured surfaces with hydroxyapatite deposition91, and in another study nanofeatures of titania presented similar, or even a tendency to enhanced, bone response as nanofeatures of hydroxyapatite92. Although, today when discussing nanofeatures it is most often as an additional structural dimension on top of the microtopography; most commercial implants proclaiming a nanofeatured topography (for example OsseoSpeed™ from Astra Tech AB, SLActive® from Straumann AG, and Nanotite from Biomet 3i inc) have a minimally or moderately rough microtopograhy93.
The dimensions of the concept of nanofeatures are similar to that of microfeatures, e.g. both the distribution and the dimensions may be of importance92. With various techniques altered structure of the features can be established, and the effect of orientation and character of nanofeatures are not yet fully evaluated.
chemistryIon incorporation of:Phosphorous ionsPhosphorous ions can be found on the TiUnite® surface (Nobel Biocare™) at the rate of approximately two atomic percentage69. Hydrothermal treatment with phosphorous ions have resulted in higher removal torque and bone in contact ratio as compared to turned, acid etched, grit blasted, grit blasted and acid etched, or spark anodized after six weeks in rabbit94.
Fluoride ionsFluoride ions have high affinity for calcium and phosphate ions, and fluoride-modifications have been found to stimulate mesenchymal bone cells29, 95. Fluoride increases the density of bone96, which may have an impact on the bone-tissue properties adjacent to the implant surface. Furthermore, in vivo models demonstrate advantages with fluoride modifications. Increased bone-tissue contact has been found through studies in dog97, 98 and rat29, together with increased biomechanical strengths in rabbit-studies evaluated for one to three months, as compared to somewhat rougher blasted surfaces99-101.
36
There is one commercial oral implant surface that is fluoride modified by etching with hydrofluoric acid, OsseoSpeed™ by Astra Tech AB. Yet, Kang et al. (2009) found F on the OsseoSpeed™ surface at very low ratio (0.3 atomic%) when using both X-ray photoelectron spectroscopy and auger electron spectroscopy69.
Magnesium ionsMagnesium implantation has been suggested to increase the hydroxyapatite nucleation and apatite growth on titanium. Magnesium ion modifications increased the adhesion and up-regulated intracellular cascades of human bone derived cells compared to non-modified Al2O3 surfaces102 and Ti6Al4V surfaces103, as well as for mouse osteoblast like cells on smooth and moderately rough surfaces104. Mg2+-implanted surfaces have also shown improved interfacial shear strength105-109, and more rapid osseointegration108,
109 as compared to control implants. In another study, Mg2+-incorporated micropatterned surfaces increased resonance frequency measurement results as compared to commercially available implants SLA®, Osseotite, TiOblast™, and Mg2+-incorporated TiUnite®; but without the micropatterned threads, the Mg-modifications did not enhance the bone-integration110.
Calcium ionsIn the thesis by Sul (2002), possible effects of incorporated Ca2+ were suggested. These are mainly that Ca2+ within the oxide layer moves towards the outer surface and the extracellular body fluids; thereafter, electrostatic interactions between the calcium ions and ions as well as adhesive bone matrix proteins arises, and calcium ions may also stimulate RGD surface receptors and prompt the recruitment of osteoblasts and osteoprogenitor cells111.
Calcium ions may be deposited through, for example, ion implantation, incorporation into the titania during electrochemical oxidation, or through plasma immersion. The dissolution of Ca2+ has been suggested a key factor for hydroxyapatite nucleation on calcium modified surfaces112. Ca2+ implantation accelerates the adsorption of phosphate ions and improves the ability of titanium to induce the formation of calcium phosphate minerals on the surface112-115, possibly, due to a more positively charged
37
surface and more hydroxyl radicals as compared to unmodified TiO2
116. When positioned in simulated body fluid the formation of octacalcium phosphate has been suggested energetically favourable as compared to hydroxyapatite, but since hydroxyapatite is more thermodynamically stable the dominating mineral crystal will shift from octacalcium phosphate to hydroxyapatite over time117. Mainly electrostatic interactions are suggested to be of importance for the mineral nucleation on a surface.
In vitro studies have generally shown that cells adhere in a lesser extent to calcium modified surfaces, meanwhile they spread more and seemingly become more activated. Nayab et al. (2005) suggest that early phosphate and calcium precipitation on calcium ion implanted surfaces explain the lesser attachment of bone cells. After a certain time (suggestively 24 hours) these particles undergo conformational changes and an increase in cell adhesion occurs118.
In previously published in vivo studies using anodized commercially pure titanium with incorporated Ca2+, calcium modification enhanced the osseointegration119-121. Possible effects of the calcium ions may be that they attract proteins and growth factors of importance for the bone cells and bone formation118, 122, that they enhance bone cell growth123, 124, and that they function as binding sites for bone mineral crystals125 through ion adsorption as earlier mentioned. Studies in the literature regarding calcium-implanted surfaces are gathered in Table 1.
Calcium ions are incorporated in the commercial Ospol AB surface at approximately two atomic percent, and was also found at somewhat over one atomic percent on the OsseoSpeed™ surface analyzed with X-ray photoelectron spectroscopy69.
Calcium phosphatesThere are a number of calcium phosphates that may form biologically as well as synthetically126. Various ions can be incorporated in the apatite crystal, for example CO3
2- and F-, which results in somewhat different qualities (e.g. solubility)127. Tricalcium phosphate (the β- form mainly used as biomaterial) is a rapidly resorbed crystal but which partly converts into more stable hydroxyapatite with the right composition of the surrounding electrolyte or when surrounded by body fluids. β-tricalcium phosphate is also sintered together with
38
hydroxyapatite to slow down the degradation rate; the ceramic is then called biphasic calcium phosphate. Octacalcium phosphate is another pre-cursor to hydroxyapatite. The composition of the calcium phosphate is of importance to control when establishing a calcium phosphate coated surface128.
One possible disadvantage with apatites is that they may resolve in a physiologic environment, or fracture from the bulk material. This was primarily a problem when hydroxyapatite was deposited using plasma-spraying methods129, 130. However, by using techniques to deposit thin layers of hydroxyapatite (sol-gel coatings or other refined methods), coat loosening may no longer be a problem. Calcium phosphates or hydroxyapatite coatings have been suggested to create a surface with bioactive characteristics and mineral nucleation occur upon the surface127.
There are a number of methods to coat a surface with calcium phosphates; for example, plasma spraying, electrophoretic deposi-tion, sputter deposition, and sol-gel coating. Calcium phosphates are commonly used within the orthopaedic research and the com-mercial dental implant surface NanoTite® (BIOMET 3i™) has a surface treated with sol-gel derived discrete crystalline deposition of calcium phosphate131, 132.
Immobilization of biofunctional molecules/proteins Proteins can either be adsorbed or covalently immobilized on surfaces. Challenges are to maintain the biofunctionality after immobilization and to control the diffusion rate. For bone-tissue interactions, mainly RGD-peptides133, collagen134, and BMP-2135, 136 have been tested. Furthermore, immobilizing other cell adhesive molecules, e.g. tetra-cell adhesion molecule, may modify and improve the osteogenesis137.
Bisphosphonates Bisphosphonates are commonly used systematic drugs to treat conditions such as osteoporosis and have an inhibitory effect on osteoclasts. Immobilized bisphosphonates on titanium138, stainless steel139, 140, and hydroxyapatite coated titanium or titanium alloy implants141, 142 have indicated enhanced bone response from various studies in rats and dogs. The application is relatively modestly explored and research within the field is ongoing.
39
29
su
rfac
es.
Maj
or fi
ndin
gs:
Sign
ifica
ntly
mor
e ce
ll ad
hesio
n an
d m
ore
spre
ad c
ells
on
CaT
iO3
surf
aces
Mos
t ce
lls a
dher
ed a
nd t
he h
ighe
st e
xpre
ssio
n of
alk
a-lin
e ph
osph
atas
e ar
ound
hyd
roxy
apat
ite, f
ollo
wed
by
Ca-
impl
ante
d sa
mpl
es. C
a-io
ns w
ere
sugg
este
d to
be
of g
reat
er im
port
ance
tha
n P-
ions
for
the
prim
ary
inte
ract
ion
with
med
ium
and
cel
ls.
Cal
cium
impl
anta
tion
had
no e
ffect
on
seed
ed c
ells
, bu
t in
crea
sed
corr
osio
n re
sist
ance
und
er s
tatio
nary
co
nditi
ons
Ca-
impl
anta
tion
decr
ease
d th
e ce
ll vi
abili
ty.
Ca-
and
P-im
plan
tatio
n im
prov
ed t
he c
orro
sion
res
is-
tanc
e bu
t di
d no
t af
fect
the
cel
ls.
Less
cel
ls ad
here
d to
Ca-
impl
ante
d su
rfac
es c
om-
pare
d to
all
othe
r. H
owev
er, m
ost
wel
l-spr
ead
cells
on
Ca-
and
K-im
plan
ted
surf
aces
.
M i
mpl
ante
d tit
aniu
mSt
udy
desi
gn:
Hum
an o
steo
blas
ts, 4
h in
cu-
batio
n; c
ell a
dhes
ion
and
mor
-ph
olog
y
Rab
bit
oste
obla
sts,
24
h in
cu-
batio
n; c
ell a
dhes
ion
and
mor
-ph
olog
y, a
lkal
ine
phos
phat
ase
activ
ity
Sim
ulat
ed b
ody
fluid
, hum
an
oste
obla
sts,
8 d
ays
incu
batio
n;
cell
viab
ility
ass
ay, a
lkal
ine
phos
phat
ase
activ
ity, c
ell m
or-
phol
ogy
Sim
ulat
ed b
ody
fluid
, hum
an
bone
der
ived
cel
ls, c
ell v
iabi
lity
assa
y, a
lkal
ine
phos
phat
ase
activ
ity, c
orro
sion
resi
stan
ce
MG
63 c
ell l
ine
(ost
eoca
rci-
nom
a), 4
h in
cuba
tion;
cel
l ad-
hesi
on a
nd s
prea
ding
in v
ivo
stud
ies o
f cal
ciu
Surf
aces
:
Hyd
roxy
apat
ite, t
rica
lciu
m-
phos
phat
e or
tita
nium
with
or
with
out
oute
r C
aTiO
3
Ca-
and
P-im
plan
ted
titan
ium
, hy
drox
yapa
tite
Ca-
impl
ante
d (1
.5 a
tom
ic%
) tit
aniu
m g
rade
2
Ca-
(10
.5 a
tom
ic%
) an
d P-
(2.
5 at
omic
%)
impl
ante
d tit
aniu
m
grad
e 2
Com
mer
cial
ly p
ure
titan
ium
im
plan
ted
with
Ca-
, K-,
and
Ar
(Sq
0.05
-0.0
8 µm
)
Tabl
e 1.
Cel
l and
R
efer
ence
s:
Cel
l stu
dies
:
Ergu
n et
al.
(200
7)14
3
Feng
et a
l. (2
004)
122
Kru
pa e
t al.
(200
1)14
4
Kru
pa e
t al.
(200
4)14
5
Kru
pa e
t al.
(200
5)14
6
Nay
ab e
t al.
(200
3)14
7
40
30
M
ajor
find
ings
:
Ca-
impl
ante
d su
rfac
es h
ad le
ss a
dher
ed b
ut m
ore
spre
ad c
ells
, with
hig
her
prol
ifera
tion
com
pare
d to
all
othe
r su
rfac
es. T
he o
ther
ion-
impl
ante
d su
rfac
es
wer
e si
mila
r to
con
trol
s. .
Cel
ls p
rese
nted
a m
ore
com
plex
mor
phol
ogy
(sim
ilar
to a
ctiv
e os
teob
last
s) o
n al
l Ca-
impl
ante
d su
rfac
es
com
pare
d to
con
trol
s. S
urfa
ces
impl
ante
d w
ith h
igh
dose
of C
a ha
d le
ss a
dher
ed b
ut m
ore
spre
ad c
ells
afte
r 2
h, h
owev
er, a
fter
24 h
mor
e ce
lls c
ompa
red
to
cont
rols
. Afte
r 24
h t
here
was
an
up-r
egul
atio
n of
a5
b1 in
tegr
in a
nd v
incu
lin p
ositi
ve a
dhes
ion
plaq
ues
with
an
incr
ease
in c
ell n
umbe
r, s
ize,
and
gra
nula
rity
on
Ca-
impl
ante
d su
rfac
es.
Ca-
impl
ante
d su
rfac
es p
rese
nted
cel
ls w
ith u
p-re
gula
ted
oste
opon
tin (
gene
act
ivat
ion)
, bon
e m
orph
ogen
etic
pro
tein
, and
bon
e si
alop
rote
in. N
o di
ffere
nces
bet
wee
n al
kalin
e ph
osph
atas
e an
d os
-te
onec
tin.
Sign
ifica
ntly
mor
e pr
olife
rativ
e an
d m
itotic
cel
ls, w
ith
a m
ore
rapi
d ce
ll cy
cle,
on
Ca-
impl
ante
d su
rfac
es.
Ca-
impl
ante
d su
rfac
es p
rese
nted
an
incr
ease
d nu
mbe
r of
cel
ls a
fter
4 da
ys. C
a-im
plan
tatio
n of
mod
erat
ely
roug
h su
rfac
es in
crea
sed
the
oste
obla
stic
gen
e-ex
pres
sion
of a
lkal
ine
phos
phat
ase,
ost
eopo
ntin
, and
os
teon
ectin
.
Stud
y de
sign
:
Har
vest
ed h
uman
bon
e ce
lls, 4
h
or 4
8/72
/96
h in
cuba
tion;
ce
ll ad
hesi
on, m
orph
olog
y, a
nd
grow
th
MG
63 c
ells
, 4 a
nd 2
4 h
incu
ba-
tion;
cel
l adh
esio
n, a
naly
sis o
f in
tegr
in a
dhes
ion
mol
ecul
es,
vinc
ulin
adh
esio
n pl
aque
s
MG
63 c
ells
, 24
h an
d 6
days
in
cuba
tion;
pro
tein
exp
ress
ion,
os
teop
ontin
gen
e ex
pres
sion
MG
63 c
ells
, 24,
48,
and
72
h in
cuba
tion;
Ki-6
7 ex
pres
sion
, nu
mbe
r of
mito
tic c
ells
Apa
tite
form
atio
n, M
CT
3T3-
E1 c
ells
; cel
l via
bilit
y (m
ito-
chon
dria
l fun
ctio
n), g
ene
ex-
pres
sion
Surf
aces
:
Com
mer
cial
ly p
ure
titan
ium
im
plan
ted
with
Ca-
, K-,
and
Ar
Com
mer
cial
ly p
ure
titan
ium
w
ith lo
w m
ediu
m a
nd h
igh
dose
s of
impl
ante
d C
a
Com
mer
cial
ly p
ure
titan
ium
, C
a-im
plan
tatio
n
Com
mer
cial
ly p
ure
titan
ium
, C
a-im
plan
tatio
n
Smoo
th t
urne
d co
mm
erci
ally
pu
re t
itani
um a
nd ,m
oder
atel
y ro
ugh
hydr
oxya
patit
e-bl
aste
d su
rfac
es, C
a-im
plan
tatio
n
Ref
eren
ces:
Nay
ab e
t al.
