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2014-06-12

Cartilage boundary lubrication and rheology of

proteoglycan 4 + hyaluronan solutions and synovial

fluid

Ludwig, Taryn Elaine

Ludwig, T. E. (2014). Cartilage boundary lubrication and rheology of proteoglycan 4 + hyaluronan

solutions and synovial fluid (Unpublished doctoral thesis). University of Calgary, Calgary, AB.

doi:10.11575/PRISM/25219

http://hdl.handle.net/11023/1577

doctoral thesis

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UNIVERSITY OF CALGARY

Cartilage boundary lubrication and rheology of proteoglycan 4 + hyaluronan solutions

and synovial fluid

by

Taryn Elaine Ludwig

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOMEDICAL ENGINEERING

CALGARY, ALBERTA

JUNE, 2014

© Taryn Elaine Ludwig 2014

ii

Abstract

Synovial fluid (SF) is the viscous fluid present within articular joints that

contributes to load bearing and lubrication functions. Proteoglycan 4 (PRG4) and

hyaluronan (HA) in SF contribute synergistically to cartilage boundary lubrication.

However, changes in SF PRG4 and HA content with osteoarthritis (OA) and associated

effects on cartilage boundary lubricating function are not fully understood. Furthermore,

the effects of PRG4+HA interaction on solution viscosity have not been thoroughly

characterized.

The objectives of this thesis were to 1) investigate the relationship between PRG4

and HA composition and boundary lubricating function of normal and OA SF, and 2) to

investigate how the concentration and structure of PRG4 contributes to interactions with

itself and HA, and subsequently the boundary lubricating and rheological properties of

SF.

Novel and previously characterized biochemical and biomechanical methods were

used to evaluate boundary lubricant composition and lubricating ability of SF. While not

all OA SF samples had low PRG4, samples that had low PRG4 concentration and

decreased HA molecular weight (MW) demonstrated decreased cartilage boundary

lubricating ability in vitro, which could be restored by addition of PRG4. SF aspirated

after a flare reaction to intra-articular injection that had low PRG4 and an approximately

normal HA MW distribution demonstrated normal cartilage boundary lubricating ability.

In purified solutions of PRG4 and HA, decreased PRG4 or decreased high MW HA

limited cartilage boundary lubricating ability. PRG4 and recombinant human PRG4

increased the viscosity of HA solutions at low concentrations, but decreased the viscosity

iii

of high concentration HA solutions. The intra- and inter-molecular disulfide bonded

structure of PRG4 was observed to be important for its contributions to both PRG4+HA

cartilage boundary lubricating ability and PRG4+HA solution viscosity.

These results demonstrate that alterations in both PRG4 and HA content in SF

may have negative effects on SF cartilage boundary lubricating and rheological function,

and are consistent with a non-covalent, crowding mechanism of interaction. They suggest

that maintaining PRG4 and HA content in SF during injury and disease, through the

development of new PRG4±HA biotherapeutic treatments, may be able to both protect

cartilage from degeneration and restore SF viscosity in vivo.

iv

Preface

This thesis is presented in manuscript based format, so there is some repetition in

the Introductions and Methods between the chapters. Chapters 2, 3, 4, and 5 have been

published, submitted, or are in preparation for submission for publication as described

below.

Chapter 2 has been published in Arthritis and Rheumatism1: Ludwig TE,

McAllister JR, Lun V, Wiley JP, Schmidt TA. Diminished cartilage lubricating ability of

human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through

proteoglycan 4 supplementation. Arthritis Rheum. 2012;64(12):3963-3971.

Chapter 3 is in preparation for submission to BMC Musculoskeletal Disorders:

Ludwig TE, McAllister JR, Lun V, Wiley JP, Schmidt TA. Effect of flare reaction to

intra-articular hyaluronan injection on cartilage boundary lubricating ability of human

synovial fluid. Submitted May 16, 2014.

Chapter 4 is in preparation for submission to the Journal of Biomechanics: Ludwig

TE, Hunter MM, Schmidt TA. Effects of concentration and structure on synergistic

proteoglycan 4 + hyaluronan cartilage boundary lubrication.

Chapter 5 is in preparation for submission to Biomacromolecules; Ludwig TE,

Cowman MK, Jay, GD, Schmidt TA. Effects of concentration and structure on

proteoglycan 4 rheology and interaction with hyaluronan.

v

Acknowledgements

I wholeheartedly thank my supervisor, Dr. Tannin Schmidt, for his support, time,

patience, and guidance over the last 5 years. He has been an outstanding role model; his

calm and logical approach to problem solving (and stressful situations), extreme

dedication to science, and diligent celebration of his trainees successes are very

motivating leadership characteristics that I will strive to develop. I especially thank him

for his support of my MD/PhD training program, being patient and involved in the assay

development processes I have gone through, and patiently going through many revisions

while I have developed my scientific writing skills. He has created a great environment

for trainees to learn and develop in. I am very fortunate and proud to have been involved

in his lab in the early days.

To all my lab mates past and present, thank you for making the lab a fun, safe,

and productive place to work, and for your help and feedback. I would like to specially

thank Saleem Abubacker for always being available as a sounding board, for reading

most of the scientific writing I have ever done, and for feeling the same (obsessive) way

about lab cleanliness as I do. I also thank Miles Hunter and Leah Peterson for being my

first experiments in mentoring.

Thank you to my committee members (Dr. Preston Wiley, Dr. Cy Frank, and Dr.

Michael Kallos), as well as my candidacy and defence examiners (Dr. Roman Krawetz,

Dr. Robert Edwards, and Dr. Braden Fleming), for your guidance and suggestions. Thank

you to the NSERC CREATE Training Program for providing a great training

environment and facilitating the start of a very productive mentorship with Dr. Wiley. Dr.

Wiley has provided invaluable feedback on this work, and has patiently but persistently

vi

encouraged me to present it to different audiences with very different (clinically relevant)

perspectives. Thank you also to Dr. Cheryl Barnabe and Dr. Laurie Hiemstra for being

mentors/career advisors and providing valuable input and advice, scientific and

otherwise. I look forward to continued relationships with these mentors throughout my

clinical training. Thank you to the Leaders in Medicine Program and my peers for

helping everyone remember the clinical applications of basic research, and vice versa,

and helping basic scientist and clinician trainees learn to talk to each other.

Thank you to Alberta-Innovates Technology Futures and Health Solutions for

support through graduate studentships, and to the OA Team, Faculty of Graduate Studies,

and the BME Graduate Program for supporting the presentation of this work at scientific

meetings at which I obtained excellent feedback.

This work would not have been possible without collaboration with the U of C

Sports Medicine Centre. I would like to thank Dr. Victor Lun, Dr. Preston Wiley, and

their OA patients who agreed that their synovial fluid could be used in this work. Thank

you also to Jenelle McAllister for making sure we actually got the synovial fluid and

helping keep track of almost 200 donors. This work would not be as impactful without

the normal human tissues provided through the University of Calgary Joint

Transplantation Program. Special thanks to Sue Miller and Dr. Roman Krawetz for

managing the acquisition of tissues from the program.

Thank you to all my friends and family, who lovingly accept my inner geek and

the fact that I am still happily in school. Thank you Ashley Tyler and Hilary Smith for

your friendship and laughter. Thank you to my parents-in-laws Harry and Denise Ludwig

for your enthusiasm towards and support of my career. Thank you most of all to my

vii

parents and sister, Laura, Hugh, and Kaley, who have been amazingly supportive

throughout my education through the “20th

grade,” so far. Thank you dad for inspiring

my scientific curiosity and helping me get started in my engineering career, which led me

to graduate school and where I am today. Thank you mom for reminding me why OA

research is important and who will ultimately benefit from new treatments. Kaley, thank

you for un-intentionally sparking my interest in orthopaedic research and biomedical

engineering, and for being a wonderful friend.

Finally, thank you to my husband, Jonathan, for encouraging me to pursue my

dreams and supporting me in the beginning of my clinician-scientist career. Your

unconditional love throughout the grumpiness induced by assay development,

unrepeatable experiments, and scientific/thesis writing, for holding down the fort while

I’ve had the opportunity to travel to conferences, and for always celebrating my

accomplishments mean the world to me.

viii

Table of Contents

Abstract ............................................................................................................................... ii Preface................................................................................................................................ iv

Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................. viii List of Tables ..................................................................................................................... xi List of Figures and Illustrations ........................................................................................ xii List of Symbols, Abbreviations and Nomenclature ......................................................... xvi

CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Overall Introduction to the Thesis .............................................................................1 1.2 Structure of Articular Cartilage .................................................................................4

1.2.1 Superficial zone .................................................................................................5 1.2.2 Middle and deep zones ......................................................................................5

1.3 Synovium and Synovial Fluid Function ....................................................................7

1.4 Osteoarthritis ............................................................................................................10 1.5 Cartilage Lubrication ...............................................................................................11

1.6 Boundary Lubricants Present in Synovial Fluid ......................................................17 1.6.1 Proteoglycan 4 (PRG4) ....................................................................................20 1.6.2 Hyaluronan (HA) .............................................................................................24

1.7 Synovial Fluid Composition ....................................................................................25 1.8 Aims .........................................................................................................................28

CHAPTER TWO: DIMINISHED CARTILAGE LUBRICATING ABILITY OF

HUMAN OSTEOARTHRITIC SYNOVIAL FLUID DEFICIENT IN

PROTEOGLYCAN 4: RESTORATION THROUGH PROTEOGLYCAN 4

SUPPLEMENTATION ............................................................................................30

2.1 Abstract ....................................................................................................................30 2.2 Introduction ..............................................................................................................32 2.3 Materials & Methods ...............................................................................................34

2.3.1 Materials ..........................................................................................................34 2.3.2 Samples ............................................................................................................35

2.3.3 hSF Biochemical Characterization ..................................................................36 2.3.4 Cartilage Boundary Lubricating Ability ..........................................................39

2.3.5 Statistical Analysis ..........................................................................................41 2.4 Results ......................................................................................................................41

2.4.1 hSF Biochemical Characterization ..................................................................41 2.4.2 Cartilage Boundary Lubricating Ability ..........................................................46

2.5 Discussion ................................................................................................................49 2.6 Acknowledgements ..................................................................................................54

CHAPTER THREE: EFFECT OF FLARE REACTION TO INTRA-ARTICULAR

HYALURONAN INJECTION ON CARTILAGE BOUNDARY

LUBRICATING ABILITY OF HUMAN SYNOVIAL FLUID: A CASE

SERIES .....................................................................................................................55

3.1 Abstract ....................................................................................................................55

ix

3.2 Introduction ..............................................................................................................57

3.3 Materials and Methods .............................................................................................60 3.4 Results ......................................................................................................................63 3.5 Discussion ................................................................................................................70

3.6 Acknowledgements ..................................................................................................76

CHAPTER FOUR: EFFECTS OF CONCENTRATION AND STRUCTURE ON

SYNERGISTIC PROTEOGLYCAN 4 + HYALURONAN CARTILAGE

BOUNDARY LUBRICATION ................................................................................77 4.1 Abstract ....................................................................................................................77

4.2 Introduction ..............................................................................................................79 4.3 Materials & Methods ...............................................................................................82

4.3.1 Materials ..........................................................................................................82

4.3.2 Sample Preparation ..........................................................................................83 4.3.3 Lubrication Testing .........................................................................................83 4.3.4 Statistical Analysis ..........................................................................................85

4.4 Results ......................................................................................................................86 4.4.1 Lubrication Testing .........................................................................................86

4.5 Discussion ................................................................................................................94 4.6 Acknowledgements ..................................................................................................99

CHAPTER FIVE: EFFECTS OF CONCENTRATION AND STRUCTURE ON

PROTEOGLYCAN 4 RHEOLOGY AND INTERACTION WITH

HYALURONAN ....................................................................................................100

5.1 Abstract ..................................................................................................................100

5.2 Introduction ............................................................................................................102

5.3 Materials and Methods ...........................................................................................105 5.3.1 Materials ........................................................................................................105

5.3.2 Viscosity of PRG4+HA Solutions .................................................................107 5.4 Results ....................................................................................................................108

5.4.1 Viscosity of PRG4+HA Solutions .................................................................108

5.5 Discussion ..............................................................................................................114 5.6 Acknowledgements ................................................................................................121

CHAPTER SIX: CONCLUSIONS ..................................................................................122 6.1 Summary of Findings .............................................................................................122

6.2 Discussion ..............................................................................................................124

6.2.1 Measurement of PRG4 Concentration in SF .................................................124

6.2.2 PRG4+HA Functional Synergism .................................................................126 6.3 Future work ............................................................................................................130

6.3.1 Measurement of PRG4 Concentration in SF .................................................130 6.3.2 PRG4+HA Functional Synergism .................................................................132

BIBLIOGRAPHY ............................................................................................................135

APPENDIX A: PROBING THE PRG4+HA INTERACTION: ISOTHERMAL

TITRATION CALORIMETRY .............................................................................161

x

A.1 Introduction ...........................................................................................................161

A.2 Materials and Methods ..........................................................................................162 A.3 Results ...................................................................................................................164 A.4 Discussion .............................................................................................................165

A.5 Acknowledgements ...............................................................................................166

APPENDIX B: PROBING THE PRG4+HA INTERACTION: SLOT BLOT FAR-

WESTERN ..............................................................................................................167 B.1 Introduction ...........................................................................................................167 B.2 Methods .................................................................................................................168

B.2.1 Materials .......................................................................................................168 B.2.2 PRG4 Bait on PVDF Membrane ..................................................................169 B.2.3 HA Bait on Hybond-N+ Membrane .............................................................170

B.3 Results ...................................................................................................................172 B.3.1 PRG4 Bait on PVDF Membrane ..................................................................172 B.3.2 HA Bait on Hybond-N+ Membrane .............................................................173

B.4 Discussion .............................................................................................................176 B.5 Acknowledgements ...............................................................................................177

APPENDIX C: TEMPORAL EFFECTS OF INTRA-ARTICULAR HA AND/OR

CORTICOSTEROIDS ON OA SYNOVIAL FLUID BOUNDARY

LUBRICANT COMPOSITION: A CASE SERIES ...............................................178

C.1 Purpose ..................................................................................................................178 C.2 Methods .................................................................................................................178

C.3 Results ...................................................................................................................179

C.4 Conclusions ...........................................................................................................184

APPENDIX D: DESCRIPTION OF CARTILAGE-CARTILAGE BOUNDARY

LUBRICATION TEST AND LUBRICANT SEQUENCES .................................185

D.1 Introduction ...........................................................................................................185

APPENDIX E: PRG4 CONCENTRATION IN ALL SF SAMPLES MEASURED ......189

APPENDIX F: FIGURE REPRINT PERMISSIONS .....................................................190 F.1 Reprint Permissions for Chapter 2, Published in Arthritis & Rheumatism ...........190 F.2 Reprint Permissions for Appendix D, Published in Osteoarthritis and Cartilage .191

xi

List of Tables

Table 1-1: Content and function of diseased human SF compared to normal human

SF 4,20,21,37,81,99-115

. ..................................................................................................... 27

Table 2-1: Patient characteristics of hSF samples identified as PRG4-deficient and

selected for lubrication testing (OA-LO). Average donor characteristics of NL

hSF (N=13). .............................................................................................................. 43

Table 3-1: Characteristics of flare patients whose SF was identified as PRG4-

deficient and were selected for lubrication testing, and normal (NL) SF from

cadaveric donors. * = significantly higher (p < 0.001) compared to normal. ........... 65

Table 6-1: Summary of boundary lubricant composition and boundary lubrication

function of SF and purified solutions tested. .......................................................... 128

xii

List of Figures and Illustrations

Figure 1-1: Zonal structure of articular cartilage showing collagen fibril orientation

and relative size (inset), aggrecan content and production of PRG4. Modified

from2,7,8

. ...................................................................................................................... 6

Figure 1-2: Formation, circulation, and removal of SF and its components.

Permeability to small molecules and proteins is limited by intercellular spacing.

Fat-soluble molecules can diffuse through cell membranes; as such their

movement is less restricted. Specialized lubricant molecules (PRG4, HA) are

secreted by synovial lining and superficial zone cartilage cells; these molecules

can accumulate at surfaces or exit the joint space by a variety of mechanisms.

Modified from9,14-16

. .................................................................................................... 9

Figure 1-3: The 3 putative modes of cartilage lubrication (modified from3,25,27

). G and

H show an adaptive, mechanically controlled lubrication mechanism at low loads

(G), and high loads (H). ............................................................................................ 14

Figure 1-4: Effects of HA concentration (A), PRG4 concentration (B), and

combination of HA, PRG4, and surface active phospholipids (SAPL) on kinetic

coefficients of friction using the previously characterized cartilage boundary

lubricating ability test described above. For C, HA was used at 3.3 mg/mL,

PRG4 was used at 450 µg/mL, and SAPL was used at 200 µg/mL34

. ...................... 18

Figure 1-5: Schematic of PRG4 structure and glycosylation pattern. Adapted from57

and65

. ......................................................................................................................... 22

Figure 1-6: Structure of the linear repeating disaccharide HA86

. ..................................... 24

Figure 2-1: Characterization of the PRG4 ELISA control by protein stain (A) and

high MW PRG4 immunoreactivity in PRG4 control, NL hSF, and OA hSF by

western blotting (B, C). PRG4 controls treated with neuraminidase and hSF

treated with hyaluronidase and neuraminidase were probed with (B) LPN and

(C) PNA-HRP. Samples were subjected to 3 – 8 % SDS-PAGE followed by

protein stain or western blotting as described in Materials and Methods. ................ 38

Figure 2-2: PRG4 concentration measured in OA hSF. This figure is not intended to

portray that a certain proportion of OA hSF is OA-LO. [PRG4] in NL samples

shown in white bars. Average [PRG4] in NL (N = 13, ) shown by black line.

OA-LO (N = 5) samples selected for friction testing shown with black bars.

Average [PRG4] in OA-LO ( shown by grey line. * = p < 0.05. ............. 44

Figure 2-3: (A) Average HA concentration in NL and OA-LO hSF. (B) HA MW

distribution in measured NL hSF (N = 8), and OA-LO (N = 5). * = p < 0.05. ......... 46

Figure 2-4: Static (μstatic,Neq) (A) and kinetic <μkinetic,Neq> at Tps = 1.2 seconds (B)

friction coefficients of PRG4 deficient OA hSF (OA-LO, N = 5), with 450 µg/ml

PRG4 and 1.0 mg/ml 1.5 MDa HA supplementation, and NL hSF. * = p<0.05. ..... 48

xiii

Figure 3-1: PRG4 concentration in flare-SF after IA HA. PRG4 concentration in

normal ( ) SF (N = 29) shown by black line, ± 95% CI shown in dashed lines.

