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Synergies in Biolubrication AKANKSHA RAJ Doctoral Thesis 2017 KTH Royal Institute of Technology School of Chemical Science and Engineering Division of Surface and Corrosion Science SE-10044 Stockholm, Sweden
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Page 1: Synergies in Biolubrication - kth.diva-portal.org1073157/FULLTEXT01.pdf · Synergies in Biolubrication AKANKSHA RAJ Doctoral Thesis 2017 ... Ytterligare information om topografi,

Synergies in Biolubrication AKANKSHA RAJ Doctoral Thesis 2017 KTH Royal Institute of Technology School of Chemical Science and Engineering Division of Surface and Corrosion Science SE-10044 Stockholm, Sweden

Page 2: Synergies in Biolubrication - kth.diva-portal.org1073157/FULLTEXT01.pdf · Synergies in Biolubrication AKANKSHA RAJ Doctoral Thesis 2017 ... Ytterligare information om topografi,

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av tekniska doktorsexamen fredagen den 17 March kl. 10:00 i Kollegiesalen, KTH, Brinellvägen 8, Stockholm. Avhandlingen försvaras på engelska Synergies in Biolubrication

Akanksha Raj ([email protected])

Doctoral Thesis

KTH Royal Institute of Technology

School of Chemical Science and Engineering

Surface and Corrosion Science

Drottning Kristinas Väg 51

SE-100 44 Stockholm

Sweden

TRITA-CHE Report 2017:8 ISSN 1654-1081 ISBN 978-91-7729-268-5 Denna avhandling är skyddad enligt upphovsrättslagen. Alla rättigheter förbehålles. Copyright © 2017 Akanksha Raj. All rights reserved. No part of this thesis may be reproduced by any means without permission from the author. The following items are printed with permission: PAPER II: © 2017 Elsevier. PAPER III: © 2015 Elsevier. PAPER IV: © 2016 Royal Society of Chemistry (ACS). PAPER V: © 2016 Elsevier. Printed at Universitetsservice US-AB, Stockholm 2017

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Mr. and Mrs. Raj,

this one’s for you.

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i

Abstract The principal objective of this thesis was to advance the level of understanding in the field of biolubrication, finding inspiration from the human synovial joints. This was addressed specifically by investigating the synergistic association of key biolubricants and the resulting lubrication performance. A range of techniques was employed during the course of this thesis work. Atomic force microscopy (AFM) was used to carry out force and friction measurements. Further information on the topography, nature, and structure of the adsorbed layers of biolubricants was obtained by using instruments such as Quartz crystal microbalance with dissipation monitoring (QCM-D), X-ray reflectivity (XRR), and AFM imaging. Key synovial fluid and cartilage components have been used as biolubricants in the investigations, namely dipalmitoylphosphatidylcholine (DPPC), hyaluronan (HA), lubricin, and cartilage oligomeric matrix protein (COMP). Focus was directed towards two lubrication couples, i.e. DPPC-hyaluronan and COMP-lubricin. DPPC-hyaluronan mixtures were probed on hydrophilic silica model surfaces whereas COMP-lubricin association structures were explored on weakly hydrophobic poly (methyl methacrylate) (PMMA) model surfaces. Both the systems included salt solution at a concentration of ≈ 150 mM. Investigations of the COMP-lubricin pair revealed that individually these components are unable to reach the desired level of lubrication. However, when they associate synergistically, COMP facilitates firm attachment of lubricin to the PMMA surface in a favourable confirmation that imparts low friction coefficient (≈ 0.06).

The interplay between DPPC and hyaluronan imparts a lubrication advantage over lone DPPC bilayers, wherein hyaluronan provides a reservoir of DPPC on the model surface and thereby imparts self-healing of the lubricating layer. The system resulted in very low friction coefficient (< 0.01). Other factors such as temperature, presence of calcium ions, molecular weight of hyaluronan, and pressure were also explored. DPPC bilayers at higher temperature (liquid disordered phase) had higher load bearing capacity due to higher flexibility (as the chains were flexible enough to patch defects and self-heal). Association between DPPC Langmuir layers and hyaluronan was enhanced in the presence of calcium ions. Further, lower molecular weight hyaluronan had a stronger tendency to bind to DPPC, and it thereby affects the packing and organisation more strongly. Finally by subjecting the model system to high pressures, it was found that DPPC-hyaluronan composite layers were more stable and robust compared to lone DPPC bilayers.

Keywords: Biolubrication, Synergies, Adsorption, Surface Force, Friction, Load Bearing Capacity, Self Healing, Phospholipids, DPPC, Hyaluronan, COMP, Lubricin, QCM-D, AFM, XRR.

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Sammanfattning Huvudsyftet med det här avhandlingsarbetet var att öka förståelsen för biosmörjning, vilket inspirerades av egenskaper hos våra synovialleder. Specifikt så angreps problemet genom studier av synergistisk association mellan några av nyckelkomponenterna och dess inverkan på den smörjande förmågan. Ett antal huvudtekniker användes vid studierna. Atomkraftsmikroskopi (AFM) användes för att mäta ytkrafter och friktionskrafter. Ytterligare information om topografi, egenskaper och struktur hos adsorberade skikt erhölls från teknieker som kvartskristallmikrovåg med dissipationsmätning (QCM-D), röntgenstråle reflektivitet (XRR), och AFM avbildning.

Några nyckelkomponenter hos synovialvätska och brosk användes som smörjande komponenter, specifikt dipalmitoyl fosfokolin (DPPC), hyaluronan (HA), lubricin och “cartilage oligomeric matrix protein” (COMP). Fokus riktades mot synergistiska par, dvs DPPC-hyaluronan och COMP-lubricin. DPPC-hyaluronan blandningar studerades på hydrofila modellytor av kiseldioxid, medan COMP-lubricin undersöktes på svagt hydrofoba poly(metyl metakrylat) (PMMA) modellytor. Båda systemen inkluderade saltlösningar med en koncentration av 150 mM.

Undersökningarna av COMP-lubricin paret visade att dessa två komponenter var för sig inte kunde uppnå önskad smörjningseffekt. Emellertid, när de associerade synergistiskt så att COMP möjliggjorde stark förankring av lubricin till PMMA ytan i gynnsam konformation erhölls en låg friktionskoefficient (≈ 0.06).

Växelverkan mellan DPPC och hyaluronan ger också upphov till förbättrade smörjningsegenskaper över enbart DPPC bilager, där hyaluronan möjliggör förankring av en reservoir av DPPC på modellytan och därigenom ger upphov till en självläkande förmåga hos det smörjande skiktet. Det här systemet gav en mycket låg friktionskoefficient (< 0.01).

Andra aspekter undersöktes också genom att studera inverkan av faktorer så som temperatur, närvaro av kalcium joner, hyaluronans molekylvikt och tryck. DPPC bilager vid högre temperatur (i flytande kristallin fas) hade högre lastbärande förmåga på grund av högre rörlighet, vilket möjliggjorde själv-läkning av defekter. Associationen mellan DPPC Langmuir skikt och hyaluronan förstärktes i närvaro av kalcium joner. Dessutom hade lågmolekylär hyaluronan en strakare tendens att associera med DPPC, vilket påverkade packning och skiktstrukur mer. Slutligen så visade vi att DPPC-hyaluronan kompositskikt var mer stabila än DPPC biskikt under höga tryck.

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List of appended papers The papers listed below have been appended to the thesis.

Paper I Molecular synergy in biolubrication: The role of cartilage oligomeric matrix protein (COMP) in surface-structuring of lubricin Akanksha Raj, Min Wang, Chao Liu, Liaquat Ali, Niclas G. Karlsson, Per M. Claesson, Andra Dėdinaitė Manuscript accepted, Journal of Colloid and Interface Science, 2017

Paper II Lubrication synergy: Mixture of hyaluronan and dipalmitoylphosphatidylcholine (DPPC) vesicles Akanksha Raj, Min Wang, Thomas Zander, D.C. Florian Wieland, Xiaoyan Liu, Junxue An, Vasil M. Garamus, Regine Willumeit-Römer, Matthew Fielden, Per M. Claesson, Andra Dėdinaitė Journal of Colloid and Interface Science 488 (2017) 225–233

Paper III The effect of temperature on supported dipalmitoylphosphatidylcholine (DPPC) bilayers: Structure and lubrication performance Min Wang, Thomas Zander, Xiaoyan Liu, Chao Liu, Akanksha Raj, D.C. Florian Wieland, Vasil M. Garamus, Regine Willumeit-Römer, Per Martin Claesson, Andra Dėdinaitė Journal of Colloid and Interface Science 445 (2015) 84–92 Paper IV Structure of DPPC–hyaluronan interfacial layers – effects of molecular weight and ion composition D. C. Florian Wieland, Patrick Degen, Thomas Zander, Soren Gayer, Akanksha Raj, Junxue An, Andra Dėdinaitė, Per Claesson, Regine Willumeit- Römer Soft Matter, 12, (2016), 729 Paper V The influence of hyaluronan on the structure of a DPPC—bilayer under high pressures Thomas Zander, D.C. Florian Wieland, Akanksha Raj, Min Wang, Benedikt Nowak, Christina Krywka, Andra Dėdinaitė, Per Martin Claesson, Vasil M. Garamus, Andreas Schreyer, Regine Willumeit-Römer Colloids and Surfaces B: Biointerfaces, 14,2 (2016) 230–238

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Contribution by the respondent:

Paper I. Major part of experimental work (except for the AFM measurements), planning, part of data interpretation and analysis, part of manuscript preparation.

Paper II. Major part of experimental work (except for the SAXS and DLS measurements), planning, major part of data interpretation and analysis, and major part of manuscript preparation.

Paper III. Part of experimental work, planning, part of data interpretation and analysis (except XRR data analysis), and part of manuscript preparation.

Paper IV. Part of experimental work, planning, part of data interpretation and analysis, and part of manuscript preparation. I did not participate in the XRR, BAM, and GID measurements.

Paper V. Part of experimental work, planning, and part of data interpretation. I did not participate in XRR data analysis.

Other papers not included in this thesis:

Influence of high hydrostatic pressure on solid supported DPPC bilayer with HA in the presence of divalent Ca2+ ions

Thomas Zander, D.C. Florian Wieland, Akanksha Raj, Paul Salmen, Susanne Dogan, Andra Dėdinaitė, Per Martin Claesson, Vasil M. Garamus, Andreas Schreyer, and Regine Willumeit-Römer

Manuscript

Lubricin binds cartilage proteins, cartilage oligomeric matrix protein, fibronectin and collagen II at the cartilage surface

Sarah A Flowers, Akanksha Raj, Chao Liu, Agata Zieba, Jessica Örnros, Liaqat Ali, Lena I Björkman, Thomas Eisler, Sebastian Kalamajski, Masood Kamali-Moghaddam, Andra Dėdinaitė, and Niclas Karlsson.

Manuscript

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v

Summary of papers

The synergistic association of COMP and lubricin on model hydrophobic (PMMA) surfaces was studied in Paper I. These biolubricants were first studied individually and then in combination. We infer from the results that COMP and lubricin associate on the model surfaces employed. Friction measurements highlighted that the friction force was high in the case of adsorbed lubricin, and it was easy to wear away the lubricin. COMP resulted in lower friction force but was still not as low as that experienced in the joints. Interestingly, when lubricin was adsorbed on the COMP-covered PMMA surface, the resulting friction was low. COMP prevents wearing away of lubricin by ensuring firm attachment in a favourable confirmation and this results in efficient lubrication.