(200
4)12
4
Nay
ab e
t al.
(200
5)11
8
Nay
ab e
t al.
(200
7a)1
23
Nay
ab e
t al.
(200
7b)1
48
Park
et a
l. (2
008)
149
41
31
Maj
or fi
ndin
gs:
Sign
ifica
ntly
mor
e bo
ne in
con
tact
to
Ca-
inco
rpor
ated
su
rfac
es w
hen
plac
ed in
tib
ia.
Ca-
impl
ante
d su
rfac
es h
ad s
igni
fican
tly h
ighe
r m
iner
-al
izat
ion
inde
x an
d os
seoi
nteg
ratio
n in
dex.
Mor
e ne
wly
form
ed b
one
and
bone
in c
onta
ct w
ith
Ca-
impl
ante
d su
rfac
es.
Ca-
impl
ante
d an
d hy
drox
yapa
tite
surf
aces
pre
sent
ed
rapi
d bo
ne fo
rmat
ion.
Gre
ater
bon
e vo
lum
es a
roun
d C
a-im
plan
ted
surf
aces
tha
n hy
drox
yapa
tite
at a
ll tim
e-po
ints
. Sig
nific
antly
less
dec
reas
e in
bon
e vo
lum
e fr
om
4 to
8 w
eeks
aro
und
Ca-
impl
ants
com
pare
d to
hy-
drox
yapa
tite.
The
am
ount
of b
one
incr
ease
d ar
ound
pu
re t
itani
um s
urfa
ces
chro
nolo
gica
lly.
The
thi
ckne
ss o
f CaT
iO3
affe
cted
the
apa
tite
form
a-tio
n. T
here
wer
e no
diff
eren
ces
rega
rdin
g th
e so
ft-tis
sue,
how
ever
, Ca-
impl
ants
stim
ulat
ed b
one
form
a-tio
n an
d pr
esen
ted
bone
dir
ectly
on
the
surf
ace.
Stud
y de
sign
:
Rab
bits
, 12
wee
ks; h
isto
logy
(b
one
in c
onta
ct, b
one
area
)
Rab
bits
, 12
wee
ks; h
isto
logy
Rat
, 2, 8
, and
18
days
; his
tol-
ogy
Rab
bits
, 2, 4
, and
, 8 w
eeks
; re
mov
al t
orqu
e, b
one
volu
me
Rat
, 7 a
nd 2
8 da
ys; s
oft
tissu
e an
d bo
ne r
espo
nse
Surf
aces
:
Smoo
th a
nodi
zed
Ca-
inco
rpor
ated
, and
min
imal
ly
roug
h an
odiz
ed a
nd b
last
ed
surf
aces
.
Tur
ned
or la
ser
etch
ed m
i-cr
oarc
oxi
date
d an
d C
a-im
plan
ted
Tita
nium
gra
de 1
and
slig
htly
ro
ughe
r C
a-im
plan
ted
surf
aces
Tita
nium
gra
de 2
, hyd
roxy
apa-
tite,
and
Ca-
impl
ante
d su
rafc
es
Tita
nium
gra
de 2
, mag
netr
on
sput
tere
d C
aTiO
3
Ref
eren
ces:
In v
ivo
Froj
d et
al.
(200
8)11
9
Guo
et a
l. (2
010)
150
Han
awa
et a
l. (1
997a
)151
Ichi
kaw
a et
al.
(200
0)15
2
Oht
su e
t al.
(200
7c)1
53
42
32
Maj
or fi
ndin
gs:
Incr
ease
d ap
atite
form
atio
n, in
crea
sed
cell
viab
ility
af
ter
4 da
ys, a
nd s
igni
fican
tly h
ighe
r re
mov
al t
orqu
e an
d m
ore
bone
in c
onta
ct w
ith C
a-im
plan
ts.
Mor
e bo
ne in
con
tact
with
Ca-
impl
ante
d su
rfac
es. N
o di
ffere
nces
reg
ardi
ng b
one
area
.
P-im
plan
ted
surf
aces
had
the
hig
hest
rem
oval
tor
que
valu
es a
nd b
one
in c
onta
ct, f
ollo
wed
by
the
Ca-
impl
ants
.
Hig
her
min
eral
app
ositi
on r
ate
afte
r 2
wee
ks, i
nitia
lly
enha
nced
ost
eobl
ast
adhe
sion
and
hig
her
met
abol
ic
activ
ity, a
s w
ell a
s, m
ore
exte
nsiv
e bo
ne in
con
tact
to
anod
ized
Ca-
and
P-im
plan
ted
impl
ants
. Ano
dize
d C
a-
and
P-im
plan
ted
impl
ants
acc
eler
ated
the
pri
mar
y os
-te
ogen
ic r
espo
nse.
Sign
ifica
ntly
hig
her
rem
oval
tor
que,
mor
e bo
ne in
co
ntac
t, an
d gr
eate
r bo
ne a
rea
arou
nd C
a-im
plan
ted
surf
aces
.
Sign
ifica
ntly
enh
ance
d os
seoi
nteg
ratio
n in
the
max
il-la
e.
Stud
y de
sign
:
Apa
tite
form
atio
n, M
CT
3T-E
1 ce
lls (1
and
4 d
ays)
, rab
bit
(6
wee
ks);
cell
viab
ility
, rem
oval
to
rque
, bon
e in
con
tact
Rab
bit,
6 w
eeks
; his
tolo
gy
(bon
e in
con
tact
, bon
e ar
ea)
Rab
bits
, 6 w
eeks
; rem
oval
to
rque
, bon
e in
con
tact
Hum
an o
steo
blas
ts, r
abbi
ts (2
an
d 4
wee
ks);
cell
mor
phol
-og
y, a
dhes
ion,
pro
lifer
atio
n,
and
met
abol
ic a
ctiv
ity, h
isto
-m
orph
omet
ry
Rab
bits
, 6 w
eeks
; rem
oval
to
rque
, bon
e in
con
tact
, and
bo
ne a
rea
with
in t
he t
hree
fir
st t
hrea
ds
Hum
an, 2
mon
ths;
his
tolo
gy
Surf
aces
:
Ti6
Al4
V, C
a-im
plan
tatio
n w
ith
diffe
rent
con
cent
ratio
ns (
all
had
smoo
th s
urfa
ces)
Blas
ted
and
acid
-etc
hed,
bl
aste
d ac
id-e
tche
d an
d C
a-im
plan
ted
roug
h su
rfac
es
Tur
ned,
aci
d et
ched
and
P-
impl
ante
d, g
rit-
blas
ted,
gri
t-bl
aste
d an
d ac
id-e
tche
d, a
no-
dize
d w
ith C
a-in
corp
orat
ion
Ano
dize
d w
ith in
corp
orat
ed
Ca-
and
P-io
ns, a
cid-
etch
ed
Mod
erat
ely
roug
h hy
drox
yapa
-tit
e an
d C
a-im
plan
ted
(40
atom
ic%
) hy
drox
yapa
tite
Tur
ned
(sm
ooth
) an
d an
o-di
zed
Ca-
and
P-in
corp
orat
ed
(min
imal
ly r
ough
) su
rfac
es
Ref
eren
ces:
Park
et a
l. (2
007b
)154
Park
et a
l. (2
009b
)155
Park
et a
l. (2
009a
)94
Rav
anet
ti et
al.
(201
0)15
6
Suh
et a
l. (2
007)
157
Shib
li et
al.
(200
7)15
8
43
33
Maj
or fi
ndin
gs:
Ca-
inco
rpor
ated
impl
ants
pre
sent
ed s
igni
fican
tly
high
er r
emov
al t
orqu
e co
mpa
red
to P
-impl
ante
d an
d tu
rned
impl
ants
, as
wel
l as,
sig
nific
antly
mor
e bo
ne in
co
ntac
t co
mpa
red
to t
urne
d im
plan
ts. A
ll an
odiz
ed
and
ion-
impl
ante
d im
plan
ts h
ad s
igni
fican
tly m
ore
bone
in c
onta
ct c
ompa
red
to t
urne
d im
plan
ts. C
a-
and
P-im
plan
tatio
n re
sulte
d in
qua
litat
ivel
y m
ore
min
-er
aliz
ed b
one.
Ano
dize
d an
d C
a-in
corp
orat
ed im
plan
ts h
ad s
igni
fi-ca
ntly
hig
her
rem
oval
toq
ue v
alue
s, m
ore
bone
in
cont
act,
mor
e ho
mog
eneo
usly
min
eral
ized
bon
e, a
s w
ell a
s ne
w b
one
spre
ad a
long
the
impl
ant
surf
ace.
Ca-
impl
ants
had
sig
nific
antly
hig
her
reso
nanc
e fr
e-qu
ency
and
rem
oval
tor
que
valu
es.
Stud
y de
sign
:
Rab
bit,
6 w
eeks
; rem
oval
to
rque
, bon
e in
con
tact
Rab
bit,
6 w
eeks
; rem
oval
to
rque
Rab
bits
, 6 w
eeks
; res
onan
ce
freq
uenc
y an
alys
is, r
emov
al
torq
ue
Surf
aces
:
Min
imal
ly r
ough
tur
ned,
ano
-di
zed
and
S-im
plan
ted
(1
atom
ic%
), an
odiz
ed a
nd P
-im
plan
ted
(8 a
tom
ic%
), an
d an
odiz
ed a
nd C
a-im
plan
ted
(11
atom
ic%
) sur
face
s.
Min
imal
ly r
ough
tur
ned
and
anod
ized
and
Ca-
inco
rpor
ated
(1
1 at
omic
%)
titan
ium
gra
de 1
Min
imal
ly r
ough
tur
ned
and
anod
ized
and
Ca-
inco
rpor
ated
(7
.4 a
tom
ic%
) tita
nium
Ref
eren
ces:
Sul (
2003
)159
Sul e
t al.
(200
2a)1
21
Sul e
t al.
(200
4)12
0
44
Surface properties of titanium implants aimed for soft tissue integrationMacro designTo secure a tight soft-tissue sealing the design of the abutment or the crossing from the implant may be modified. Platform switching160 or contoured abutments, with a shape similar to an hourglass, are examples of available commercial products. The modifications are made to allegedly enhance the aesthetics, prevent the progression of biofilms associated with pathology, and avoid crestal bone loss.
Fibroblasts and epithelial cells seem to be influenced by surface orientations and grooves. Circular grooves may inhibit epithelial downgrowth, and grooved surfaces have been suggested to counteract encapsulation of implants161.
Micro topographySmooth surfaces are most commonly used on the abutment part of dental implants62. However, theoretically some surface structure may be needed in order to have proper soft tissue attachment, which may further on be of importance for the defence against bacterial biofilm progression44.
In vitro studies indicate advantages with minimal surface roughness for the adhesion and differentiation of fibroblasts and epithelial cells162-165. The literature of in vivo studies presents unclear results on the importance of surface roughness. Oxidized and acid-etched surfaces have shown longer connective tissue seal and less epithelial down-growth compared to machined/turned surfaces after eight weeks in human39. Whether this was due to surface chemistry, topography, or physics could not be concluded. Furthermore, micro-arc oxidation of Ti-6Al-4V has revealed significantly higher soft tissue attachment compared to machined/turned surfaces after eight weeks in goats166. Surface texture was shown of importance for the collagen fibre orientation and the connective tissue-implant interface in human, when machined/turned, oxidized, and acid-etched surfaces was compared42. On the other hand, no differences in soft-tissue response was found between rough sandblasted, fine sandblasted, and polished commercially pure titanium surfaces, with similar lengths of direct connective tissue contact and stable tissue seal, after three months in dogs40. Abrahamsson et al. (2002) indicated no differences between turned and dual acid-etched surfaces considered
45
“smooth” and “rough” when placed in dogs167; however, they were both classed as smooth according to Albrektsson and Wennerberg (2004) with an average height deviation (Sa) of 0.22 µm and 0.45 µm. Wennerberg et al. (2003), compared as-received (Sa 0.26 µm), turned (Sa 0.40 µm), and Al2O3 blasted surfaces with different topography (Sa 0.76 µm, 1.00µm, 1.87 µm) in human volunteers, and found no differences regarding clinical or histological quantitative or qualitative parameters between the surfaces were reported41. However, removal torque of oxidized abutments decreased with 20 percent compared to turned surfaces after at least 30 days in human mucosa168. The importance of microtopography for the soft tissue response is, thereby, uncertain.
nano topographyEtched surfaces and anodized surfaces, both characterized of nanotopographical outer surfaces, have been used as abutment surfaces. Sol-gel derived nanoporous TiO2 coatings, established a direct contact with rat soft-tissue in contrast to uncoated titanium used as control169, 170. There are also suggestions of less capsule-like connective tissue surrounding the former surface170, 171. Table 2 presents the studies reported in the literature on sol-gel derived nanoporous TiO2 surfaces. In addition, study III in the present thesis found some advantages for nanoporous TiO2 surfaces compared to turned surfaces in an experimental study in human41.
chemistryRegarding surface chemistry, most studies indicate no differences in the soft-tissue response either in humans172, or in animals173-
175 for materials used as oral implant abutments or for soft tissue adaptation, e.g. titanium/titania and zirconia. However, lower grade of inflammatory response has been found to ziconia as compared to titanium176. Similar findings with generally no differences in tissue response, further apply to Al2O3 in comparison with titania in human177 and in animals178. For hydroxyapatite coated surfaces, there are findings of similar179 or enhanced soft-tissue/mucosa adaptation to such surfaces compared with titanium oxide surafaces; when Ti6Al4V were coated with hydroxyapatite together with bioglass, and compared to machined/turned Ti6Al4V there were a thinner capsule formed together with appreciated improved tissue quality180.
46
36
Maj
or fi
ndin
gs:
Stro
nger
adh
esio
n, g
reat
er c
ell a
ctiv
ity/m
ore
effic
ient
gr
owth
, and
mor
e fla
tten
ed a
nd e
long
ated
cel
ls on
na
nopo
rous
TiO
2 su
rfac
es
Nan
opor
ous
TiO
2 su
rfac
es w
ere
firm
ly a
ttac
hed
to a
th
ick
laye
r of
org
aniz
ed c
onne
ctiv
e tis
sue
prop
er w
ith
mat
ure
fibro
blas
ts, a
nd d
iffic
ult
to r
emov
e. D
irec
tly
atta
ched
col
lage
n fib
res
wer
e oc
casi
onal
ly s
een;
num
-be
r of
fibr
es in
crea
sed
from
2 t
o 7
days
. The
re w
ere
no c
olla
gen
fibre
s an
d on
ly fe
w fi
brob
last
s on
con
trol
s p
ecim
ens.
The
re w
ere
no d
iffer
ence
bet
wee
n m
oder
atel
y ro
ugh
rutil
e an
d sm
ooth
ana
tase
sur
face
s
Day
3: i
mm
atur
e ce
lls a
nd t
issu
e in
con
tact
with
bot
h im
plan
ts.
Day
7: n
on-in
flam
ed c
onne
ctiv
e tis
sues
clo
se t
o th
e im
plan
t s.