PRG4-deficient samples selected for friction testing are circled. “L” and “R”

denotes SF that was obtained from the left and right knee of the same patient.

“1” and “2” denotes the 1st and 2

nd aspiration of the same knee after a flare

reaction to HA. .......................................................................................................... 64

Figure 3-2: (A) HA concentration in flare-SF after IA HA. HA concentration in

normal ( ) SF (N = 29) shown by black lines, ± 95% CI shown in dashed lines.

PRG4-deficient samples selected for friction testing are circled. “L” and “R”

denotes SF that was obtained from the left and right knee of the same patient.

“1” and “2” denotes the 1st and 2

nd aspiration of the same knee after a flare

reaction to HA. (B) HA MW distribution in N = 5 PRG4-deficient flare-SF

samples selected for friction testing and N = 15 normal (NL) SF (* represents p

< 0.05). Values are mean ± 95% CI. ......................................................................... 67

Figure 3-3: Effect of HA and PRG4 supplementation on the cartilage boundary

lubricating ability of PRG4-deficient flare-SF samples, as determined by in vitro

cartilage-on-cartilage friction testing. Two friction coefficients, static (μstatic,Neq)

(A) and kinetic (<μkinetic,Neq>; at Tps = 1.2 seconds) (B) were calculated in PBS

(negative control lubricant), PRG4-deficient flare-SF alone, flare-SF plus PRG4,

flare-SF plus PRG4 and HA, and normal SF (NL; positive control lubricant).

Values are mean ± 95% CI. ...................................................................................... 69

Figure 4-1: μstatic,Neq (A), and <μkinetic,Neq> at Tps = 1.2 seconds (B) for PRG4 high and

low dose response + constant [HA] = 3.3 mg/mL (TESTS 1A, 1B). * =

significantly higher than SF (p < 0.05). .................................................................... 87

Figure 4-2: μstatic,Neq (A, B, C) for HA dose responses + constant [PRG4] = 45 µg/mL

(TEST 2A) (A), 150 µg/mL (TEST 2B) (B), and 450 µg/mL (TEST 2C) (C).

<μkinetic,Neq> at Tps = 1.2 seconds (D) for all doses of HA in [PRG4] = 45, 150,

450 µg/mL (TEST 2A, 2B, 2C). Average <μkinetic,Neq> in PBS and SF shown for

reference. # = significantly higher than [PRG4] = 450 µg/mL (p < 0.05). ^ =

significantly higher than [PRG4] = 150 µg/mL (p < 0.05). ...................................... 89

Figure 4-3: μstatic,Neq (A), and <μkinetic,Neq> at Tps = 1.2 seconds (B) for hylan G-F20 ±

[PRG4] = 450 µg/mL (TEST 3). * = p < 0.05. ......................................................... 91

Figure 4-4: μstatic,Neq (A), and <μkinetic,Neq> at Tps = 1.2 seconds (B) for HA, HA +

[R/A PRG4] = 450 µg/mL, and HA + [PRG4] = 450 µg/mL (TEST 4). * = p <

0.05. ........................................................................................................................... 93

Figure 5-1: Characterization of PRG4 (A), reduced and alkylated (R/A) PRG4 (A),

recombinant human (rh) PRG4 (B), and R/A rhPRG4 (B) by protein stain after 3

– 8% SDS-PAGE. * denotes an ~460 kDa monomeric species, and ** denotes

higher MW species of ~1 MDa and higher MW aggregates54

................................ 106

xiv

Figure 5-2: Shear rate dependent viscosity at 25°C (A) and 37°C (B) of PRG4 alone

at 45, 150, 450 µg/mL, and R/A PRG4 at 450 µg/mL. ........................................... 109

Figure 5-3: Shear rate dependent viscosity at 25°C of HA at 0.3 (A, D, G), 1.0 (B, E,

H), and 3.3 (C, F, I) mg/mL alone and with 45 (A, B C), 150 (D, E, F) and 450

µg/mL (G, H, I) PRG4. ........................................................................................... 111

Figure 5-4: Shear rate dependent viscosity at 25°C of HA at 0.3 (A), 1.0 (B), and 3.3

(C) mg/mL alone and with R/A PRG4 450 µg/mL ................................................ 112

Figure 5-5: Shear rate dependent viscosity at 25°C of rhPRG4 alone at 4.5, 45, 150,

450 µg/mL, and R/A rhPRG4 at 450 µg/mL. ......................................................... 113

Figure 5-6: Shear rate dependent viscosity at 25°C of HA at 0.3, 1.0, and 3.3 mg/mL

alone and with 45 and 450 µg/mL rhPRG4. ........................................................... 114

Figure A-1: SDS-PAGE of non-reduced PRG4* used for ITC experiments (right lane)

showing ~1 MDa (top arrow) and 460 kDa species (bottom arrow), and reduced

PRG4* (Red PRG4* - left lane) showing lower MW species for comparison. ...... 163

Figure A-2: Power required to maintain temperature in sample cell (top panel) and

integrated heat plot (bottom panel) for (A) HA injected into PBS and (B) HA

injected into PRG4. Note the values on the y-axes are very small. ........................ 164

Figure B-1: Determination of concentrations for HA on PVDF slot blot using HA

alone. Detection with biotinylated HABP and streptavidin-HRP. Concentrations

outlined in green box (0.0003, 0.003, 0.03 mg/mL) selected for subsequent

experiments. ............................................................................................................ 171

Figure B-2: Far-western blot of HA on PRG4-blotted PVDF membrane. Detection

with biotinylated HABP and streptavidin-HRP. ..................................................... 173

Figure B-3: Far-western blot of PRG4 onto HA-blotted Hybond-N+ membrane. (A)

Detection with PRG4 antibody H140. (B) Detection with PRG4 antibody 9G3,

reprobe. ................................................................................................................... 175

Figure C-1: PRG4 concentration in OA SF over time during treatment with IA HA or

corticosteroid. Each line represents 1 knee, and circular markers denote knee SF

from the left (filled circles) and right (open circles) knee of 1 patient. SF was

aspirated prior to therapeutic injection. Red markers denote an IA injection was

received after aspiration, all other markers are corticosteroid injections. Grey

shaded area shows average [PRG4] in normal SF ± 95% confidence interval. ...... 180

Figure C-2: HA concentration in OA SF over time during treatment with IA HA or

corticosteroid. Each line represents 1 knee, and circular markers denote knee SF

from the left (filled circles) and right (open circles) knee of 1 patient. SF was

aspirated prior to therapeutic injection. Red markers denote an IA injection was

xv

received after aspiration, all other markers are corticosteroid injections. Grey

shaded area shows average [HA] in normal SF ± 95% confidence interval. .......... 181

Figure C-3: HA MW distribution (high MW 3 – 6 MDa and low MW < 0.5 MDa) in

OA SF over time during treatment with IA HA or corticosteroid. Each line

represents 1 knee, and circular markers denote knee SF from the left (filled

circles) and right (open circles) knee of 1 patient. SF was aspirated prior to

therapeutic injection. Red markers denote an IA injection was received after

aspiration, all other markers are corticosteroid injections. Grey shaded area

shows average HA MW in normal SF ± 95% confidence interval. ........................ 183

Figure D-1: Schematic depicting location osteochondral samples are harvested from

(A), annulus and core shaped samples (B), sample immersion overnight in

lubricant bath (C), sample orientation, applied load, and rotation during testing

(D), and test sequence schematic showing compression, stress relaxation, and

order of pres-spin durations (Tps) over the duration of the tests (E). ..................... 186

Figure D-2: Lubricant sequences used in Chapters 2 and 3 to evaluate boundary

lubricating ability of various human SF. ................................................................. 187

Figure D-3: Lubricant sequences used in Chapter 4 to evaluate boundary lubricating

ability of various PRG4+HA solutions. .................................................................. 188

Figure E-1: PRG4 concentration measured in all SF samples that were measured in

this thesis work. Grey bars indicate that those samples were identified as having

low PRG4 and were selected for friction testing. The average normal value in N

= 29 cadaveric SF samples (±95% confidence interval) is shown in the black

horizontal lines. (Please note the average normal changed between Chapters 2, 3,

and Appendix C, as more normal samples were acquired and measured.) ............. 189

Figure F-1: Reprint permissions for Chapter 2, published in Arthritis & Rheumatism,

2012; 64 (12): 3963-3971 ....................................................................................... 190

Figure F-2: Reprint permission for Appendix C, published in Osteoarthritis and

Cartilage 2014; Supplement 22: S481-S482. ......................................................... 191

xvi

List of Symbols, Abbreviations and Nomenclature

Symbol Definition

Maximum axial torque in the first 20° of

rotation

Equilibrium axial load

Normal average

Effective radius

Axial torque averaged over the last 360° of

rotation

OA-LO average

[HA] Hyaluronan concentration

[PRG4] Proteoglycan 4 concentration

<μkinetic,Neq> Kinetic coefficient of friction calculated using

equilibrium load

μstatic,Neq Static coefficient of friction calculated using

equilibrium load

µ Coefficient of friction

9G3 Anti-PRG4 antibody, mucin domain

ACL Anterior cruciate ligament

ANOVA Analysis of variance

BCA Bicinchoninic acid assay

BHCl Benzamidine hydrochloride

bSF Bovine synovial fluid

CACP Camptodactyly-arthropathy-coxa vara-

pericarditis

CI Confidence interval

CST Corticosteroid

DEAE Diethylaminoethyl

DZ Deep zone

ELISA Enzyme linked immunosorbent assay

F Female

F Friction force

GAG Glycosaminoglycan

Gal Galactose

GalNac N-Acetylgalactosamine

H140 Anti-PRG4 antibody, C-terminal

HA Hyaluronan

HA’se Hyaluronidase

HABP Hyaluronan binding protein

HRP Horseradish peroxidase

hSF Human synovial fluid

IA Intra-articular

IgG Immunoglobulin G

IL-1α Interleukin -1α

ITC Isothermal titration calorimetry

xvii

L Left

LGP-1 Lubricating glycoprotein 1

LPN Anti-PRG4 antibody, C-terminal

M Male

mRNA Messenger ribonucleic acid

MSF Megakaryocyte stimulating factor

MW Molecular weight

MZ Middle zone

N Normal force

N One synovial fluid sample

N One friction testing replicate

Na2-EDTA Disodium ethylenediaminetetraacetate

NEM N-Ethylmaleimide

NeuAc N-Acetylneuraminic acid (sialic acid)

NL Normal

OA Osteoarthritis

OA-LO Osteoarthritic synovial fluid deficient in PRG4

PBS Phosphate buffered saline

PBST Phosphate buffered saline with Tween

PI Protease inhibitor

PMSF Phenylmethanesulfonyl fluoride

PNA Peanut agglutinin

PRG4 Proteoglycan 4

PRG4* Size exclusion column-purified PRG4

PVDF Polyvinylidene fluoride

R Right

R/A Reduced and alkylated

RA Rheumatoid arthritis

rhPRG4 Recombinant human PRG4

SAPL Surface active phospholipid

SD Standard deviation

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

SEM Standard error of the mean

Ser Serine

SF Synovial fluid

SZ Superficial zone

SZP Superficial zone protein

TAE Tris acetate ethylenediaminetetraacetate

TBS Tris buffered saline

TBST Tris buffered saline with Tween

TGF-β1 Transforming growth factor β1

Thr Threonine

TMB Tetramethylbenzidene

Tps Pre-spin duration

1

Chapter One: Introduction

1.1 Overall Introduction to the Thesis

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid (SF)

and at the surface of articular cartilage. Along with hyaluronan (HA), PRG4 contributes

to boundary lubrication of articular surfaces. These lubricant molecules are critical for

normal joint function, and alterations in biochemical composition of SF during joint

injury and disease may result in compromised boundary lubricating function. However,

changes in SF PRG4 content in osteoarthritis (OA) remain to be fully understood. A

synergistic cartilage boundary lubricating functional interaction has been observed

between PRG4 and HA in vitro at a cartilage-cartilage interface, and some observations

of interaction of PRG4 and HA in solution and at model surfaces have also been reported.

However, the mechanism of these interactions and dependence on PRG4 and HA

structure and concentration remain poorly understood.

The hypotheses of this thesis work were:

1. OA SF can have diminished PRG4 content and associated impaired

cartilage boundary lubricating ability, which can be at least partially

restored by supplementation with PRG4 and/or HA.

2. The concentration and structure of PRG4 mediates interactions with itself

and other SF constitutes, such as HA, in solution; these interactions

contribute to the boundary lubricating and rheological properties of SF

These hypotheses were investigated through a combination of both novel and

previously characterized biochemical, biomechanical, and rheological techniques.

2

This work has contributed to the understanding of a fundamental joint lubrication

mechanism and progress towards understanding the effects and mechanisms of new and

improved SF biotherapeutics. Aspects of these contributions will be discussed as outlined

below. This thesis is presented in manuscript based format, so there is some repetition in

the Introductions and Methods between the chapters. Chapters 2, 3, 4, and 5 have been

published, submitted, or are in preparation for submission for publication as described in

detail in the Preface.

Chapter 1 provides an overall introduction to the structure and function of cartilage

and synovium, SF composition and function, boundary lubrication, and PRG4 and HA

structure and functions as boundary lubricants within articular joints.

Chapter 2, which has been published in Arthritis and Rheumatism1, describes the

development of a sandwich enzyme linked immunosorbent assay (ELISA) and its use to

quantify PRG4 in SF from normal donors and patients with chronic OA, and investigates

the cartilage boundary lubricating function of PRG4-deficient OA SF compared to that of

normal SF, with and without supplementation with PRG4±HA.

Chapter 3, which is in preparation for submission to the BMC Musculoskeletal

Disorders, investigates the effect of an inflammatory flare reaction to intra-articular (IA)

HA injection on boundary lubricant composition of OA SF, and investigates if the

cartilage boundary lubricating ability of flare-SF deficient in PRG4 is diminished.

Chapter 4, which is in preparation for submission to the Journal of Biomechanics,

evaluates the effects that PRG4 and HA concentration have on their functional synergism

as cartilage boundary lubricants. The effects of PRG4 and HA structure on this

synergistic relationship are also investigated.

3

Chapter 5 describes the characterization of the shear rate dependent viscosity of

bovine PRG4 and recombinant human PRG4 (rhPRG4) over a range of concentrations

with and without disruption of PRG4 intra- and inter-molecular disulfide bonded

structure (tertiary and quaternary structure) by reduction and alkylation (R/A); the effects

of addition of PRG4 and rhPRG4 to HA solutions are also discussed. This chapter is in

preparation for submission to Biomacromolecules.

Finally, Chapter 6 summarizes the major findings of this work, suggests future

directions of work, and discusses overall implications for improvements in current

biotherapeutic treatments for OA. Other collaborations the candidate has been involved in

that are related to the goal of understanding how alterations in boundary lubricant content

and structure affect SF function are also discussed. These include metabolomic and

glycosylation analysis of PRG4 in OA, and characterization of boundary lubricant

content of early OA, late OA, rheumatoid arthritis (RA), and normal SF (to be correlated

with lipidomics).

The 6 appendices included in this thesis discuss related supplementary work or

information. Several methods were attempted by the candidate to probe the mechanism of

the PRG4+HA interaction but failed to provide clear evidence of such an interaction; the

use of isothermal titration calorimetry and far-Western dot blot will be briefly discussed

as supplementary material in Appendix A and Appendix B, respectively. The PRG4

sandwich ELISA was also used to measure PRG4 concentrations in SF aspirated from

patients receiving repeated courses of corticosteroid and/or HA injections; this abstract is

included as Appendix C. Appendix D includes schematics and lubricant sequences for the

cartilage boundary lubrication test used in Chapters 2, 3, and 4. Appendix E contains a

4

graph showing the PRG4 concentration measured in all OA SF samples described in this

thesis, including those not selected for lubrication testing. Appendix F contains

permission from publishers to reprint the published works presented in Chapter 2 and

Appendix C.

1.2 Structure of Articular Cartilage

Articular cartilage is the smooth tissue covering the ends of long bones in

synovial joints. The 3 key mechanical functions of articular cartilage are to bear load and

provide low wear, low friction motion2. These functions are required to be met at a wide

range of loads and speeds, over millions of cycles of joint motion per year3. The

viscoelastic properties of cartilage, its zonal structure, and its interactions with other joint

tissues such as subchondral bone, synovial fluid (SF) and synovium allow these functions

to be achieved. This thesis will focus on the friction-reducing function of articular

cartilage and SF.

Cartilage is generally less than 5 mm thick in human joints, however thickness

can vary within and between joints4, and it is a hierarchically organized tissue. Cartilage

is 68 – 85% water, which is essential to its function; the flow of interstitial fluid through

the porous tissue during loading creates frictional drag forces that dominate the

viscoelastic behaviour of normal tissues5. Collagen II, a fibrillar matrix protein, makes up

10 – 20% of the wet weight of articular cartilage, and the remaining 5 – 10% is

proteoglycans (large molecules consisting of a core protein with covalently attached

linear un-branched polysaccharides called glycosaminoglycans, or GAGs)5. The specific

5

biochemical composition and structure of each zone contributes to the overall function of

the tissue.

1.2.1 Superficial zone

The superficial zone (SZ) or tangential zone of articular cartilage generally makes

up the most superficial 10 – 20% of the total cartilage thickness2 (Figure 1-1).

Chondrocytes in this zone are small, elongated and flat in shape, and oriented parallel to

the surface as single cells at a relatively high density. Collagen fibrils in this zone are

parallel to the surface, and there is relatively less matrix proteoglycan present in this zone

compared to the deeper zones2. The orientation of the collagen fibrils parallel to the

surface imparts high tensile strength to this region5. A unique feature of superficial zone

chondrocytes of importance in the context of this thesis is their production of PRG4 (also

known as lubricin and described in detail in section 1.6.1)6. PRG4 is expressed to a much

lesser degree by middle and deep zone cells, likely due to the different loading

conditions7.