It has previously been shown that DPPC and hyaluronan build composite layers upon sequential injections onto a model hydrophilic (silica) surface. However under physiological conditions it is more likely that these biolubricants first self assemble in bulk and then attach to the surface. Paper II investigated the structure, adsorption and lubrication performance of these aggregates from a premixed solution of DPPC vesicles and hyaluronan. We observed that in the bulk solution, hyaluronan binds to the outer shell of the DPPC vesicles. When the mixture solution was injected on the silica surface, a DPPC bilayer was rapidly formed followed by a slow adsorption of DPPC hyaluronan aggregates. Overall, the layer formed was heterogeneous in nature, and in spite of this, it resulted in low friction force. The aggregates are easily compressed and disrupted and the layers are transformed to structures facilitating low friction and high load bearing capacity. Even though disruption of aggregate structures causes transient high friction peaks, the accumulation of DPPC (build-up provided by hyaluronan) imparts a self-healing ability and therefore the return to very low friction force. This self-healing ability is a clear synergistic advantage over lone DPPC bilayers.

Results from Paper III highlight DPPC bilayer morphology and lubrication performance at different temperatures, ranging from 25 °C to 52 °C. This was done by the XRR technique, AFM imaging, and AFM force and friction measurements. The DPPC bilayer was in the gel phase at low temperature and fluid phase at higher temperature. The structural changes associated with the phase transition were noticed at a relatively lower temperature in the case of AFM as compared to XRR. This is believed to be due to the kinetic energy that is transferred by the tapping

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vi

AFM tip. The friction force generated by the DPPC bilayers was low in both the phases. However, it was observed that bilayers in the fluid phase were more stable under the combined action of load and shear, and therefore the load bearing capacity was higher. This is because increased flexibility of the chains and higher surface mobility at higher temperature allows for repair of defects and therefore provides a self-healing ability.

Paper IV discusses (i) the effect of molecular weight of hyaluronan on DPPC Langmuir layers and (ii) the influence of calcium ions on the association structures. The data suggest that hyaluronan binds to the DPPC Langmuir layers and this interaction is influenced by the molecular weight of hyaluronan. Strong interaction is observed in the case of lower molecular weight hyaluronan, and the packing and organisation of the structures are affected most in this case. The association between DPPC and hyaluronan was further enhanced in the presence of calcium ions.

The influence of hyaluronan on DPPC bilayers subjected to high pressures was investigated in Paper V. The structure of the hyaluronan layer that adsorbed to solid-supported DPPC bilayers was characterised by the XRR technique. It was found that hyaluronan mostly adsorbs to the DPPC headgroup. Thereafter the response of the system to high pressures was evaluated. Data reveal that at high pressure, addition of hyaluronan to DPPC bilayers results in a composite layer, which is more stable than lone DPPC bilayers. Phase transitions associated with changes in pressure were also observed and it was found that the pressure-induced phase transition in DPPC-hyaluronan composite layers was fully reversible. This is in contrast to lone DPPC bilayers, wherein the pressure-induced phase transitions were not completely reversible. This is of importance in the case of joint lubrication as only unimpaired layers ensure low friction.

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Table of contents

Abstract i

Sammanfattning ii

List of appended papers iii

Summary of papers v

List of abbreviations

1. Introduction 1

1.1 The synovial joint 1

1.2 Lubrication synergies 4

1.3 Model systems 5

1.3.1 COMP-lubricin model system 5

1.3.2 Phospholipid-hyaluronan model system 8

1.4 Surface interaction and friction forces 11

1.4.1 Van der Waals force and electrical double layer force (DLVO-theory)

11

1.4.2 Attractive forces 14

1.4.3 Repulsive forces 15

1.4.4 Friction force 16

1.4.5 Hydration lubrication 18

2. Materials 20

3. Instruments 23

3.1 Atomic force microscopy (AFM) 23

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3.1.1 Force measurements 24

3.1.2 Friction measurements 26

3.2 Quartz crystal microbalance with dissipation monitoring (QCM-D)

27

3.3 Table of other instruments 29

4. Key results and discussion 30

4.1 COMP-lubricin synergy system 30

4.1.1 COMP on PMMA 30

4.1.2 Lubricin on PMMA 32

4.1.3 COMP-lubricin synergy on model surfaces 32

4.2 Phospholipid-hyaluronan synergy system 34

4.2.1 DPPC bilayer morphology at different temperatures

34

4.2.2 Lubrication performance of DPPC bilayers at different temperatures

36

4.2.3 Lubrication synergy: Mixture of hyaluronan and DPPC vesicles

39

4.2.3.1 DPPC and hyaluronan association structures in bulk

40

4.2.3.2 Adsorption process of DPPC-hyaluronan association structures

41

4.2.3.3 Morphology of adsorbed layer from DPPC- hyaluronan mixture

43

4.2.3.4 Surface force measurements 44

4.2.3.5 Lubrication performance of DPPC-hyaluronan association structures

46

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4.3 Effect of molecular weight and ions on DPPC-hyaluronan interactions

48

4.3.1 Effect of molecular weight of hyaluronan 49

4.3.2 Effect of Ca2+ ions 53

4.4 Effect of high pressures on DPPC bilayers and DPPC-hyaluronan structures

54

5. Conclusions and impact 57

6. Acknowledgements 60

7. References 62

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List of abbreviations

1. DPPC Dipalmitoylphosphatidylcholine

2. COMP Cartilage oligomeric matrix protein

3. HA Hyaluronan

4. PBS Phosphate buffered saline

5. PMMA Poly (methyl methacrylate)

6. AFM Atomic force microscopy

7. QCM-D Quartz crystal microbalance with

dissipation monitoring

8. XRR X-ray reflectivity

9. BAM Brewster angle microscopy

10. DLS Dynamic light scattering

11. SAXS Small-angle X-ray scattering

12. CMC Critical micelle concentration

13. Mw Weight average molecular weight

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Chapter 1

1

1. Introduction

The mammalian synovial joints are remarkable constructs of nature that

allow free movement under varying pressures with friction coefficients in

the range of 0.001–0.01 [1]. These are elegantly designed to last for, in

best case, over a century. This is illustrated by the likes of athletes

Harriette Thompson, Fauja Singh, Stanislow Kowalski running well past

their ninth to tenth decades. The optimal performance of the synovial

joints relies on an effective lubrication system. Failure of the lubrication

system leads to acute pain, disabilities, reduced quality of life, and is an

economic burden on the healthcare system. For example, according to the

World Health Organisation, ten years after the onset of rheumatoid

arthritis, at least 50% of patients in the developed countries are unable to

retain a full-time job [2]. Considered estimates by the European League

against Rheumatism suggest that rheumatic and musculoskeletal diseases

cost upwards of € 200 billion of public spending in Europe alone [3]. All

this along with the fact that we are veering towards an ageing population,

adds impetus to study and better understand biolubrication and

biolubricants, which work together to achieve healthy functioning and

admirable nanotribological performance, specifically in the context of the

human synovial joints. In this, nature also provides us with a template

that continues to inspire and which may be extended to other cutting-

edge technologies and environmentally friendly lubricants.

1.1 The synovial joint

The excellent lubrication of the synovial joints is attributed to the

cartilage that lines the synovial joints, the surrounding synovial fluid, key

biolubricants, and associating self-assembly structures.

The cartilage is a dynamic tissue that bestows the joint surfaces with low

friction, high load bearing capacity, shock absorption, and wear

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Chapter 1

2

resistance. In spite of being ≈ 2 mm in thickness [4] and regularly

subjected to high loads, it has lasting durability.

The cartilage is avascular and aneural. It has very low stiffness with a

Young’s modulus in the range of a few MPa [5]. The cartilage comprises

both cellular and extracellular components. The cellular components are

called chondrocytes. They are present at 2 vol% in the cartilage and are

responsible for producing, organising, and maintaining [6] the other 98%

of the extracellular material. The extracellular matrix of the cartilage

primarily consists of water (≈ 80%), collagen (50–75% dry weight)

proteoglycans (15–30% dry weight) and lipids (10% dry weight) [7].

Collagen fibres impart tensile strength to the cartilage and enclose

proteoglycans that confer elasticity and load distribution.

The structure of the cartilage is best described (refer figure 1) by four

zones; superficial, transitional, radial, and calcified. In the superficial

zone the collagen fibres are oriented parallel to the surface. The

outermost part of the superficial zone consists of lamina splendens,

which face towards the synovial fluid (200-500 nm thick, devoid of

collagen fibres) [8] and can be easily peeled off from the rest of the

cartilage [9]. Chondrocytes are of an elongated shape and lie parallel to

the surface. The proteoglycan content here is at its lowest, whereas the

water and collagen content is at its highest. In the transitional zone the

collagen fibres are hemispherical, ill defined in shape, and chondrocytes

are more round. The thickness of the collagen fibres increases towards

the bone and in the radial zone is the thickest and perpendicularly

oriented. Chondrocytes appear spherical and align themselves

perpendicularly in a columnar pattern. The calcified zone is separated

from the radial zone by a tidemark. This transitional area helps anchor

cartilage to the bone.

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Chapter 1

3

Figure 1: Schematic of the knee joint (above) and cartilage (below)

Synovial fluid

Bone

Synovial membrane

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Chapter 1

4

Given that the cartilage is avascular, the neighbouring synovial fluid is

responsible for providing nourishment with nutrients and removing

waste products. The synovial fluid (ionic strength ≈ 150 mM [10], pH 7.8

[11]) surrounds the joints and provides lubrication by virtue of reduced

friction and wear. It is composed of key biolubricants such as hyaluronan,

lubricin, and phospholipids. The cartilage surface forms a boundary layer

with these key molecules, which when depleted, may be replenished via

the synovial fluid and diffusion from the cartilage surface. Several modes

of lubrication are considered to play a role in cartilage lubrication but

boundary lubrication has been the focus of this study. It relies on

characteristic molecular and biochemical properties of the lubricant layer

in preventing solid-solid contact and consequently wear of the cartilage

surface.

1.2 Lubrication synergies

The ability of joint surfaces to self-repair is limited. These surfaces

respond to various factors including load and shear. While they are

constructed of molecules, which themselves are soft and compliant, the

pressures borne by them can be continual, abrupt, and high. It is hard to

believe that a single component, and one which works individually, is

capable of performing all the necessary functions and utilising all

necessary features to impart the required level of lubricity to the joints for

extended periods of time. Rather, we believe that meaningful insights can

be gained for better understanding, by exploring the association of these

components and the resulting synergism in lubrication. Therefore, we

have pursued the pathway of investigating possible synergies between the

key synovial fluid components. In this work we have focused on two

lubrication couples (i) Cartilage Oligomeric Matrix Protein (COMP) and

lubricin (ii) Dipalmitoylphosphatidylcholine (DPPC) and hyaluronan.

Nonetheless we would like to impress on the fact that the natural

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Chapter 1

5

biolubrication system is far more complex, and there are believed to be

other biolubricants, mechanisms, and factors, which come into play but

are beyond the subject of this work.