Day
14:
mat
ure,
den
se c
onne
ctiv
e tis
sue
caps
ule
with
ce
lls in
con
tact
with
bot
h su
rfac
es.
poro
us T
iO2 s
urfa
ces.
Stud
y de
sign
:
Hum
an g
ingi
val f
ibro
blas
ts;
adhe
sion
, pro
lifer
atio
n
Rat
s (3
and
12
days
); pu
ll-ou
t fo
rce,
hist
olog
ical
eva
luat
ion
Rat
(3,
7, a
nd 1
4 da
ys);
hist
ol-
ogy
(ligh
t m
icro
scop
y, S
EM,
TEM
)
d in
viv
o st
udie
s on
nan
o
Surf
aces
:
Ti g
rade
2, t
urne
d vs
. na
nopo
rous
TiO
2 (R
a 0.1
5 µm
)
Ti g
rade
2, t
urne
d vs
. na
nopo
rous
TiO
2 (R
a 0.1
5 µm
)
Ti g
rade
2, n
anop
orou
s T
iO2
+/-
CO
2 la
ser
trea
tmen
t (a
na-
tase
, iso
trop
ic, R
a 0.2
6 µm
) vs
. la
ser
trea
ted
(rut
ile, a
niso
t-ro
pic,
Ra 1
.48
µm)
Tabl
e 2.
In v
itro
an
Ref
eren
ces:
In v
itro
Mer
etoj
a et
al.
(201
0)18
1
In v
ivo
Are
va e
t al.
(200
4)16
9
Ros
si et
al.
(200
7)18
2
37
Maj
or fi
ndin
gs:
Non
-cap
sule
like
con
nect
ive
tissu
e pr
oper
adj
acen
t to
na
nopo
rous
TiO
2 su
rfac
es a
fter
11 d
ays,
whi
le c
apsu
le
like
tissu
e w
ere
loca
ted
at a
dis
tanc
e fr
om t
urne
d su
r-fa
ces.
Thi
n ca
psul
e lik
e co
nnec
tive
tissu
e w
ere
firm
ly
atta
ched
to
nano
poro
us T
iO2
surf
aces
afte
r 90
day
s,
no c
onta
ct a
t co
ntro
ls. H
ighe
r fo
rce
need
ed fo
r na
nopo
rous
TiO
2 su
rfac
es, b
ut n
o si
gnifi
cant
diff
er-
ence
with
pul
l out
tes
ts. T
est
impl
ants
wer
e co
vere
d w
ith c
onne
ctiv
e tis
sue
prop
er b
ut n
ot c
ontr
ols.
Sig
-ni
fican
tly e
nhan
ced
quan
titat
ive,
qua
litat
ive,
and
inte
r-fa
ce a
naly
ses
for
nano
poro
us T
iO2
surf
aces
, with
m
ore,
thi
cker
, mat
ure
cells
con
tain
ing
mor
e an
d di
f-fe
rent
org
anel
les
in t
he c
ytop
lasm
; the
re w
ere
inti-
mat
e co
ntac
t be
twee
n th
e pl
asm
a m
embr
ane
and
the
oxid
e la
yer.
Sign
ifica
ntly
sho
rter
dis
tanc
e fr
om g
ingi
val m
argi
n to
al
veol
ar b
one
cres
t ar
ound
nan
opor
ous
TiO
2 su
r-fa
ces.
No
othe
r si
gnifi
cant
diff
eren
ces,
how
ever
, a
proc
laim
ed t
ende
ncy
of le
ss in
flam
mat
ory
resp
onse
, le
ss d
ense
and
cap
sule
-like
con
nect
ive
tissu
e, a
nd
firm
er s
oft
tissu
e in
tegr
atio
n fo
r na
nopo
rous
TiO
2 su
rfac
es c
ompa
red
to u
nmod
ified
sur
face
s.
The
re w
as s
igni
fican
tly le
ss b
one-
loss
aro
und
stab
le
impl
ants
, sig
nific
antly
mor
e or
al m
ucos
a in
con
tact
, an
d a
clin
ical
impr
essi
on o
f hea
lthie
r an
d fir
mer
sof
t tis
sue
seal
ing
of n
anop
orou
s T
iO2 su
rfac
es. N
o ot
her
sign
ifica
nt d
iffer
ence
s.
Stud
y de
sign
:
Rat
(3,
11,
and
90
days
or
14
and
21 d
ays)
; pul
l-out
forc
e,
hist
olog
y
Dog
s (8
wee
ks);
X-r
ay, h
isto
l-og
y/hi
stom
orph
omet
ry
Hum
an (
14 ±
2 w
eeks
); cl
ini-
cal,
and
hist
olog
ical
eva
luat
ion
usin
g lig
ht m
icro
scop
y, S
EM,
and
TEM
Surf
aces
:
Ti g
rade
2, t
urne
d vs
. na
nopo
rous
TiO
2 (R
a 0.1
5 µm
)
ITI®
(w
ithou
t bl
astin
g an
d ac
id
etch
ing)
impl
ants
for
bone
in-
tegr
atio
n, w
ith im
plan
t ne
cks:
un
mod
ified
vs.
nan
opor
ous
TiO
2 (R
a 0.2
6 µm
)
Ti g
rade
4, t
urne
d (S
a 0.1
7 µm
)vs
. nan
opor
ous
TiO
2 (S
a 0.
16
µm)
Ref
eren
ces:
Pald
an e
t al.
(200
8)17
0
Ros
si et
al.
(200
8)17
1
Wen
nerb
erg
et a
l. (2
009)
183
47
37
Maj
or fi
ndin
gs:
Non
-cap
sule
like
con
nect
ive
tissu
e pr
oper
adj
acen
t to
na
nopo
rous
TiO
2 su
rfac
es a
fter
11 d
ays,
whi
le c
apsu
le
like
tissu
e w
ere
loca
ted
at a
dis
tanc
e fr
om t
urne
d su
r-fa
ces.
Thi
n ca
psul
e lik
e co
nnec
tive
tissu
e w
ere
firm
ly
atta
ched
to
nano
poro
us T
iO2
surf
aces
afte
r 90
day
s,
no c
onta
ct a
t co
ntro
ls. H
ighe
r fo
rce
need
ed fo
r na
nopo
rous
TiO
2 su
rfac
es, b
ut n
o si
gnifi
cant
diff
er-
ence
with
pul
l out
tes
ts. T
est
impl
ants
wer
e co
vere
d w
ith c
onne
ctiv
e tis
sue
prop
er b
ut n
ot c
ontr
ols.
Sig
-ni
fican
tly e
nhan
ced
quan
titat
ive,
qua
litat
ive,
and
inte
r-fa
ce a
naly
ses
for
nano
poro
us T
iO2
surf
aces
, with
m
ore,
thi
cker
, mat
ure
cells
con
tain
ing
mor
e an
d di
f-fe
rent
org
anel
les
in t
he c
ytop
lasm
; the
re w
ere
inti-
mat
e co
ntac
t be
twee
n th
e pl
asm
a m
embr
ane
and
the
oxid
e la
yer.
Sign
ifica
ntly
sho
rter
dis
tanc
e fr
om g
ingi
val m
argi
n to
al
veol
ar b
one
cres
t ar
ound
nan
opor
ous
TiO
2 su
r-fa
ces.
No
othe
r si
gnifi
cant
diff
eren
ces,
how
ever
, a
proc
laim
ed t
ende
ncy
of le
ss in
flam
mat
ory
resp
onse
, le
ss d
ense
and
cap
sule
-like
con
nect
ive
tissu
e, a
nd
firm
er s
oft
tissu
e in
tegr
atio
n fo
r na
nopo
rous
TiO
2 su
rfac
es c
ompa
red
to u
nmod
ified
sur
face
s.
The
re w
as s
igni
fican
tly le
ss b
one-
loss
aro
und
stab
le
impl
ants
, sig
nific
antly
mor
e or
al m
ucos
a in
con
tact
, an
d a
clin
ical
impr
essi
on o
f hea
lthie
r an
d fir
mer
sof
t tis
sue
seal
ing
of n
anop
orou
s T
iO2 su
rfac
es. N
o ot
her
sign
ifica
nt d
iffer
ence
s.
Stud
y de
sign
:
Rat
(3,
11,
and
90
days
or
14
and
21 d
ays)
; pul
l-out
forc
e,
hist
olog
y
Dog
s (8
wee
ks);
X-r
ay, h
isto
l-og
y/hi
stom
orph
omet
ry
Hum
an (
14 ±
2 w
eeks
); cl
ini-
cal,
and
hist
olog
ical
eva
luat
ion
usin
g lig
ht m
icro
scop
y, S
EM,
and
TEM
Surf
aces
:
Ti g
rade
2, t
urne
d vs
. na
nopo
rous
TiO
2 (R
a 0.1
5 µm
)
ITI®
(w
ithou
t bl
astin
g an
d ac
id
etch
ing)
impl
ants
for
bone
in-
tegr
atio
n, w
ith im
plan
t ne
cks:
un
mod
ified
vs.
nan
opor
ous
TiO
2 (R
a 0.2
6 µm
)
Ti g
rade
4, t
urne
d (S
a 0.1
7 µm
)vs
. nan
opor
ous
TiO
2 (S
a 0.
16
µm)
Ref
eren
ces:
Pald
an e
t al.
(200
8)17
0
Ros
si et
al.
(200
8)17
1
Wen
nerb
erg
et a
l. (2
009)
183
48
Complications with titanium implant treatmentsNature of the complicationsLack of tissue sealing may be considered the main complication with titanium implants. Microbial biofilms on the implant surface may during certain conditions provoke an inflammatory reaction in the soft-tissue and the condition is then referred to as peri-mucositis. Peri-mucositis can be defined as: a reversible inflammation of the soft tissues around implants in function with no occurring bone resorption184. When bone resorption occurs alongside bleeding on probing at the same site, the condition has been termed peri-implantitis185. However, at the same time bleeding on probing and pus may occur without ongoing bone resorption186. There are indications of peri-implantitis being more aggressive considering the grade of inflammation in the surrounding tissues and progression of bone loss compared to periodontitis187. Possibly the lacking of periodontal ligaments or the suggested lack of vessels in close vicinity to the bone-implant interface contributes to the scenario. Furthermore, the bacterial phenotype may be of more aggressive nature in biofilms associated with pathological conditions. It is not known whether certain implant surfaces affect the bacteria in ways that could impact the pathogenesis of the established biofilm.
In addition, overload and parafunction on the supraconstructions of dental implants have been suggested as cause for marginal bone resorption188, 189. However, as with peri-implantitis, there are clinical studies not validating a correlation190, 191. If bone resorption occurs around dental implants, for whatever reason, the biological environ-ment might alter for surfaces supposed to be integrated in bone, when they are exposed to the oral cavity and its extensive microbiota.
Another complication that has been addressed as a risk, mainly for rough plasma sprayed surfaces, has been ion leakage192. However, for example, blasting with variously sized particles resulting in moderately rough and rough surfaces has not indicated an increased risk for ion release; although, slightly more ions were detected up to a distance of 400 µm from the roughest surface193.
ExtentComplications with titanium implants within the dental field are reported to various extents. Evaluations of implant treatment are
49
commonly separated into implant survival and implant success. Success criteria were suggested by Albrektsson et al. (1986) and included absence of mobility, pain, and neuropathy, and no more than 1 mm bone loss during the first year and thereafter no more than 0.2 mm per year194. The definition of success versus survival as well as clear definition of peri-implantitis does, however, differ, and the prevalence or incidence is therefore inexplicit. Furthermore, one may consider the extent on an implant-level and also on a patient-level195. Fransson (2009) has compiled what is reported on the “prevalence, extent and, severity of peri-implantitis” and discussed this in his thesis. One of the studies from the same thesis, reported that 28 percent out of 662 participants had implants with progressive bone loss, and on the implant level the corresponding number was 12.4 percent out of 3413 implants195. A study by Roos-Jansåker el al. (2006) presents prevalence of peri-implantitis for 16 percent of the patients evaluated and 6.6 percent of implants evaluated196. However, when the data presented by Roos-Jansåker et al. (2006) was re-calculated using another definition of peri-implantatis and presented in a review by Zitzmann and Berglundh (2008) the prevalence of peri-implantatis on a subject level was more than 56 percent and 25 percent on an implant level197. This defines the conflicting or problematic situation with diverse definitions of peri-implantits. It must be pointed out that peri-implantitis as such, at least in a primary form, remains controversial198, 199. Marginal bone loss may be explained by the healing/adaptation theory200 with the entity peri-implantitis possibly existing in a secondary form when bone resorption has occurred.
However, irrespective if peri-implantitis occur in a primary or a secondary form we need to treat the condition by cleaning the surface. From this follows that we may need to develop particular surfaces that are less prone to extensive biofilm formation, as well as, easier to clean compared to many surfaces today.
Biofilms and their accumulation on titanium surfacesAll biomaterials are almost instantly covered with a conditioning film when inserted into mammalians201. For exposed surfaces in the oral environment, short after the formation of a saliva pellicle primary colonizer will firmly adhere to the surface mainly through
50
receptors presented in the pellicle202-204; titanium surfaces exposed to the oral cavity are colonized with bacteria, without there being an association with pathological conditions205. The transition of biofilms towards having a pathogenic potential may be explained by an ecological hypothesis as mainly discussed regarding dental caries206, 207. The conditioning film affects bacteria differently depending on, for example, their hydrophobicity. In what extent the hydrophobicity of a surface is of importance for the bacterial adhesion is debated; nevertheless, it may have an impact202. Most microorganisms, furthermore, exhibit a negative surface charge at physiological pH, and would thereby be attracted to positively charged surfaces. Acidic proteins may increase the net negative charge of the surface, and, thereby, increase the potential energy barrier between the bacteria and the surface, which prevents the bacterial approach and attachment; basic proteins would create a positive net charge that together with van der Waals forces would attract bacteria and facilitate the adhesion. When absorption of non-polar proteins, attachment of bacteria occurs through hydrophobic interactions201. Furthermore, in the presence of saliva various bacterial species may express various genes encoding surface proteins; as an example, streptococci may bind to agglutinin in saliva and enhance the expression of sspA/B (surface protein that bind to salivary agglutinin). Oral bacteria that have the possibility to bind to a receptor and thereafter utilizing it as a nutrient have diverse mechanisms for attachment and are optimal initial adherers. Initial adherers may bind to other surface receptors as well as other bacteria. Bacteria with similar adhesins (may be different species) compete for binding to a receptor on a surface208
Colonization of oral implantsThe microbiota surrounding titanium oral implants and teeth have been suggested to be of similar character, foremost during healthy conditions209, 210. Furthermore, the proceedings associated with biofilm formation on implant surfaces, as well as the bacterial consortium constituting the biofilm (analyzed using checkerboard DNA-DNA hybridization, cultural techniques, real-time polymerase chain reaction, or in another case phase contrast microscopy) have been suggested alike as when established around teeth from
51
experimental studies in human211, 212. Streptococci and actinomyces are early colonizers of teeth213, 214, and titanium surfaces are, as well, during the first hours mainly colonized by streptococci205, 215. Streptococci are the main genus of oral bacteria showing extensive intra- as well as intergeneric coaggregation213. Other early adherers do not co-aggregate with many other early adheres and generally not with late adherers either. Fusobacterium nucleatum is the most numerous gram-negative bacteria within dental biofilms. They have been suggested a position at the border zone, linking early and late adherers, which may explain their high frequency in oral biofilms.213 However, the initial adherers enable adhesion of other bacteria, resulting in coaggregation and biofilm formation216. When a multispecies consortium is established in a biofilm, synergetic effects and various interactions occur214, 216, 217.