1.2.2 Middle and deep zones

The middle (MZ) and deep (DZ) zones make up 40 – 60% and ~30% of the total

cartilage thickness respectively2 (Figure 1-1). Collagen fibrils in the MZ are of a wide

range of sizes and randomly oriented, and there is higher aggrecan content (a GAG

molecule that attaches to HA to form aggregates) in this region compared to the SZ2.

Chondrocytes in the MZ are randomly distributed, single round cells, whereas those in

the DZ are round and arranged in columns2. Collagen fibrils in the DZ are thick and

6

perpendicular to the bone and cross into the underlying calcified bone to anchor the

cartilage. The DZ also has a high concentration of aggrecan2. The high aggrecan content

in the MZ and DZ contributes to the compressive stiffness of cartilage due to the swelling

pressure caused by attraction of positive ions in solution towards the highly negatively

charged GAG side chains5.

Figure 1-1: Zonal structure of articular cartilage showing collagen fibril

orientation and relative size (inset), aggrecan content and production of

PRG4. Modified from2,7,8

.

7

1.3 Synovium and Synovial Fluid Function

The dense, fibrous capsule of diarthrodial joints is lined by the synovium which

includes a thin, intimal lining 2-3 cells thick and a sub-intimal layer of connective tissue

that supports the lining and blood vessels4 (Figure 1-2). The physiological functions of

the synovium are to9:

1. Provide a low friction lining for the joint, especially during expansion and

contraction over cartilage surfaces that are not in contact with each other

to prevent pinching between the moving surfaces

2. To transport nutrients to, and waste from, the joint cavity

3. To secrete synovial lubricant molecules

As cartilage is an avascular, aneural and alymphatic tissue, nutrients and

metabolites must be exchanged via diffusion first across the synovium and into the SF,

through SF, and then through the cartilage matrix to chondrocytes4. (Diffusion of

nutrients from subchondral bone vasculature is not considered to be a major source of

nutrition for chondrocytes10

, however recent studies have suggested that small molecules

may be able to diffuse between cartilage and subchondral bone11

.) In the context of

lubrication, the function of the synovium as a “blood-joint” barrier is of interest, as this

contributes to the regulation of SF content.

SF is an ultra-filtrate of blood plasma present in diarthrodial joints4. SF functions

to:

8

1. Provide shock absorption through the distribution of loads provided by its

viscoelastic properties12

and its ability to behave as an elastic fluid at high

frequencies13

2. Circulate nutrients and soluble mediators between the blood stream and

cartilage3

3. Provide joint lubrication3

After diffusion across the synovial membrane, nutrient and metabolite circulation

in SF is achieved by diffusion and the “stirring” effect of joint motion. Exchange of small

solutes between SF and cartilage is achieved primarily by diffusion, and bulk flow

contributes to the transport of macromolecules14

. Small molecules (<~10kDa), such as

oxygen, glucose, carbon dioxide, water, and electrolytes are able to be transported freely

in and out of the joint cavity4 (Figure 1-2).

SF composition is dynamic, with constituent molecules exchanging between

tissues and entering and exiting the joint via several mechanisms:

1. Exchange between plasma and SF (capillaries)

2. Secretion by local cells

3. Diffusion out of SF to lymphatic vessels

4. Cellular uptake, accumulation at surfaces, and degradation within the joint

cavity

Regulation of SF composition may be altered during inflammatory conditions,

possibly due to increased permeability of the synovium15

.

9

Figure 1-2: Formation, circulation, and removal of SF and its

components. Permeability to small molecules and proteins is limited by

intercellular spacing. Fat-soluble molecules can diffuse through cell

membranes; as such their movement is less restricted. Specialized

lubricant molecules (PRG4, HA) are secreted by synovial lining and

superficial zone cartilage cells; these molecules can accumulate at surfaces

or exit the joint space by a variety of mechanisms. Modified from9,14-16

.

10

1.4 Osteoarthritis

Osteoarthritis (OA) is the most common type of arthritis and is a major cause of

pain and functional limitation. OA of the knee alone affects over 250 million people

worldwide, and the number of years living in disability due to OA have increased

considerably in the past 20 years17

. One hallmark of OA is degradation of articular

cartilage, however it is a disease of the whole joint and also involves changes in the

subchondral bone, synovium, ligaments, menisci, and joint capsule18

. OA is a multi-

factorial disease, with risk factors ranging from age, sex, prior joint injury, obesity, and

genetics to abnormal joint mechanics18

. It is estimated that the direct costs of OA in the

US is $270 billion, and indirect costs in the population aged 18 – 64 is $28 billion

annually; this is in the range of 3% of the US gross domestic product19

.

In the context of this thesis the link between joint injury and OA, through altered

SF composition and SF boundary lubricating function, is of interest. Concentrations of

PRG4 have been shown to decrease after ACL tear20

, and compromised boundary

lubrication has been associated with increased wear of the articular surface21

. HA

composition has also been shown to be altered in OA, and intra-articular HA injections

are currently used as viscosupplements to treat pain in OA13

. However, currently

available treatments are unable to consistently or considerably modify or stop disease

progression22,23

, and the search for better early diagnosis and treatment strategies

continues.

11

1.5 Cartilage Lubrication

Low friction and low wear motion throughout the wide range of loads and speeds

experienced by joint surfaces is achieved through a combination of lubrication

mechanisms. This combination of mechanisms is required to overcome the start-up,

steady state, rolling, and sliding conditions present. Friction is quantified using the

coefficient of friction, a dimensionless quantity calculated as the ratio of frictional to

normal forces using Amontons Law:

Where: F = Friction force (N)

µ = Coefficient of friction (dimensionless)

N = Normal force (N)

Fluid film lubrication occurs when the cartilage surfaces are separated by a layer

of fluid, resulting in a low coefficient of friction (reflecting decreased resistance to

motion) due to low viscous shear stresses3. Several types of fluid film lubrication have

been proposed to be active in joints, primarily at high speeds and low loads (i.e. swing

phase of gait). Hydrodynamic lubrication (Figure 1-3A) occurs when a fluid wedge is

dragged through a gap between articulating surfaces, however this mechanisms requires

continuous high speed motion and light loads3. In squeeze-film lubrication (Figure 1-3B)

the fluid layer is created when the viscous SF resists being forced out of the gap between

cartilage surfaces as they approach each other, however the squeeze-film will disappear

during prolonged loading3. Weeping lubrication (Figure 1-3C) or self-pressurized

hydrostatic lubrication occurs when pressurized fluid is exuded from the cartilage tissue

during loading and generates a fluid film, whereas boosted lubrication (Figure 1-3D)

12

occurs as fluid is forced into the cartilage, leaving concentrated pools of lubricants

behind3.

As interstitial fluid pressure and fluid load support is decreased over time due to

interstitial fluid depressurization, or with higher joint loads or lower speeds, the solid

fraction of cartilage bears more load and a boundary mode of lubrication becomes

dominant (Figure 1-3E). In boundary lubrication, friction is mediated by interactions

between molecules adsorbed to the cartilage surface, and this cartilage-cartilage contact

creates a high coefficient of friction. A mixed mode of lubrication exists when both fluid

film and boundary mode lubrication are operational (Figure 1-3F), however the

boundary mode can become dominant if there is a large number of contact asperities.

Coefficients of friction are usually high in the boundary lubrication regime24

, and while

friction and wear are not necessarily correlated at cartilage surfaces25

, boundary layers

may act as a “last line of defense” against high friction and wear at cartilage surfaces26

.

Recently an “adaptive mechanically controlled” lubrication mechanism has been

proposed for articular cartilage25

. This differs from mixed modes, where multiple modes

are simultaneously active, in that the active mode is changed with changing conditions24

.

In this mechanism under low loads, a layer of PRG4 that is adsorbed to the surface and

entangled HA provides boundary lubrication (Figure 1-3G). At higher loads, HA gets

trapped (effectively bound to the surface) by re-alignment of collagen fibrils, and the

trapped PRG4+HA acts as an “emergency” boundary lubricant25

(Figure 1-3H).

For conventional engineering materials, the variation of the coefficient of friction

with load and sliding speed throughout various lubrication regimes are often depicted on

a Stribeck curve. However, this is difficult for biological materials due to their non-

13

homogeneous/visco-elastic surfaces that are often covered with layer(s) of

macromolecules, and which are often lubricated by complex, non-Newtonian solutions of

macromolecules24

; these factors are not accounted for in a Stribeck curve.

14

Figure 1-3: The 3 putative modes of cartilage lubrication (modified

from3,25,27

). G and H show an adaptive, mechanically controlled

lubrication mechanism at low loads (G), and high loads (H).

15

While it is thought that interstitial fluid pressurization supports up to 95% of

applied load at the onset of loading2, opposing cartilage surfaces are in contact over only

~10% of the total area, making this area vulnerable to high friction28

. To ensure that

friction remains low at the articular surface in areas of cartilage-cartilage contact that

develop when fluid pressurization decreases, the boundary lubricants present in SF are

critical. Friction at the articular surface may be associated with cartilage wear21

, and

alterations in SF composition may have implications for SF boundary lubricating ability,

as will be discussed below.

The work presented in this thesis uses a previously characterized in vitro cartilage-

on-cartilage boundary lubrication test to evaluate the coefficients of friction of lubricants

in a boundary mode. Coefficients of friction were calculated as the ratios of friction

forces to normal forces:

μ

Where:

= Resistance to the onset of motion

= Resistance to steady state motion

= Maximum axial torque in the first 20° of rotation

= Axial torque averaged over the last 360° of rotation

= Effective radius

= Equilibrium axial load

16

Coefficients of friction in this test were constant with changes in speed and load,

indicating that boundary lubrication is achieved29

. The test uses a stationary contact area

under compression and, as such, is independent of bulk viscosity of the solution being

tested; there is minimal or no entrainment of fluid during articulation. The compression to

18% of the total cartilage thickness used in this test is slightly higher than estimates of

cartilage deformation based on MRI imaging before and after activity (7%)30

; however

the protocol generates loads of 0.1 – 0.2 MPa, slightly lower than those experienced in

the knee (1 – 1.5 MPa) or hip (0 – 5 MPa) during walking28,31

. Schematics of sample

harvesting, the test sequence, and lubricant sequences used for each test presented in this

thesis are presented in Appendix D. For Chapters 2 and 3, cartilage from macroscopically

normal areas of human distal femurs were used, and for Chapter 4 cartilage from

macroscopically normal areas of mature (22 – 26 months) bovine stifle joints were used.

Preliminary work by another student has shown that the overnight rinsing in PBS

removes the majority of PRG4 from the cartilage surface, and that soaking in PRG4 or SF

does indeed replenish PRG4 at the surface of the cartilage. During test characterization it

was shown that testing in PBS and then SF did not affect the coefficients of friction in

SF, and a preliminary study investigating the lubricating ability of HA and

dipalmitoylphosphatidylcholine (a surface active phospholipid found in SF32

)

demonstrated that the order of the lubricants of interest did not affect the coefficients of

friction. However, order effects were not explicitly tested in the studies presented in this

thesis.

17

1.6 Boundary Lubricants Present in Synovial Fluid

Boundary lubrication is essential, although not sufficient, to maintain cartilage

integrity. This is evident in camptodactyly-arthropathy-coxa vara-pericarditis (CACP)

syndrome in humans, an autosomal recessive disease where SF is void of PRG4 and fails

to lubricate21

. These patients suffer early joint failure. Furthermore, PRG4 knockout mice

demonstrate earlier cartilage wear and higher whole joint friction than wild-type mice21

.

Alterations in boundary lubricant composition with injury and disease (summarized in

Table 1-1) may also have effects on the boundary lubricating ability of SF.

Boundary lubricants by definition must be adsorbed to the lubricated surface, and

boundary films can be formed by either physical or chemical adsorption33

. In cartilage,

the 2 key boundary lubricants are HA and PRG4; both act to reduce friction in the

boundary mode at a cartilage-cartilage interface in a dose-dependent manner (Figure 1-

4A and B). These lubricants have been shown to work synergistically to reduce friction at

a cartilage-cartilage interface towards that of whole SF34

(Figure 1-4C). Though these

molecules are known to functionally interact at cartilage interfaces, the mechanism of

their interaction remains to be confirmed. Current evidence supports the idea that the

PRG4+HA interaction is physical in nature (possibly electrostatic, crowding or

entanglement) rather than chemical25,35,36

. Some indirect biophysical evidence of their

interaction does exist37

(and is more thoroughly discussed in Chapter 5).

18

Figure 1-4: Effects of HA concentration (A), PRG4 concentration (B),

and combination of HA, PRG4, and surface active phospholipids (SAPL)

on kinetic coefficients of friction using the previously characterized

cartilage boundary lubricating ability test described above. For C, HA was

used at 3.3 mg/mL, PRG4 was used at 450 µg/mL, and SAPL was used at

200 µg/mL34

.

There are several mechanisms by which boundary lubricant films can operate,

including38

:

1. Low shear interlayers (lubricant molecules slide between intra-molecular

low-shear planes)

2. Friction modifying layers (reaction products form ordered structures at

surfaces, and sliding between the adsorbed layers)

3. Shear resistant layers (strongly adhered bonded layer which is shear

resistant)

4. Sacrificial layers (a layer is removed instead of the surface being worn)

19

There is some evidence that a sacrificial layer mechanism of boundary lubrication

is active at the cartilage surface. The rate at which the coefficient of friction and

boundary layer thickness were replenished by soaking in SF or PRG4 was faster than the

rate at which the coefficient of friction increased/boundary layer thickness decreased

during constant sliding, suggesting that the rate of repletion was faster than that of

depletion26

. However, gastric mucin on a model surface has been proposed to reduce

friction through viscous boundary lubrication39

.

As boundary lubricants at least one of PRG4 or HA must be adsorbed to the

surface, but the order in which they adsorb or the mechanism by which this occurs

remains to be elucidated. The method of adsorption to the cartilage surface may have

implications for friction and wear resistance. On mica surfaces, chemically adsorbed HA

with PRG4 in solution provides friction and wear reduction, however wear reduction is

not seen if HA is the only molecule physically adsorbed to the surface36

. In contrast, it

has also been observed that PRG4 adsorbs to and generates repulsive interactions

between both hydrophilic and hydrophobic model surfaces, to which HA was unable to

adsorb or generate repulsive interactions; adsorption was improved by adding HA to

PRG440

. PRG4 has also been observed to adsorb and mediate friction between collagen II

functionalized surfaces41

. While relatively little is known about the PRG4+HA

interaction mechanism, the boundary lubricants themselves have been fairly well

characterized. More details of SF boundary lubricant composition in health and disease

will be discussed in the introduction for Chapter 2.

Spurred by the limited availability and high cost of producing PRG4, synthetic

boundary lubricant analogs are another active area of research. In vitro, the synthetic

20

biolubricant “2M TEG” has been shown to reduce friction in the boundary mode at a

previously-worn cartilage-cartilage interface42

. PRG4-mimetic copolymers have

demonstrated the ability to bind to cartilage surfaces and reduce boundary mode friction

in vitro43

, and also to prevent cartilage degeneration in post traumatic animal models in

vivo44

. Finally, scaffolds chemically modified to be HA-binding have shown promise in

in vivo cartilage repair models45

. Recombinant human PRG4 has recently become

available46

, and the application of these synthetic and natural molecules for treatment of

cartilage is promising.

1.6.1 Proteoglycan 4 (PRG4)

PRG4 is a mucin-like glycoprotein present in SF47 and at the surface of articular

cartilage48. PRG4 proteins (also known as lubricin49

, lubricating glycoprotein (LGP-1)50

,

superficial zone protein (SZP)6 and megakaryocyte stimulating factor (MSF) precursor

51)

are expressed by synovial fibroblasts, superficial zone chondrocytes, and tendon and

meniscal cells52

through the PRG4 gene53

. Characterization of PRG4 secreted by bovine

cartilage explants using multi-angle laser light scattering demonstrated the existence of

monomeric species of 239, 379, and 467 kDa MW as well as an ~1 MDa disulfide

bonded multimer54

. The PRG4 core protein is 1404 amino acids in length55

and is

estimated to be approximately 200 nm in length49

. Six isoforms of PRG4 produced by

alternative splicing have been observed in human samples56

. PRG4 has a number of

functions within the joint:

21

1. Preventing protein deposition on cartilage57

2. Controlling adhesion-dependent synovial cell growth (preventing

synovial hyperplasia and inhibiting the adhesion of synovial cells to

the cartilage surface)57

3. Possibly regulating pathways affecting chondrocyte hypertrophy and

catabolism58

4. Boundary lubrication of cartilage surfaces34

PRG4 exhibits 2 key properties of mucins: disulfide bonded multimerization59

and

extensive glycosylations in its central domain. The glycosylations are present as O-linked

galactosamine-galactose chains attached to serine and threonine residues, incompletely

capped with sialic acid (Ser/Thr-GalNAc-Gal±NeuAc)60,61

(Figure 1-5). These

glycosylations are essential to lubricating ability; enzymatic digestion/removal of the

sialic acid cap and GalNAc-Gal has been observed to reduce lubricating ability, as has

removal of the penultimate galactose62

. It is thought that the lubricating ability conferred

by the glycosylations is due to generation of repulsive hydration forces63,64

or charge

repulsion64

. Alterations in glycosylation of PRG4 between OA and RA has previously

been observed; this could make it an interesting candidate for identification of

inflammatory “signatures” in early stage disease65,66

. While it is a mucin-like

glycoprotein, PRG4 differs from other mucins in that its terminal globular domains are

slightly smaller and it has a slightly positive charge at neutral pH (due to lysine and

arginine residues in the core)67

.

22

Figure 1-5: Schematic of PRG4 structure and glycosylation pattern.

Adapted from57

and65

.

PRG4 expression in joint tissues has been demonstrated to be regulated by both

biochemical and biomechanical signals. There are several possible mechanisms by which

PRG4 concentration/expression by joint cells may be altered:

1. Increased degradation (possibly due to increase elastase activity68

)

2. Decreased synthesis (inhibition by inflammatory cytokines69

)

3. Increased loss from the joint due to increased synovial permeability15

23

Dynamic shear stimulation has been shown to up-regulate PRG4 expression in

cartilage explants70,71

. Similar trends have been observed in organ culture of intact

joints72

, and in the joints of developing mice where PRG4 expression is initiated by joint

motion57

. In a mouse model of acute joint exercise it was observed that SF composition,

including PRG4, can change very quickly (on the order of minutes) after joint loading. In

this model, PRG4 expression increased with increasing intensity of exercise up to a

plateau73

. However in a rat model, high intensity running has also been associated with

increased cartilage degeneration, which correlated with decreased PRG4 mRNA

expression74

. While there appears to be increased expression of PRG4 after injury in

some animal models, there does not appear to be increased accumulation at the cartilage

surface (though this is difficult to quantify), possibly due to inflammation/proteolysis75

.