For the COMP-lubricin system, hydrophobic PMMA model surfaces were

used, whereas for the DPPC-hyaluronan system hydrophilic silica model

surfaces were used. The fact that the respective surfaces allow strong

adsorption of the corresponding molecules, reasons their selection.

The overall objective of this work was to advance understanding in the

field of biolubrication pertaining to human synovial joints. As mentioned,

this has specifically been addressed by studying the synergy between key

synovial components and its effect on the lubricity of model surfaces.

This has been elucidated and detailed in papers I and II. Further, other

aspects of joints were tapped into by investigating the effect of

temperature, high pressure, calcium ions, and molecular weight of a key

component on the model system. This has been described in papers III,

IV, and V.

1.3 Model Systems

1.3.1 COMP-lubricin model system

As mentioned in the previous section, lubricin is postulated to be a key

biolubricant for the knee joint [12-14]. It is known to impart very good

lubricity at the cartilage surface, however, to be able to do so it is equally

important for the boundary lubricant to prevent wear (which it has been

able to achieve modestly on model surfaces [12, 15]). The boundary

lubrication conditions experienced by the cartilage surface under the

combined action of load and shear, require the surface lubricin layer to be

capable of strongly adhering to the cartilage surface. COMP and lubricin

are known to associate in bulk and in one of the recent works of my

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Chapter 1

6

collaborators [16], both were found to co-localise at the cartilage surface.

In light of these novel findings, I wished to investigate the interaction of

this lubrication couple.

COMP

COMP has piqued the interest of many researchers, especially in the

recent past. It plays a role in maintaining the integrity of the knee joint

[17]. Mutations in the COMP gene induce skeletal disorders such as

Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia

(MED) [18]. Even though the concentration of COMP in the cartilage is

relatively low [19], the crucial functions it has and it’s ability to form

synergistic structures, can not in the least be ignored.

Figure 2: Schematic of a native COMP molecule

COMP is a non-collagenous, extra cellular matrix protein found in the

cartilage, ligaments, tendons, synovium, meniscus, and osteoblasts [20].

It is a 524 kDa pentameric glycoprotein having multiple domains, and is

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Chapter 1

7

stabilised by disulphide bonds [21] as shown in figure 2. The chains of

this bouquet-like protein are connected at its amino terminus by a coiled-

coil domain. Further, it comprises four small globular epidermal growth

factor (EGF) like domains, type III (calcium binding) domain, and is

terminated by a large carboxyl globular domain [21].

Fun facts: COMP mutation associated with disorders such as dwarfism

and early onset osteoarthritis, mainly occurs in the type III domain of

COMP molecule [18].

Lubricin

As the name implies, lubricin is a mucinous glycoprotein, which

contributes to the lubricating ability of the cartilage. The absence and

mutation of the lubricin gene have been linked to decreased joint

lubrication, increased cartilage wear [22, 23], and the rare

camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome

[23]. Lubricin is found in the superficial zone of the articular cartilage,

synovial membrane, and synovial fluid [24]. The concentration of lubricin

is 0.052–0.450 mg/mL in healthy synovial fluid [25]. In humans, the

proteoglycan 4 (PRG4) gene encodes it.

Figure 3: Schematic of a lubricin molecule

The average length of lubricin first observed by Swann et al. was 222 nm

and a diameter of 1-2 nm. It has a molecular weight of 227.5 kDa [26].

Lubricin is primarily a flexible rod [26] constituting a large mucin-like

domain. This central domain has anionic side chains that participate in

O-linked glycosylation (nearly 50% w/w) with NeuAc-Gal-GalNAc. Two-

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Chapter 1

8

thirds of these sugar groups are capped by charged sialic acid [12, 13].

This region accumulates most of the negative charge of the protein, and

due to heavy glycosylation renders it hydrophilic. Two non-glycosylated

end domains lie adjacent to the central domain. These subdomains at the

left and right flanks are similar to two globular proteins; Somatomedin-B

(SMB) at the N terminus, and Hemopexin (PEX) at the right terminus.

Positive charge is mainly located in these domains. Due to the presence of

cysteine residues in the end domains lubricin can form intra and

intermolecular disulphide bonds, thereby enabling it to form

supramolecular structures [22].

Fun facts: When 54% or less of β (Gal-GalNAc) was removed from

human lubricin, the lubricating ability was reduced by almost 80% [13].

1.3.2 Phospholipid-hyaluronan model system

Hyaluronan has long been implied to play a major role in the lubrication

of the synovial joints. However, it has been shown that hyaluronan alone

can not provide low friction and therefore, is a rather poor boundary

lubricant [27]. Phospholipids so far are the only key biolubricant to have

provided both low friction and high load bearing capacity on artificial

surfaces [28]. It has been concluded after assessment of the lubricating

abilities of DPPC and hyaluronan on an osteoarthritis (OA) damaged

cartilage, that greatest improvements are seen when both are

administered together [29]. Both DPPC and hyaluronan are in plenty in

the knee joint, and are known to form association structures [30, 31].

Given these reasons, we link the appreciable lubrication performance of

this model system to the interplay between these components.

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Chapter 1

9

DPPC

The total amount of phospholipids in human synovial fluid ranges from

0.1–0.2 mg/mL [32]. Three major classes of phospholipids have been

identified from bovine articular cartilage surfaces. These were

phosphatidylcholine (41%), phosphatidylethanolamine (27%), and

sphingomyelin (32%). Modern lipodimic analysis also showed that

phosphatidylcholine was the predominant phospholipid [33]. Among the

phosphatidylcholines, unsaturated acyl chains were predominant. At 11%

and 15%, DPPC was the most abundant among the saturated lipids

obtained from analysis of the respective polymeric and metallic

component of retrieved implants [34]. As DPPC is saturated and not

prone to degradation and oxidation, it is easier to handle. These reasons,

along with the fact that it has been well-characterised in the literature,

has helped me choose DPPC to conduct various investigations for the

better part of my studies.

Figure 4: Sketch of a (C 16:0) DPPC molecule

DPPC consists of a hydrophilic zwitterionic headgroup (comprising a

positively charged choline group and a negatively charged phosphate

group) linked by a glycerol group to two saturated hydrocarbon chains as

shown in figure 4. It has limited solubility in water with a CMC of 0.46

nM [35]. DPPC molecules tend to assemble spontaneously and form

bilayer structures in aqueous solutions and on surfaces, where the

hydrophilic head group interacts freely with water and hydrocarbon

chains are facing inwards, away from water. In the course of this work

DPPC bilayers were formed on silica surfaces from DPPC vesicles

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Chapter 1

10

solution via spontaneous vesicle rupture. Details on preparation of the

vesicle solution can be found in the papers appended to the thesis. Above

≈ 41 °C the bilayers are in the liquid disordered phase where the chains

are fluid-like, and below this temperature they are in the gel phase where

the chains are solid-like. In the subsequent sections of this thesis we shall

visit this aspect further and discuss its relevance to our model system.

Hyaluronan

Out of all the components that I have considered for my doctoral studies,

hyaluronan is the most abundantly available in the synovial joints. In

healthy knee joints, it is present both at the cartilage surface and in the

synovial fluid at a concentration of 1.45–3.12 mg/mL [36]. Hyaluronan

was used across a range of molecular weights in my work; 10 kDa to 1500

kDa. As shown in figure 5 hyaluronan is a linear polysaccharide

consisting of repeating units of D-glucuronic acid and N-acetyl-

glucosamine. The pKa of D-glucuronic acid is about 3.3 [37] and

dissociation of the acid makes hyaluronan negatively charged in

physiological solution. Hyaluronan has an intrinsic persistence length of

about 9 nm [38]. It has a unique viscoelastic nature; wherein it can form

a viscoelastic gel at higher concentrations, which is broken down under

high shear forces and then reversibly restored as shear is reduced [39].

Figure 5: Molecular structure of hyaluronan

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Chapter 1

11

Hyaluronan is one of the most highly hydrated molecules in nature [40]

and capable of retaining water. However it is important to note that in its

secondary structure, hyaluronan has a hydrophobic patch [41]. This

property allows hyaluronan to associate with itself and other molecules

that are of relevance in my model system, such as DPPC. Thus,

hyaluronan is highly hydrophilic with hydrophobic patches, making it

amphiphilic in nature.

Fun facts: The consistency, shear thinning, biocompatibility and water

retaining ability (up to 1000 times its own weight) of hyaluronan make it

a popular choice for skin care products like moisturisers [40] and for

achieving anti-wrinkle effects in injectable fillers [42].

1.4 Surface interaction and friction forces

1.4.1 Van der Waals force and electrical double layer force (DLVO-theory)

In order to analyse our model system, it is important to interpret the

surface forces that come into play when two opposing surfaces interact.

This sheds light into the behaviour of the system and can complement

dynamic interactions such as those that have been studied by conducting

friction measurements. The following section describes friction,

lubrication, and (at the outset) the main characteristics of the surface

forces that are of primary importance to the model systems investigated

in this work. These surface forces, depending on their nature, have been

classified as attractive or repulsive.

Van der Waals force

The van der Waals force originates due to interaction between fluctuating

electromagnetic waves extending from the surface of a material. It is a

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Chapter 1

12

result of (i) Orientation force (ii) Induction force and (iii) London force,

with London force being the dominant one as it acts between all

molecules and atoms [43].

The van der Waals force is attractive between similar surfaces and may be

repulsive between dissimilar ones. It is always present and relatively

short ranged in nature. It is not simple to calculate this force, but it can

be best described by the Lifshitz theory [44]. This theory ignores the

molecular nature of the interacting bodies and the intervening medium;

rather it employs a continuum approach.

The van der Waals interaction of two media across a third intervening

medium, can be calculated depending on the geometry being considered.

In my work, we consider the case of a sphere against a flat surface. By

convention, negative sign denotes attraction and a positive sign denotes

repulsion. The van der Waals force in this case is given as follows [45]:

𝐹 (𝑑)𝑣𝑑𝑊 = − 𝐴𝐻𝑅6𝐷2

(1)

Where 𝑅 is the radius of the sphere, 𝐷 is the distance between the surface

and the sphere, 𝐴𝐻 is the Hamaker constant and can be calculated by

[45]:

𝐴𝐻 = 34

𝑘𝐵𝑇 (𝜀1 − 𝜀3𝜀1 + 𝜀3

) (𝜀2 − 𝜀3𝜀2 + 𝜀3

) +

3ℎ𝑣𝑒8√2

(𝑛12 − 𝑛32)(𝑛22 − 𝑛32)√(𝑛12 + 𝑛32)√(𝑛22 + 𝑛32)[√(𝑛12 + 𝑛32)+ √(𝑛22 + 𝑛32)]

(2)

Where 𝜀𝑖 is the static dielectric constant of medium 𝑖, 𝑛𝑖 is the refractive

index of medium 𝑖, ℎ is Planck’s constant, 𝑣𝑒 is the dominant electronic

absorption frequency, 𝑘𝐵 is Boltzmann’s constant, and 𝑇 is the absolute

temperature.

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Chapter 1

13

Electrical double layer force

Most surfaces in water obtain a surface charge either by surface

dissociation or adsorption of charged species. This surface charge is

countered by oppositely charged ions distributed outside the surface.

Together the surface charge and these ions form the electric double layer.