In cases with periodontitis, Treponema denticola and Porphyromonas gingivalis are considered pathogenic bacteria, and these have been suggested to be involved in cases with peri-implantitis as well218. Significantly higher mean counts of P. gingivalis, T. denticola, and Tannerella forsythia were observed in biofilms around implants with peri-implantitis, both supra- and subgingivally, as compared to around healthy implants; host-compatible beneficial microbial complexes were also reduced. In general, the microbiota associated with peri-implantitis was comprised of more periodontal pathogenic bacterial species, including the supragingival biofilm219. A significant relationship between periimplant probing depth and the total anaerobic cultivable microbiota as well as the frequency of detection of P. gingivalis has also been detected44. The suggested pathogens probably establishes in the biofilm as late adherers when they lack good adhesion properties.
Microorganisms in a biofilm are encased in a primarily bacterial derived matrix, the extracellular polymeric substance, composed of polysaccharides, nucleic acids, and proteins220. However, once a multiphenotypic microbial biofilm has been established, the structure and organization may change significantly214.
Theoretical models of bacterial-biomaterial interactionsTheories for bacterial attachment represent a rather complex concept. Partly due to the fact that a pellicle or conditioning film
52
always will be present at a solid surface installed in the body and that bacteria are living cells that may communicate and undergo conformation changes, for example, when sensing a surface202, 221. The DLVO (Derjaguin-Landau-Vermek-Overbeek) theory was one of the original ones; the result is the net interaction between generally attractive van der Waals interactions and repulsive (because of a mostly negatively charged substratum) Coulomb interactions derived from the electrical double layer between the cell and the substratum. Another theory is the thermodynamic one, in which the forces involved are gathered and considered as free energy; adhesion would be favoured and occur spontaneously if the Gibbs free energy would be negative. The mentioned theories have further been gathered and modified into the extended DLVO theory, which involves the Gibbs free energy of the van der Waals interactions, the double-layer/electrostatic interactions, and also acid-base interactions (correlated to hydrophobicity/hydrophilicity)204.
The initial bacterial attachment to solid surfaces may also be presented in two steps202, with the first corresponding to the extended DLVO theory. First, bacteria are positioned at a distance to the surface that enables attraction via van der Waals forces (interactions between permanent or induced dipoles), electrostatic forces (depending on the bacteria and surface charges; may be attractive or repulsive), and hydrophobic interactions (due to water molecules avoiding non-polar components)222. Once the initial forces, stated above, have brought the bacteria in close vicinity to the surface, hydrophobic interactions as well as covalent and hydrogen bonding are retaining the bacteria on the surface. This stage may also involve an electron exchange between the bacteria and the surface223, 224. Bacteria could both donate or accept electrons from the surface, and when they donate electrons they may adhere more firmly; titanium may due to its native oxide layer, not be considered a conductive material; however, it may still be considered semi-conductive when free electrons are possible on the surfaces224. Secondly, bacteria may attach to the surface via specific adhesins (ligands, pili, or fimbriae) that may complex with the surface, or via exo-polysaccharides. Bacteria may due to their complex surface structure (with, for example, flagellae, fimbriae, pili, glycoproteins, carbohydrates, teichoic acids) have regions on the surface that are hydrophobic
53
and other that are hydrophilic; furthermore, the surface charge may differ between different region, although, the net surface charge is generally negative202.
Impact of surface characteristics on bacterial adhesion and biofilm formationThe bacterial adhesion, as well as the tissue response, is most probably influenced by the surface characteristics of the implants 225. The first colonization of a surface is of importance for the implant outcome. Gristina coined the term “the race for the surface” in 1987 226, representing the importance of tissue cells to take possession of a tissue integrated implant surface prior to bacteria. A surface should, for that reason, stimulate tissue-cells, but not bacteria and their adhesion. Although, being eukaryotic and prokaryotic, both are living cells and possibly stimulated by certain similar stimuli.
The present thesis has not focused on antibacterial surface properties, being for example silver-ion deposition227, 228, ultraviolett irradiation of TiO2-coated surfaces229 or bonding of antibiotics to titanium surfaces230. In addition, albumin coated titanium have reduced bacterial attachment in vitro compared to untreated titanium231.
With the earlier mentioned modifications of implant surfaces, the physico-chemical properties of the surfaces are being altered. Surface orientation may be of importance and bacteria have been found to form larger aggregates on isotropic micro/nano-oriented gold surfaces, but hindered on anisotropic surfaces with defined orientation232. Furthermore, in vitro and in vivo studies have indicated that bacterial adhesion increases with surface roughness and surface free energy233, 234. Regarding surface roughness in height, an average height deviation (Sa or Ra) of 0.2 µm has been suggested a threshold below that decreased surface roughness does not affect the bacterial adhesion235. However, studies in humans did not reveal differences in plaque accumulation or number of inflammatory cells to minimally and moderately rough blasted surfaces positioned in the oral mucosa, when compared to smooth, turned surfaces41, 236, whilst other studies in humans, yet again, indicated bacterial adhesion and biofilm formation to increase with increased roughness, for smooth and minimally rough titanium surfaces237, 238. Having said
54
this, dual acid-etched surfaces on abutments placed in humans for one year showed increased plaque accumulation, but no significant differences regarding bleeding on probing or histological analyses compared to turned surfaces239. Animal studies on ligature induced peri-implantitis showed progression to a greater extent for rough, sandblasted acid etched implants, compared to smooth, polished implants240, and, in another study, for moderately rough, anodized implants, compared to rough, sandblasted acid etched implants241. The correlation between the progression of induced peri-implantitis and the clinical corresponding situation is uncertain and there are five year retrospective studies on 593 participants presenting 94 percent survival rates for both smooth (turned) and moderately rough anodically oxidized implants242. Furthermore, there is a clinical study indicating insignificant differences between minimally rough and rough surfaces regarding crestal bone alteration after one year of loading; however, in the same study crater formed defects was found surrounding some of the implants with rough surfaces243. As a summary, the impact of surface roughness in height on complications remains uncertain.
Nanofeatured surfaces have been found both to decrease244, 245 and increase246-249 bacterial adhesion using single species models in vitro. Regarding surface chemistry, varying the surface chemical composition (Ti, ZrN) alter the bacterial adhesion250, 251. Furthermore, for example, calcium adsorption into the oxide layer of titanium has been found to increase the bacterial adhesion252, while fluoride ions have been found to decrease bacterial adhesion253. Although, studies have been performed investigating the importance of surface properties for bacterial adhesion and biofilm formation on biomedical implants, there is still a lack of evaluations and understanding of the phenomenon.
55
AIMS
The main aim of the present thesis was to evaluate the relation between surface chemistry and surface topography regarding the biological outcome. Implants need to perform in three biological arenas: in relation to bone tissue, soft tissue, and microbial biofilms. An implant should be properly osseointegrated and have a tight sealing of surrounding soft tissues but it should at the same time not be prone for extensive biofilm formation or be difficult to clean. Hence, the aims of this thesis were:
Bone:• To evaluate the importance of anodic oxidation and Ca2+
incorporation/surface chemistry of commercially pure titanium implants regarding osseointegration, and whereas the chemical modification may compensate for a minimal surface roughness.
Oral mucosa:• To evaluate the effect of sol-gel derived nanoporous TiO2 coating
of commercially pure titanium for the sealing of oral mucosa.
Bacterial adhesion and biofilm formation:• To investigate bacterial adhesion, as well as biofilm formation
and retention of oral bacteria in vitro on smooth and moderately rough, anodized and Ca2+ incorporated, as well as, nanoporous surfaces.
56
57
MATERIALS AND METHODS
Surface processingTitanium grade 4 has been used in all five studies. In the animal studies, evaluating the bone response, turned threaded implants with a length of 8 mm and a diameter of 3.5 mm were used. In the human experimental study, evaluating the response of the oral mucosa, micro-implants with diameter 2.2 mm and lengths of 10 mm or 13 mm, with an oral mucosa penetrating investigational part of 3.4 mm or 6.4 mm, respectively, were used. For the in vitro bacteria studies, discs with a diam-eter of 8 mm with a central hole were used.
All implants were rinsed and cleaned in an ultrasonic bath with diluted Extran MA01® (Merck, Darmstadt, Germany) and rinsed in absolute alcohol before use in any study. All implants were, in addition, sterilized using an autoclave before inserted into humans or rabbits.
Anodic oxidationWhen anodizing a material a current is applied to a system where the material to be processed constitutes the anode. The electrolytes constituted of either sodium glycerophosphate hydrate (C3H6(OH)2PO4Na2 × H2O), which constituted the basic electrolyte, or a mixture of sodium glycerophosphate hydrate and calcium acetate
Figure 2. Illustration of the implants installed in rabbits.
58
(Ca(CH3COO)2) when aiming for Ca2+ incorporation. The micro-arc oxidation process was done in accordance to the prescription of Sul et al. 68, 121. However, Sul et al. used titanium grade 1 as bulk material and, therefore, the resulting surfaces may vary.
sol-gel derived nanoporous tiO2 coatingThe solution, constituting the future surface coat, gradually evolves into a biphasic system with both a solution (sol) and a gel phase. Specimens are dipped in the sol-gel, thereafter dried, where the rate determines the porosity of the established layer that also undergoes shrinking at this stage. Lastly the samples are heat-treated to enhance the mechanical properties.
Prior to dip coating procedure, the discs used in Study V were cleaned in a basic hydrogen peroxide solution, extensively rinsed in distilled water, and dried in flowing N2. The sol was prepared by mixing and stirring of two solutions: tetraisopropylorthotitanate (Ti(CH3)2CHO4, Merck Hohenbrunn, Germany) dissolved in 15 ml 99.8% ethanol and 99.8% ethanol, 170µl H2O, together with 840 µl HNO3. The solutions were mixed for one-hour then 100 µl PEG 400 (Merck, Hohenbrunn, Germany) was added. The clear sol was kept at room temperature during aging. The dip coating procedure was performed with computer controlled stepper motor stage with a dipping speed of 30 mm/min, and the TiO2 sol-gel coated discs were sintered in an oven at 500°C (air) for 30 minutes. After heating the discs were cleaned ultrasonically in ethanol for four minutes and finally dried in flowing N2. Model silica surfaces, was prepared with similar coating and used for topographical analyses.
Blasting process75 µm, 100 µm, or 120 µm sized Al2O3 particles were used when blasting the implants in all studies. The technique results in impressions from the blasted particles, and, as a side effect, the chemical composition may be altered by the blasting particles. 3.2 kg air pressure from 3 cm distance was used. 75 µm particles were used in study I, 100 µm particles were used in study II, and 120 µm particles were used in study IV. The particle size was altered to achieve somewhat rougher surfaces.
59
Al2O3 as blasting particles has shown equal bone response as TiO2 particles in a previously published rabbit study 254.
Surface characteristics measurementsOptical interferometryOptical interferometry uses white light (λ 550 nm) and is based on the principles of interference: light is directed towards the surface and interference fringes between the incident and reflected light are detected at different positions due to the level of the investigated area. The pattern of the registrations reflects on the surface topography. The MicroXamTM (PhaseShift, Tucson, USA) has a lateral resolution of 0.3 µm and a vertical resolution of 0.05 nm. The maximal measuring area is 5.3x4 mm and the vertical range is 5 mm. Three screw shaped implants of each surface were measured on nine sites of the threaded area: three tops, three valleys and three flanks 255. Three discs of each surface were measured at ten sites evenly distributed. Each measurement was performed over a 200×260 µm area. Images were produced using SPIP™ (Scanning Probe Image Processor, Image Metrology, Denmark).
Atomic force microscopyAn atomic force microscopy has a higher resolution than an interferometer and are, therefore, of greater use to characterize nanofeatured materials. In study V, the topography of nanoporous TiO2 coating (on Si) was characterized by atomic force microscope (AFM 3100, Nanoscope III, Digital Instruments).
Gaussian filterGaussian filters combines a signal or a structure detected as a function with a Gaussian distribution that has a symmetric shape of a bell curve. Using a Gaussian filter is suitable for a screw-shaped geometry, and the size 50×50 µm has been found suitable for surfaces with rich microfeatures 255. By adding the
Figure 3. Illustration of a Gaussian distribution.
60
filter, the surface structures are considered concentric circles with a Gaussian distribution from the centre point. By coordinating the values from the distributions after the convolution, the new “image” is produced. The original registration receives the highest Gaussian value and neighbouring registrations receive smaller values as their distance to the original structure increases. The size of the filter decides the width of the “bell curve”.
Surface structures can be divided into form, waviness, and roughness 255. Depending on what filter used, a low- or high-pas s Gaussian filter as well as the filter size, the desired structures can be extracted. A low pass filter removes the high frequency components, derived from the smaller structures, leaving the form, while a high pass filter removes the low frequency components, thus extracting the roughness. The size (cut-off length) of the high pass filter decides what is considered as the roughness, the waviness, or form.
Surface parametersThere are more than a hundred available surface parameters. Of the ones used, average height deviation, the Sa value, is the most frequently reported to describe surface topography. The parameters are divided into groups depending on what sort of characteristics they reflect upon. All S-parameters are three-dimensional parameters. The evaluation was performed with the Surfascan software (Somicronic Instrument, Lyon, France) and the surface parameters used were:
Height parameters: Sa (µm) = average roughness in height, an amplitude parameter; average height deviation from a mean plane within the measuring area. An Sa value of 1.5 µm have been found by Wennerberg to give the firmest bone fixation 255. Ra represents the two-dimensional parameter.Ssk = surface skewness/distribution of peaks and valleys. A positive value correlates to more structures above the mean plane, and a negative value larger surface below the mean plane.
61
Spatial parameter: Sds (µm-2) = summit density, a spatial parameter; the number of summits (higher than its eight nearest neighbours) per unit area. The summits may act as binding sites for the initial mineral crystal growth and, also, their density or established patterns may have varying effect on surrounding cells and proteins.
Hybrid parameters: Sdr (%) = developed interfacial area ratio, a hybrid parameter; additional surface area contributed by the roughness compared to a totally flat plane. If the surface area increases there may be a greater possibility for interactions between the surface and its surrounding tissues. The Sdr value 1.5/50% have been suggested optimal for bone integration255.
Functional parameters: Sci = core fluid retention index, a functional parameter; the volume of fluid (e.g. blood and nutrients) that the surface would be able to contain. Arvidsson et al. found a low core fluid retention index to be favourable for biological outcome256.
scanning electron microscopyWhen a focused electron beam, generated from an electron source (a “gun”) is rastered across a surface, electrons from the surface are scattered in all directions; these are called secondary electrons and are detected in order to show the topography of a sample. Backscattered electrons (illustrating composition contrast), diffracted backscattered electrons (determination of structure and orientation of crystals), x-rays (elemental identification), visible light, and heat is generated as well. The amount of electrons generated at the surface, and their energy, depend on the acceleration voltage, topography and the elements of the material. Thereby, both topographic and elemental information can be obtained.