The observation that PRG4 concentrations in human SF are decreased after ACL injury20

and that forced joint exercise in a rat ACL transaction model, where PRG4 concentrations

are decreased, exacerbates cartilage degradation76

further support the critical role of

PRG4 and the importance of normal mechanical loading on its regulation. Transforming

growth factor beta-1 (TGF-β1) has been observed to increase PRG4 expression in

chondrocyte explants, while interleukin 1 alpha (IL-1α) inhibited PRG4 expression; this

increase may be due to an increased number of PRG4-secreting chondrocytes in the SZ77

.

It is thought that the up-regulation of PRG4 expression by mechanical loading occurs

through the TGF-β1 signaling pathway78

, however recent evidence suggests that there are

other unknown mechanisms involved in the mechanoregulation of PRG4 expression79

.

24

1.6.2 Hyaluronan (HA)

HA (also known as hyaluronic acid or hyaluronate) is a linear repeating

disaccharide (D-glucuronic acid and D-N-acetlyglucosamine)5; each HA disaccharide

repeat is approximately 1 nm in length80

. HA is present in SF in a wide range of

molecular weights (MW) and concentrations in healthy and diseased/injured SF (Figure

1-6, Table 1-1). While HA content may decrease with age81

its composition appears to be

relatively stable in joints over time82,83

. HA is produced by chondrocytes and

synoviocytes via the HA synthase enzyme family84

, and is present in SF4 and at the

articular surface85

.

Figure 1-6: Structure of the linear repeating disaccharide HA86

.

Within the joint, HA provides both viscous and elastic properties to SF13

, and acts

as a cartilage boundary lubricant34

. Chapter 5 will discuss previous rheological

characterization of HA solutions. The cartilage boundary lubricating ability of purified

25

solutions of HA is dependent upon MW35,87

and concentration87

. However, when

combined with PRG4 this size-dependency is no longer observed35

. Combined, PRG4

and HA work synergistically to provide cartilage boundary lubricating ability

approaching that of SF34

.

Intra-articular (IA) HA injection is frequently used as a viscosupplement

treatment for patients with OA. Current commercially available formulations vary in both

their MW and duration of action88

. IA HA injection is performed after aspiration of knee

joint effusion, and can provide pain relief and improve function for up to 6 months. Local

pain relief is the only clinically proven effect of HA viscosupplementation89

; it has to

date failed to show any structure modifying effects. The mechanism of action of IA HA is

somewhat unclear, as it remains in the joint for a shorter time than its duration of

action90

; this may be due to inhibition of inflammatory mediators91-95

or stimulation of

endogenous HA production94,96

. Local analgesia may be provided through protection of

nociceptive nerve endings by the elasto-viscous HA97,98

, as identical concentrations of

lower MW HA do not have the same analgesic effects89

.

1.7 Synovial Fluid Composition

SF volume present in a normal human knee joint is approximately 1 – 4 mL,

however this can be considerably increased during injury and disease4. The total protein

concentration in normal SF is ~18 mg/mL; this concentration is normally lower than that

of blood plasma, as large proteins (such as fibrinogen and macroglobulins) are unable to

26

cross into SF from blood plasma as easily as smaller proteins (such as albumin)4. The

permeability of the synovial membrane (effective pore size 20 – 90 nm15

) can be altered

during disease, thus the total protein concentration can be increased in conditions such as

OA and RA4.

In addition to contributions from the filtration of blood plasma, specialized

lubricant molecules are also produced by the synovium itself. In addition to PRG4,

synoviocytes also secrete HA96

. SF is a shear-thinning (viscosity decreases with

increasing shear rates) and time-dependent fluid that is able to behave as a viscous fluid

at low frequencies and as an elastic gel at high frequencies to impart protection to

cartilage and surrounding tissues during joint movement13

. The composition and function

of normal and diseased SF are summarized in Table 1-1 below and more details about

changes in HA and PRG4 content with disease are discussed in Chapter 2. Rheological

properties of SF and its components are discussed in Chapter 5.

27

Table 1-1: Content and function of diseased human SF compared to

normal human SF 4,20,21,37,81,99-115

.

Normal

SF

OA SF

Acute

Injury

CACP SF RA SF

Volume (mL) 1 – 4 1.4 – 90^ ↑ ↑ ↑

[Protein]

(mg/mL)

15 – 25 29 – 39 ↑ ? 36 – 54

[PRG4] (µg/mL) 35 – 250 ↑ ↑, ↓, normal Void ↑, ↓

[HA] (mg/mL) 1.8 – 3.3 Normal ↓, normal Normal ↓

HA MW (kDa) 27 – 10000 Normal, ↓ ↓ ? ↓

Boundary

lubricating

ability

--- Normal, ↓ ↓ ↓ ↓

Viscoelastic

behaviour

--- ↓ viscosity,

↓ elasticity

↓ elasticity ↑

viscosity,

VE ≈ HA

↓ viscosity,

↓elasticity

SF = synovial fluid, OA = osteoarthritis, CACP = camptodactyly-

arthropathy-coxa vara-pericarditis, RA = rheumatoid arthritis, [PRG4] =

proteoglycan 4 concentration, [HA] = hyaluronan concentration, MW =

molecular weight, VE = visco-elasticity, ^ based on SF processed during

the completion of this thesis work, ? = unknown.

28

1.8 Aims

This thesis investigates the relationship between boundary lubricant composition

and function in normal and diseased SF, and investigates how PRG4 contributes to the

boundary lubricating and rheological properties of SF through concentration and

interactions with itself and other SF constituents in solution through the following aims:

o Aim 1-i: Quantify PRG4 and HA composition in normal (NL) and chronic OA

SF, post intra-articular injection flare SF, and repeat donor OA human SF

(Chapters 2 and 3, Appendix D).

o Aim 1-ii: Assess the human articular cartilage boundary lubricating ability of

OA SF deficient in PRG4 and NL SF (Chapters 2 and 3).

o Aim 1-iii: Determine if normal human articular cartilage boundary lubricating

function can be restored to OA hSF deficient in PRG4 with supplementation

of PRG4 and/or HA (Chapters 2 and 3).

o Aim 2-i: Evaluate the cartilage boundary lubricating ability of HA with

increasing concentrations of PRG4 (from pathological to physiological and

super-physiological), and of PRG4 with increasing concentrations of HA

(again from pathological to physiological and super-physiological, Chapter 4).

o Aim 2-ii: Evaluate the ability of PRG4 to contribute to cartilage boundary

lubricating ability of cross-linked HA, and the ability of reduced and alkylated

PRG4 to contribute to the cartilage boundary lubricating ability of HA

(Chapter 4).

29

o Aim 2-iii: Investigate intermolecular interaction, entanglement and gel

formation via characterization of viscous behaviour of PRG4 alone and PRG4

+ HA using rheological methods (Chapter 5).

30

Chapter Two: Diminished cartilage lubricating ability of human osteoarthritic

synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4

supplementation

2.1 Abstract

Objectives: (1) Quantify proteoglycan 4 (PRG4) and hyaluronan (HA) content in

normal (NL) and chronic osteoarthritic (OA) human synovial fluid (hSF). (2) Assess the

human cartilage boundary lubricating ability of PRG4-deficient OA hSF compared to NL

hSF, with and without supplementation of PRG4±HA.

Methods: OA hSF was aspirated from 16 patients with symptomatic chronic knee

OA, prior to therapeutic injection. PRG4 concentration was measured using a custom

sandwich enzyme linked immunosorbent assay (ELISA). HA concentration was

measured using a commercially available ELISA, and HA molecular weight (MW)

distribution by agarose gel electrophoresis. Human cartilage boundary lubricating ability

of OA hSF deficient in PRG4 (“OA-LO”), OA-LO hSF supplemented with PRG4±HA,

and NL hSF was assessed using a previously characterized cartilage-cartilage friction

test. Static, μstatic,Neq, and kinetic, <μkinetic,Neq>, friction coefficients were calculated.

Results: NL hSF PRG4 concentration averaged 287.1 ± 31.8 μg/mL. OA hSF

samples deficient in PRG4 compared to NL (OA-LO, 146.5 ± 28.2 μg/mL, p < 0.05)

were identified and selected for lubrication testing. HA concentration in OA-LO hSF

31

(0.73 ± 0.08 mg/mL) was not significantly different from NL hSF (0.54 ± 0.09 mg/mL, p

= 0.26). In OA-LO, HA MW distribution was shifted towards the lower range. Human

cartilage boundary lubricating ability of OA-LO was significantly diminished compared

to NL (<μkinetic,Neq> = 0.043 ± 0.008 vs. 0.025 ± 0.002, p < 0.05), and restored when

supplemented with PRG4 (OA-LO+PRG4 <μkinetic,Neq> = 0.023 ± 0.003, p < 0.05).

Conclusion: These results indicate that some OA hSF may have decreased PRG4

levels and diminished cartilage boundary lubricating ability compared to normal, and that

PRG4 supplementation can restore normal cartilage boundary lubrication function to

these OA hSF.

32

2.2 Introduction

The proteoglycan 4 (PRG4)53

gene encodes for mucin-like O-linked glycosylated

proteins including lubricin49

and superficial zone protein6. PRG4 proteins, collectively

referred to as PRG4, are synthesized and secreted by cells within articular joints

including superficial zone articular chondrocytes6 and synoviocytes

116. PRG4 is present

in synovial fluid (SF)47

and at the articular cartilage surface48

. PRG4 acts as a boundary

lubricant; it mediates friction during cartilage-on-cartilage contact between the articular

surfaces where lubrication is provided by molecular interactions at the surface3. While

PRG4 alone is an effective boundary lubricant, it also acts synergistically with

hyaluronan (HA) to further reduce friction to levels approaching that of whole SF34

. HA,

a linear polymer of repeating disaccharides composed of D-glucuronic acid and D-N-

acetlyglucosamine115

, is another boundary lubricant present in SF34

. It appears that both

PRG4 and HA are critical to the boundary lubricating function of human SF (hSF).

Changes in the PRG4 composition of hSF after acute injury and in osteoarthritis

(OA) have been observed. Average concentrations of PRG4 in normal (NL) hSF between

35 and 250 μg/mL20,103-107

have been reported. PRG4 concentrations have been observed

to decrease significantly after anterior cruciate ligament (ACL) injury, returning to

normal within approximately 1 year20

. Concentration has been observed to increase after

intra-articular fracture103

, remain normal after internal derangement105

, and be elevated in

late stage OA104,106

. However, animal models have suggested that PRG4 concentration in

SF and presence in the superficial zone can decrease in secondary OA117,118,119

. Along

with altered lubricant composition, compromised boundary lubricating ability was

observed after intra-articular fracture103

. However, no difference between the steady state

33

boundary lubricating ability of OA and NL hSF has been observed106,110

. Mutations in the

PRG4 gene cause an autosomal recessive disorder in humans, camptodactyly-

arthropathy-coxa vara-pericarditis (CACP) syndrome120

. hSF from these patients is void

of PRG4 and fails to lubricate21

. Collectively these findings in normal, injured, and

diseased hSF suggest that hSF deficient in PRG4 lacks normal boundary lubricating

ability.

HA composition of hSF has also been observed to change with injury and disease.

Average normal concentrations of HA in hSF range between 1.8 and 3.33

mg/mL21,102,103,105,106,110

. HA concentration in hSF has been observed to remain normal in

internal derangement injuries105

, to significantly decrease with intra-articular fracture103

,

effusive joint injury, and arthritic disease102,111,121

, and to remain normal during

OA106,110,81

and CACP21

. HA concentration has also been observed to be correlated with

patient age81

. HA molecular weight (MW) distribution has been shown to range

continuously between 27 kDa and 10 MDa in normal hSF, peaking between 6 – 7

MDa81,112-114

. HA MW distribution has been observed to shift to the lower range during

injury105

and OA106

, but has also been observed to remain constant between NL and OA

hSF81

. HA MW distribution in hSF is of interest for the potential difference in lubricating

ability and interaction with PRG4 of different MW HA species35

. It has been observed

that HA supplementation of HA deficient equine SF after acute injury was able to restore

compromised boundary lubricating ability87

.

Intra-articular (IA) HA is currently used to treat OA. Commercially available

formulations of IA HA range from 0.5 – 6 MDa and 8 – 15 mg/mL88,122

. It has been

demonstrated that IA injection of PRG4 using rat injury models of OA protects against

34

cartilage degeneration75,123,124

. The potential application of PRG4 as a new and improved

therapy for treatment of post-injury and OA knee joints, and maintenance of healthy

joints, is promising. However, it is unclear if PRG4 concentrations remain normal in OA

hSF, and the biomechanical effects of supplemental PRG4 on the boundary lubricating

ability of hSF, especially that deficient in PRG4, on normal human cartilage are

unknown.

The objectives of this study were therefore to: (1) quantify PRG4 and HA content

in NL and chronic OA hSF and (2) assess the human cartilage boundary lubricating

ability of PRG4-deficient OA hSF compared to NL hSF, with and without

supplementation of PRG4±HA. The hypothesis was that OA hSF can have diminished

PRG4 content and associated impaired cartilage lubricating ability, which can be at least

partially restored by supplementation with PRG4 and/or HA.

2.3 Materials & Methods

2.3.1 Materials

Materials for the PRG4 enzyme linked immunosorbent assay (ELISA)59

and

PRG4 preparation and lubrication testing34

were obtained as described previously. In

addition, disodium ethylenediaminetetraacetate (Na2-EDTA), Benzamidine hydrochloride

(BHCl), N-Ethylmaleimide (NEM), and bicinchoninic acid (BCA) protein assay kit were

from Thermo Fisher Scientific (Rockford, IL, USA). Phenylmethylsulfonyl fluoride

(PMSF) was from Bio Basic (Amherst, NY, USA). Costar enzyme immunoassay high

35

binding plates were from Corning Inc (Corning, NY, USA). Horseradish peroxidase

conjugated peanut agglutinin (PNA-HRP), 3,3′,5,5′-Tetramethylbenzidine (TMB) tablets,

dimethyl sulfoxide, hydrogen peroxide (30%), dibasic sodium phosphate, citric acid,

sulfuric acid (95.0-98.0%) and Stains-All were from Sigma-Aldrich (St. Louis, MO,

USA). Hyaluronan DuoSet ELISA Development kit was from R&D Systems

(Minneapolis, MN, USA). Proteinase K was from Roche Applied Science (Laval, QC,

CAN). MegaLadder and HiLadder HA MW markers were from Hyalose LLC (Oklahoma

City, OK, USA). Sodium hyaluronate (1.5 MDa) was from Lifecore Biomedical (Chaska,

MN, USA). Materials and equipment for SDS-PAGE Western blotting and protein

staining were obtained from Invitrogen (Carlsbad, CA, USA).

2.3.2 Samples

Collection of all human tissues and fluids was approved by the University of

Calgary Conjoint Health Research Ethics Board. OA hSF was aspirated from patients

with symptomatic chronic knee OA requiring aspiration prior to therapeutic injection.

Patients were diagnosed with knee OA by 2 sport medicine physicians (co-authors VL

and PW) following a review of patient symptoms, physical examination, and plain-film

radiographs. OA hSF was aspirated using standard sterile knee aspiration technique. As

much fluid as possible was aspirated with each attempt. NL hSF (N = 13) and normal

human distal femurs (N = 3) were obtained through the Joint Transplantation Program at

the University of Calgary and were harvested within 4 hours of donor death. Femurs were

stored at -80°C until use. Articular cartilage was macroscopically normal (International

Cartilage Repair Society grade 1 – 2), as assessed at time of use. NL and OA hSF were

36

clarified by centrifugation (3000g, 30 minutes, 4°C20,103,110

) prior to storage at -80°C with

protease inhibitors (PIs), as well as without PIs for HA MW analysis (when sufficient

volume available). “N” represents 1 hSF sample. Sixteen OA hSF samples were screened

for PRG4 concentration. Samples with low PRG4 (OA-LO, defined as average PRG4

concentration below the average PRG4 concentration in NL hSF, N = 5) were selected

for lubrication testing and assessed as a distinct group. Patients had no history of

therapeutic injection or injury within 4 months of aspiration.

2.3.3 hSF Biochemical Characterization

Biochemical characterization was performed on N = 16 OA and N = 13 NL

samples. As this is an ongoing study, PRG4-deficient samples were selected for

lubrication testing as they were identified. PRG4-deficient samples were selected if

patients had no recent history of injury or prior therapeutic injection, sufficient volume

for lubrication testing, and no visible contamination with blood after clarification. The

number of PRG4-deficient samples selected is not intended to reflect a proportion of the

OA population that is OA-LO. Total protein concentration was measured by BCA assay

on hSF samples in duplicate diluted 30 and 60X in distilled water.

PRG4 Concentration. PRG4 concentration in hSF was measured, in triplicate, by

custom sandwich ELISA. An anti-peptide capture antibody (LPN) recognizing AA1356 –

1373 at the C- terminal of full length PRG459

was used, followed by detection with PNA-

HRP125

. hSF was digested with S. Hyaluronidase (1 U/mL, 3hrs at 37°C) and

subsequently with Sialidase A-66 (neuraminidase, overnight at 37°C) prior to

37

quantification. Purified PRG4 controls (described below) were also treated with

Sialidase.

Purified control PRG4 for the ELISA was prepared from culture medium

conditioned by bovine cartilage explants as described previously34

. PRG4 standards used

to determine hSF PRG4 concentrations were purified by DEAE-Sepharose anion

exchange chromatography and Superose 6 size exclusion chromatography, verified for

purity by western-blot analysis and quantified by BCA. An appropriate diluent was used

so that the slopes of the control and sample absorbance curves were equivalent in the

linear range of the sigmoidal curve.