When another such charged surface is brought close together, the

concentration of charged ions in the gap between the surfaces increases.

This generates an osmotic repulsion and thus a repulsive force, which at

large separations decays exponentially. The interaction free energy at

large separations is given by:

𝑊 (𝑑)𝐸𝐷𝐿 = 𝐶𝑒−𝜅𝐷 (3)

Here, 𝐷 is the distance between the two surfaces, and 𝐶 is a constant that

depends on surface charge density and solution conditions. The range of

the interaction is characterised by the Debye length, 𝜅−1, and depends on

the salt concentration. The Debye length decreases with an increase in

salt concentration. In water at 25 °C, for a monovalent electrolyte the

Debye length is [43]:

𝜅−1 = 0.304

√𝑐

(4)

Here, 𝑐 is the salt concentration and given in molar, and the Debye length

in nm. In my experiments, a salt concentration of 155 mM was used.

Plugging this in the equation above results in a Debye length of 0.77 nm.

This means that the double layer force is of short range and consequently

contribution of other forces (mentioned subsequently) would be more in

my studies.

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Chapter 1

14

I note that in my studies similar surfaces were always considered, and in

this case the double layer force is repulsive. However, for surfaces with

opposite charge the double layer force is attractive.

The van der Waals force and the electrical double layer force together are

considered in the DLVO theory. The interplay of these forces describes

the net interaction between two surfaces. However, in a system several

other forces may contribute significantly. Some of these non-DLVO forces

include hydration, protrusion, and hydrophobic forces and these will now

be discussed.

1.4.2 Attractive forces

Hydrophobic attraction

Hydrophobic interactions are prevalent and of importance in nature. For

example they are of relevance to cell membranes, protein folding, and

DNA double helix stabilisation. When water molecules come in contact

with a hydrophobic molecule, the water molecules undergo

rearrangement and restructuring to maintain hydrogen bonding [46].

However, such a reorientation and restructuring of water molecules is

entropically unfavourable. Thus, in order to minimise this disturbance

hydrophobic molecules tend to cluster, and the resultant force between

them is known as the hydrophobic interaction [47]. In water, as a result

of the hydrophobic interactions, hydrophobic surfaces attract each other

as the coming together of such surfaces is entropically favourable.

In my work hydrophobic interaction explained self-assembly structures

formed by DPPC bilayers. It also possibly causes the association of DPPC

and hyaluronan, wherein the hydrophobic patches of hyaluronan

associate with the hydrophobic chain of the DPPC molecules. Another

example would be the adsorption of COMP and lubricin driven by

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Chapter 1

15

hydrophobic interaction between these molecules and the PMMA model

surface.

Bridging attraction

Bridging forces can influence interaction of two surfaces coated with a

polymer. When opposing surfaces are close enough and have vacant

binding sites, the polymer chains may bridge both the surfaces. This

makes it possible for the polymer chain to adopt many favourable

conformations, which leads to an increase in entropy. Upon separation of

these surfaces, detachment is resisted and attractive bridging interaction

persists. In my work I have encountered such forces in the case of

adsorption of lubricin on the PMMA surface. Interestingly, this

phenomenon is witnessed in other biological systems as well. Examples

include polymer bridges that were found to connect myelin membranes

and prevented them from escaping by keeping them at an appropriate

distance [48], and surface bound polymers containing ligand that exposes

the binding sites to bring it to the receptor [49].

1.4.3 Repulsive forces

While the important attractive forces that are most relevant to my work

have been briefly discussed above, one can imagine what would follow if

these alone were to exist, especially in the physiological context.

Repulsive forces help to keep a system stable and safely separated.

Protrusion force

As two opposing amphiphilic surfaces approach each other, protrusion

forces become important. Thermal motion of the molecules drives this

force. The protruding segments are forced into the surface and their

motion is perpendicular to the surface in an in and out manner [50].

When two surfaces are close enough, motion of protruding segments is

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Chapter 1

16

restricted, which leads to a decrease in entropy and thus this short-range

steric force is repulsive in nature. Protrusion forces have been

encountered in my work in relation to DPPC phospholipid bilayers and

that too at different temperatures.

Hydration force

Water molecules are known to bind strongly to surfaces with hydrophilic

groups. When opposing surfaces approach each other, it costs energy to

dehydrate the surfaces, and thus short-range repulsive hydration forces

arise. The strength of this hydration force depends on the energy required

to dehydrate the surface. In my studies, these forces were seen at < 5 nm

(between 1-3 nm) in the case of both silica and DPPC-coated surfaces.

Steric force

When two surfaces covered with polymers/polyelectrolytes approach

each other and their segments begin to overlap, a repulsive force is

generated due to the unfavourable entropy associated with the confining

of the chains. Further, this force depends on factors such as coverage of

polymer on the surface, type of adsorption of polymer to the surface, and

quality of the solvent [50]. This force has often been seen in my studies.

Examples include the steric repulsive force resulting from the adsorption

of COMP on the PMMA surface, and that of premixed DPPC-hyaluronan

on the silica surface.

1.4.4 Friction force

Friction is the resistance to relative motion of surfaces sliding against

each other. Depending on the requirement of a system, friction may be

regulated. In gripping things or applying brakes of a vehicle, friction is

desirable, whereas in knee joints, implants or contact lenses, it needs to

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Chapter 1

17

be small. Friction is often described by Amonton’s first rule, which is

mathematically defined as:

𝐹𝑓 = 𝜇𝐹𝑛 (5)

Here 𝐹𝑓 is the friction force, 𝐹𝑛 the applied load, and 𝜇 is the friction

coefficient. This is an empirical rule, wherein the friction coefficient

depends on the system, and the friction force does not depend on the

macroscopic area. In some of the systems that I investigated Amonton’s

first rule applied, whereas in others it did not. In cases when it did not

apply, it was due to new energy dissipative mechanisms (e.g. adhesion)

coming in to play. Such attractive interactions then modify the rule to:

𝐹𝑓 = 𝜇𝐹𝑛 + 𝐶 (6)

Where 𝐶 is the friction force at zero load. Furthermore, when both these

rules do not hold, it is appropriate to define an effective friction

coefficient as:

𝜇𝑒𝑓𝑓 = 𝐹𝑓

𝐹𝑛

(7)

Load bearing capacity is a recurrent term in this thesis. We define it as

the load at which the lubricating layer starts to wear off. This is seen by a

superlinear increase in friction force with load. For polymer-coated

surfaces several energy dissipation mechanisms can contribute to friction

[51]. These include formation and breakage of attractive bonds between

segments on opposing surfaces, formation and breakage of polymer-

surface bonds, and polymer sliding past polymer in the interpenetration

zone. The energy dissipated, 𝑊, due to sliding a distance 𝑙 is then given

by:

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Chapter 1

18

𝑊 = 𝐹𝑓𝑙 (8)

Friction measurements have been conducted in my work using the AFM

technique, which will be discussed further in section 3.1.2.

Biolubricants and biolubrication helps in maintaining low friction,

characteristic to our biological model system. This low friction was

attained by virtue of various different mechanisms and phenomena. In

my studies lubricin provided low friction by (i) being well anchored to

COMP-coated PMMA surface and thus avoiding breakage and formation

of segment-surface attachment points (ii) having side chains that reduced

interpenetration and (iii) having side chains that interact favourably with

water and thus avoid segment-segment attraction. Phospholipid bilayers

have demonstrated very low friction upon shearing. The reduction in

friction is attributed largely to hydration lubrication and this mechanism

of significance will be discussed below.

1.4.5 Hydration lubrication

Hydration lubrication has been of critical importance in my studies.

Repulsive hydration/protrusion forces are able to withstand high loads

and large pressures. At the same time, the fluid-like manner in which the

surrounding fluid behaves when the opposing surfaces are sheared,

allows for the fluid to be easily sheared and thereby accounts for the low

friction and excellent lubrication. This is particularly relevant in

biological systems as in ours, where the natural medium is water. High

hydration in case of phospholipid bilayers and lubricin in my model

systems, are able to bear high normal loads and easily shear the

surrounding water layers. This may reason for the very low friction

witnessed in both the systems. The plane of shear between two opposing

bilayers surrounded by water is depicted below. In case there is a water

layer present between the phospholipid headgroup and substrate, an

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Chapter 1

19

additional shear plane will exist there. This is a possibility even though

we could not find evidence for water between the silica surface and the

DPPC bilayer in our X-ray reflectivity data.

Figure 6: Interpretation of opposing phospholipid bilayers indicating position of shear plane

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Chapter 2

20

2. Materials The following materials were used for the investigations carried out in my thesis work:

Material Purchased/Received Comments

DPPC Avanti Polar Lipids, Inc.,

Alabama, USA

Powder form

Hyaluronan Mw 10 kDa, 250 kDa, 750

kDa, 1500 kDa, 2500 kDa

Creative PEG works, USA

Mw 620 kDa kindly gifted by

Novozymes, Denmark

Powder form

Recombinant

COMP

R&D Systems, Inc., USA COMP used in this work

was in monomeric form

(Mw 81.8 kDa), while

native COMP is in

pentameric form.

Lubricin and

reduced

lubricin

Kind gift from Dr. Niclas

Karlsson, Gothenburg

University, Sweden

Collected and isolated

from synovial fluid from a

pool of patients diagnosed

with rheumatoid arthritis

and spondyloarthritis (for

reduced lubricin).

Dialysed and lyophilised

protein powder was

provided and used.

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Chapter 2

21

Sodium

chloride

Sigma Aldrich ACS reagent, assay > 99%

Phosphate

Buffered

Saline

Sigma Aldrich Sigma P4417

Calcium

chloride

dihydrate

Sigma Aldrich ACS reagent, assay > 99%

Water Purified using Millipore

system comprising Milli-Q

system with 0.22 μm

Millipak filter.

Resistivity of purified

water was 18.2 MΩcm at

25 °C and total organic

carbon content < 3 ppb.

QCM crystals AT cut silica crystals,

AT cut PMMA crystals

Q-sense, Gothenburg,

Sweden

QSX 303

QSX 999

Silica wafer Wafer net, Inc., USA In paper 3, wafer surfaces

were roughened to

amplify difference in

friction measurement

between bare and coated

surfaces

Cantilevers Mikromasch, Germany Rectangular tipless

cantilever for force and

friction measurements,

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Chapter 2

22

silicon nitride tip for AFM

imaging

Probes Silica colloidal probes, Bang

Laboratories, Fishers, USA

PMMA colloidal probes,

Kisker, Germany

All chemicals were used without further purification while colloidal

probes and surfaces were cleaned prior to use as specified in each

publication.

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Chapter 3

23

3. Instruments

3.1 Atomic force microscopy (AFM)

AFM is a type of scanning probe microscopy technique, which provides

high resolution imaging of the topography of a sample surface. Sample

surface interactions can also be measured using an AFM. I have used the

AFM to measure both normal and friction forces. The key elements of an

AFM include a sample surface, laser beam probe, cantilever spring

(rectangular), piezo scanner, and a photo diode. The working of an AFM

for normal force and friction measurements in my project is such that the

probe is glued at the end of the cantilever and scans the sample surface,

which is mounted on a scanner (as shown in figure 7). The scanner allows

for movement in the x-y-z direction. A laser beam is focused on the

backside of the cantilever and reflected off to the detector system, i.e. the

photodiode. The photodiode is split into four quadrants and detects

changes associated with the deflection of the cantilever. Vertical shifts are

related to force measurements and horizontal shifts are associated with

friction measurements. In this work for normal force and friction

measurements, the colloidal probe microscopy technique was employed

[52]. A spherical silica or PMMA particle (size range 5 to 20 μm) was

glued to the end of the cantilever using a micromanipulator connected to

an optical microscope. This imparts advantages over a sharp tip

including, a well-defined geometry, Derjaguin’s approximation being

valid, larger forces between probe and surface and therefore more

accurate measurement.