62
transmission electron microscopyWhen using a transmission electron microscope electrons pass through a sample and are detected on the other side of the sample from the electron source. The image created contains contrast differences, due to for example, sample thickness and crystal orientation. The instrument allows high-resolution information but requires electron transparent samples (< 100 nm in thickness). Different sample techniques exists such as polishing, broad and focused ion milling and ultramicrotome cutting. Sample content, quality and size are dependent on the sample preparation technique.
X-ray photoelectron spectroscopy The instrument analyses the kinetic energy distribution of emitted photoelectrons by using photo-ionization, under vacuum conditions. An electron energy analyzer registers the kinetic energies from the emitted electrons and a photoelectron spectrum can hence be recorded. Each surface element is associated with a certain binding energy and will give rise to a characteristic set of peaks in the photoelectron spectrum. The intensity of the peaks is related to the concentration of the element within the sampled region; the technique, thereby, provides a quantitative analysis of the surface composition. The technique is essentially surface sensitive. The analyses in the studies involved were made with an instrument (PHI 5000 ESCA system, Perkin Elmer Wellesley, USA) using an operating angle of 45° at 150 W, using an Al excitation source.
Ellipsometry With ellipsometry the metrology of thin films can be investigated by the polarization of light reflected off a surface. The method is capable of investigating layers from a few Ångström up to micrometers. The thickness of the sol-gel coating used in study V was measured with null ellipsometry at l=632 nm (Auto-El-III, Rudolph Research, USA). The assumed refractive index of TiO2 in anatase crystal structure was n=2.49.
In vivo evaluations – rabbit model Animals and surgical technique Ten female or ten male New Zealand White rabbits were used in
63
each experiment. The animals were adult (nine months of age) and weighed between four to five kg. The rabbits received one implant in each distal femoral metaphysis and two in each proximal tibial metaphysis, randomizely positioned. The skin and fascial layers were opened and closed separately. The fascial layers were sutured with resorbable sutures. The implantation holes were drilled with a low rotary speed and profuse saline cooling was used. One operator inserted all implants. The animals were allowed to bear their full body weight immediately after surgery. Twelve weeks after the implants were inserted the animals were sacrificed with Pentobarbital vet (Apoteket AB, Uppsala, Sweden) after sedation with 1.0 ml Hypnorm Vet.
Biomechanical evaluationThe peak loosening torque was evaluated with removal torque measurements. It may be considered a three-dimensional test as it reflects upon the interfacial shear strength between the bone tissue and the implant 51. The static torque was applied to the implant at a linear rate of 9.5 Ncm/s and the device core ensured that it was fixed. The removal torque was stopped when the implants started to turn in order to have the implant site as intact as possible for histological sampling.
Preparation of histological specimen The implants and their surrounding tissues were removed en bloc and immersed in four percent neutral buffered formaldehyde. The specimens were dehydrated in graded series of ethanol and embedded in light curing resin (Technovit 7200 VLC, Kültzer & Co, Germany). Undecalcified ground sections were ground ad modum Donath (1988) using a cut and grinding machine (Exakt Apparatebau, Hamburg, Germany) to a thickness of about two cell layers (15-20 mm). The slides were then stained with Toluidine blue mixed with pyronin G.
light microscopy evaluationsA computer-connected microscope was used to measure bone to implant contact and bone area surrounding the implants. A qualitative observation, regarding grade of inflammation, released material
64
particles, or other deviating appearances, was always performed for every sample. To evaluate the bone contact the distances with bone in contact to the implant were divided with the total length of the implant and presented as a percentage. The surrounding bone area was evaluated by measuring the bone area within a region of interest that was outlined at a distance of the thread depth, about 330 mm, from the implant. For histological investigations of implants turned loose (study II), appreciations of the length of bone that had been in contact with the implants were measured. These lengths showed no other tissue between the bone and the fracture line, however, blood cells, e.g. platelets, could be present. Measuring the bone area was performed the same way. All measurements were made with a magnification of ×100, in a blinded manner. However, when there were uncertainties a higher magnification was chosen for visualization. The specimens were divided into two evaluation groups depending on whether they had been placed in femur or tibia.
In vivo evaluations – human model
Investigation design and patient selectionThe study was a single-centre, randomized controlled clinical investigation with intra-subject comparison of two different surfaces. The participants (15 subjects) were sought among subjects coming to the clinic for a standard dental implant treatment. They were consecutively enrolled in the study as they passed the inclusion and exclusion criteria. The investigation was conducted in accordance with the ethical principles in the Declaration of Helsinki and other applicable regulatory requirements.
The implants were randomly positioned and the bone quality was assessed during installation with a flapless surgical technique. Each subject received two micro-implants penetrating the oral mucosa: one test and one comparator implant. With this intra-subject comparison each subject acted as its own control. The majority of the implants were positioned in posterior location. Primary stability was clinically estimated and recorded.
X-ray imagingRadiographs were taken at the time of implant installation and at the time for implant retrieval. Measurements were done directly from digital radiographs using computerized calibrations.
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sample retrievalFourteen weeks (±two weeks) after implant installation the two experimental micro-implants were removed after clinical evaluation. A cut was made around the micro-implant through the mucosa and 2-3 mm into the bone, in most cases using a 5 mm trephine drill with sterile irrigation, or a very small bone chisel. The implant-gingiva interface was protected from rupturing forces at the retrieval. Both micro-implants from each subject were retrieved using the same proceeding.
Immediately after retrieval, the samples were put in modified Karnovsky fixation (2 % paraformaldehyde, 2.5 % glutaraldehyde in 0.05 M sodium cacodylate buffer), kept cool, and sent to the laboratory within 72 hours.
Preparation of histological specimen Samples were dehydrated in increasing ethanol concentrations and subsequently infiltrated and polymerized in heat-curing resin (Agar 100 Resin). Resulting blocks with the micro-implant and its surrounding tissues were sawed in half along the long axis of the micro-implant in bucco-palatinal direction. If the direction could not be identified, the laboratory selected an area rich in tissue.
light microscopy evaluationsGround sectionsOne half of each block was prepared for ground sectioning using a cut and ground machine (Exakt Apparatebau, Hamburg, Germany). All sections were then stained with 1% Toluidine-blue dissolved in 1% borax, mixed in 4:1 proportion with 1% Pyronin-G.
A quantitative light microscopy histological analysis provided an evaluation of the soft tissue to metal contact as percentage of the distance along the entire abutment part of the micro-implant. In addition, depth of crevice/sulcus of the marginal mucosa, area of the sulcus, height of the marginal mucosa, total thickness of mucosa, and length of the abutment part was measured.
Semi-thin sectionsThe other half of the block was prepared using an electrolytic dissolution technique to remove the bulk part of the metal.
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The sample was then cut horizontally to remove the bone and re-embedded for preparation of semi-thin sections (about 1.5 µm). The sections were then stained with Richardson. Total number of the inflammatory cells (lymphocytes, plasma cells, macrophages, and polymorphonuclear cells) and the number of fibroblasts in a region of interest were calculated in a light microscope by placing a grid over the histological sample. One region of interest per sample side was chosen. Furthermore, a qualitative light microscopy histological analysis provided an evaluation of inflammation, tissue repair, and adherence of the oral mucosa to the surface of the device.
transmission electron microscopy analysis The ultrastructure of cells at the tissue-implant interface was studied using transmission electron microscopy on ultra-thin sections. All specimens were taken from the lower one third of the abutment part of the implants. Four test and five control samples were analyzed. Cell membrane contact to material, focal adhesion contact points, presence of collagen attachment, and the interaction of inflammatory cells with the material surface or material fragments were evaluated.
In vitro modelsBacterial strains and cultureThe oral strains used for biofilm assays were Streptococcus sanguinis ATCC 10556, Actinomyces naeslundii isolated from dental plaque258, and Lactobacillus salivarius isolated from a root canal259. All strains were routinely maintained on blood agar or Todd-Hewitt broth at 37° C in 5 % CO2. Aliquots of bacterial at the mid-exponential growth phase (OD600 nm≈0.6) were added to six-well plates containing the titanium surfaces and the incubation took place at 37° C on a rotary shaker in 5 % CO2 for two or 14 hours. For experiments using human whole saliva, unstimulated whole saliva was collected from a healthy volunteer with good oral health and discs were then incubated at 37° C in 5 % CO2 for 14 hours with gentle shaking. When discs were incubated with bacteria suspended in sterile saliva, unstimulated whole saliva was collected from a healthy volunteer and filter sterilized using filters with 0.22 µm pore-size. The adherent bacteria were then fixed in 4 % paraformaldehyde overnight at 4° C.
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16s rrnA fluorescence in situ hybridization16S rRNA fluorescence in situ hybridization enables detection and quantification of bacteria in their natural environment as well as a specific bacteria specie or cell 260. The 16S rRNA exists in the smaller subunit, 30S, of prokaryotic ribosomes. By hybridization of oligonucleotide probes marked with fluorescents specific sequences can be marked. Cells are first fixed in paraformaldehyde according to the protocol used. Thereafter, cells were permealized using a lys-ozyme to allow for entrance of the oligonucleotide probes together with a hybridizing buffer. The hybridizing buffer enables anneal-ing of the oligonucleotide probe to the specific single stranded rRNA sequence. By using various lasers the different fluorescence can be made visible. To avoid problems with background stain-ing, an additional washing period was added to the fluorescence in situ hybridization protocol. For two-species biofilms and bio-films derived from whole saliva, the streptococcal probe STR493 (5’-GTTAGCCGTCCCTTTCTGG-3), fluorescently labelled green with ATTO-488, and the red-labelled ATTO-565 general bacte-rial probe EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) were used. For three-species biofilms, the probe STR493 (green), the red-labelled lactobacilli probe LAC722 (5’-YCACCGCTACA-CATGRAGTTCCACT-3’), and the probe IF201 (5’-GCTACCGT-CAACCCACCC-3’) labelled with Pacific blue for identification of actinomyces.
Disadvantages with 16S rRNA fluorescence in situ hybridization may be detachment of biofilm during the washing procedures. Furthermore, the method is rather sensitive and certain important steps are of great importance, such as the permeabilization of the cell wall as well as the hybridization of the oligonecleotide probes. Lysozyme and hybridizing buffers were never more than a week old when used in the experiments, and the washing
Figure 4. Bacteria on a titanium surface detected with fluorescent oligonucleotide probes and confocal laser scanning microscopy.
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buffer never more than four weeks old. The probes used are all well documented261-263 and their specificity can be considered accurate.
confocal laser scanning microscopyConfocal laser scanning microscopy enables high-resolution three-dimensional imaging of a fluorescent or reflective specimen in a non-destructive manner. The focus plane can be set at different depths within a sample and, thereby, scanning through a sample is possible. By the pinhole size of the microscope the focus depth can be adjusted, the smaller the size the thinner the focus plane and the greater the resolution. The resolution is, furthermore, depending on the numerical aperture of the objective lens, the refractive indices of the immersion media, the specimen itself, as well as the wavelengths of the excitation and emission light, and the electronic system that digitalizes and samples the fluorescent signals. 264
An Eclipse TE2000 inverted confocal scanning laser microscope (Nikon Corporation, Tokyo, Japan) were used in study IV and V. Green fluorescence was provided by an Ar laser (488 nm laser excitation), red fluorescence was given by a G-HeNe laser (543 nm laser excitation), and blue fluorescence was provided by an UV laser (390 nm laser excitation). Images were obtained from a total of 15 sites/disc, which were randomly selected with the MultiPoint series macro in the confocal interface software EZ-C1 version 3.40, build 691 (Nikon Corporation, Tokyo, Japan). An oil immersion objective (x60) with a numerical aperture of 1.4 and the confocal pinhole set to a diameter of 30 µm was administered. Images were obtained with a zoom factor of 1.0, a pixel resolution of 0.42 µm/pixel, and a field resolution of 512 by 512 pixels or 1024 by 1024 pixels for images for publication. Each stack had a substratum coverage field area of 215 by 215 µm. In three-dimensional section analyses, the vertical sectioning step was of 2 µm. The number of layers varied between the surfaces due to the surface roughness but always included all bacteria present on the surface. The image stacks were serially transformed from the confocal laser scanning microscope format Image Display Subsystem to the tiff format using a macro in the EZ-C1 software.
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Biofilm biovolume quantificationsThe tiff-images were analyzed with the image analysis MATLAB software bioimage_L 265 to quantify the biovolumes. The detected bacteria could be analyzed and quantified with different colour modalities.
StatisticsData from the topographical evaluations were treated with one-way ANOVA followed by the post-hoc test Games-Howell. For study I and II, data from the histomorphometrical evaluations were treated with the nonparametric Wilcoxon matched pairs signed ranks test for the femur group and the nonparametric Kruskal-Wallis test for the tibia group. If significant difference was found with Kruskal-Wallis, further investigations were performed with the nonparametric Mann-Whitney U test to clarify between which groups. For study III, paired t-test was applied. If assumptions of paired t-test were violated Wilcoxon matched pairs signed ranks test was used. Wilcoxon matched pairs signed ranks test was used for X-ray derived data. For study IV, three independent experiments of each setup were performed. To compare biofilms within one experiment, the nonparametric Friedman´s test was used. For paired investigations between the surfaces within one experiment, Wilcoxon signed ranks test was used. In study V, the nonparametric Mann-Whitney U test was used to detect differences between test and control surfaces A confidence interval of 95 % was chosen, p-values below 0.05 were considered statistically significant.
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RESULTS
Surface propertiestopographyAll implants are grouped according to Albrektsson and Wennerberg (2004) into smooth (Sa <0.5 µm), minimally rough (Sa 0.5-1.0 µm), and moderately rough (Sa 1.0-2.0 µm). All anodized surfaces had a porous appearance. Further, the Ca2+ incorporated surfaces generally had smaller, more densely positioned outer porous structures compared to the non- Ca2+ incorporated surfaces as can be seen in Figure 5. All surface variables are gathered in Table 3 and 4.
Surfaces tested for osseointegration:• Study I - The Ca implants were smooth, densely peaked
implants, with the smallest interfacial surface area compared to the other implants. The Ox and Bl implants were minimally rough with similar summit density but the blasted implants had somewhat smaller surface area. There were no differences in core fluid retention index.
• Study II – The Ca surface used in study I are in study II called OxCa. It once again had the smallest average height deviation and interfacial surface area. The OxCa and the Ox surfaces were smooth. The Ox surface used in the present study had a smaller average roughness compared to the one used in study I. However, it was still slightly rougher than the OxCa surface. The summit density was similar between the Ox and the OxCa surface; however, the Ox surface had slightly larger surface area. The other surfaces, the BlOx, BlOxCa, and Bl surfaces, were found moderately rough. The Bl surface was vaguely
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smoother than the other and the BlOxCa was the roughest. The summit density was similar for the anodized moderately rough surfaces but lesser for the solely blasted surface. The same relation pertains to the developed interfacial surface area then the BlOxCa had somewhat smaller area compared to the BlOx surface. There were some differences between the surfaces regarding the core fluid retention index.