High binding ELISA plates were coated with capture antibody (50 μL LPN at 2

μg/mL) overnight at 4°C. Plates were then washed and blocked with 5% milk in PBS for

1 hour at 37°C. After the block was removed, hSF samples diluted to 4X and PRG4

controls at 320 μg/mL were loaded, in triplicate, serially diluted (2X) and incubated for 1

hour at 37°C with nutation. The plates were then washed and incubated with detection

PNA-HRP (50 μL at 5 μg/mL) for 1 hour at 37°C. Plates were washed, developed with

TMB, and stopped with 2M sulfuric acid. Plates were read at 450 nm and 540 nm;

readings at 540 nm were subtracted from those at 450 nm to correct for optical properties

of the plastic (as per manufacturer recommendation).

The assay was able to detect PRG4 to 10 μg/mL in 90 μL of hSF diluted to 4X.

The coefficient of variation for triplicates averaged 12 ± 9 % (mean ± SD). Variation

between plates averaged 17 ± 9% (mean ± SD). ELISA specificity for high MW PRG4

immunoreactive to both LPN and PNA-HRP was confirmed by western blot on purified

38

PRG4 and hSF following 3 – 8% TrisAcetate SDS-PAGE and transfer to polyvinylidene

fluoride (PVDF) membrane (Figure 2-1).

Figure 2-1: Characterization of the PRG4 ELISA control by protein stain

(A) and high MW PRG4 immunoreactivity in PRG4 control, NL hSF, and

OA hSF by western blotting (B, C). PRG4 controls treated with

neuraminidase and hSF treated with hyaluronidase and neuraminidase

were probed with (B) LPN and (C) PNA-HRP. Samples were subjected to

3 – 8 % SDS-PAGE followed by protein stain or western blotting as

described in Materials and Methods.

39

HA Concentration. HA concentration in hSF was measured, in triplicate, using a

commercially available sandwich ELISA which provided recombinant human aggrecan

as a capture reagent and biotinylated recombinant human aggrecan for detection. hSF

samples were diluted 1:40000 in 5% Tween 20 in PBS. Intra-assay variation averaged 18

± 10% (mean ± SD) and inter-assay variation 13 ± 12% (mean ± SD).

HA MW Distribution. HA MW distribution in hSF samples, stored without PIs

and treated with Proteinase K, was measured in duplicate by 1% agarose gel

electrophoresis, as described in a previous study112

. HA MW distribution was measured

in 8 NL hSF samples, and the 5 OA-LO. Briefly, Hi- (0.5 – 1.5 MDa) and MegaLadder

(1.5 – 6.1 MDa) MW markers were used as HA controls. One blank lane was left

between samples for background measurement. After electrophoresis for 3 hours at 50 V,

gels were stained with 0.005% Stains-All in 50% ethanol and de-stained in 10% ethanol.

The migration of HA was assessed by densitometric analysis with Image J (NIH,

Bethesda, MD).

2.3.4 Cartilage Boundary Lubricating Ability

Human cartilage boundary lubricating ability of hSF was tested in a cartilage-on-

cartilage friction test in the boundary lubrication regime using normal human

osteochondral cores as described previously29

. Briefly, annulus and core shaped

osteochondral samples were harvested from macroscopically normal areas of the

patellofemoral groove of human distal femurs (3 donors, age 64 ± 4 (mean ± SD)).

Samples were shaken vigorously overnight at 4°C in 40 mL of PBS to rinse residual hSF

from the articular surface (previously confirmed by lubrication testing29,34

). Samples were

40

bathed overnight in the subsequent test lubricant at 4°C prior to lubrication testing; the

cartilage surface was completely immersed in 0.1 mL (annulus) and 0.2 mL (core).

Boundary lubrication tests were performed on an ELF 3200 (Bose EnduraTEC,

Minnetonka, MN) as described previously29

. Samples were first compressed at 0.002

mm/s to 18% of the total cartilage thickness followed by a 40 minute stress relaxation

period to allow for interstitial fluid depressurization period. Using an exponential decay

curve fit for load during stress relaxation confirmed that approximately 63.2% of the

equilibrium load was reached after an average time constant of 6.7 minutes, and 98.1%

was reached at 27 minutes. Furthermore, predicted values of load at 40 minutes and 60

minutes were within 0.002 N of one another. This indicates that fluid depressurization

was achieved at 40 minutes, nearly 6 times the time constant. Without removing

compression, samples were rotated +2 and -2 revolutions at 0.3 mm/second with pre-

sliding durations (Tps: duration of time the samples are stationary prior to rotation) of

120, 12, and 1.2 seconds. The test sequence was then repeated in the opposite direction of

rotation. This friction test has been shown to maintain boundary lubrication at a

depressurized cartilage-cartilage interface29

.

In all experiments, each osteochondral pair (annulus and core from the same

donor but not necessarily the same joint) was tested sequentially in each of the 5 test

lubricants. Each OA hSF sample found to be deficient in PRG4 (OA-LO, N = 5 of the

initial 16 samples) was tested in triplicate (n = 3 annulus and core pairs) in the following

sequence: 1) PBS (negative control lubricant), 2) OA-LO, 3) OA-LO+PRG4, 4) OA-

LO+PRG4+HA, 5) NL (positive control lubricant). NL hSF from 1 of the 13 donors (left

and right knee, average [PRG4] 254.7 ± 118.5 μg/mL, average [HA] 0.23 ± 0.12 mg/mL,

41

age 59) was used for all cartilage boundary lubricating ability experiments. OA-LO hSF

was supplemented with PRG4 and HA at concentrations based on preliminary ELISA

measurements in NL hSF. Purified PRG4 at 450 µg/ml (obtained as described above) and

1.5 MDa HA at 1 mg/ml were dried and re-suspended in OA-LO hSF. Static, μstatic,Neq

(representing resistance to the onset of motion), and kinetic, <μkinetic,Neq> (representing

resistance to steady motion), friction coefficients were calculated29

.

2.3.5 Statistical Analysis

Data is presented as mean ± SEM unless noted otherwise. Repeated measures

ANOVA was used to assess effects of lubricant solution and Tps (as repeated factors) on

μstatic,Neq and <μkinetic,Neq>. The effect of test lubricant on <μkinetic,Neq> at Tps = 1.2 seconds

was assessed by ANOVA with Tukey post hoc testing. ANOVA was used to assess

differences in PRG4 and HA composition. Arcsine-square root transformation was used

to improve uniformity of the variance for HA MW proportional (%) distribution126

.

Statistical analysis was performed with Systat 12 (Systat, Richmond, CA).

2.4 Results

2.4.1 hSF Biochemical Characterization

Samples identified as OA-LO and selected for friction testing were similar to NL

samples in terms of donor characteristics, as shown in Table 2-1. There was no

significant difference between the ages of the OA-LO patients and NL donors (p = 0.29).

42

Total aspirate volume was significantly higher in OA-LO (17.2 ± 6.2 mL vs. 4.5 ± 1.3

mL, p < 0.01), as was total protein concentration (28.8 ± 2.0 mg/mL vs. 15.6 ± 1.3

mg/mL, p < 0.001).

43

Table 2-1: Patient characteristics of hSF samples identified as PRG4-

deficient and selected for lubrication testing (OA-LO). Average donor

characteristics of NL hSF (N=13).

All data shown as mean ± SEM. Normal (NL), PRG4-deficient (OA-LO),

male (M), female (F). a: significantly higher than NL hSF, p < 0.05.

Note: Of the 13 NL hSF donors, 1 sample was used as a positive control

lubricant for the cartilage boundary lubricating ability tests. Osteochondral

samples were harvested from distal femurs obtained from 3 of the 13

donors.

Sample Age Sex Aspirate

Volume (mL)

Total Protein

(mg/mL)

OA-LO 1 56 M 9 22.2

OA-LO 2 79 M 12 33.2

OA-LO 3 54 M 42 30.8

OA-LO 4 62 M 10 31.4

OA-LO 5 66 F 13 26.6

OA-LO

Avg

63 ± 4 17. 2 ± 6.2a 28.8 ± 2.0

a

NL Avg 58 ± 3 10M, 3F 4.5 ± 1.3 15.6 ± 1.3

44

PRG4 Concentration. PRG4 concentration showed variability across both NL and

OA samples (Figure 2-2, this figure is not intended to portray that a certain proportion of

OA hSF is OA-LO). PRG4 concentration in NL hSF averaged 287.1 ± 31.8 μg/mL.

Samples identified as OA-LO and selected for lubrication testing averaged 146.5 ± 28.2

μg/mL and were significantly deficient in PRG4 relative to NL (p < 0.05, Figure 2-2).

(Samples measured but not identified as low in PRG4 are shown in Appendix E).

Figure 2-2: PRG4 concentration measured in OA hSF. This figure is not

intended to portray that a certain proportion of OA hSF is OA-LO.

[PRG4] in NL samples shown in white bars. Average [PRG4] in NL (N =

13, ) shown by black line. OA-LO (N = 5) samples selected for friction

testing shown with black bars. Average [PRG4] in OA-LO (

shown by grey line. * = p < 0.05.

45

HA Concentration. HA concentration did not vary between NL and OA hSF

(Figure 2-3A). HA concentration in NL hSF averaged 0.54 ± 0.09 mg/mL (range 0.11 –

0.96 mg/mL). OA-LO samples were not significantly different from NL (0.73 ± 0.08

mg/mL, p = 0.26).

HA MW Distribution. HA MW distribution was shifted towards the lower MW

range in OA-LO compared to NL hSF (Figure 2-3B). Relative HA concentration (as a

percentage of total concentration) in the > 6.1 MDa range tended to be lower in OA-LO

(0.7 ± 0.4%) compared to NL hSF (2.8 ± 1.0%, p = 0.05). In the 3.1 – 6.1 MDa range,

OA-LO (33.6 ± 2.9%) was significantly lower compared to NL hSF (49.1 ± 3.6%, p <

0.05). In the 1.1 – 3.1 MDa, 0.5 – 1.1 MDa, and < 0.5 MDa ranges, OA-LO hSF was

significantly higher than NL hSF (31.1 ± 1.7 vs. 24.7 ± 1.2%, 21.7 ± 1.1 vs. 13.4 ± 1.3%,

12.9 ± 2.0 vs. 7.1 ± 0.8%, all p < 0.05.)

46

Figure 2-3: (A) Average HA concentration in NL and OA-LO hSF. (B)

HA MW distribution in measured NL hSF (N = 8), and OA-LO (N = 5). *

= p < 0.05.

2.4.2 Cartilage Boundary Lubricating Ability

In all experiments, friction was modulated by test lubricant and Tps. In all test

lubricants, μstatic,Neq decreased with decreasing Tps and appeared to approach <μkinetic,Neq>

asymptotically as Tps decreased from 120 seconds towards 0 seconds. Values of μstatic,Neq

were consistently highest in PBS, ranging from 0.143 ± 0.011 at Tps = 1.2 seconds to

0.242 ± 0.013 at Tps = 120 seconds; values were lower and similar for NL and

supplemented hSF samples, ranging from 0.026 ± 0.002 at Tps = 1.2 seconds to 0.096 ±

0.007 at Tps = 120 seconds for NL hSF. In all test lubricants, values of <μkinetic,Neq>

increased only slightly with increasing Tps, with mean ± SD values at Tps = 1.2 seconds

47

being on average within 13 ± 1% of values at Tps = 120 seconds. Therefore, as presented

previously34

and for brevity and clarity, <μkinetic,Neq> data are shown at Tps = 1.2 seconds

only. Average equilibrium stress for all tests was 0.209 ± 0.026 MPa.

OA hSF deficient in PRG4 failed to lubricate as well as NL hSF. Both μstatic,Neq

and <μkinetic,Neq> varied with test lubricant and Tps, with an interaction effect (all p <

0.001) (Figure 2-4A). <μkinetic,Neq> at Tps = 1.2 seconds also varied with test lubricant (p

< 0.001) (Figure 2-4B). <μkinetic,Neq> for OA-LO was significantly higher than NL hSF

(0.043 ± 0.008 vs. 0.025 ± 0.002, p < 0.05).

Friction coefficients in OA-LO samples were restored to levels of NL hSF with

PRG4 supplementation (Figure 2-4). <μkinetic,Neq> in OA-LO (0.043 ± 0.008) was

significantly reduced in OA-LO+PRG4 (0.023 ± 0.003, p < 0.05). <μkinetic,Neq> in OA-LO

(0.043 ± 0.008) was also significantly reduced in OA-LO+PRG4+HA (0.024 ± 0.002, p <

0.05).

In general, no additional effect on lubricating ability was provided by subsequent

HA supplementation. <μkinetic,Neq> in OA-LO+PRG4 and OA-LO+PRG4+HA did not

differ from each other, nor from NL hSF (p = 0.996-1).

48

Figure 2-4: Static (μstatic,Neq) (A) and kinetic <μkinetic,Neq> at Tps = 1.2

seconds (B) friction coefficients of PRG4 deficient OA hSF (OA-LO, N =

5), with 450 µg/ml PRG4 and 1.0 mg/ml 1.5 MDa HA supplementation,

and NL hSF. * = p<0.05.

49

2.5 Discussion

This study provides insight into the molecular basis for altered cartilage boundary

lubricating ability of OA hSF. These results are consistent with the notion that PRG4

concentrations can vary considerably within OA patients, and also within NL donors.

Furthermore, they indicate that normal PRG4 levels may not be present in all chronic OA

hSF, and suggest a sub-population of OA patients who demonstrate PRG4 deficiency,

associated with diminished cartilage boundary lubricating ability, in their hSF may exist.

These results further emphasize that PRG4 is a critical boundary lubricant and is required

for normal joint lubrication.

This ELISA extends upon previous PRG4 quantification methods. In this assay,

hSF was treated with neuraminidase prior to quantification. PNA-HRP has previously

been used as a capture reagent for an hSF sandwich ELISA104,20

without neuraminidase

digestion. Due to ~46% capping of human PRG4 glycosylations with sialic acid62

, PRG4

concentration measured with and without neuraminidase digestion may differ. Digestion

of hSF and control PRG4 with neuraminidase prior to ELISA measurement increased

both PRG4 control and hSF signal strength. PRG4 concentration in samples not treated

with neuraminidase could not be accurately determined from similarly treated controls

due to the very low signal obtained, as the assay is optimized for controls and samples

treated with neuraminidase. Potential PRG4+HA interactions that may interfere with

antibody recognition of PRG4 were disrupted using hyaluronidase, as previously

performed in a quantitative western blot method103,105,106,

. Several antibodies have been

used amongst previous PRG4 quantification methods103,105,127,

. This ELISA recognizes

high MW PRG4 species (> 345 kDa, including multimers, identified by LPN capture59

)

50

with glycosylations (identified by PNA-HRP detection)125

, both of which are important

for functionality62

. Finally, hSF was stored with PIs before quantification; sample storage

without PIs may result in an underestimate if PRG4 has degraded during storage.

Addition of PIs had no effect on ELISA-measured PRG4 signal (data not shown).

PRG4 concentrations obtained for NL hSF in this study are in agreement with

those measured in previous studies. Furthermore, the range of PRG4 concentrations in

NL hSF measured (129 – 450 μg/mL) reflects the previously reported wide range of

PRG4 concentrations in normal hSF20,103-107

. Large variability within diseased hSF has

been reported (276 – 762 μg/mL105-107

), and was also observed (range in all OA hSF

measured 95 – 426 μg/mL). It should be noted that none of the OA-LO donors had

history of recent injury, which is known to affect PRG4 concentration20

. PRG4

concentration has previously been observed to increase with OA104,106,107

, and several

samples with normal to elevated PRG4 concentrations were also identified in this study

(data not presented). While a decrease in PRG4 with OA has not previously been

reported in humans, a decrease in SF PRG4 with secondary OA has been observed in

guinea pigs117,118

, as has decreased PRG4-positive chondrocyte presence in the superficial

zone after meniscectomy in an ovine model119

. A decrease in lubricating ability of hSF

from patients with rheumatoid arthritis (RA) has been observed110

, as has a classification

of RA patients based on high and low expression of PRG4 in synovium109

. Possible

mechanisms for decreased PRG4 concentration in the OA-LO patients identified in this

study include decreased PRG4 expression/synthesis, increased degradation of PRG420

, or

increased loss of PRG4 from the joint capsule through an inflamed synovium4,101

. Further

investigation into characteristics of the patients studied here would contribute to the

51

understanding of the mechanism underlying PRG4 deficiency. Increased friction due to

PRG4 deficiency is a clinically relevant issue, as friction and wear are thought to be

coupled at the articular surface21

.

The normal HA concentration and shift to lower MW observed in the OA-LO

samples is consistent with previous studies105,106,110,81

. The HA concentrations measured

are lower than observed in previous studies of hSF. Concentrations ranged from 0.11 –

0.96 (NL) and 0.23 –2.69 mg/mL (OA, not friction tested), and in the literature from 1.8

– 3.33 mg/mL (NL11,13-14,19,21-22

) and 0.1 – 1.3 mg/mL (diseased102,111

). There was no

statistically significant difference between OA and NL hSF HA concentration, as

previously reported106,110,81

. HA concentrations measured for bovine SF (bSF, 0.32 – 0.79

mg/mL, data not shown) agree with previously measured values (~0.5 mg/mL128

).

Both PRG4 deficiency and a shift towards lower MW of HA in some chronic OA

hSF were observed in the current study. Previous studies have demonstrated that the

boundary lubricating ability of HA alone increases with increasing MW87

, however the

synergistic boundary lubricating ability of PRG4+HA is not dependent on MW35

. These

studies together suggest that treatment with PRG4 could negate the deleterious effects of

a shift to low HA MW in OA hSF and prevent alterations in boundary lubricating

ability35

. Completing biochemical and biomechanical characterization on hSF samples

with normal and elevated PRG4 concentrations (identified but not described) will help

clarify this relationship. In this study a statistically significant effect of additional

supplementation with HA on boundary lubricating ability of PRG4 deficient OA hSF

samples was not observed. However as PRG4 supplementation of PRG4-deficient

samples was of interest and performed first, the effect of HA supplementation alone in

52

hSF remains to be fully elucidated. Other studies have shown that HA supplementation of

acute injury equine SF deficient in HA restored compromised boundary lubricating

ability87

. Alterations in boundary lubricating ability of hSF is of great interested, as small

increases in friction have been observed to be associated with increased wear at articular

surfaces21

.