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Chapter 3

24

The AFM can be used in different modes according to the information

required. Peak force tapping mode has been used in this work to perform

imaging. In this mode, the sharp tip and sample are in contact

intermittently and due to the short period of contact, lateral forces are

reduced. There is a feedback loop that keeps the maximum force

imparted on the tip constant, thereby protecting both the tip and sample

from damage. This is beneficial for soft samples such as ours, which are

easy to damage.

Figure 7: A schematic illustration of the main components of an AFM. The colloidal probe is

used for normal force and friction measurements, while a sharp tip is used for imaging.

3.1.1 Force measurements

Surface force interactions were investigated with the help of AFM

colloidal probe force measurements. These measurements are made by

ramping the probe (normal direction) with respect to a surface and

monitoring the cantilever deflection as a function of piezo expansion. The

probe and surface approach each other from a large distance where no

sample surface

probe

piezo scanner

laser

photodiode

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Chapter 3

25

force acts on the probe. At shorter distances, attractive and repulsive

forces are experienced depending on the type of interactions. Once the

probe and surface come into physical contact, the probe deflects the same

amount as the surface moves. This corresponding linear region of the

force-displacement curve gives us the constant compliance region. The

surfaces are then snapped apart as they are retracted. In actuality it is the

sample that is moved. The force curve consists of an approach and

retraction cycle. The deflection of the cantilever as a function of piezo

expansion is converted to force as a function of probe-sample separation.

This is done by the AFM Force IT software (Force IT, Sweden). In theory:

(i) The cantilever deflection measured in volts 𝑍 (𝑉) is converted to

metres 𝑍 (𝑚) by multiplying it with deflection sensitivity 𝛿 (inverse of

slope corresponding to constant compliance region) as [53]:

𝑍 (𝑚) = 𝑍 (𝑉) × 𝛿 (𝑚𝑉 ) (9)

(ii) This deflection in metres is then converted to force (𝑁) by Hooke’s

law:

𝐹𝑛 = 𝑘𝑛 𝑍 (𝑚) (10)

Where 𝑘𝑛 is the normal spring constant that is determined from a

separate calibration step. The normal spring constant was calibrated

using the Sader method [54]. In order to make comparisons of two

different experiments, the force 𝐹 is normalised by radius of the probe 𝑅

with the help of Derjaguin’s approximation using [55]:

𝐹(𝐷)𝑅 = 2𝜋𝑊(𝐷)

(11)

Where 𝑊 (𝐷) is the interaction free energy per unit area between two

surfaces separated by a distance 𝐷.

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Chapter 3

26

(iii) The separation of surfaces is then obtained from the combined

movement of the piezo and cantilever. It should be noted though that the

constant compliance provides only a relative zero of separation especially

in the case of soft samples where physical contact occurs before constant

compliance is reached. Thus to minimise error, the deflection sensitivity

used is measured between hard substrates before the adsorption of soft

layers.

3.1.2 Friction measurements

While normal force measurements are done by the vertical movement of

the probe with respect to the sample, friction measurements are done by

sliding the probe laterally with respect to the sample surface. During a

friction measurement a certain load is applied and the probe and sample

are brought into contact. The generation of friction between the probe

and surface during the lateral movement, results in the twisting of the

cantilever. This twisting of the cantilever (both back and forth) is

recorded by the lateral deflection of the signal. The lateral deflection is

converted to friction force using the following equation:

𝐹𝑓 = ∆𝑉𝑙𝑎𝑡𝑘𝑡

2ℎ𝑒𝑓𝑓𝛿 (12)

Where 𝑘𝑡 is the torsional spring constant (determined by the hybrid

method [56]), ℎ𝑒𝑓𝑓 is the effective height of the probe (diameter of probe

plus half the thickness of the cantilever), 𝛿 is the torsional detector

sensitivity, and ∆𝑉𝑙𝑎𝑡 is the lateral output voltage.

The data was processed using the Friction (IT) software (Force IT,

Sweden) mentioned earlier. The friction force can then be measured at

different loads, and for a predetermined number of loops at the same

load (typically ten in my work), which can then be averaged. The average

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Chapter 3

27

is then plotted as a function of the applied normal load. When converting

the applied load to pressure, the following formula is used:

𝑃 = 𝐹𝑛

𝐴 (13)

Here 𝐹𝑛 is the applied load and 𝐴 is the contact area that can be calculated

by using the JKR or Hertz theory [57]; whichever is more appropriate to

the given system. The JKR method is preferred for soft and adhesive

surfaces, and the Hertz method for hard and non-adhesive surfaces.

3.2 Quartz crystal microbalance with dissipation (QCM-D)

The QCM-D is able to detect both frequency and dissipation changes due

to e.g. adsorption and is therefore a good tool to characterise samples of

adsorbed layers. The QCM-D consists of a quartz crystal sensor

sandwiched between electrodes. An AC voltage is applied to the crystal,

which causes it to oscillate at its resonance frequency. In case of

adsorption of material on the quartz surface, there will be an associated

change in mass. This change in mass is in the simplest case directly

related to the accompanied change in frequency of the oscillating crystal.

It should be noted that the mass we refer to, is actually the sensed mass,

which includes the mass of the absorbed material and the coupled water.

When the driving voltage is shut off, energy from the oscillating crystal is

dissipated from the system. Dissipation is measured by monitoring the

amplitude decay profile of the oscillator. It is defined as [58]:

𝐷 = 𝐸𝑙𝑜𝑠𝑡

2𝜋𝐸𝑠𝑡𝑜𝑟𝑒𝑑

(14)

𝐸𝑙𝑜𝑠𝑡 is the energy dissipated per oscillation, and 𝐸𝑠𝑡𝑜𝑟𝑒𝑑 is the total energy

stored in the system.

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Chapter 3

28

The change in frequency and dissipation were measured using the

programme Q-tools (from Q sense, Gothenburg). These were then used to

calculate changes in mass. There are a few models that can be adopted to

do so. The Sauerbrey equation is used to calculate changes in mass for

films that are rigid, and sufficiently thin. The linear relationship between

changes in frequency, ∆𝑓 and mass, ∆𝑚 is given by [59]:

∆𝑚 = − 𝐶 ∆𝑓𝑛

𝑛 (15)

Where 𝐶 is a constant based on the crystal property, and for the crystal

used in this work it is 17.7 ng cm-2 Hz-1, and 𝑛 is the overtone number.

However, if the adsorbed layer is viscoelastic in nature, the Sauerbrey

relation underestimates the mass, and another model is then used to fully

characterise the film to take into account the energy losses. In my studies

the Voigt model [60] was used. The viscoelastic response of the film was

represented by an elastic component coupled in parallel to a viscous

component. Again the software Q-tools (Q sense) was used to analyse the

data using Voigt viscoelastic modelling. The programme uses data from

different overtones together to make the calculations. In my work the

third, fifth, and seventh overtones were used. The following equations are

used to calculate changes in frequency and dissipation:

∆𝑓 = −

12𝜋𝜌0ℎ0

{𝜂2

𝛿2+ ℎ1𝜌1𝜔 − 2ℎ1 (

𝜂2

𝛿2)

2 𝜂1𝜔2

𝜇12 + 𝜂1

2𝜔2} (16)

∆𝐷 =

1𝜋𝑓𝜌0ℎ0

{𝜂2

𝛿2+ ℎ1𝜌1𝜔 + 2ℎ1 (

𝜂2

𝛿2)

2 𝜂1𝜔2

𝜇12 + 𝜂1

2𝜔2} (17)

Where 𝜌 is density of quartz, ℎ is thickness of quartz, 𝜂 is the shear

viscosity, 𝛿 is the viscous penetration depth, 𝜇 is the shear elasticity, and

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Chapter 3

29

𝜔 is angular frequency of oscillation. The subscripts correspond to the

respective layers, i.e. 0 for the crystal, 1 for the adsorbed layer, and 2 for

the bulk solution.

3.3 Table of other instruments

Other techniques that provided useful information in this work are listed

below. The relevant information obtained in the context of this thesis has

also been summarised and included in the following table.

Instrument and measurement Information obtained

X-ray reflectivity (XRR) [61] Vertical structure of adsorbed bilayers and composite layers at silica - water interface (normal to the surface), and Langmuir layers at water - air interface. One can also learn of the structural variations, thickness, and roughness of a film.

Small angle X-ray scattering (SAXS) [61]

The self-assembly structure at the nano scale formed in mixed hyaluronan and DPPC vesicle solutions, presence of double bilayer vesicles, repeat distance of lamellae.

Electrophoretic mobility [62] The sign of the charge of the aggregates and the mobility with which they travel towards oppositely charged electrodes.

Structural organisation of Langmuir monolayers

Lateral interactions between and phase behaviour of the molecules in the monolayers

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Chapter 4

30

4. Key results and discussion

As mentioned earlier, it is of importance to learn if COMP and lubricin

associate favourably in our model system, what impact COMP has in

retaining lubricin on the surface, and consequently on the lubrication

performance. Studying the biolubricants in question on hydrophobic

PMMA model surfaces, first individually and then synergistically followed

this line of thought.

4.1 COMP-lubricin synergy system

4.1.1 COMP on PMMA

As seen from QCM-D measurements (figure 8), COMP adsorbs on the

PMMA surface from a 100 μg/mL solution of COMP in a 150 mM PBS

solution. The resulting layer is about 23 nm thick and has a sensed mass

of 26.5 mg/m2. Additionally we know from the AFM normal force

measurements, that when COMP adsorbs to two opposing surfaces that

slide against each other, the force generated is purely repulsive in nature.

Figure 8: Adsorption of COMP on PMMA at 25°C from 100 μg/mL solution dissolved in 150 mM PBS, pH 7.5. Rinsing was done with 150 mM PBS (marked by ↓ arrows). Solid and open symbols are frequency change and dissipation change, respectively. For clarity, only every 20th point is plotted.

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Chapter 4

31

This means that the lubricating layer should be sheared rather easily, and

would likely result in low friction. Compared to PMMA-PMMA and

lubricin-PMMA surfaces, the resulting friction for COMP covered PMMA

surfaces is low and the friction coefficient is ≈ 0.3 (figure 9 bottom).

Figure 9: Friction force as a function of applied load between (top) PMMA surfaces carrying a weakly adsorbed lubricin layer on loading (solid squares) and unloading (open squares) (bottom) PMMA surfaces coated with COMP (solid circles) and COMP-lubricin (solid triangles).