Surfaces evaluated for human oral mucosa adhesion: • Study III – The surface of the threaded part of the implants were
smooth, as was the abutment or mucosa penetrating part. The test and control groups had similar surface roughness, summit density, and developed interfacial area. The surface roughness as well as the developed area was minor. The surface porosity of the Test surface was 21 %, with outer porous structures between 15-50 nm. Also, the nanoporous TiO2 coating contributed to an average height deviation (Sa) in the nanometre range of 0.88 nm.
Surfaces used to analyze biofilm formation and bacterial retention: • Study IV – The surfaces were similar to surfaces used in other
studies. A turned surface constituted a control and had the lowest surface roughness, summit density and interfacial surface area. It was also the only anisotropic surface with remaining shallow impregnations from the turning process. The turned surface, as well as anodized surfaces with (OxCa) our without Ca2+ modification (Ox) were smooth surfaces. The Ox surface, however, had a larger average roughness as well as interfacial surface area compared to the two other smooth surfaces. The OxCa surface, in addition, had a higher summit density and a somewhat larger interfacial area compared to the turned surface. The Bl and BlOxCa surfaces were moderately rough and had similar surface parameter results; the topographical difference was mainly the surface microporosity.
• Study V – All surfaces evaluated were smooth with similar topography; however, the anodized calcium incorporated presented slightly larger developed surface area ratio, summit density, and average height deviation. In addition, the nanoporous surfaces had a featured topography in the nanometre level of resolution.
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Table 3. Surface parameter values for all surfaces used in the studies.
Sa (µm) Sds (µm-2) Sdr (%) Sci Ssk
Ox (I) 0.64 ± 0.16 0.136 ± 0.009 44 ± 21 1.33 ± 0.16Ca (OxCa)(I) 0.27 ± 0.06 0.208 ± 0.024 17 ± 3 1.33 ± 0.13Bl (I) 0.90 ± 0.13 0.118 ± 0.009 31 ± 4 1.38 ± 0.07
Ox (II) 0.41 ± 0.06 0.215 ± 0.027 44 ± 14 1.17 ± 0.12
OxCa (II) 0.30 ± 0.13 0.233 ± 0.024 19 ± 4 1.36 ± 0.28
BlOx (II) 1.35 ± 0.18 0.500 ± 0.014 173 ±16 1.31 ± 0.08
BlOxCa (II) 1.40 ± 0.28 0.496 ± 0.017 149 ± 27 1.27 ± 0.16
Bl (II) 1.25 ± 0.12 0.156 ± 0.006 67 ± 13 1.24 ± 0.10
Test (SG)(III) 0.16 ± 0.01 0.125 ± 0.004 4 ± 1 0.58 ± 0.50
Control (III) 0.17 ± 0.01 0.116 ± 0.009 3 ± 1 0.92 ± 0.41
Threads (III) 0.21 ± 0.02 0.157 ± 0.011 9 ± 2 0.98 ± 0.34
turned (IV) 0.18 ± 0.02 0.131 ± 0.022 4 ± 1
Ox (IV) 0.40 ± 0.07 0.235 ± 0.011 43 ± 8
OxCa (IV) 0.22 ± 0.01 0.229 ± 0.003 15 ± 2
BlOxCa (IV) 1.54 ± 0.14 0.178 ± 0.003 88 ± 9
Bl (IV) 1.52 ± 0.11 0.164 ± 0.019 76 ± 9
SG (V) 0.16 ± 0.04 0.130 ± 0.005 3 ± 1REF (V) 0.16 ± 0.02 0.115 ± 0.007 3 ± 1TU (turned)(V) 0.18 ± 0.02 0.131 ± 0.022 4 ± 1OC (OxCa)(V) 0.22 ± 0.01 0.229 ± 0.003 15 ± 2
Figure 5. The figure present the surfaces used in Study IV. Upper images are derived from interferometry and produced with SPIP™. Scanning electron microscopy produces images below.. The surfaces are, from left to right, turned, anodic oxidized, anodic oxidized and Ca2+-incorporated, blasted, and blasted anodic oxidized and Ca2+-incorporated.
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Table 4. Surface characteristics of the nanoporous TiO2 surfaces.
Sa/Ra (nm) Oxide thickness (nm) Porosity (%)
Test (SG; III) (0.88) (380) (21.0)
SG (V) 1.58 90 ± 10
chemistryThe surfaces used in study II and IV were evaluated with X-ray photoelectron spectroscopy. TiO
2 mainly constituted the outermost
surface for all implants used. Calcium constituted about two atomic percent of the Ca2+ incorporated surfaces. Aluminium was detected on all blasted surfaces, and phosphor mainly on the anodized surfaces without Ca2+ modification. Carbon constituted about 30 atomic percent of all surfaces used in study IV, probably impurities due to handling of the specimens. The surfaces used in study II were analyzed directly after their manufacturing.
Table 5. Elemental composition of surfaces used in Study II and IV from X-ray spectroscopy analysis.
Ti O Ca P Na Al C
Ox (II) 26.3 68.9 4.6 0.1
OxCa (II) 31.0 67.6 1.2 0.3
BlOx (II) 23.8 68.5 5.3 0.1 2.2
BlOxCa (II) 28.9 66.7 1.8 0.3 2.3
Bl (II) 28.6 65.7 0.1 5.6
turned (IV) 14.5 48.0 37.5
Ox (IV) 5.3 46.5 0.4 8.9 37.8
OxCa (IV) 16.0 48.9 1.7 0.7 32.7
BlOxCa (IV) 7.5 52.8 1.9 3.6 34.2
Bl (IV) 4.8 49.8 1.8 7.7 35.9
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Table 4. Surface characteristics of the nanoporous TiO2 surfaces.
Sa/Ra (nm) Oxide thickness (nm) Porosity (%)
Test (SG; III) (0.88) (380) (21.0)
SG (V) 1.58 90 ± 10
chemistryThe surfaces used in study II and IV were evaluated with X-ray photoelectron spectroscopy. TiO
2 mainly constituted the outermost
surface for all implants used. Calcium constituted about two atomic percent of the Ca2+ incorporated surfaces. Aluminium was detected on all blasted surfaces, and phosphor mainly on the anodized surfaces without Ca2+ modification. Carbon constituted about 30 atomic percent of all surfaces used in study IV, probably impurities due to handling of the specimens. The surfaces used in study II were analyzed directly after their manufacturing.
Table 5. Elemental composition of surfaces used in Study II and IV from X-ray spectroscopy analysis.
Ti O Ca P Na Al C
Ox (II) 26.3 68.9 4.6 0.1
OxCa (II) 31.0 67.6 1.2 0.3
BlOx (II) 23.8 68.5 5.3 0.1 2.2
BlOxCa (II) 28.9 66.7 1.8 0.3 2.3
Bl (II) 28.6 65.7 0.1 5.6
turned (IV) 14.5 48.0 37.5
Ox (IV) 5.3 46.5 0.4 8.9 37.8
OxCa (IV) 16.0 48.9 1.7 0.7 32.7
BlOxCa (IV) 7.5 52.8 1.9 3.6 34.2
Bl (IV) 4.8 49.8 1.8 7.7 35.9
Osseointegration - study I and IIBone-implant-contact/estimated bone to implant contact after 12 weeks in rabbits
Smooth calcium-incorporated anodized implants had significantly more bone contact (47 ± 9 %) compared to minimally rough anodized implants without calcium modifications (30 ± 10 %) as well as minimally rough Al2O3 blasted implants (34 ± 6 %) implants when placed in rabbit tibia (p=0.002). The calcium incorporated implants also had a higher percentage of bone contact (32 ± 9 %) compared to anodized implants (20 ± 6 %) when placed in femur but the difference was not significant.
The mean length of bone estimated to have been in contact with the implants placed in femur was 8.18 ± 1.80 mm for smooth anodized implants and 7.66 ± 2.23 mm for smooth calcium incoporated anodized implants. For implants placed in tibia, moderately rough anodized implants (8.39 ± 2.10 mm) had significantly more bone in close vicinity to the implant compared to moderately rough
Figure 6. There was a significantly greater (*) proportion of bone in contact with the Ca2+-incorporated implants placed in rabbit tibia in Study I. The lower diagrams display the lengths of bone estimated to have been in contact with the implants that had been turned out in Study II; the blasted and anodic oxidized implants had significantly longer lengths of bone estimated to have been in contact.
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blasted (6.26 ± 1.64 mm) and moderately rough anodized calcium incorporated implants (6.47 ± 1.26 mm) (p=0.026). Smooth anodized calcium-incorporated implants had a mean value of 7.81 ± 1.59 mm.
Surrounding bone area after 12 weeks in rabbitsMinimally rough anodized implants had the highest percentage of surrounding bone area when placed in the tibia (47 ± 10 %), followed by the smooth anodized calcium-incorporated implants (40 ± 8 %) and the minimally rough blasted (37 ± 8 %) implants. When placed in femur the anodized implants again had a higher percentage (46 ± 11 %) compared to the anodized calcium-incorporated implants (43 ± 15 %). There were no significant differences between the three surfaces in the tibia or the two in the femur.
In study II, mean percentage of bone surrounding the implant was 50.8 ± 11.0 % for smooth anodized implants and 38.6 ± 9.6 % for smooth anodized calcium-incorporated implants placed in femur. When placed in tibia smooth anodized calcium-incorporated implants (33.5 ± 4.8 %) and moderately rough anodized implants (37.0 ± 9.4 %) had a significantly higher percentage of bone surrounding the implant compared to moderately rough anodized calcium-incorporated implants (26.8 ± 8.1 %) (p=0.047). There were no significant differences between the implant groups when only the three best threads were evaluated. Smooth anodized calcium-incorporated implants had a mean percentage of 70.9 ± 9.5 %, moderately rough anodized implants 70.8 ± 7.4 %, moderately rough anodized calcium-incorporated implants 64.2 ± 16.2 %, and moderately rough blasted implants 63.1 ± 13.5 %.
Qualitative evaluation of the implant surroundings after 12 weeks in rabbitsBone conduction had taken place at various places around all implant surfaces. There were no noticeable differences with respect to quality or presence of inflammatory cells surrounding the different implant surfaces in any of the two studies.
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Interfacial shear strength/RTQ values after 12 weeks in rabbitsThere were no significant differences between the smooth implants inserted in femur. The mean peak removal torque value and mean shear force value for anodized implants was 109.9 ± 20.6 Ncm respectively 8.34 ± 1.90 N/mm2 and 85.3 ± 25.6 Ncm respectively 6.75 ± 1.25 N/mm2 for anodized calcium incorporated implants.
Between the tibial implants there were significant differences. The moderately rough calcium incorporated implants had significantly greater mean peak removal torque value (90.7 ± 23.3 Ncm) compared to smooth anodized calcium incorporated implants (64.6 ± 18.2 Ncm) and moderately rough anodized implants (69.7 ± 17.5 Ncm) (p = 0.029). Moderately rough blasted implants had a mean value of 79.9 ± 17.4 Ncm. Both moderately rough calcium-incorporated implants and moderately rough blasted implants had significantly higher shear force values, mean values 8.55 ± 1.40 N/mm2 and 8.09 ± 2.41 N/mm2, compared to smooth anodized calcium-incorporated and moderately rough anodized implants with mean values of 5.23 ± 2.17 N/mm2 and 5.21 ± 1.38 N/mm2 (p < 0.001).
Figure 7. Blasted anodized and Ca2+-incorporated implants needed significantly higher (*) removal torque force to be turned loose, and, together with solely blasted implants, had the highest interfacial shear strength when placed in rabbit tibia.
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Interaction with oral mucosa – study IIIClinical investigationAt the time of removal (14 ± 2 weeks after implant installation) neither erythema, expulsion of fluids nor tenderness were reported for the nanoporous TiO2 implants while two polished demonstrated mild erythema and expulsion of fluids and one of those also showed tenderness. The overall clinical impression was that the nanoporous TiO2 implants demonstrated healthier and firmer attachment of soft tissue compared to the polished surfaces.
Marginal bone level from routine X-ray imagesWhen only stable implants were included in an intersubject statistical analysis of the median marginal bone loss there was significantly less bone loss around nanoporous TiO2 surfaces compared to unmodified surfaces. When all implants were evaluated, an intrasubject comparison did not reveal any statistically significant difference between the implants in the change of bone level from week 0 to week 14.
histological investigationLight microscopy - Ground sectionsHistomorphometrical measurements revealed significantly higher proportion of oral mucosa in contact with the nanoporous TiO2 implants. Other parameters indicated some advantages for the nanoporous TiO2 surfaces compared to the polished.
Light microscopy - Semi-thin sectionsThe analysis revealed lower numbers of inflammatory cells and higher number of fibroblasts in vicinity to the nanoporous TiO2 surfaces; however, these were not statistical differences.
Light microscopy - Qualitative histological descriptionNo differences were found from the qualitative histological examination. What could be distinguished was that micro-implants, in general, were not well osseointegrated. Oral epithelium down-growth to the implant threads was found in two nanoporous TiO2 implants and in seven polished implants. All implants were mostly surrounded by capsule-like dense fibrous connective tissue with
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elongated fibroblasts and parallel fibre orientation to the implant surface. The degree of inflammatory reaction was low around both implant surfaces. However, typical chronic subepithelial inflammation with lymphocytes, plasma cells, macrophages, and mast cells was observed. Polymorphonuclear leucocytes were rarely observed.
Transmission electron microscopyAt the interface, a layer of proteinacous material with a relatively dense appearance was seen. This layer varied in thickness from approximately 50 nm to 100 nm in most cases but in some cases the layer even exceeded 5 µm. At some instances cells were seen close to the surface but without cellular attachment directly to the oxide layer. Some of these cells were of epithelial origin and desmosomes were frequently seen in these sections. In addition, fibroblasts close to the surface were seen showing signs of activation, i.e. an expanded rough endoplasmic reticulum and mostly euchromatin in the nuclei. In all sections bundles of collagen fibres could be seen in all possible directions, relatively close to the surface or at a 1-5 µm distance. There were no ultrastructural differences between the surfaces.
bacterial adhesion and biofilm formation - study IV and VBiofilm accumulation on titanium surfacesThe bacterial adhesion was different between the surfaces after two hours incubation of S. sanguinis and A. naeslundii as well as S. sanguinis, A. naeslundii, and L. salivarius (p=0.022 respectively p=0.022). A similar pattern was seen with blasted surfaces presenting the largest biofilm biovolume, while the turned and smooth calcium-incorporated surfaces presented fewest adhered cells. No intra-experiment paired statistical comparisons revealed significant differences but when compiling the results from two- and three-species two hours biofilms, blasting seven-fold the biofilm accumulation compared to turned surfaces (p=0.028), an effect that was diminished for calcium-incorporated smooth and moderately rough surfaces. Calcium modification of smooth and moderately rough surfaces, on the other hand, significantly decreased the bacterial adhesion after two hours when compared to surfaces of similar topography (p=0.028 respectively p=0.043). There were no significant differences between the turned and the anodic oxidized
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surfaces (p=0.075). There was a predominance of S. sanguinis on all test surfaces for two-species biofilms.
