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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

Akademisk avhandling som med tillstnd av Kungliga Tekniska Hgskolan i Stockholm framlgges till offentlig granskning fr avlggande av tekniska doktorsexamen fredagen den 17 March kl. 10:00 i Kollegiesalen, KTH, Brinellvgen 8, Stockholm. Avhandlingen frsvaras p engelska Synergies in Biolubrication

Akanksha Raj (rajaka@kth.se)

Doctoral Thesis

KTH Royal Institute of Technology

School of Chemical Science and Engineering

Surface and Corrosion Science

Drottning Kristinas Vg 51

SE-100 44 Stockholm

Sweden

TRITA-CHE Report 2017:8 ISSN 1654-1081 ISBN 978-91-7729-268-5 Denna avhandling r skyddad enligt upphovsrttslagen. Alla rttigheter frbehlles. 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

Mr. and Mrs. Raj,

this ones for you.

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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 hr avhandlingsarbetet var att ka frstelsen fr biosmrjning, vilket inspirerades av egenskaper hos vra synovialleder. Specifikt s angreps problemet genom studier av synergistisk association mellan ngra av nyckelkomponenterna och dess inverkan p den smrjande frmgan. Ett antal huvudtekniker anvndes vid studierna. Atomkraftsmikroskopi (AFM) anvndes fr att mta ytkrafter och friktionskrafter. Ytterligare information om topografi, egenskaper och struktur hos adsorberade skikt erhlls frn teknieker som kvartskristallmikrovg med dissipationsmtning (QCM-D), rntgenstrle reflektivitet (XRR), och AFM avbildning.

Ngra nyckelkomponenter hos synovialvtska och brosk anvndes som smrjande 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 p hydrofila modellytor av kiseldioxid, medan COMP-lubricin undersktes p svagt hydrofoba poly(metyl metakrylat) (PMMA) modellytor. Bda systemen inkluderade saltlsningar med en koncentration av 150 mM.

Underskningarna av COMP-lubricin paret visade att dessa tv komponenter var fr sig inte kunde uppn nskad smrjningseffekt. Emellertid, nr de associerade synergistiskt s att COMP mjliggjorde stark frankring av lubricin till PMMA ytan i gynnsam konformation erhlls en lg friktionskoefficient ( 0.06).

Vxelverkan mellan DPPC och hyaluronan ger ocks upphov till frbttrade smrjningsegenskaper ver enbart DPPC bilager, dr hyaluronan mjliggr frankring av en reservoir av DPPC p modellytan och drigenom ger upphov till en sjlvlkande frmga hos det smrjande skiktet. Det hr systemet gav en mycket lg friktionskoefficient (< 0.01).

Andra aspekter undersktes ocks genom att studera inverkan av faktorer s som temperatur, nrvaro av kalcium joner, hyaluronans molekylvikt och tryck. DPPC bilager vid hgre temperatur (i flytande kristallin fas) hade hgre lastbrande frmga p grund av hgre rrlighet, vilket mjliggjorde sjlv-lkning av defekter. Associationen mellan DPPC Langmuir skikt och hyaluronan frstrktes i nrvaro av kalcium joner. Dessutom hade lgmolekylr hyaluronan en strakare tendens att associera med DPPC, vilket pverkade packning och skiktstrukur mer. Slutligen s visade vi att DPPC-hyaluronan kompositskikt var mer stabila n DPPC biskikt under hga 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 Ddinait 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-Rmer, Matthew Fielden, Per M. Claesson, Andra Ddinait Journal of Colloid and Interface Science 488 (2017) 225233

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-Rmer, Per Martin Claesson, Andra Ddinait Journal of Colloid and Interface Science 445 (2015) 8492 Paper IV Structure of DPPChyaluronan interfacial layers effects of molecular weight and ion composition D. C. Florian Wieland, Patrick Degen, Thomas Zander, Soren Gayer, Akanksha Raj, Junxue An, Andra Ddinait, Per Claesson, Regine Willumeit- Rmer Soft Matter, 12, (2016), 729 Paper V The influence of hyaluronan on the structure of a DPPCbilayer under high pressures Thomas Zander, D.C. Florian Wieland, Akanksha Raj, Min Wang, Benedikt Nowak, Christina Krywka, Andra Ddinait, Per Martin Claesson, Vasil M. Garamus, Andreas Schreyer, Regine Willumeit-Rmer Colloids and Surfaces B: Biointerfaces, 14,2 (2016) 230238

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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 Ddinait, Per Martin Claesson, Vasil M. Garamus, Andreas Schreyer, and Regine Willumeit-Rmer

Manuscript

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

Sarah A Flowers, Akanks