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Chapter 4

32

4.1.2 Lubricin on PMMA

The lubrication performance of the lubricin layer formed on PMMA

surface was worse than that offered by COMP alone. From surface

interaction measurements, repulsive surface forces were seen (on

approach) at large separations. However upon compression, structural

rearrangements ensued. Some bridging was also seen in the force curves

on retrace. The adsorption of lubricin on PMMA was seemingly driven by

hydrophobic interactions between the non-glycosylated parts of lubricin

and the hydrophobic PMMA surface. Due to the weak attachment of

lubricin to PMMA, the resulting friction is significantly higher as

compared to that of COMP on PMMA (figure 9). Also there is clear

removal of some lubricin from the surface due to the combined action of

shear and load, since friction is higher on unloading compared to loading.

This is in agreement with what has been reported in literature about

lubricin being able to adsorb to model surfaces, but unable to individually

impart lubrication close to that experienced in the joints [15, 22].

4.1.3 COMP-lubricin synergy on model surfaces

As can be seen from the results above, it is evident that while COMP is

more resistant than lubricin to failing under the combined action of load

and shear, the friction levels attained are still not close to those

experienced physiologically in the joints. Thus we probed into the

lubrication performance resulting from the combined effect of COMP and

lubricin, i.e. with COMP forming the underlying layer and attached

lubricin being exposed to the aqueous solution. The results were notably

better. The resulting structure imparts very low friction with a friction

coefficient of 0.06, and load bearing capacity of minimum 7 MPa (refer

once again figure 9). Additionally, the combined COMP-lubricin layer has

high resistance to wear. COMP and lubricin associate specifically by non-

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Chapter 4

33

covalent and covalent interactions via cysteine residues present at the C

terminal of COMP and the N terminal of lubricin. This in turn leads to

exposure of the glycosylated region of lubricin to the aqueous solution.

Figure 10: Adsorption of lubricin on COMP-coated PMMA at 25°C from 100 μg/mL solution in 150 mM PBS, pH 7.5. Rinsing was done with 150 mM PBS (marked by ↓ arrows). Solid and open symbols are frequency change and dissipation change, respectively. For clarity, only every 30th point is plotted.

QCM-D studies give insights into the nature of the formed layer (figure

10). 100 μg/mL human lubricin dissolved in 150 mM PBS was injected on

COMP-covered PMMA surface, and the lubricin layer on top of the COMP

layer was found to be viscoelastic with a thickness of about 20 nm and

sensed mass of 23 mg/m2.

These results underscore the importance of synergism when discerning

mechanisms that govern joint lubrication. The desirable lubrication

performance, which lubricin and COMP are unsuccessful in achieving

individually, is attained through their joined efforts. Lubricin associates

with COMP specifically and requires it as an underlying support. Zappone

et al. have also demonstrated favourable resistance to shear offered by

the tenacious anchoring of end domains of lubricin boundary layer [22].

As a consequence of the firm attachment, the structure in this system is

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Chapter 4

34

better able to resist wear and the specific association allows lubricin to

expose parts of its structure in a manner that renders superb lubricity to

sliding model surfaces covered with these biolubricants.

4.2 Phospholipid-hyaluronan synergy system

The other lubrication couple that has been investigated at length in my

thesis is DPPC and hyaluronan. Naturally, before I began to look into

their association structures, it was imperative to characterise DPPC

bilayers. Adsorption of DPPC on a hydrophilic silica support and the

subsequent formation of bilayers in fluid phase have been elaborated by

our group earlier [30, 63]. Free hydrated DPPC bilayers undergo a main

phase transition at about 41 °C [64]. It is improbable that in physiological

scenario the complex mixture of phospholipids (including unsaturated

phospholipids) found in the synovial joints is in the gel state with frozen

acyl chains. To elucidate how the state of the DPPC layer affected

lubrication, I have studied this lipid across a range of temperatures and

phase transition on silica surfaces [65].

4.2.1 DPPC bilayer morphology at different temperatures

The DPPC bilayer structure was investigated by the XRR technique.

Figure 11 depicts the modelled electron density profiles, which were fitted

to experimental data using a six-layer model. The figure also illustrates

the corresponding structure and orientation of the bilayer; the left side of

the bilayer is next to the silicon wafer and the right side is oriented

towards the aqueous phase. It can be inferred from the profiles, that at 25

°C (and also 39 °C) the thickness of the bilayer is 5.6 nm, whereas at 55

°C it is 4.8 nm. This decrease in bilayer thickness indicates that a

transition occurred from the gel phase to the liquid disordered phase.

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Chapter 4

35

Figure 11: Electron density profiles of DPPC bilayers at the silica liquid interface at different temperatures, ‘z’ denotes the distance from the silicon surface. A sketch of the bilayer structure is also shown.

Information on lateral variation across bilayer adsorption on the silica

surface was obtained using AFM imaging. DPPC bilayers were adsorbed

on silica surface at 52 °C and temperature variations were made by

following a sequence of 52 °C o 47 °C o 37 °C o 32 °C o 25 °C, and

then reversed to 32 °C o 37 °C o 47 °C o 52 °C.

Figure 12: AFM PeakForce height images of a DPPC bilayer on a silica surface, taken at different temperatures. The images were recorded in 150 mM PBS buffer solution, and flattened to remove tilt. The image size is 1x1 µm2, and the height scale bar is 3 nm in each case. The arrows show the order in which the images were recorded, and the temperature is provided below each image.

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Chapter 4

36

Images recorded at 52 °C, 47 °C, and 37 °C were similar and in the liquid

disordered phase. A visible change in the form of small and grainy

structures started to occur at 32 °C, below which the bilayer was in gel

phase. This difference in the phase transition occurring at a lower

temperature of 32 °C in the case of AFM imaging and at a higher

temperature of 39 °C in the case of XRR, may be accounted to the kinetic

energy transferred by the tapping AFM tip, which resultantly causes a

shift in phase transition temperature.

4.2.2 Lubrication performance of DPPC bilayers at different temperatures

Force and friction measurements were performed on DPPC bilayer

covered silica surfaces at varying temperatures to get insights into the

lubrication performance of DPPC bilayers in the different phases.

There were no long-range forces observed when force measurements

were conducted on DPPC covered silica surfaces. Only short-range

repulsive forces arising from hydration and protrusion forces were

observed. Further, these repulsive forces increased with temperature.

Friction forces were measured at different temperatures on silica surfaces

covered with the DPPC bilayer, and it was seen that low friction forces

prevailed across the range of temperatures. The friction coefficient at <

47 °C is low (< 0.03) and nearly independent of temperature (figure 13).

The appreciable lubrication performance is ascribed to protrusion and

hydration forces, which makes shearing of the water layers - separating

the opposing bilayers easy.

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Chapter 4

37

Figure 13: Friction force as a function of load between DPPC bilayer-coated silica surfaces immersed in 150 mM PBS at different temperatures.

The maximum load that the bilayer is subjected to in this system is 20

nN. When converted by the JKR theory [57] this corresponds to 42 MPa

(nearly double that which the cartilage can sustain [66]). However, it was

also noted that in some cases the bilayer was destroyed, particularly at

lower temperatures. An example of a friction versus load cycle where this

was observed at 25 °C is presented in figure 14.

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Chapter 4

38

Figure 14: Example of a friction vs. load cycle measured between DPPC bilayers at 25 °C in 150 mM PBS. In this case the bilayer structure was compromised at a load of about 18 nN. Friction forces measured on loading and unloading are shown with filled (●) and unfilled (○) symbols, respectively.

The bilayers were intact and able to withstand loads up to about 18 nN.

As the load is increased further, friction increases significantly and the

bilayers give in. This is followed by the advent of a new energy dissipative

mechanism that increases the friction force. One such energy dissipative

process may arise from attractive hydrocarbon-hydrocarbon contacts of

phospholipids chains between the sliding surfaces. As the load was

decreased, the friction force failed to return to its original value indicating

that the DPPC bilayers do not heal.

Since the load bearing capacity at a given temperature differed between

different experiments, a statistical evaluation was used to assess the load

bearing capacity. At 25 °C half of the experiments showed bilayer failure

at < 20 nN (figure 15), whereas no such failure was seen at 52 °C.

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Chapter 4

39

Figure 15: The percentage of friction experiments performed at a given temperature where the load bearing capacity was found to be less than 20 nN. The numbers in the bar columns indicate the total number of experiments performed at the respective temperatures.

This evaluation leads us to draw the inference that the stability of the

bilayer under the combined action of load and shear increases with

temperature. Increased fluidity seems to impart flexibility to the bilayer

and thereby induces a self-healing ability wherein the chains are flexible

enough to heal the defects. This results in the high load bearing capacity

that is witnessed at higher temperatures.

4.2.3 Lubrication synergy: Mixture of hyaluronan and DPPC vesicles

That DPPC and hyaluronan associate on surfaces upon sequential

injections, has been shown in one of the earlier works of our group [30].

A composite layer was formed whereby phospholipids and hyaluronan

were accumulated on model surfaces. However, in a physiological

environment the likelihood is that these biolubricants, which are present

in synovial fluid, form self-assembly aggregates and then attach to the

surface. Therefore, in order to study association of hyaluronan and DPPC

vesicles in a premixed solution, and consecutively its adsorption and

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Chapter 4

40

lubrication performance on model surfaces, we have carried out the

investigations summarised below [31].

4.2.3.1 DPPC-hyaluronan association structures in bulk

SAXS data of DPPC-hyaluronan structures associating in bulk indicates

the presence of unilamellar and double bilayer vesicles. Electron density

profiles (figure 16) were obtained from the fitting of scattering curves,

and a satisfactory fit was achieved with an additional layer of hyaluronan.

As vesicles are closed structures, additional material would only adsorb to

one side of the bilayer (as depicted by the hump in the figure below). The

head-to-head distance of the bilayers remained the same in the presence

and absence of hyaluronan; this evidently indicates that hyaluronan

binds to the outer shell of the DPPC vesicles. Furthermore,

complementary to our current findings, previously reported DLS

measurements suggested that addition of hyaluronan to a DPPC vesicle

solution resulted in an increase in hydrodynamic size of the vesicles [30].

Figure 16: Electron density profiles of the single DPPC bilayer structure (top) and DPPC double bilayer structure (bottom) in absence (blue) and presence (green) of hyaluronan.

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Chapter 4

41

4.2.3.2 Adsorption process of DPPC-hyaluronan association structures

A mixed hyaluronan/DPPC vesicle solution was injected on a silica

surface, and adsorption of the associating structures was studied with the

QCM-D technique (figure 17 a). Following the first injection, the initial

peak in frequency and dissipation change indicates rupturing of DPPC

vesicles and formation of a DPPC bilayer, consistent with that observed in

our previous studies [30]. This part of the adsorption step incurred small

changes in dissipation and thus was modelled using the Sauerbrey model.

Thereafter, adsorption was allowed to continue and the magnitude of Δf

and ΔD continued to increase in nearly a linear manner. Surface

saturation was not observed even after 100 minutes. This was followed by

rinsing and limited desorption was seen. A second injection again

resulted in a linear change in Δf and ΔD with time and limited desorption

upon rinsing. As indicated in the figure, the last two parts of the

adsorption were modelled using the Voigt model due to large changes in

ΔD.