Biofilm formation after 14 hours revealed differences between the surfaces for two- and three-species biofilms (p=0.022 respectively p=0.027). Two-species biofilms accumulated to greatest extent on blasted surafces, however, three-species biofilms accumulated to greatest extent on the calcium-incorporated surfaces. In three-species biofilms the amount of S. sanguinis and L. salivarius was similar, meanwhile A. naeslundii only made up a small proportion within the consortium.
Figure 8. All experiments were performed as triplicates from three independent consortium and are displayed as three marks respectively in the diagrams. The upper diagrams present the bacterial adhesion after two hours. Blasted surfaces generally adhered more bacteria, while turned and smooth Ca2+-incorporated anodized surfaces adhered least bacteria, Ca2+-incorporation and anodization tended to decrease the bacterial adhesion. The diagrams below presents the biofilm biovolumes formed after 14 hours. Two-species biofilms accumulated to a greater extent on blasted surfaces. However, three species biofilms increased on calcium-incorporated surfaces.
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In the fifth study, where surfaces were incubated with similar two-species cultures as in the forth study, more bacteria tended to adhere to anodized calcium incorporated surfaces, compared to the other surfaces after two hours (p=0.05); however, the slight difference was diminished after 14 hours. A. naeslundii were in these experiments the dominating bacteria.
Mechanical removal of adhered bacteriaBacteria remained on all surfaces after mechanical removal. However, moderately rough blasted surfaces retained the largest amounts of bacteria after brushing and there were significant differences between the surfaces (p=0.017). Noteworthy, A. naeslundii had been detached from all surfaces.
Biofilm accumulation in presence of salivaThere were no differences between the smooth surfaces in study V after incubation for two or 14 hours in sterile saliva. Presence of saliva, however, on average eleven-folded the adherence of bacteria to all surfaces, and the bacteria appeared more aggregated than when cultivated in broth.
Fourteen hour biofilms derived from whole human saliva accumulated in greatest extent on blasted surfaces. All other surfaces adhered significantly smaller biofilms to similar extent. The proportion of streptococci, identified by 16S rRNA fluorescence in situ hybridization, was 39 % for the blasted surfaces and 20-29 % for the other surfaces.
Figure 9. Remaining bacteria after mechanical removal with a soft toothbrush without dentifrice. Greater amounts of bacteria remained on blasted surfaces.
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Figure 10. S. sanguinis and A. naeslundii biofilms after two and 14 hours incubation in broth or sterile human saliva. There were no significant differences between the surfaces but the presence of saliva in general eleven-fold the biofilm biovolume.
Figure 11. Greatest saliva derived biofilm biovolumes accumulated on blasted surfaces. Biofilms of saliva derived bacteria did not accumulate in as great extent as biofilms formed in broth.
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DISCUSSION
Surface processingIn all studies, some of the surfaces were aimed to have either similar topography or similar chemistry while the other parameter varied. This aim was not entirely met since it is particularly difficult to alter the chemistry without affecting the topography. Mainly the smooth anodized implants varied somewhat regarding average height deviation and developed interfacial area ratio. The studies performed in the present thesis were of an applied nature. If, on the other hand, they would have aimed at investigating the mechanism of a specific surface characteristic on the tissue-response or the bacterial adhesion, other combinations of surfaces (as well as additional methods) would have been more appropriate.
Blasting with Al2O3 adds both topographical and chemical alterations. To evaluate the effect of surface chemistry represented by calcium-modification, surface-embedded aluminium particles may, theoretically, have influenced the results. However, Al2O3
blasted implant surfaces performed similar to TiO2 blasted surfaces in an earlier rabbit study254.
Ca2+ incorporated surfaces were used, sometimes with various roughness, in all studies regarding bone and bacterial adhesion. The sol-gel derived nanoporous TiO2 surface was used when evaluating soft-tissue and bacterial adhesion. Its effect on bone may not be as relevant when such a smooth surface have more implications regarding soft tissue. However, if one considers the necessity of an implant to be “tri-functional”, able to perform in three biological arenas: in relation to bone-tissue, soft-tissue, and the microbial community, it would have been interesting to evaluate the soft-tissue
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response to all surfaces even those only aimed for bone integration. If one considers the circumstances following any complication with an implant, the surface supposed to be integrated in the bone may need to interact with the soft-tissue and the extensive oral microbiota.
Surface characteristics measurementsEvaluation with interferometry is appropriate for the surfaces used in all studies. For nanofeatured surfaces, atomic force microscope or another high-resolution equipment should be used to characterize the nanotopography. The interferometer may have certain limits regarding transparency of the outermost layer (the oxide) and undercuts or highly vertical planes in the surface.
In the studies involving nanostructural modification (III and V) the other surfaces were not characterized with atomic force microscope, which may lead to restricted possibility for conclusions regarding the topographical modifications. Evaluations of chemical compositions should preferably have been performed for the surfaces used in all studies, as well as oxide thickness and oxide crystallinity evaluations of the anodized implants.
In vivo evaluations – rabbit modelsThe rabbit model is commonly used and well documented. Implants placed in femur may be considered positioned in trabecular bone, while implants placed in tibia are placed in mainly lamellar or cortical bone. The smooth implants investigated risked lack of proper osseointegration, as other studies have indicated for such smooth implants84. Therefore, the evaluation needed to be of the integration of mature bone. The turnover for rabbit bone is six weeks6 and after 12 weeks, the time-period chosen for the present studies, mature bone has formed around the implants and the most active remodelling phase has most probably finished. To investigate the osseointegration of the implants, and not mainly the healing capacity of the bone, the chosen evaluation time can be considered appropriate. Next step when evaluating the effect of the Ca2+ may be after shorter healing periods when the main advantage of surfaces with characteristics of suggestively bioactive nature has been suggested in the direct and early tissue-response.
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Other conditions to consider before making parallels with the clinical situation are that the implants were not loaded and that rabbit bone is of endochondral origin while the human maxilla is of intramembranous origin.
The histological investigation with undecalcified ground sections is a commonly used method since presented by Donath et al. (1988). However, it reveals only a two-dimensional, finite version of the implant performance within the tissues, and it is a rather time-consuming method. To get more information out of the samples the separated parts can each be separated at least once more266. A strength with histological evaluations is that qualitative as well as quantitative evaluations can be performed from histological sections. Other advantages are, for example, further grinding or other chosen embedding methods/materials, that enables additional investigations of the samples using e.g. transmission electron microscopy or enzyme-histochemical staining267.
The biomechanical evaluation using the removal torque apparatus performs a test of the interfacial shear strength and may to a greater extent than histology represent the “whole picture” of the implant performance in the tissue, since it is reflecting on the three dimensional-integration.
A combination of histological and biomechanical evaluations could be advantageous; on the other hand, to have sections with intact tissue-implant interface is of great value. Study planning that allows for proper statistical evaluations with sufficient sample size is then of importance. Moreover, combination of in vitro and in vivo investigations on the same material and presented together could provide explanatory models for biological responses (e.g. Cooper et al. 2006, Sawase et al. 2008).
Figure 12. Further sectioning of embedded samples may provide additional information. Modified after Sennerby et al. (1992).
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In vivo evaluations – human modelThe threaded part of the micro-implants was probably too smooth to be properly osseointegrated, which possibly had a negative impact on the study outcome since not all implants were stable to allow for optimal soft-tissue or bone healing. The control of the patients regarding oral hygiene was not complete which may have impacted the results further. However, since intrasubject, paired statistical evaluations were performed, intersubject differences are being compensated for.
The quality of the sections allowed a detailed quantitative and qualitative analysis. Nevertheless, some sections revealed either lack of tissues or signs of trauma (for example fresh microfractures in the bone and/or bleeding) that were most likely due to trauma during retrieval.
In vitro evaluationsTo hypothesize how a material would perform in the body we need pre-clinical and basic scientific experiments. Therefore, although the biological situation may be difficult to resemble in vitro, experiments may give us a clue whether a material would present major disadvantages when installed in humans (e.g. toxicity or extreme biofilm adhesion).
The bacterial species used in the studies within this thesis was chosen on the basis of that they are common oral organisms, and S. sanguinis and A. naeslundii are, in addition, suggested primary colonizers of teeth213. The colonization of oral implants may be expected to be similar to the colonization of teeth, with certain species representing the initial colonizers. Streptococci have been found to be the initially dominant species colonizing titanium surfaces placed in the oral cavity215. Coaggregation constitutes the initial development of a biofilm, and microbial interactions are established within the multi-phenotypic microbial community within a biofilm208. It is, therefore, of importance to have multi-species inoculums when evaluating biofilm formation on titanium surfaces. At the same time, it may be of interest to investigate the effect of certain surface characteristics on a specific bacterial specie. If single-species cultures would then be used, the question whether the species would act the same in a multi-species community would still be present. However,
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usage of 16S rRNA fluorescence in situ hybridization together with confocal microscopying (as used in the present thesis), enables investigation of a specific specie: its proportion within the biofilm, its position within the biofilm three-dimensional structure, and its existence within the biofilm at various time-points.
Lentz and Uzodinma (1989) proposed titanium plates possible as substrates for biofilms for scanning electron microscopy evaluation268, and the method is commonly used in studies today. To refine the analytic capacity, 16S rRNA fluorescence in situ hybridization together with confocal microscopy was an appropriate model, as well as informative and useful for future studies. Problems with background staining, especially related to anodized implants, when using e.g. LIVE/DEAD® BacLight™ (Invitrogen Corporation) disappeared after somewhat more extensive washing. Since the bacteria had been fixated, detachment did probably not occur in any notable extent. The need to have a three-dimensional evaluation of the accumulated biofilms is, furthermore, evident with the high complexity of many implant surfaces.
Another strength with the fourth study performed in the framework of this thesis, was that the bacteria inoculums were independent, i.e. all incubation consortia were from separate colony forming units. Although, a limited number of species were used and these were derived from the same isolate, the biological variance within one bacterial specie was encountered for.
Regarding the number of surfaces evaluated, it is common to work with triplicates within the field of microbiology. Furthermore, as we evaluated 15 sites at each disc, which all had a homogenous appearance, at the same time as bacteria presents microenvironments the evaluation of each surface can be considered sufficient. However, as we gathered all quantifications from one disc and presented that as one result, concurrently as we aimed at evaluating the surfaces, it may have been appropriate to evaluate at least six discs in each experiment.
Surface characteristicsThe surfaces used in study II, III, IV, and V had characteristics that allowed for evaluation of the importance of surface chemistry respectively surface topography for the bone- and soft-tissue response
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as well as bacterial adhesion and biofilm formation in vitro. In study I, the fact that the smoothest surface revealed more bone-tissue in contact with the implant surface was of interest since such smooth surfaces have been indicated not to osseointegrate as well as rougher.
Both the smooth and moderately rough Ca2+ incorporated anodized surfaces had fairly smaller interfacial surface areas, compared to the anodized surfaces without Ca2+. This was probably a consequence of the affected surface topography (e.g. smaller porous structures) due to altered chemistry. Since the interferometer cannot detect areas hidden by other surface structures in a vertical aspect, such as undercuts, the interfacial area of surfaces with high topographical complexity may be revealed as smaller than what it really is.
It is useful to present certain well-chosen parameters for implant surfaces in publications from different groups, to be able to, at least to some extent, correlate the performances of surfaces with similar characteristics. Following the recommendation by Wennerberg and Albrektsson74, at least one height (Sa), one spatial (Sds), and one hybrid parameter (S dr) have been presented in all studies; furthermore, a functional parameter (Sci) was applied in study I and II. The mentioned parameters are commonly found in the literature and reflect upon surface characteristics that may impact its outcome. When considering the total span of the surface topographical height, as can be presented with the parameter St, a somewhat more than ten-fold of the Sa value may be a landmark. The suggested parameters may, however, not be optimal for all surface types, e.g. Lamolle et al. (2009), analyzing surfaces treated with cathodic reduction using hydrofluoric acid, found the parameters surface skewness Ssk, kurtosis Sku, and core fluid retention Sci positively correlated to the implant’s retention in bone in vivo, and, at the same time, oxide crystallinity and average height deviation were not so correlated; however, they only used smooth implants269.
Osseointegration - study I and IISurface chemistry and, of special interest for this thesis, calcium-modification of anodized titanium surfaces, may compensate for a minimal surface roughness (study I and II). Similar positive effects of calcium have been found in other in vivo studies presented earlier (e.g. Sul et al. from 2009). Whether chemical bonding does exist or not remains unproven. Perhaps bonding in lower strength range is
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established and dissolved in a dynamic manner at the tissue-implant interface. To have a constituent chemical bonding, with ionic/covalent bonds, would probably require a more stable interface.
Furthermore, what cannot be out-ruled is that there may be different optimal surface roughness depending on the characteristics of the surface. Sul et al. (2005) suggested a Sa value of 0.8 µm to be optimal for anodized surfaces within the limits of the surfaces used in that study270.
Sealing of oral mucosa – study IIINanoporous TiO2 surfaces indicate advantages for soft-tissue adhesion as presented in earlier literature, as well as in the study within the present thesis (study III). Still, the results are not evident enough to leave out the possibility that the hypothesis of an advantage for nanoporous TiO2 coating may be incorrect.
The orientation of fibres or appearance of capsule like encapsulation was similar between the two surfaces and such responses has been found effected by the nanoporous TiO2 surface in other studies170, 171. Since a number of the microimplants used in study III did not osseointegrate well, the healing of the mucosa may have been disrupted and does not reflect the optimal healing situation. However, the number of unstable implants was not significantly different and the negative effect should, therefore, have affected both groups.
bacterial adhesion and biofilm formation - study IV and VWhether surface characteristics impact bacterial adhesion and biofilm accumulation in the clinical situation is debated. Indeed the only effect of the surface may be relative to the protein adsorption. There is no clear evidence of the surface impact from clinical studies. What can be addressed is that there is a lack of clinical studies investigating the effect of specific surfaces on biofilm transition and the health of the surrounding tissues. Furthermore, the literature mainly consists of descriptive data and not comparable, quantified data, as concluded by Subramani et al. (2009)225. However, most experimental studies indicate increased biofilm accumulation with increased surface roughness, but there are clinical studies indicating no correlation41. What can be addressed is that three-dimensional visualization, as used in the studies within this thesis, clearly showed
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that biofilm formation extended from the bottom of the pits to above the peaks of the surface. It is most likely that surface characteristics have an impact that extends further than on the protein adsorption. For instance, initially adhered bacteria will be more closely positioned when adhered to the structures of moderately rough or rough surfaces, thereby, possibly enabling microbial interactions to a greater extent (illustrated in the figure below).
Once a biofilm is established, various bacterial species can adhere to it214. In the oral cavity, a biofilm will be present at an exposed surface at all times. What is of more central importance is the transition into a fully mature biofilm, at times associated with pathological conditions. Suggestively, surface characteristics are of importance for the early bacterial adhesion, while microbial interactions majorly impact the proceeding biofilm establishment. Yet again, biofilm formation starts with initial bacterial adhesion and co-aggregation, which may be influenced by the surfaces properties.
On the other hand, colonization of commensal oral bacteria is highly associated with oral health. Therefore, microbial balance is of major importance since the oral cavity holds enormous amounts of bacteria that will colonize intraoral surfaces.