This study is unique in that both normal human cartilage and SF were obtained for

controls. Normal human cartilage was obtained from macroscopically normal areas of

femurs from donors not taking anti-inflammatory drugs. The coefficients of friction for

boundary lubrication obtained for NL hSF on normal human cartilage (<μkinetic,Neq> =

0.025) agree with coefficients of friction measured for bSF on bovine cartilage in an

identical test (<μkinetic,Neq> = 0.02534

); this supports the use and description of the normal

cartilage. Furthermore, total protein concentrations measured in NL hSF were consistent

with previously reported values (18 – 28 mg/mL)4,101

and were lower than that measured

in OA. NL hSF volumes obtained were generally within the normal range of 0.5 – 4 mL4.

OA volumes were significantly higher, as expected. It should be noted that in this study,

no correlation between aspirated volume and [PRG4] was observed. Previous studies

using this in vitro cartilage-cartilage friction test confirmed that up to 5 sequential tests

could be conducted on a single osteochondral pair over 5 days, with overnight storage at

4°C between tests, without sample degradation29

. To account for any potential carryover

effect of test lubricants, and to isolate the effect of PRG4 supplementation, the test

sequence used was chosen in order of presumed increasing lubricity. The HA and PRG4

used in this study were representative of those in native hSF and have been used in other

studies35

. The concentration for PRG4 supplementation was selected based on values

53

previously observed to provide boundary lubrication34

, previously reported values in

hSF20,103-107

, and preliminary measurements of NL hSF by ELISA (as additional NL hSF

samples are obtained and characterized on an ongoing basis). HA concentration for

supplementation was selected based on preliminary measurements of NL hSF by ELISA,

and a MW of 1.5 MDa was selected as it is in the range of commercially available IA HA

formulations88,122

. Furthermore, 1.5 MDa HA has previously been shown to provide

boundary lubrication35

.

This study supports and significantly extends the observation that hSF deficient in

PRG4 demonstrates decreased boundary lubricating ability. OA-LO hSF samples

identified had normal HA concentration, altered HA MW distribution, and decreased

lubricating ability. This suggests that HA MW distribution may be important, that low

MW HA alone is not sufficient to provide normal boundary lubrication, and provides

further motivation to study PRG4+HA interactions in SF. PRG4 has been observed to

exist in both a disulfide bonded multimeric form and monomeric form, which may affect

lubricating function59

. Future work determining the multimer:monomer composition of

PRG4 in NL hSF and alterations with OA will provide further insight into this

fundamental joint lubrication mechanism. Altered glycosylation patterns in OA, as

observed between RA and OA, could be another source of variation in boundary

lubricating ability125

. The observations of this study are supported by in vivo studies by

other research groups demonstrating that IA injection of PRG4 into an injury-induced OA

model in rats can prevent cartilage degeneration75,123

. Collectively these results taken

together with those of the present study suggest that in addition to post-injury patients,

some chronic OA patients who have PRG4-deficient SF may benefit from PRG4

54

supplementation as a biotherapeutic treatment to restore lubrication and maintain healthy

joints.

2.6 Acknowledgements

This chapter, in full, is published in Arthritis & Rheumatism volume 64, no 12,

December 2012, page 3963-3971. The candidate is the primary author and thanks co-

authors Jenelle McAllister, Dr. Victor Lun, Dr. J. Preston Wiley, and Dr. Tannin A

Schmidt. Study conception and design was performed by TL and TS. Acquisition of data

was performed by TL (biochemical and biomechanical data), JM (SF acquisition), VL

(SF acquisition), and PW (SF acquisition). Analysis and interpretation of data was

performed by TL, PW, and TS. All authors were involved in revising the article and

approved the final submitted version.

The authors also thank the University of Calgary Joint Transplantation Program

for access to the normal human tissue, the Sports Medicine Centre at the University of

Calgary for collection of OA hSF, Mrs. Sue Miller and Dr. Roman Krawetz for assistance

with collection of NL hSF (and RK for assistance with the HA ELISA). This work was

supported by funding from the National Science and Engineering Research Council of

Canada, Canadian Arthritis Network, Alberta Innovates-Technology Futures, Alberta

Innovates Health Solutions OA Team Grant, Faculty of Kinesiology and Schulich School

of Engineering’s Center for Bioengineering Research and Education at the University of

Calgary.

55

Chapter Three: Effect of flare reaction to intra-articular hyaluronan injection on

cartilage boundary lubricating ability of human synovial fluid: A case series

3.1 Abstract

Background: Proteoglycan 4 (PRG4) and hyaluronan (HA) are key constituents

of synovial fluid (SF) that contribute to boundary lubrication. Decreased boundary

lubricating ability of SF may be associated with increased wear at the articular surface,

making SF composition of boundary lubricants an important clinical consideration. The

effect of a flare reaction to intra-articular (IA) HA injection on SF boundary lubricant

composition and function is currently unknown.

Purpose: The objectives of this study were to 1) quantify PRG4 and HA content

in OA SF after flare reaction IA HA injection and 2) assess the cartilage boundary

lubricating ability of PRG4-deficient flare-SF, with and without supplementation with

PRG4±HA.

Study Design: Retrospective case series. Seven SF samples from 5 OA patients

who returned to the clinic (for treatment of swelling or persistent pain after injection)

within 11 days of initial IA HA were included in this study. One patient was aspirated on

both day 5 and day 7 after the initial injection, and the left and right knees of another

patient were both aspirated on day 11 after initial injection.

56

Methods: PRG4 and HA concentration were measured by sandwich enzyme

linked immunosorbent assay, and HA molecular weight (MW) was measured by 1%

agarose gel electrophoresis. Five flare samples that were identified as having low PRG4

concentration were selected for cartilage-cartilage boundary lubricating ability tests.

Results: Flare-SF samples contained PRG4 and HA concentrations ranging from

below normal to super-physiological. HA MW in these samples was shifted towards the

lower range in the 3.1 – 6.1 and 0.5 – 1.1 MDa ranges (p = 0.02, 0.005) only. The kinetic

coefficient of friction in PRG4-deficient flare-SF was not altered compared to normal SF,

and no changes were observed with PRG4 or PRG4+HA supplementation (p = 0.70 –

1.0).

Conclusions: SF can exhibit altered boundary lubricant composition after a flare

reaction to IA HA. Despite a decrease in PRG4 concentration in some samples, normal

cartilage boundary lubricating ability was retained, possibly due to sufficient high MW

HA content.

Clinical Relevance: Understanding changes in PRG4 and HA content in SF from

acutely injured and chronically diseased joints will contribute to the development of

biotherapeutic treatments to restore/maintain boundary lubricating ability and possibly

prevent wear of the articular surface.

57

3.2 Introduction

Lubrication of articular cartilage is achieved by a combination of lubrication

mechanisms. Fluid film lubrication occurs at high speeds and low loads when cartilage

surfaces are separated by a layer of synovial fluid (SF). Boundary lubrication occurs at

low speeds and high loads when cartilage surfaces are in contact and lubrication is

provided through molecular interactions at the surface3. This surface-to-surface contact is

thought to occur over approximately 10% of the cartilage area, exposing these contact

areas to high friction28

. The hydrostatic pressure that supports load during fluid film

lubrication dissipates over time with loading, causing the cartilage surfaces to bear more

load129

. Whole SF has been shown to be an effective boundary mode lubricant29

, and this

boundary lubricating ability is provided primarily by its constituents proteoglycan 4

(PRG4) and hyaluronan (HA)34

. SF boundary lubricant composition and function are of

clinical interest, as friction has been observed to be associated with wear at the articular

surface21

.

The PRG453

gene encodes for mucin-like O-linked glycosylated proteins,

collectively referred to as PRG4. PRG4 is synthesized by cells within articular joints,

including superficial zone articular chondrocytes6 and synoviocytes

116, and is present in

SF47

and at the articular cartilage surface48

. PRG4 is important for normal boundary

lubricating function; SF from patients with a genetic autosomal recessive disorder caused

by mutations in the PRG4 gene, camptodactyly-arthropathy-coxa vara-pericarditis

(CACP) syndrome120

, is void of PRG4 and fails to lubricate compared to normal SF21

.

Furthermore, PRG4 knock-out mice demonstrate earlier cartilage wear and higher total

joint friction21

, and in human SF PRG4 concentrations are decreased for approximately 1

58

year after ACL tear20

. While PRG4 is necessary for normal joint function, other SF

constituents and their interactions with PRG4 are also required to maintain normal

boundary lubricating ability.

HA, a linear polymer of repeating disaccharides composed of D-glucuronic acid

and D-N-acetlyglucosamine115

, is another boundary lubricant present in SF34,104

. It

appears that both PRG4 and HA are critical to the boundary lubricating function of SF, as

PRG4 acts synergistically with HA to reduce friction to levels approaching that of whole

SF34

. Decreases in HA concentration and/or molecular weight87

in post-injury equine SF

and PRG41 concentration in chronic OA human SF have been observed to be linked to

decreased boundary lubricating ability at a cartilage-cartilage interface in vitro;

lubricating ability could respectively be restored with HA or PRG4 supplementation.

While the mechanism of the PRG4+HA interaction is currently unknown, both are

required to approach the boundary lubricating ability of SF in in vitro cartilage-cartilage

boundary lubrication tests.

HA of various molecular weight (MW) is routinely used, over long-terms, as a

safe and effective (though this has come under criticism lately by the American Academy

of Orthopaedic Surgeons23

) intra-articular (IA) viscosupplement for pain relief in

osteoarthritis (OA)92

. It has been shown to provide pain relief for up to 6 months88

despite

a comparatively short residence time in the joint (8.8 days for hylan G-F 20, a high MW

~6 MDa partially cross-linked product130

). An inflammatory or flare reaction is an

adverse event associated with all IA injections131

, including viscosupplementation with

HA132

. There appears to be several types of flare reactions that can occur in response to

IA injections. First, some flare reactions are associated with injection site pain and

59

swelling133

24 – 72 hours after injection134

; these reactions are usually mild, do not recur,

and subsequent injections can be performed133

. Secondly, injection of cross-linked HA

products may be associated with infrequent severe acute inflammatory reactions, or

pseudosepsis; these reactions often require clinical intervention and tend to occur after

exposure to more than one injection133

. Some flares occurring after hylan G-F 20

injection are thought to be cell-mediated hypersensitivity reactions135

.

Through the natural course of disease, OA patients can experience “flare-ups” in

symptoms; these flare-ups are characterized by sudden aggravation of knee pain, with an

identifiable onset, that causes nocturnal awakening, and evidence of effusion136

. SF from

flare-up patients has been observed to have lower HA concentration (with higher MW),

increased protein size and concentration, and decreased viscosity compared to OA

patients without flare-up136

; decreased viscosity could have an effect on the fluid film

lubricating function of SF.

This case series focuses on SF from patients that have had a flare reaction to IA

HA injection, as the effects of a flare reaction to IA injection on SF boundary lubricant

composition and function are unknown. The objectives of this study were to: 1) quantify

PRG4 and HA content in OA SF after flare reaction to IA HA injection and 2) assess the

cartilage boundary lubricating ability of PRG4-deficient flare-SF, with and without

supplementation with PRG4±HA. These results were then compared to SF from non-

arthritic knees. The hypothesis was that flare-SF can have diminished PRG4 content and

associated impaired cartilage boundary lubricating ability, which can be at least partially

restored by supplementation with PRG4 and/or HA.

60

3.3 Materials and Methods

This was a retrospective case series study approved by the Conjoint Health

Research Ethics Board at the University of Calgary. Informed consent was obtained from

patients prior to aspiration. In a larger ongoing study, SF was aspirated from patients with

symptomatic chronic knee OA requiring aspiration prior to therapeutic IA HA or

corticosteroid injection. Patients of both sexes and all ages were included in this larger

study if they were diagnosed with knee OA by a sport medicine physician after physical

examination and review of patient symptoms and plain-film radiographs; concurrent

pathologies in addition to OA (chondral damage, meniscal tears, previous joint injury

etc.) were noted, and these samples were not selected for this case series. For this case

series, patients who returned to the clinic for subsequent treatment within 2 weeks of

receiving an IA HA injection (hylan G-F 20) and whose knee was aspirated again at this

time were included. (While the exact mechanism of these flare reactions is unknown, the

patients were not identified as having severe pseudoseptic reactions, and likely returned

for treatment of swelling or persistent pain after injection.) OA SF was aspirated using

standard sterile knee aspiration technique, and as much fluid as possible was aspirated

with each attempt. Normal SF and normal human distal femurs were obtained through the

Joint Transplantation Program at the Institution and were harvested within 4 hours of

donor death. Femurs were stored at -80˚C until use. Normal and flare-SF were clarified

by centrifugation20,110

prior to storage at -80˚C with protease inhibitors, as well as without

protease inhibitors for HA MW analysis when sufficient was volume available.

Seven flare-SF samples from 5 knee OA patients requiring aspiration ≤ 11 days

after HA injection were included in this study. One patient had both the left and right

61

knees aspirated after a flare reaction to IA HA in both knees, and one patient was

aspirated on day 5 and day 7 after the initial injection (biochemical data for aspiration at

day 5 and 7 are presented for completeness, however only the SF aspirated at day 7 was

examined for lubricating ability). Samples were first screened for PRG4 concentration to

select samples of interest for lubricating ability testing. Five samples from 4 patients were

identified as having PRG4 concentration lower than the average normal SF concentration

(cadaveric, N = 29) and were selected for boundary lubricating ability tests. The number

of PRG4-deficient samples is not intended to reflect a proportion of the flare population

with low PRG4.

Total protein concentration in SF was measured by bicinchoninic acid assay. HA

and PRG4 concentration were measured using sandwich enzyme linked immunosorbent

assays (ELISA) as previously described1. Briefly, PRG4 concentration was measured in

triplicate using a custom sandwich ELISA1 with antibody LPN used to capture the C-

terminal of full-length PRG459

and peanut agglutinin-horseradish peroxidase to detect

glycosylations in the mucin domain of PRG4125

. HA concentration was measured in

triplicate using a commercially available kit from R&D Systems®. HA MW distribution

was determined using 1% agarose gel electrophoresis112

followed by staining with Stains-

All and densitometric analysis using ImageJ (NIH, Bethesda, MD). HA MW distribution

in each SF sample was quantified as relative abundance (%) in each of 5 MW ranges

(based on size of control HA markers) spanning very high to low MW: > 6.1, 3.1 – 6.1,

1.1 – 3.1, 0.5 - 1.1, and < 0.5 MDa.

Human cartilage boundary lubricating ability of flare-SF deficient in PRG4,

supplemented flare-SF, and normal SF was measured in a previously characterized in

62

vitro cartilage-on-cartilage boundary mode friction test on a Bose ELF 320029

. Annulus

and core shaped osteochondral samples were harvested from the patellofemoral groove of

5 macroscopically normal human distal femurs (age 57 – 82 years). Samples were shaken

overnight in phosphate buffered saline (PBS) at 4°C to remove any residual SF from the

cartilage surface, and then soaked overnight at 4°C in the subsequent test lubricant prior

to testing1. The cartilage surfaces of the annulus and core were opposed against each

other, compressed to 18% of the total cartilage thickness, and allowed to stress-relax for

40 minutes. Without removing compression, samples were then rotated at 0.3 mm/s +2

and -2 revolutions with pre-sliding duration (Tps) of 120, 12, and 1.2 seconds; Tps is the

duration of time the samples are opposed and stationary prior to rotation. The test was

then repeated in the opposite direction of rotation. Static (μstatic,Neq, resistance to the onset

of motion) coefficients of friction were calculated using the peak torque within 10° of the

start of rotation. Torque from the second revolution was averaged to calculate the kinetic

(<μkinetic,Neq>, resistance to steady motion) friction coefficients.

In all experiments, each osteochondral pair was tested sequentially over 5 days in

each of the 5 test lubricants. Each flare-SF deficient in PRG4 selected for friction testing

(N = 5) was tested in triplicate (n = 3, total n = 15) in the following sequence: 1) PBS

(negative control), 2) flare-SF deficient in PRG4, 3) flare-SF+PRG4, 4) flare-

SF+PRG4+HA, 5) normal SF (positive control). Three normal SF samples were used as

positive controls for boundary lubricating ability tests (age 56 – 59, PRG4 concentration

136 – 373 μg/mL). Flare-SF was supplemented with normal concentrations (based on

ELISA measurements in normal SF) of PRG4 and PRG4+HA. PRG4 at 450 µg/mL

(obtained from bovine cartilage explants culture as described previously34

) and 1.5 MDa

63

HA at 1.0 mg/mL (obtained from Lifecore Biomedical, LLC) were used for

supplementation.

Data are presented as mean with 95% confidence interval (CI, lower limit, upper

limit) unless otherwise noted. Differences in PRG4 and HA composition were assessed

by ANOVA. Arcsine square root transformation was used to improve uniformity of the

variance for the proportional distribution of HA MW126

. Repeated measures ANOVA

was used to assess the effects of lubricant solution and Tps (as a repeated factor) on

μstatic,Neq and <μkinetic,Neq>. The effect of lubricant solution on μstatic,Neq at each Tps and

<μkinetic,Neq> at Tps = 1.2 seconds was assessed by ANOVA with Tukey post-hoc testing.

Statistical analysis was performed with Systat 12.

3.4 Results

Flare-SF samples contained PRG4 and HA at concentrations ranging from below

normal to super-physiological. PRG4 concentration in normal SF averaged 281.4 (241.6,

321.3) µg/mL (Figure 3-1). PRG4 concentration in flare-SF samples selected for friction

testing were below the average normal concentration and ranged from 102.8 to 231.0

µg/mL; PRG4 concentrations in all flare samples ranged from 102.8 to 348.7 µg/mL.

Table 3-1 summarizes the characteristics of the PRG4-deficient flare-SF selected for

friction testing as well as the normal donors. (Flare-SF samples measured but not

included in this chapter, including flares to IA corticosteroid injection, are shown in

Appendix E).

64

Figure 3-1: PRG4 concentration in flare-SF after IA HA. PRG4

concentration in normal ( ) SF (N = 29) shown by black line, ± 95% CI

shown in dashed lines. PRG4-deficient samples selected for friction

testing are circled. “L” and “R” denotes SF that was obtained from the left

and right knee of the same patient. “1” and “2” denotes the 1st and 2

nd

aspiration of the same knee after a flare reaction to HA.

65

Table 3-1: Characteristics of flare patients whose SF was identified as

PRG4-deficient and were selected for lubrication testing, and normal (NL)

SF from cadaveric donors. * = significantly higher (p < 0.001) compared

to normal.