Figure 17 b reveals the nature of the resulting layer after initial formation

of a DPPC bilayer. The close to linear relation between ΔD and Δf

indicates that there was no significant stiffening of the outer layer, and

the structures formed by adsorption from the DPPC-hyaluronan mixture

are extended and viscoelastic. When this is compared to previous work

where hyaluronan was added sequentially to a DPPC bilayer, the relation

between ΔD and Δf was seen to be sublinear. This means that the layer

formed in case of sequential addition was stiffer than that formed by

adsorption from a DPPC-hyaluronan mixture.

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Chapter 4

42

Figure 17: (a) Adsorption from a solution containing 0.5 mg/mL DPPC and 0.5 mg/mL hyaluronan (start marked by ↑) on a silica surface in 155 mM NaCl solution at 55°C monitored by QCM-D for the 3rd overtone (frequency (black line) and dissipation (grey line) change). Rinsing (start marked by ↓) was done with 155 mM NaCl solution at 55 °C. For clarity every 30th point is plotted. (b) Dissipation change versus frequency change during the first adsorption step illustrated in a (black line) and data for hyaluronan adsorption on a pre-adsorbed DPPC bilayer (grey line) re-plotted from ref [30]. For clarity, every 7th point is plotted.

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Chapter 4

43

4.2.3.3 Morphology of adsorbed layer from DPPC-hyaluronan mixture

Figure 18: AFM topography image of the adsorbed layer formed after 40 minutes adsorption from solutions containing 0.5 mg/mL DPPC and 0.5 mg/mL hyaluronan in 155 mM NaCl on a silica surface. After 40 minutes the solution was exchanged with pure 155 mM NaCl solution, and the resulting layer was imaged in this solution at a temperature of 47 °C. Image size 1 x 1 μm2. Image given in reference [30] from sequential adsorption of hyaluronan on DPPC bilayers on a silica surface can be used for comparison.

The morphology of the adsorbed layer from the DPPC-hyaluronan

mixture can give insights into the structures discussed above. The

adsorbed layer was not homogeneous and aggregates of varying sizes

were present. Some were smaller than the hydrodynamic diameter of

vesicles found in bulk solution [30], whereas others would correspond to

the size of flattened vesicles. It may be suggested that DPPC-hyaluronan

aggregates form on top of the DPPC bilayer, and these also include intact

vesicles. This is further evidenced by the force curves that report the

presence of vesicle-like structures on the surface.

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Chapter 4

44

4.2.3.4 Surface force measurements

As the layer imaged above was heterogeneous, the force curves measured

on this layer also resulted in variations from spot to spot. Typical surface

force curves demonstrated long-range repulsion that increased

monotonically with decreasing separation as depicted in figure 19 a. This

is different from the short-range repulsion observed between lone DPPC

bilayers. This suggests that hyaluronan constitutes the outermost layer

and complements earlier findings of hyaluronan binding to the outer

surface of DPPC vesicles.

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Chapter 4

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Figure 19: Force normalised by radius as a function of separation between silica surfaces carrying an adsorbed layer formed from solutions containing 0.5 mg/mL DPPC and 0.5 mg/mL hyaluronan in 155 mM NaCl at 47 °C. The adsorption was allowed to proceed for 40 minutes before rinsing with 155 mM NaCl, and the forces were recorded after rinsing. (a) Typical force curves recorded before friction (approach (■) and separation (□)). (b) Very long range repulsive forces noted on the first approach on one spot (■) before friction measurements. The forces recorded on first separation (□) are also shown. The inset shows the 10th force curve measured on approach (■) and separation (□) on this spot.

The presence of aggregates observed in the AFM image above affects

surface interactions. An example is shown in figure 19 b. The presence of

a large aggregate was indicated by a very long-range repulsive force,

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Chapter 4

46

which was observed on the approach curve of the first force measurement

(up to 300 nm). The force continued to increase and then suddenly

returned to zero. This can be interpreted as deformation of the aggregate,

which is followed by structural rearrangements. Ten consecutive force

measurements were performed and the final force measurement is shown

in the inset of figure 19 b. Following compressions, a few small steps were

observed; each step size roughly corresponding to the thickness of a

DPPC bilayer. Thus it can be understood from the results above that large

aggregates, which are present on the surface are disrupted upon

compression and transformed into multilayer structures. It can be

proposed that the presence of these DPPC-hyaluronan aggregates on the

surface allows build-up of a reservoir of biolubricants.

4.2.3.5 Lubrication performance of DPPC-hyaluronan association structures

It is impressive that the heterogeneous nature of the adsorbed layer does

not compromise its lubrication performance. Friction forces measured

between the inhomogeneous layers formed from the DPPC hyaluronan

mixture were very low. Friction measurements were carried out at three

different spots. The best fit, which corresponds to the red line in figure 20

a resulted in a friction coefficient value of 0.006. Such low friction force is

comparable to that reported for synovial joints [1]. The inhomogeneous

layers rearrange under the combined action of load and shear into

phospholipid bilayer or multilayer structures, which are separated by

easily sheared water layers. As discussed in an earlier section of this

thesis it is the presence of strong hydration and entropic protrusion

forces between DPPC bilayers [67] that facilitates sliding with a low

friction force. The maximum load used in this study corresponded to a

pressure of 23 MPa, close to the critical pressure that a cartilage surface

can withstand before it breaks under axial load [66].

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Figure 20: (a) Friction force as a function of load between two silica surfaces carrying an adsorbed layer formed after 40 minutes adsorption from a 155 mM NaCl solution containing 0.5 mg/mL DPPC and 0.5 mg/mL hyaluronan, followed by rinsing (■). The temperature was 47 oC. The solid red line (follows Amonton’s rule) has a slope of 0.006 and the slope of the dashed line (reference line) is 0.01. (b) Illustration of one of the three friction measurements depicting high friction force upon loading at a normal force of 10 nN.

An observation of interest that occurred in some cases is illustrated in

figure 20 b. At a load of 10 nN, there was a sharp and transient increase

in the value of the fiction force. This may be attributed to the energy

dissipation arising from structural rearrangements induced by load and

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shear. It is of importance to note that the layer continued to provide low

friction even after the friction peak. Thus, the DPPC hyaluronan mixture

provides lubrication synergy; permits accumulation of large quantities of

phospholipids at the interface and furthers the return to a low friction

state even after the initial layer structure is disrupted. The same does not

hold good in the case of lone DPPC bilayers (as we saw earlier, figure 14),

which are compromised under high loads [65].

The interplay of DPPC and hyaluronan presents a clear lubrication

advantage over that of lone DPPC bilayer. Hyaluronan is not essential for

achieving low friction force, yet it provides build-up of a reservoir of

phospholipid lubricant on the model surface, and consequently the

DPPC-hyaluronan structures possess a self-healing ability. Clearly this

excellent lubrication and self-healing ability will be advantageous if such

layers are present on joint surfaces. Considering B.A. Hills had assumed

and managed to demonstrate through morphological evidence that

phospholipids form the outermost lubricating lining of the surface of

joints [68], such a conjecture does not seem far-fetched.

4.3 Effect of molecular weight and ions on DPPC-hyaluronan interactions

Ca2+ ions are present in the synovial fluid [69] and are also known to bind

to DPPC [70]. Thus, they may have an influence on the structure and

interactions of DPPC and hyaluronan, and resultantly affect the system

under investigation. In the context of joint diseases it would be

compelling to also examine the effect of molecular weight of hyaluronan

on this model system. To the best of our knowledge, no direct

measurements have been conducted to study lubrication performance of

cartilage by varying the molecular weight of hyaluronan. However low

molecular weight hyaluronan has been associated with rheumatoid

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arthritis [36, 71]. Langmuir monolayers of DPPC were chosen as model

system in this part of the study as they were more suitable for the

required techniques of the investigations.

4.3.1 Effect of molecular weight of hyaluronan

Information on lateral interactions between molecules and phase changes

in Langmuir monolayers was obtained from the surface pressure versus

mean molecular area isotherms (π/A isotherms) [72]. As can be seen

from figure 21 a, the π/A isotherm for DPPC with low molecular weight

hyaluronan in the subphase is more expanded than the other subphases.

This implies that low molecular weight hyaluronan affects lateral

interactions greatly and therefore the packing of monolayers is most

severely influenced in its presence.

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Figure 21: (top) Π/A-isotherms of DPPC on aqueous subphases containing 155 mM sodium chloride. The concentration of HA was 0.5 mg/mL. (bottom) Π/A-isotherms of DPPC on subphases containing 155 mM sodium chloride and 10 mM calcium chloride. The concentration of HA was 0.5 mg/mL.

BAM images complement the findings above and provide information on

changes in the domain structures at the micrometre scale. Again, addition

of low molecular weight hyaluronan caused the most apparent

morphological changes in the domain structures; wherein clearly

separated small-condensed regions with a round circumference were

seen. However, the morphology in presence of high molecular weight

hyaluronan and that in the absence of hyaluronan remained similar, with

bigger multilobed-structures.

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Figure 22: Brewster angle microscopy images of DPPC monolayers at the liquid air interface on salt solutions with (a) HA of MW 10 kDa (left) 155 mM sodium chloride (right) 155 mM sodium chloride with 10 mM calcium chloride (b) HA of MW 1500 kDa (left) 155 mM sodium chloride (right) 155 mM sodium chloride with 10 mM calcium chloride.

The images of subphase containing low molecular weight hyaluronan also

point to the presence of some material in between the condensed regions,

which is presumably due to the presence of two phases at the interface; a

condensed phase and another more disordered and thin layer. In the case

of the disordered phase, a possible route of interaction of hyaluronan is

through it’s hydrophobic patches to the alkyl chains of DPPC [73, 74] as

sketched in figure 23. In the case of the ordered phase, it is likely that

hyaluronan interacts with the DPPC headgroup, via its negatively charged

carboxylic acid groups and the positively charged region of the DPPC

headgroup [75, 76].

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This was further explored through XRR measurements. It was concluded

from the XRR findings that there was significant accumulation of low

molecular weight hyaluronan in the hydrophilic headgroup region of

DPPC, which prevented the headgroups from closely packing together.

On the other hand, higher molecular weight hyaluronan mostly resided

underneath (away from the liquid air interface) the polar DPPC

headgroup.

It has previously been shown in our group that the addition of

hyaluronan to supported DPPC bilayers somewhat reduces the load

bearing capacity [30]. It can be speculated from the above-mentioned

results that the increase in distance between headgroups resulting from

the addition of low molecular weight hyaluronan, disturbs the lateral

packing of the layers. This results in poorer load bearing capacity as only

well ordered bilayers should be capable of providing appreciable load

bearing capacity. This may provide insights into an aspect of lubrication

failure in the event of arthritis.

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Figure 23: Schematic of the interaction of HA with DPPC at the water-air interface

4.3.2 Effect of Ca2+ ions

Electrophoretic mobility measurements indicated that the positive value

of electrophoretic mobility of DPPC increased with an increase in calcium

chloride concentration, which confirmed adsorption of divalent ions to

the DPPC phospholipid headgroup [77].

From the π/A isotherms it was noted that addition of 10 mM CaCl2 affects

both DPPC monolayer, and DPPC-hyaluronan interactions significantly.