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In this thesis, blasted surfaces, in general, tended to adhere larger biofilms, as well as retaining them. The relation between the surfaces was similar for the accumulated biofilms when inoculated with independent incubation medium. The experimental design called for a statistical test that related the samples; therefore Friedman’s ranks test was used. Reasons for increased biofilm formation and maturation on moderately rough surfaces may be that bacteria can more easily adhere and not so easily detach, when they are protected from disrupting forces by the surface structures; another aspect may be closer cell-distance and, thereby, mechanisms such as e.g. microbial interactions may occur in greater extent. Indications of a synergetic effect of surface roughness, i.e. not only an increased surface area but also the position of the bacteria, may be suggested when the surface area, derived from the Sdr values, are compensated for. The relation between the surfaces remained.
The effect of surface nanoporosity did not seem to impact the biofilm formation. Of interest with nanofeatured surfaces may be the molecular regulation of cells, as suggested earlier, and the possibility to impact the primary colonizers allowing for co-aggregations of other bacterial species.
Figure 15. The relation between the surfaces remained after that the additional surface area contributed by the surface structures, derived by the surface parameter developed interfacial area ratio, were compensated for (red marks in the figures).
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The effect of surface chemistry on bacterial adhesion is controversial. Calcium-adsorbed surfaces was found to have increased adhesion of the periodontal-pathogens P. gingivalis and A. actinomycetemcomitans 252. However, we found calcium-modifications to decrease biofilm accumulation on both smooth and moderately rough surfaces, except for L. salivarius, S. sanguinis, and A-naeslundii-biofilms after accumulation for 14 hours (Study IV). Of interest was the changed pattern observed after certain time with a specific biofilm-consortium. The bacteria specie constituting the major biofilm biovolume was L. salivarius; furthermore, the proportion of A. naeslundii, was minor. This may indicate that the surface composition may impact specific species within a consortium, but the effect may also have been due to interactions within the bacterial consortium217. In addition, the visualization of the biofilm organization revealed that A. naeslundii were positioned mainly in the upper layers.
Study IV showed that there were remaining bacteria on all surfaces, but to a minor extent on smooth ones, after brushing with a soft toothbrush without dentifrice. Moderately rough blasted surfaces retained large biofilms. The possibility to clean a surface is of great importance for the treatment of a pathological condition with an established pathogenic biofilm271, 272. Mechanical removal is a treatment-form superior to antibiotics since antibiotics are not as efficient on bacteria within a biofilm, and may increase the extent of multiresistant bacteria. Other methods to clean a titanium implant surface may be, for example, using laser, rinsing with chlorhexidin273, or ultraviolet irradiation for a photocatalytic effect274.
With improved results of osseointegration, for example, due to more biologically stimulating surfaces with increased roughness or certain chemistry, implant therapies may be more frequently applied for compromised patients. That could possibly affect the future prevalence of bone resorption and biofilms associated with pathological conditions. The surfaces of various part of an implant varies; however, it should be kept in mind the possibility for changed relations for an implant leaving the surface supposed to be integrated in bone exposed to the oral microbiota. In that case it is, as earlier discussed, of importance to have a tight soft-tissue sealing to this part of the implant as well.
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SUMMARY, FUTURE PROSPECTIVES, AND DERIVED HYPOTHESIS
Oral implants need to perform in three biological arenas: in relation to bone, soft-tissue, and microbial biofilms. What characteristics that may make a surface less prone to accumulate large biofilms and/or that do not stimulate the bacteria to co-adhere or assume a pathogenic phenotype, cannot be completely stated. However, smooth surfaces tend to accumulate and retain less biofilm biovolume in comparison to moderately rough surfaces. The optimal surface for osseointegration has been suggested to be of moderate roughness, whereas surfaces used in relation to oral mucosa generally have a smooth surface. Regarding the roughness, the part of the implants aimed to be bone integrated provides the most precarious surface for biofilm accumulation.
Anodization and calcium ion incorporation of a titanium surface could possibly compensate for a minimal surface roughness regarding the performance in bone-tissue. Furthermore, the anodized and calcium ion incorporated smooth surfaces did not seem prone for extensive biofilm formation. To have a smooth surface performing comparatively to moderately rough surfaces, through altered chemistry or other altered properties, could be a new focus for oral implants when smooth surfaces possibly reduce the risks for biofilm infections. Furthermore, although surfaces aimed for soft-tissue integration most commonly are smooth, there are surface modifications (topographical and chemical) that aim for a stimulation of the cells and biological structures within the soft-tissue and since bacteria also are cells the stimulation and/or
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adhesion of such may as well increase. Modification with sol-gel dip coating, resulting in a nanoporous TiO2 surface, indicates advantages for soft-tissue integration in relation to turned/polished surfaces. The same did not show increased biofilm accumulation in relation to its controls.
The in vivo studies in the framework of this thesis have been of an applied nature, not aiming at describing or investigating a biomulecular effect of the specific surface modifications. Therefore, what will be investigated further include the molecular and cellular mechanisms of calcium and nanoporous TiO2 modifications, along with the clinical performance of the specific surface modifications in the long-term regarding maintenance of bone-tissue levels, soft-tissue adhesion, and biofilm formation. Regarding biofilm formation, all test surfaces should, as a next step, be positioned in the in situ environment as surfaces of temporary abutments on stable installed implants.
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CONCLUSIONS
Bone:• Surface chemistry/anodic oxidation and Ca2+ incorporation of
titanium surfaces may enhance the osseointegration and could possibly compensate for a minimal surface roughness.
Oral mucosa:• Nanoporous TiO2 coating indicate some advantages in relation
to unmodified titanium regarding the sealing of oral mucosa.
Bacterial adhesion and biofilm formation:• A tendency of increased biofilm accumulation of oral bacteria
in vitro was found for moderately rough (Sa 1-2 µm) blasted surfaces compared to smooth ones (Sa <0.5 µm). Moderately rough surfaces, in addition, retained more bacteria after mechanical removal of adhered biofilms compared to smooth. Nanoporous TiO2 modifications or Ca2+ incorporation did not affect the bacterial adhesion or biofilm formation compared to turned surfaces.
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POPULäRVETENSkAPLIg SAMMANFATTNINg
Dentala implantat är en väletablerad behandling för att ersätta för-lorade tänder med långsiktigt goda resultat. Det finns en uppsjö av implantat med olika ytor på marknaden och bakom dessa ligger stora forskningssatsningar. Aktuell forskning syftar till att leda fram till nya ytor med fördelaktiga egenskaper jämfört med dagens ytor avse-ende inläkningen i benet och kontakten med den orala slemhinnan, för att även kunna behandla patienter med sämre läkförmåga samt för att förkorta behandlingstiden för samtliga patienter. Forskning syftar dessutom till att kartlägga mekanismerna för osseointegration och till att förstå inläkningen av implantat på en molekylär och cel-lulär nivå. En moderat rå yta är vanligen förekommande på kom-mersiella implantat då djurstudier har visat att en råare yta stimulerar benceller i större utsträckning, har mer ben i kontakt samt retineras hårdare av benet vid urvridningsförsök i större utsträckning än en slät yta. Ytorna är även i flertalet fall kemiskt modifierade och fram-förallt en modifierad kemi har föreslagits ge en yta egenskaper av en bioaktiv natur. Bioaktivitet är ett frekvent använt uttryck vilket kan spegla en kemisk bindning mellan exempelvis en implantatyta och omgivande vävnader (vilket idag ej har kunnat påvisas) eller en effekt på omgivande vävnader (exempelvis attraktion av specifika proteiner eller benceller). Ytegenskaperna syftar, sammanfattningsvis, till att påskynda läkningsprocessen och eventuellt förbättra inläkningen av implantaten. Något att alltid ha i åtanke vid patientrelaterad forsk-ning eller behandling är eventuella konsekvenser med en negativ influens på patienten. I fallet med dentala implantat är en konsekvens bennedbrytning kring implantaten och därmed en större blottlagd
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implantatyta exponerad för munhålans bakterierika miljö. Biofilmer etableras i stort sett direkt på ytor om möjligt, då det finns flera för-delar för majoriteten av bakterier att leva i en biofilm. Friska tillstånd är starkt kopplade till en mikrobiologisk balans, det vill säga att det inte är en dominans av bakterier med sjukdomsorsakande egenska-per. Då balansen av någon anledning rubbas, förslagsvis på grund av en okontrollerbart stor mängd bakterier, stimulering av bakterier att utveckla sjukdomsframkallande egenskaper eller ett nedsatt försvar hos värden, kan biofilmer orsaka sjukdomstillstånd. Att en implan-tatyta inte ökar risken för att biofilmer med sjukdomsframkallande karaktär etableras på ytan är viktigt och bör utredas. Det är idag inte säkerställt vilka eller överhuvudtaget om det finns ytegenskaper med positiva eller negativa effekter på biofilmsetableringen. Ökad ytenergi och framförallt ökad ytråhet har dock kopplats till en ökad bakterie-vidhäftning.
Den aktuella avhandlingen har syftat till att utreda hur kalciumjo-ner i ett förtjockat titanoxidlager påverkar inläkningen i ben genom att installera implantat i kaninben och sedan utvärdera dessa genom histologiska snitt samt genom biomekanisk urvridning. Resultaten blev att kalciummodifieringar ledde till mer benkontakt med ytan samt en starkare retention vid urvridning. Det var heller ingen skillnad i retention mellan släta kalciummodifierade implantat och moderat råa implantat. Inom avhandlingen har även effekten av nanoporosi-tet för inläkning i munslemhinna studerats genom en experimentell studie i människa. En klinisk uppskattning framhöll fördelar för den nanoporösa ytan jämfört med den ickemodifierade kontrollytan och histologiskt hade den nanoporösa ytan mer vävnad i kontakt. För att utvärdera olika ytor avseende bakterievidhäftning och biofilmbild-ning sattes bakterielösningar till ytorna och sedan fick dessa fästa till ytan under två eller 14 timmar. Även filtersteriliserad saliv och hel-saliv från en frisk individ användes i studierna. Resultatet blev att en ökad ytråhet tenderade till att öka bakterievidhäftningen och biofilm-bildningen på ytan samt att en större mängd bakterier fanns kvar på moderat råa ytor jämför med släta efter rengöring.
Sammanfattningsvis kan möjligen en specifik ytkemi kompensera för en minimal ytråhet, vilket eventuellt minskar risken för etablering av sjukdomsframkallande biofilmer på ytorna om dessa exponeras i munhålan. Nanoporösa ytor har möjligen en positiv influens på anslutningen av mjukvävnad mot implantatytan, vilket är av bety-delse för en god estetik samt för skydd mot utbredningen av biofilmer.
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Acknowledgements
Many persons have contributed and inspired along the way of this work. I feel lucky to be in all of yours presence. I would especially like to thank:
My superb supervisor, Professor Ann Wennerberg. You are an inspiration and role model, both as a researcher and as a person. I am more than happy to have had your supervision and I am extremely thankful for you tutoring me. Thank you for caring and supporting, at the same time as encouraging and urging for progression.
My co-supervisor professor Gunnel Svensäter, a great and inspiring researcher and person, who has taken her time to discuss biofilms along with other matters. You have clearly influenced my way of thinking.
My co-supervisor, Victoria Franke-Stenport, for being really helpful, to the point, and skilful, in addition to fun and caring. I have learnt a lot from you in various ways.
Luiz Chávez de Paz, whom I really enjoy working with. I very much appreciate your intelligence, kindness, and great sense of humour.
Professor Tomas Albrektsson for always being helpful and for sharing his great knowledge, furthermore, for creating a very pleasant atmosphere at the department.
The Professors Peter Thomsen, Carina Johansson, Lars Sennerby, Ulf Nannmark, and Young-Taeg Sul, for sharing their knowledge and freely discussing various matters during lab hours as well as coffee breaks.
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My greatest thanks to everyone at the departments of Prosthodontics and Oral Biology in Malmö, and the department of Biomaterials in Gothenburg who have made these years really enjoyable. A special thanks to: Petra, Maria, and Ann, for contributing to this work, for teaching me the techniques in the lab, and for just being great, Ulf for handling the administration, Julia for improving the work, interesting discussions, and English expertise, Luiz: for from the start being helpful, Lena: for great support, Anna W: for being such a lovely, inspiring, and helpful person, Felicia and Carina: for being inspirations, Bertil: for being The Mac-expert, Kostas and Ryo: for brightening up both Malmö and Gothenburg, Sara and Anders: for being good friends, for making it fun, and for many interesting discussions. Dear Marjan, so happy to have gained your friendship, you are just fabulous.
Great thanks to Professor Lars Rasmusson for direct response to my interest in research and for helping along the way.
Thanks to Lennart Carlsson for all help, as well as fun and interesting discussions.
Oscar, your complete understanding and pure encouragements were great inspiration to progression and future challenges.
Thanks to all mentioned above for adding that little something extra to the fantastic world of research.
Thanks to Jan Wennström, Sanjiv Kanagaraja, Patrick Palacci, Iain Hutchison, and Bertil Friberg, for letting me experience their brilliance in the clinic. It has been significant and great fun to get a clinical insight.
Thanks to all my great colleagues at the clinic in Frölunda, my friends at the Swedish Dental Society, and, not the least, my personal friends who make my life really joyful. Of special importance during these years have been: Malin – always there and just extraordinary brilliant, smart, and wise. Marta and Malin - you are the best of friends. Sandra – you are so important to me. Ami - my stunning friend, always making me burst into laughter. Elisabeth – I admire your controlled manners and professionalism, at the same time as you are full of life and warm at heart. Erika – you are just amazingly supporting and inspiring. Carl-Martin - I really appreciate your
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wisdom since it so often is striking to me. Sofia – bright, enterprising, fun, and great inspiration in many ways. Camilla – so easy-going and relaxing. Per - I am inspired by your way of making the most out of your life. Emma – you are just a lovely person. Tania and Lisa - great to have shared some South American adventures with you. Dear Erik, David, Brandon, Emil, and Hanna - thank you for the security you have provided in addition to all hilarious memories.
Margaretha, I have really treasured our regular lunches and all discussions during the time I have known you, as well as your support.
Thanks to my dear uncles and their loved ones, Håkan, Bosse, Tobbe, Sune, Margaretha, Marcella, and Inga-Lill, and my cousins Cecilia, Kjell, Henke, Eva, Johan, Åsa, John, Carl-Johan, Erik, and Johan with their families for always being joy-bringing when I see you. Johann – I have always had the greatest respect for you and you have influenced me greatly. Camilla, you have inspired me incredibly much regarding school and research, at the same time as you have always provided guidance - you are truly amazing.
My grandmother, Ulla, whom I love and appreciate. Your advice, support, and exhortations, have been significant.
My dear mother Carina - thank you for giving me the inborn feeling that anything is possible. I have always admired your strength and capacity, as well as appreciated your love.
My always supporting beloved father Pelle, who I really enjoy sharing thoughts with. Thank you for making me secure enough to never doubt go testing my wings.
My brilliant brother Dan, whom I adore and admire. You simplify things for me and I really enjoy discussing various matters with you.
Grants from the Swedish Research Council, the Hjalmar Svensson Research Foundation, the Wilhelm and Martina Lundgren Research Foundation, Biomaterials Research Center, and the Swedish Dental Society allowed this work and are gratefully acknowledged.
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