Sample Age Sex Days post

injection

Aspirate

Volume (mL)

Total Protein

(mg/mL)

Flare 1 L 47 M 11 27 37.2

Flare 1 R 47 M 11 32 35.3

Flare 2 53 M 4 55 32.0

Flare 3 48 F 7 60 46.8

Flare 4 47 F 1 22 25.5

Flare Avg 48 (46, 51) 39.2 (24.2,

54.2)*

35.4 (28.5,

42.2)*

NL Avg

(N = 29)

55 (51, 59) 6F,

12M

3.6 (2.2, 4.9) 15.4 (13.0,

17.8)

Data are presented as mean with 95% confidence interval (lower limit,

upper limit). L = left, R = right, M = male, F = female, Avg = average, NL

= normal. * = significantly higher than NL, p < 0.05.

66

HA concentration in normal SF averaged 0.53 (0.41, 0.64) mg/mL (Figure 3-2A).

HA concentration in all flare-SF samples ranged from 0.16 to 0.68 mg/mL. HA MW in

PRG4-deficient flare samples was shifted towards smaller sizes in 2 MW ranges (Figure

3-2B). Relative HA concentration (as a percentage of total concentration) in PRG4-

deficient flare SF was significantly lower in the 3.1 – 6.1 MDa (p = 0.02) and 0.5 – 1.1

MDa (p = 0.005) ranges, and not significantly different from normal in the > 6.1 MDa (p

= 0.095), 1.1 – 3.1 MDa (p = 0.16), and < 0.5 MDa (p = 0.20) ranges.

67

Figure 3-2: (A) HA concentration in flare-SF after IA HA. HA

concentration in normal ( ) SF (N = 29) shown by black lines, ± 95% CI

shown in dashed lines. PRG4-deficient samples selected for friction

testing are circled. “L” and “R” denotes SF that was obtained from the left

and right knee of the same patient. “1” and “2” denotes the 1st and 2

nd

aspiration of the same knee after a flare reaction to HA. (B) HA MW

distribution in N = 5 PRG4-deficient flare-SF samples selected for friction

testing and N = 15 normal (NL) SF (* represents p < 0.05). Values are

mean ± 95% CI.

68

Friction was modulated by test lubricant and Tps. In each test lubricant, μstatic,Neq

decreased with decreasing Tps and appeared to approach <μkinetic,Neq> as Tps decreased

from 120 seconds towards 0 seconds. Values of μstatic,Neq were consistently highest in

PBS; values were lower and similar for flare, supplemented, and normal SF. In all test

lubricants, values of <μkinetic,Neq> increased only slightly with increasing Tps, with mean

values at Tps = 1.2 seconds being on average within 10 ± 1% of values at Tps = 120

seconds. Therefore, as presented previously34

and for brevity and clarity, <μkinetic,Neq>

data are shown at Tps = 1.2 seconds only. Average equilibrium stress for all tests was

0.165 (0.151, 0.178) MPa.

Lubricating ability of flare-SF deficient in PRG4 did not differ from that of

normal SF. μstatic,Neq varied with test lubricant and Tps, with an interaction effect (all p <

0.001) (Figure 3-3A). Values of μstatic,Neq were similar in flare and normal SF at all Tps (p

= 0.83 – 1). <μkinetic,Neq> at Tps = 1.2 seconds also varied with test lubricant (p < 0.001)

(Figure 3-3B). <μkinetic,Neq> for flare-SF was not different than normal SF (0.033 (0.027,

0.039) vs. 0.030 (0.025, 0.034), p = 0.70).

Friction coefficients in flare-SF samples were not altered with PRG4 or

PRG4+HA supplementation. Values of μstatic,Neq were similar in flare-SF, flare-SF+PRG4,

and flare-SF+PRG4+HA at all Tps (p = 0.72 – 1, Figure 3-3A). <μkinetic,Neq> at Tps = 1.2

seconds was also similar in flare-SF, flare-SF+PRG4, flare-SF+PRG4+HA, and normal

SF (p = 0.76 – 1.0, Figure 3-3B).

69

Figure 3-3: Effect of HA and PRG4 supplementation on the cartilage

boundary lubricating ability of PRG4-deficient flare-SF samples, as

determined by in vitro cartilage-on-cartilage friction testing. Two friction

coefficients, static (μstatic,Neq) (A) and kinetic (<μkinetic,Neq>; at Tps = 1.2

seconds) (B) were calculated in PBS (negative control lubricant), PRG4-

deficient flare-SF alone, flare-SF plus PRG4, flare-SF plus PRG4 and HA,

and normal SF (NL; positive control lubricant). Values are mean ± 95%

CI.

70

3.5 Discussion

These results demonstrate that OA SF can exhibit altered boundary lubricant

composition after flare reaction to IA HA injection, but also retain normal cartilage

boundary lubricating ability. The range of PRG4 concentrations observed in this study

are consistent with previous observations that PRG4 concentrations can vary

considerably within both normal donors and OA patients1. The normal concentration and

partially altered MW of HA observed also agree with previous work81,106

. These results

provide insight into the molecular basis of cartilage boundary lubricating ability, and

suggest that maintenance of HA at physiologically normal concentration and size is

important for interaction with PRG4, even at diminished levels, and normal joint

lubrication.

The PRG4 concentration measured in flare-SF ranged from below normal to

super-physiological. There did not appear to be a consistent response in PRG4

concentration to flare reaction, which may suggest that other factors in addition to the

flare response are affecting SF PRG4 composition. Joint loading/exercise73

and

inflammation66,136

are known to affect PRG4 composition and glycosylation in SF, and

could have varied between the patients included in this study. PRG4 concentrations have

been reported to both increase103,104

and decrease1 in chronic OA, suggesting individual

baseline levels and responses to external stimuli may vary. In addition, there did not

appear to be a consistent response in HA concentrations or HA MW distribution. While

HA concentrations measured here are somewhat lower than previous measurements in

human SF, the approximately normal concentration and partial shift to lower MW are

consistent with previous observations81,106

.

71

The normal human cartilage-cartilage boundary lubrication test used in this study

has previously been used for bovine cartilage and SF29

, bovine cartilage and ovine SF137

,

and normal human cartilage and SF1. The normal human cartilage used in this study was

harvested from macroscopically normal areas of distal femurs from donors who were not

taking anti-inflammatory medications at time of death. The total protein concentration

and volumes of the normal SF used here (Table 3-1) is within previously reported ranges

for normal SF4, and both aspirated volumes and total protein concentration for OA SF

were significantly higher as expected4. Coefficients of friction obtained here for normal

human SF on normal human cartilage (<μkinetic,Neq> = 0.030) are consistent with previous

measurements of bovine cartilage and bovine (<μkinetic,Neq> = 0.02529

) and ovine

(<μkinetic,Neq> = 0.034 – 0.041137

) SF, suggesting that the boundary lubricating function of

the human cartilage used here is representative of normal cartilage. Characterization of

this test demonstrated that testing in PBS and then SF does not affect the values measured

in SF29

, and lubricants in this study were selected in order of presumed increasing

lubricating ability; however, order effects were not explicitly evaluated with these

lubricants.

Previous in vitro studies have demonstrated that if composition of either PRG4 or

HA is altered it can affect the lubricating ability of whole SF, possibly through alterations

in PRG4+HA synergism. Decreased boundary lubricating ability has been observed in

equine SF with decreased HA concentration, low HA MW, and increased PRG4

concentration after acute injury; boundary lubricating ability was restored with

supplementation with high MW HA (4 MDa)87

. In human chronic OA SF, decreased

boundary lubricating ability was observed in SF with decreased PRG4 concentration,

72

normal HA concentration, and HA MW shifted towards the lower range in all MW

ranges from 0.5 – 6 MDa; boundary lubricating ability was restored with addition of

PRG4, and subsequent addition of HA had no additional effect1. Together these studies

suggest that the composition of both PRG4 and HA are important for normal SF function.

In solutions of purified HA, boundary lubricating ability improves slightly with

increasing MW, but this MW dependence is not observed in purified PRG4+HA

solutions where even very small HA (20 kDa) with physiologically normal

concentrations of PRG4 (450 μg/mL) lubricates similar to large HA (5MDa) with PRG4,

as well as to whole SF35

. The flare-SF samples tested here had decreased PRG4, HA

concentration similar to normal, and HA MW significantly shifted towards lower sizes in

only 2 MW ranges: 3.1 – 6.1 and 0.5 – 1.1 MDa. PRG4-deficient chronic OA SF that

exhibited decreased lubricating ability discussed above had HA MW shifted significantly

towards lower sizes in all MW ranges from 0.5 to 6.1 MDa, suggesting that the flare-SF

presented here had sufficient amounts of adequately high MW HA to either provide

lubricating ability, or interact with PRG4 to provide lubricating ability. These results are

consistent with those obtained in purified solutions, where decreased PRG4 concentration

or decreased high MW HA concentration can limit cartilage boundary lubricating ability

of PRG4+HA solutions (see Chapter 4).

The flare-SF used in this study was obtained between 1 – 11 days after injection,

which is slightly longer than the aforementioned flare timeline of 24 – 72 hours after

injection. An acute exercise model in murine knee joints demonstrated that PRG4

concentrations in SF can change very quickly (on the order of minutes) after joint

loading73

, and previous modeling work has predicted that PRG4 and HA concentrations

73

would return to equilibrium within hours to days (respectively) after joint lavage138

.

While decreased PRG4 was not associated with altered lubricating ability in the flare-SF

samples in this study, which also had normal HA concentrations and a normal amount of

high MW HA, it is possible that changes in the very acute stages (on the order of minutes

– hours) occurred prior to SF aspiration. The immediate effect of a flare reaction to IA

HA on boundary lubricant composition would be difficult to measure, but could have

deleterious implications on SF function. The duration of diminished boundary lubrication

required to cause cartilage wear is currently unknown. While there is evidence that

friction and wear are linked at the articular surface21

, there is also evidence that PRG4

can act to prevent wear even when it no longer reduces friction139

and that increasing

PRG4 concentrations may contribute to wear protection36

of model surfaces. While

friction was measured in this study, the effects of altered lubricant composition (e.g.

decreased PRG4 and/or HA) on cartilage wear are also clinically important and remain to

be elucidated.

The mechanisms of the flare reactions included in this study are unknown, and

large SF aspirations, which may indicate underlying inflammatory processes, were not

excluded in this study as has been done previously94

. Inflammation in the joint may affect

the glycosylation pattern of PRG465

, which is essential for lubricating function62

.

Furthermore, PRG4 is present in SF in both monomeric and disulfide-bonded multimeric

forms, and recent evidence has suggested that PRG4 solutions enriched in multimers

have increased lubricating ability compared to solutions deficient in multimers140

.

Differences in both the glycosylation of PRG4 and relative multimer/monomer

abundance in normal and diseased or injured SF remain to be investigated, as both may

74

have implications on whether or not boundary lubricating ability is altered. This would be

of particular interest in situations where both PRG4 and HA composition are decreased,

and there is insufficient high MW HA available to provide boundary lubricating

ability/interaction with PRG4.

Given that PRG4 and HA are critical contributors to the boundary lubricating

ability of SF, and that PRG4 and HA concentrations in SF seem to be variable, the

potential application of PRG4 combined with HA as a biotherapeutic treatment for

restoration of altered SF lubricant content is intriguing. While IA HA is generally an

effective, well-tolerated treatment that can provide pain relief for OA patients, it does not

appear to protect the cartilage surface or act as a disease-modifying agent98

.

Furthermore, recent in vitro evidence has suggested that diminished boundary lubrication

may be associated with increased chondrocytes apoptosis, and that hylan G-F 20 alone is

unable to lubricate and prevent apoptosis as well as SF141

. PRG4 has been used as an IA

therapeutic in rat ACL-transection models of OA and has been observed to reduce

cartilage degeneration123

, and also to counteract the additional cartilage damage caused

by forced joint exercise76

. Furthermore, over-expression of PRG4 in mouse articular

cartilage protects against development of both age-related and post-traumatic OA58

.

Given that IA PRG4 may stimulate endogenous production of PRG475

, its potential as a

cartilage-preserving biotherapeutic is promising, especially in conjunction with the pain

relief already provided by HA.

While flare reactions to IA therapies are a relatively rare occurrence (reported at

5.7% for a single injection of hylan G-F 20132

), the response of joint tissues to treatment

is important for both pain relief provided to the patient and for maintenance of SF

75

boundary lubricating function. In addition to the post-IA flare, post-ACL tear, and

chronic OA patients that have been discussed here, other populations who may be at risk

for altered lubricant composition and boundary lubricating ability are patients who have

had a reaction to IA corticosteroid injection, patients who have received a treatment

course of IA HA or corticosteroid over a long period of time, or patients who have had

SF lubricants washed away during joint surgery. In addition to quantifying PRG4 and HA

concentration, HA MW, and boundary lubricating ability of these normal, post-injury,

and diseased SFs, it will also be important to evaluate PRG4 glycosylation patterns, and

PRG4 multimer-monomer distribution to fully understand the relationship between SF

boundary lubricant composition and function. This study provides further motivation for

elucidating the synergistic mechanism of interaction of PRG4 and HA, so that

optimization of concentrations and size distributions of each for new biotherapeutic

treatments can be evaluated.

In this study, quantification of PRG4 and HA content revealed that post-IA HA

flare SF can exhibit decreased PRG4 content after flare reaction to IA injection. Possibly

due to 1) sufficient amounts of high MW HA being retained or 2) maintenance of the

PRG4+HA interaction, this SF was able to retain normal cartilage boundary lubricating

ability. Supplementation with PRG4 and PRG4+HA had no additional effect on

lubricating ability. These findings support and extend the concept that the concentration

and structure of both PRG4 and HA in SF are important to maintain normal joint

lubrication. Previous studies have demonstrated that SF PRG4 and HA can be altered in

acute and chronic conditions, and that in vitro restoration of lubricant levels can negate

the deleterious effects of lubricant alterations1,87

. Collectively, this and previous studies

76

suggest that maintaining normal composition of both PRG4 and HA through

biotherapeutic treatment may preserve SF lubricating function and therefore contribute to

joint preservation and health.


This chapter, in full, is in preparation for submission to BMC Musculoskeletal

Disorders. The candidate is the primary author and thanks co-authors Jenelle McAllister,

Dr. Victor Lun, Dr. J. Preston Wiley, and Dr. Tannin A Schmidt. Study conception and

design was performed by TL and TS. Acquisition of data was performed by TL

(biochemical and biomechanical data), JM (SF acquisition), VL (SF acquisition), and PW

(SF acquisition). Analysis and interpretation of data was performed by TL, PW, and TS.

All authors were involved in revising the article and approved the final submitted version.

The authors also thank the University of Calgary Joint Transplantation Program

for access to the normal human tissue (Mrs Sue Miller and Dr. Roman Krawetz), the

Sports Medicine Centre at the University of Calgary for collection of OA hSF. This work

was supported by funding from the National Science and Engineering Research Council

of Canada, Canadian Arthritis Network, Alberta Innovates-Technology Futures, Alberta

Innovates Health Solutions OA Team Grant, Faculty of Kinesiology and Schulich School

of Engineering’s Center for Bioengineering Research and Education at the University of

Calgary.

77

Chapter Four: Effects of concentration and structure on synergistic proteoglycan 4 +

hyaluronan cartilage boundary lubrication

4.1 Abstract

Objectives: Evaluate cartilage boundary lubricating ability of 1) constant

hyaluronan concentration ([HA]) in solution with a range of proteoglycan 4

concentrations ([PRG4]), 2) constant [PRG4] with a range of [HA], 3) hylan G-F

20+PRG4, and 4) HA+reduced and alkylated (R/A) PRG4.

Design: Static and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were

calculated using a cartilage-cartilage boundary mode friction test. Test 1: HA (1.5 MDa,

3.3 mg/mL) +PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) +HA

(1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) +PRG4 (450

μg/mL). Test 4: HA (3.3 mg/mL) +R/A PRG4 (450 μg/mL).

Results: In [HA] = 3.3 mg/mL, <μkinetic,Neq> for 4.5 and 45 µg/mL PRG4 were

significantly higher than SF. At [HA] = 0.3, 1.0, and 3.3 mg/mL, <μkinetic,Neq> for 45

µg/mL PRG4 was significantly higher than 450 µg/mL, and was also significantly higher

than 150 µg/mL for [HA] = 0.3 mg/mL. Addition of R/A PRG4 to HA failed to

significantly reduce <μkinetic,Neq> compared to HA alone. Addition of PRG4 to hylan G-F

20 significantly reduced <μkinetic,Neq> compared to hylan G-F 20 alone.

78

Conclusion: These results demonstrate that decreased levels of PRG4 and/or

decreased high MW HA can limit the cartilage boundary lubricating ability of PRG4+HA

solutions. The reduction of friction by adding PRG4 to cross-linked HA, but not with

addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction

where disulfide-bonded protein structure is important. Both PRG4 and HA are important

contributors to cartilage boundary lubrication.

79

4.2 Introduction

Friction between articular cartilage surfaces in motion is mediated through a

combination of lubrication mechanisms. During fluid film lubrication, cartilage surfaces

are separated by a fluid layer, while during boundary lubrication friction is mediated by

interactions between lubricant molecules adsorbed to the surface3. The boundary

lubrication mode becomes increasingly dominant as loading time is increased and

interstitial fluid is depressurized129,142

. Opposing cartilage surfaces make contact over

only approximately 10% of the total area, making these areas of contact vulnerable to

high friction28

. Synovial fluid (SF) constituents proteoglycan 4 (PRG4) and hyaluronan

(HA) are the primary contributors to its cartilage boundary lubricating ability34

. PRG453

is a mucin-like O-linked glycosylated protein present in SF47

and at the articular cartilage

surface48

. HA, a linear polymer of D-glucuronic acid and D-N-acetylglucosamine115

, is

also present in SF. Alone, solutions of PRG4 or HA reduce friction at a cartilage-cartilage

biointerface in a boundary mode of lubrication compared to phosphate buffered saline

(PBS). When combined, PRG4+HA further reduce friction synergistically towards that of

whole SF34

. Both PRG4 and HA are critical to the cartilage boundary lubricating function

of SF, and decreased boundary lubricating ability of SF has been linked with increased

wear at the articular surface21

.