Take for instance the isotherm of DPPC without hyaluronan in figure 21

b. The surface pressure at a given area per molecule is lower when

calcium ions are present (especially for high surface pressures). This

means that the lipids pack better in the presence of calcium ions [78]. The

interactions between DPPC and hyaluronan, when calcium was present in

the subphase, were influenced greatly as the isotherms changed for both

low and high molecular weight hyaluronan. In the absence of calcium

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ions the monolayer expanded only with low molecular weight hyaluronan

in the subphase, whereas in the presence of calcium ions, it always

expanded. This suggests enhanced interactions take place between DPPC

and hyaluronan owing to the presence of calcium ions. The observations

mentioned above were once again supported by BAM images. In all the

different subphases, condensed regions were more densely packed in the

presence of calcium ions.

Further, XRR measurements highlighted that when calcium ions were

present in the subphase hyaluronan was amassed next to DPPC, which

explains the significant changes that occured in π/A isotherms in

presence of calcium ions.

4.4 Effect of high pressures on DPPC bilayers and DPPC-hyaluronan structures

Human joints are subjected to different loads throughout the day

depending on activities that are partaken. For daily activities joints have

been reported in vivo to bear physiological loads up to 18 MPa (180 bar)

[79]. In light of improved lubrication performance, which is imparted by

the synergistic effect of important biomolecules in the joint, it will be of

relevance to examine the behaviour of DPPC-hyaluronan composite

layers under high pressures. As a first step towards this we investigated

pressure effects on supported DPPC layers in absence of hyaluronan

using XRR [80].

XRR studies were performed at different pressures ranging from those

experienced during every day activities and during extreme conditions.

As discussed in an earlier section of this thesis silica-supported DPPC

bilayers are in the fluid phase at 55°C. Phase transitions can also be

induced by pressure and for DPPC bilayers at 55 °C it occurs at 500 bar

[81]. Pressure cycle measurements were performed from 60 bar to 2 kbar

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by increasing the pressure in steps. In order to verify the reversibility of

pressure-induced changes, the final measurement was brought down and

conducted at 60 bar. In case of lone DPPC bilayers the reflectivity curves

indicate that the position of the first minima shifted to lower q-values

(increase in bilayer thickness) when there was an increase in pressure

from 100 bar to 1 kbar; indicating a phase transition from fluid to gel

phase. Thereafter on return from a pressure of 2 kbar to 60 bar the final

reflectivity curve was found not to be identical to the first measurement

at 60 bar. This demonstrates that the shift was not reversible, and that

DPPC bilayers by themselves are vulnerable to high hydrostatic

pressures.

The same pressure cycle measurements were then performed for DPPC-

hyaluronan composite layers as were done for lone DPPC bilayers and

striking differences were noticed. It was found that unlike DPPC bilayers,

structural changes induced by high pressures in DPPC-hyaluronan

composite layers were reversible (depicted in fig 24). This stresses the

fact that with addition of hyaluronan the stability and robustness of

mixed DPPC-hyaluronan composites is improved over that of DPPC alone

at high hydrostatic pressures.

These results point to the likelihood that hyaluronan contributes to

preserving the integrity of the lipid bilayers when subjected to high

pressures. This is of importance while considering boundary lubrication

conditions in the joints, as only unimpaired lipid layers would ensure low

friction.

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Figure 24: Fresnel normalized reflectivity curves of (top) DPPC at 55 °C and different pressures up to 2 kbar (b) DPPC/HA1500 composite layers at 55 °C and different pressures up to 2 kbar. Solid black lines show the fits. Blue bars indicate the position of the first minimum (A: 1 & 2kbar, B: 60, 100 & 1bar).

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5. Conclusions and impact

The optimal functioning of joints necessitates low friction, high load

bearing capacity, wear protection, and a self-healing ability. This is

ensured by several mechanisms and biolubricants, i.e. not by any one

component working alone. With this work, we have achieved a better

understanding of the association, layer structure, and resulting

lubrication performance of some major biolubricants implicated in

biolubrication.

From the first lubrication couple (COMP-lubricin) we learnt that even

though the favourable confirmation of one component (lubricin) could

result in low friction, it was imperative that it stayed strongly attached to

the model surface to have a load-bearing capacity of significance. This

was achieved with the help of the other component (COMP). COMP

associates specifically with lubricin, ensures lubricin’s anchoring, and

exposes its structure in a favourable confirmation that results in low

friction. To the best of our knowledge this synergistic behaviour has not

been experimentally studied elsewhere.

We have progressed from our previous work by building a more

physiologically relevant model of the second lubrication couple (DPPC-

hyaluronan), wherein DPPC and hyaluronan were injected from a

premixed solution. The friction force generated was extremely low. More

importantly, it was inferred that even though hyaluronan is not important

for reaching low friction force values, it bestows an important self-healing

ability by providing a reservoir of the DPPC biolubricants at the sliding

surfaces. This inference holds importance in the understanding of a few

biological scenarios such as, the depletion of biolubricants in certain

areas following trauma/suffering. Components like hyaluronan can

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complement its synergy partner and may replete the required

biolubricants and hence provide self-repair.

By studying additional factors (effect of temperature, pressure, molecular

weight of hyaluronan, presence of calcium ions) a deeper understanding

of the topic was gained and advances were made to the existing

knowledge.

It was found that fluidity (and therefore flexibility and mobility) of the

DPPC chains was needed to repair and heal areas that included defects.

This was seen to occur only when the phospholipid was present in the

fluid phase and not in the more rigid gel phase.

Low molecular weight hyaluronan is linked to rheumatoid arthritis. Our

studies on the effect of molecular weight of hyaluronan on DPPC-

hyaluronan interfacial layers, shed light on the fact that low molecular

weight hyaluronan affects the packing and organisation of such layers

more than high molecular weight hyaluronan. If such layers exist on the

joint surfaces, then a disturbance of this sort may unfavourably affect the

intactness of the layers, and consequently its load bearing capacity. The

same study examined the presence of calcium ions and concluded that

interactions between DPPC and hyaluronan were enhanced by the

presence of these ions. Given that calcium ions are present in the synovial

fluid, this information is consequential to gain insights on the influence

of calcium ions on DPPC-hyaluronan structures and interactions.

Finally, by subjecting the DPPC-hyaluronan system to high pressures, it

was found that hyaluronan helps in stabilising DPPC bilayers. DPPC

bilayers by themselves are vulnerable to such high pressures. As we

constantly subject our knees to varying and also high pressures, a better

understanding of this aspect of joint lubrication was achieved with the

help of such a synergy study.

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The above synergy studies potentially layout the basis for future work,

which can build on existing knowledge with additional biolubricants.

Successful findings in this field can also be related to biolubrication in the

eyes and lungs. Further, such studies would also be of valuable clinical

relevance for joints, possibly resulting in better repair strategies, longer

lasting prosthesis, artificial cartilage, and synthetic lubricants.

Additionally, it would be useful in the context of tissue engineering,

biomedical devices, and environmentally friendly lubricants.

We are still far from grasping the ways of Nature with regards to joint

lubrication. Nevertheless, encouraging progress is being made in this

field. The quest for a magic bullet molecule has been veered towards key

biolubricants and their synergistic workings. This field holds the potential

for contribution to alleviating the suffering of millions, and at the same

time has tremendous business potential. I will be eagerly awaiting

advancements and strides that will be made in this field.

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6. Acknowledgements

It is the gentle guidance, constant support, and unflinching faith of so many people that has led me to this new milestone in life. I am grateful for the opportunities that were provided to me at the right time and right place. This has helped me realise my endeavours, and thus acknowledgements are in order.

Before I begin to thank members of this PhD journey, I would like to thank extended family, friends, teachers, supervisors, and colleagues, whose guidance and motivation I have received in the past, well before my PhD days. Especially the ones in India and Switzerland. I am ever so indebted for your generous help during my stay, without which, reaching this far would have been out of question.

Dr. Andra Dėdinaitė and Prof. Per Claesson it has been an honour and my good fortune to have known and worked with you. You have always encouraged an atmosphere that is conducive to learning and questioning. Andra, I have been inspired by your dedication to and vast knowledge of this field. I ardently hope for continued progress and success in this field. No question was ever a silly question for you. I have been amazed fairly regularly in the past four years by how you have always made time for me. Always! Additionally, I could freely discuss any problem outside the work front with you (very helpful when one is thousands of miles away from home). You have fostered team spirit, and that makes me feel we have achieved this together. All this has been invaluable for my scientific and personal growth. Per, seldom does one come across consideration and kindness to other people’s needs to the degree you have shown. Your constructive criticism, keen eye for details, clinical approach and reassuring manner, has taught me to deconstruct complex problems and helped me improve. I have walked out of your office having learnt something new every time, and enjoyed intellectually stimulating moments. You hold a genuine interest in the well being of your student. I am glad I had both of you to learn from as one of my early teachers, and would like to carry these work ethics henceforth with me.

I thank financial support from the People Programme of the European Union’s Seventh Framework Programme (Marie Curie Actions). Collaborations have been instrumental to my studies and so I take this opportunity to thank all of my collaborators at the University of Gothenburg, Lund University, HZG Hamburg, and SP Stockholm. Particularly, Florian and Thomas, your commitment to work, well past the end of the project has been commendable! In hindsight, I will fondly look back at the long beam times and night shifts at the synchrotron facilities. It has been a pleasure to work with you. The entire Marie Curie network is appreciated. I also give my thanks to all my co-authors. Thanks to Master’s student, Yushi for working so hard when the situation demanded it.

To all the past and current members of this division, thank you! I have made some friends for life. Your warm welcome helped me settle in. Your friendships have helped me enjoy the good times and endure the challenging ones. Thanks (in no particular order) to Marie, Katerina, Jie, Rachel, Jonathan, Yousef, Golrokh, Maziar, Chao, Min, Junxue, Olga, Eleonora, Mattias, Krishnan, Xiaoyan, Tingru, Maria (SP), Elizaveta, Neda, Eric, Erik, Hui, Sara, Celine, Angelika, Georgia, and

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61

Illia. Forgive me in case I have forgotten someone. Working late in the lab was more fun when there was company, and there is something exclusive about such labmate-ship. Thank you Laetitia, Adrien, Deb, Elin, and Rasmus.

All badminton partners are acknowledged for helping me smash away my worries.

Indebted to the very talented-Ana Filipa for the cover page illustration.

The Chauhan’s, Sulena and family, Ashu and family, Anu and family, Divya and family, Richa, Zahra, Yamini, Neha, Deeksha, Ramya, you have been home away from home. Grateful to Param, Sukhda, Mitra, Shweta, Sonal, Ramaa, Gautami, Kateryna, Floor, Leila, Roya, Mina and Bhavya.

Gajendra, thank you for everything. This would not have been possible without you. To those who know you, also know that I mean this quite literally!

Mamma and papa, it is not becoming to thank you in words. Nevertheless, I would like you to know that I have deep gratitude and admiration for the both of you. I have been very lucky with all the opportunities you have provided, and for the happy, healthy, stimulating, and enterprising environment you have always maintained at home. Anu, when I fall short of strength I lean on you, and Amrit, when I begin to lose perspective I tap into your wisdom.

It couldn’t have been better!

Despite everyone’s generous contribution, I take full responsibility for any inadvertent errors that may have escaped my attention.

आकाांक्षा राज AKANKSHA RAJ जनवरी २०१७ JANUARY 2017 स्टाक्होम, स्वीडन STOCKHOLM, SWEDEN

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