Compromised cartilage boundary lubricating ability of SF with diminished PRG4

or diminished high molecular weight (MW) HA content can be restored in vitro by

addition of the deficient lubricant. Some PRG4-deficient SF from patients with

osteoarthritis (OA) had normal HA concentration, an HA MW distribution shifted

towards the lower range over all sizes from 6 MDa to 0.5 MDa, and failed to lubricate as

80

well as normal SF. Normal cartilage boundary lubricating ability could be restored with

addition of PRG4 to the SF1, as evidenced by a measured reduction in friction. A similar

decrease in SF HA concentration and HA MW, although with an increase in PRG4

concentration, has been observed in an equine acute injury model; this SF also failed to

lubricate, though the cartilage boundary lubricating ability could be restored by

supplementation with high MW HA (4 MDa), but not low MW HA (800 kDa)87

.

Conversely, some PRG4-deficient SF aspirated from OA patients after a “flare” reaction

to intra-articular HA injection demonstrated normal HA concentration, an HA MW

distribution shifted towards the lower range in only the 3 – 6 and 0.5 – 1 MDa ranges,

and lubricated as well as normal SF – even with moderately decreased levels of PRG4143

.

While the mechanism of this functional, friction-reducing PRG4+HA synergism

at a cartilage-cartilage biointerface in a boundary mode of lubrication remains to be fully

understood, characterization of some potential factors affecting the synergism in vitro has

previously been performed. In solutions of HA alone, friction coefficients were reduced

by increasing HA concentration34,87

, and slightly by molecular weight (MW) ranging

from 20 kDa to 5 MDa, at a concentration of 3.3 mg/ml35,87

. However, upon addition of

PRG4 at 450 µg/mL this HA MW dependence was no longer observed35

and friction was

reduced by addition of PRG4 over the range of MW of the 3.3 mg/ml HA solutions.

These studies suggest that both PRG4 and HA, particularly high MW HA, are necessary

contributors to the cartilage boundary lubricating function of SF, yet the potential

concentration dependence of high MW HA, which can be diminished in diseased SF, on

the functional friction-reducing PRG4+HA synergism at a cartilage-cartilage biointerface

remains to be clarified.

81

The MW of HA has also been linked to its efficacy as an intra-articular

viscosupplement. Intra-articular HA injections are currently used to treat pain in OA

patients, and it is thought that increasing the MW of HA by cross-linking increases joint

residence time13

. Increased MW may also contribute to pain relief by increased protection

of nerve endings via increased viscosity97

. Hylan G-F 20 (“Synvisc”, Genzyme) is a

currently available treatment consisting of cross-linked HA of ~6 MDa; it is composed of

80% water soluble hylan A molecules formed by cross-linking with formaldehyde, and

20% insoluble hylan B viscoelastic gel molecules formed by cross-linking with

vinylsulfone144

. The effect that the cross-linking process has on hylan G-F 20’s ability to

interact with PRG4 to reduce friction in a boundary mode at a cartilage biointerface,

towards that of whole SF, is currently unknown.

PRG4’s disulfide-bonded structure has previously been observed to affect its

cartilage boundary lubricating ability. The lubricating ability of PRG4 is decreased after

it is reduced and alkylated to break both inter- and intra-molecular disulfide bonds145

.

Preparations of PRG4 enriched in disulfide-bonded multimeric species provide enhanced

lubricating ability compared to preparations enriched in monomeric PRG4140

,

demonstrating the functional importance of inter-molecular disulfide bonds specifically,

as reduced preparations of monomers appear to lubricate as well as non-reduced

monomers146

. Furthermore, reduction and alkylation decreases the ability of PRG4 to

adsorb to cartilage surfaces147

. However, the effect of loss of disulfide-bonded structure

by reduction and alkylation (R/A) on PRG4’s ability to interact with HA and

synergistically reduce friction in a boundary mode at a cartilage-cartilage biointerface is

also unknown.

82

The objectives of this study were to evaluate cartilage boundary lubricating ability

of 1) PRG4+HA in solution at constant HA concentration in a range of PRG4

concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) hylan

G-F 20 + PRG4, and 4) HA + R/A PRG4. The hypothesis was that PRG4 contributes to

the boundary lubricating ability of PRG4+HA solutions through concentration and

structurally mediated effects with itself and HA.

4.3 Materials & Methods

4.3.1 Materials

Materials for lubrication testing were obtained as described previously35

. HA of 1.5

MDa was obtained from Lifecore Biomedical LLC (Chaska, MN, USA), and bovine SF

was obtained from Animal Technologies (Tyler, TX, USA). Hylan G-F 20 was from

Sanofi Canada (Laval, QC, Canada). PRG4 was purified from culture media conditioned

by mature bovine cartilage explants, as described previously34

. Purity of the PRG4

preparation was confirmed by 3-8% Tris-Acetate SDS-PAGE followed by protein stain

and Western blotting with anti-PRG4 antibody LPN59

with Invitrogen’s NuPAGE

system. Concentration of the purified PRG4 was determined by bicinchoninic acid assay.

Lubricants were prepared by combining the required volumes of PRG4 (prepared in

PBS) and HA (prepared in PBS) at the appropriate concentrations. Hylan G-F 20

(initially 8.0 mg/mL) was diluted to 3.3 mg/mL in PBS. R/A PRG4 was prepared in PBS

by incubation with 10 mM dithiothreitol for 2 hours at 60°C and then 40 mM sodium

83

iodoacetate for 2 hours at room temperature in the dark59

, followed by dialysis against

PBS overnight at 37°C. R/A was confirmed by protein stain after SDS-PAGE (not shown

here, see Figure 5-1 for example).

4.3.2 Sample Preparation

Osteochondral samples (N = 42) were harvested from the patellofemoral groove

of 11 skeletally mature bovine stifle joints as described previously29

. Samples were rinsed

vigorously overnight in ~40 mL of PBS at 4°C to remove residual SF from the cartilage

surface. Samples were then stored at -80°C in PBS with protease inhibitors until the day

prior to testing, at which time they were thawed and again rinsed vigorously overnight in

PBS. Samples were then bathed in the next day’s test lubricant (0.2 mL for core, 0.1 mL

for annulus), such that the cartilage surface was completely immersed, at 4°C overnight

prior to lubrication testing.

4.3.3 Lubrication Testing

Lubrication tests were performed on a Bose ELF 3200 using a previously

characterized in vitro cartilage-on-cartilage boundary mode lubrication test29

. Briefly,

annulus and core shaped osteochondral samples were opposed against each other,

resulting in a stationary contact area, compressed to 18% of the total cartilage thickness

at 0.002 mm/s, and an interstitial fluid depressurization period of 40 minutes was

allowed. Without removal of this equilibrium load (Neq) samples were then rotated +2

revolutions and -2 revolutions at 0.3 mm/s with pre-sliding durations (Tps; duration of

time samples are stationary prior to rotation) of 1200, 120, 12, and 1.2 seconds. This test

84

sequence was then repeated in the opposite direction of rotation. Using the Neq, static

(μstatic,Neq), and kinetic (<μkinetic,Neq>) coefficients of friction were calculated29

,

representing the resistance to the onset of motion and steady motion, respectively.

In all experiments, each osteochondral pair was tested sequentially over 4 – 5

days in each of the 4 – 5 test lubricants. Lubricants were selected in order of predicted

increasing lubricating ability to minimize carryover effects. In all tests, PBS served as the

negative control lubricant and bovine SF served as the positive control lubricant. PRG4

and HA concentrations were selected to represent values lower, similar, and higher, to

those observed in human SF1. Four sets of tests were performed to evaluate cartilage

boundary lubricating ability of varying concentrations of PRG4 and HA, as well that of

hylan G-F 20 ±PRG4 and HA +R/A PRG4.

PRG4 Dose Response in HA. To determine the effect of PRG4 concentration on

PRG4+HA cartilage boundary lubricating ability in a constant [HA] = 3.3 mg/mL, two

tests were performed: Test 1A (PRG4 high dose, N = 6): PBS, HA +150 µg/mL PRG4,

HA +450 µg/mL PRG4, HA +1500 µg/mL PRG4, SF; and Test 1B (PRG4 low dose, N =

4): PBS, HA +4.5 µg/mL PRG4, HA +45 µg/mL PRG4, HA +150 µg/mL PRG4, SF.

HA Dose Response in PRG4. To determine the effect of HA concentration on

PRG4+HA cartilage boundary lubricating ability in constant [PRG4] of 450, 150 or 45

µg/mL, three tests were performed: Test 2A (HA dose, [PRG4] = 450 µg/mL, N = 8):

PBS, PRG4 +0.3 mg/mL HA, PRG4 +1.0 mg/mL HA, PRG4 +3.3 mg/mL HA, SF; Test

2B (HA dose, [PRG4] = 150 µg/mL, N = 4): PBS, PRG4 +0.3 mg/mL HA, PRG4 +1.0

mg/mL HA, PRG4 +3.3 mg/mL HA, SF; and Test 2C (HA dose, [PRG4] = 45 µg/mL, N

= 4): PBS, PRG4 +0.3 mg/mL HA, PRG4 +1.0 mg/mL HA, PRG4 +3.3 mg/mL HA, SF.

85

Partially Cross-linked HA. To determine the cartilage boundary lubricating ability

of PRG4 combined with cross-linked HA, the following test sequence was performed:

Test 3 ([hylan G-F 20] = 3.3 mg/mL, N = 8): PBS, hylan G-F 20, hylan G-F 20 +450

µg/mL PRG4, SF.

R/A PRG4. To determine the cartilage boundary lubricating ability of HA

combined with R/A PRG4 (disruption of tertiary and quaternary structure, inter- and

intra-molecular disulfide bonds are broken), the following test sequence was performed:

Test 4 ([HA] = 3.3 mg/mL, [R/A PRG4] and [PRG4] = 450 µg/mL, N = 8): PBS, HA,

HA +R/A PRG4, HA +PRG4, SF.

4.3.4 Statistical Analysis

Unless otherwise indicated, data are presented as mean ± 95% confidence interval

(upper limit, lower limit). The effects of test lubricant and Tps (as a repeated factor) on

friction coefficients, μstatic,Neq and <μkinetic,Neq>, were assessed by repeated measures

analysis of variance (ANOVA). To compare lubricants within test sequences, the effect

of test lubricant on <μkinetic,Neq> at Tps = 1.2 seconds between test lubricants and SF was

assessed by ANOVA, with Tukey post-hoc testing. To compare lubricants between test

sequences (i.e. between the HA dose responses in 3 three PRG4 concentrations), the

effect of test lubricant on <μkinetic,Neq> at Tps = 1.2 seconds was assessed by ANOVA

with Fishers post-hoc testing. Statistical analysis was performed using Systat12 (Systat

Software, Inc., Richmond, CA).

86

4.4 Results

4.4.1 Lubrication Testing

In all tests, friction was affected by test lubricant and Tps. In all test lubricants,

μstatic,Neq was highest at Tps = 1200 seconds and asymptotically approached <μkinetic,Neq>

as Tps decreased to 1.2 seconds. Values of μstatic,Neq at Tps = 1200 seconds were 71 ± 13%

(mean ± SD) higher than those at Tps = 1.2 seconds. <μkinetic,Neq> increased only slightly

with Tps, with values at Tps = 1.2 seconds being on average within 18 ± 8% (mean ± SD)

of those at Tps = 1200 seconds. Therefore, for brevity and clarity, only <μkinetic,Neq> at

Tps = 1.2 seconds will be presented. Average equilibrium compressive stress across all

tests was 0.09 ± 0.02 MPa (mean ± SD).

PRG4 Dose Response in HA. In constant [HA] = 3.3 mg/mL, coefficients of

friction appeared to decrease towards that of SF as [PRG4] increased, decreasing towards

a plateau between 45 and 150 µg/mL. μstatic,Neq varied with test lubricant and Tps (p <

0.0001), without an interaction (p = 0.17, Figure 4-1A). <μkinetic,Neq> at Tps = 1.2 seconds

also varied with test lubricant (p = 0.015). Values of <μkinetic,Neq> in [PRG4] = 4.5 (0.074

(0.107, 0.057) and 45 (0.072 (0.084, 0.066) µg/mL were significantly higher than those in

SF (p = 0.025, 0.041). <μkinetic,Neq> in [PRG4] = 150 (0.059 (0.070, 0.054)), 450 (0.054

(0.067, 0.047)), and 1500 (0.055 (0.071, 0.046)) µg/mL were similar to each other and to

SF (p = 0.14 – 1.0, Figure 4-1B).

87

Figure 4-1: μstatic,Neq (A), and <μkinetic,Neq> at Tps = 1.2 seconds (B) for

PRG4 high and low dose response + constant [HA] = 3.3 mg/mL (TESTS

1A, 1B). * = significantly higher than SF (p < 0.05).

88

HA Dose Response in PRG4. In [PRG4] = 450 µg/mL, μstatic,Neq varied with test

lubricant and Tps (both p < 0.0001) with an interaction (p = 0.05, Figure 4-2A). In

[PRG4] = 150 µg/mL, μstatic,Neq varied with test lubricant and Tps (p = 0.005, p < 0.0001)

without an interaction (p = 0.92, Figure 4-2B). In [PRG4] = 45 µg/mL, μstatic,Neq also

varied with test lubricant and Tps (p = 0.001 and p < 0.0001) without an interaction (p =

0.37, Figure 4-2C).

When compared between test sequences, values of <μkinetic,Neq> were higher in

[PRG4] = 45 µg/mL compared to those in [PRG4] = 450 µg/mL at [HA] = 0.3 mg/mL

(0.152 (0.182, 0.122) vs. 0.073 (0.101, 0.045)), 1.0 mg/mL (0.126 (0.155, 0.096) vs.

0.072 (0.103, 0.042)), and 3.3 mg/mL (0.084 (0.107, 0.06) vs. 0.044 (0.064, 0.025), p =

0.003, 0.04, 0.03, respectively, Figure 4-2D). At [HA] = 0.3 mg/mL, [PRG4] = 45

µg/mL was also higher than 150 µg/mL (0.078 (0.101, 0.054), p = 0.01). There was no

difference between <μkinetic,Neq> for [PRG4] = 150 and 450 µg/mL at [HA] = 0.3 or 1.0

mg/mL (p = 0.80, 0.74), however the difference was appreciable (though not significant)

at [HA] = 3.3 mg/mL (p = 0.11).

89

Figure 4-2: μstatic,Neq (A, B, C) for HA dose responses + constant [PRG4]

= 45 µg/mL (TEST 2A) (A), 150 µg/mL (TEST 2B) (B), and 450 µg/mL

(TEST 2C) (C). <μkinetic,Neq> at Tps = 1.2 seconds (D) for all doses of HA

in [PRG4] = 45, 150, 450 µg/mL (TEST 2A, 2B, 2C). Average

<μkinetic,Neq> in PBS and SF shown for reference. # = significantly higher

than [PRG4] = 450 µg/mL (p < 0.05). ^ = significantly higher than

[PRG4] = 150 µg/mL (p < 0.05).

90

Partially Cross-linked HA. Addition of PRG4 at 450 µg/mL to hylan G-F 20 at

3.3 mg/mL decreased friction compared to hylan G-F 20 alone. μstatic,Neq varied with test

lubricant and Tps with an interaction (p < 0.0001, p < 0.0001, p = 0.001 Figure 4-3A).

<μkinetic,Neq> at Tps = 1.2 seconds also varied with test lubricant (p = 0.017, Figure 4-3B).

Hylan G-F 20 alone (0.074 (0.083, 0.065)) failed to lubricate as well as SF (p = 0.001).

Hylan G-F 20 +PRG4 (0.048 (0.055, 0.042)) was significantly lower than hylan G-F 20

alone (p = 0.04), and provided boundary lubricating ability equivalent to that of SF (p =

0.29).

91


hylan G-F20 ± [PRG4] = 450 µg/mL (TEST 3). * = p < 0.05.

92

R/A PRG4. Addition of R/A PRG4 at 450 µg/mL to 1.5 MDa HA at 3.3 mg/mL

appeared to slightly, but not significantly, lower friction compared to HA alone. μstatic,Neq

varied with test lubricant and Tps with an interaction (p < 0.0001, p < 0.0001, p = 0.42,

Figure 4-4A). <μkinetic,Neq> at Tps = 1.2 seconds also varied with test lubricant (p = 0.002,

Figure 4-4B). HA alone (0.080 (0.088, 0.072)) was significantly higher than SF (p =

0.001). Addition of R/A PRG4 to HA (0.061 (0.069, 0.052) did not significantly reduce

<μkinetic,Neq> compared to HA alone (p = 0.30), but HA +R/A PRG4 was not significantly

different from SF (p = 0.07). Addition of PRG4 to HA (0.050 (0.057, 0.044)) improved

lubricating ability significantly compared to HA alone (p = 0.04), and there were no

significant differences between HA +PRG4 and HA +R/A PRG4 or SF (p = 0.74, 0.44).

93


HA, HA + [R/A PRG4] = 450 µg/mL, and HA + [PRG4] = 450 µg/mL

(TEST 4). * = p < 0.05.

94

4.5 Discussion

The results described here demonstrate that concentration of both PRG4 and high

MW HA can have an effect on the ability of PRG4+HA solutions to reduce friction in the

boundary mode at a cartilage-cartilage biointerface. The lubricating ability provided by

the PRG4+HA solutions tested here approached that of whole SF except for very low

PRG4 (4.5, 45 µg/mL) concentrations in physiologically normal HA concentrations. This

diminished cartilage boundary lubricating ability was exacerbated when low PRG4

concentrations (45, 150 µg/mL) were added to low HA concentrations (0.3, 1.0 mg/mL);

in this case physiological levels of PRG4 reduced friction, but not to the same level as

when combined with higher HA concentrations. These results indicate that both PRG4

and high MW HA concentration can be limiting in achieving reduction of friction in the

boundary mode at a cartilage-cartilage biointerface, and suggest that both are necessary

contributors to the cartilage boundary lubricating ability of SF. PRG4+hylan G-F 20

demonstrated improved lubricating ability compared to hylan G-F 20 alone, suggesting

that the PRG4+HA cartilage boundary lubrication synergism was also maintained with

cross-linked HA. The addition of R/A PRG4 to HA was unable to significantly reduce

friction, suggesting that PRG4s tertiary and quaternary protein structure is important in

its friction reducing synergism with HA at a cartilage-cartilage biointerface.

The cartilage-cartilage boundary lubrication test used here is able to quantify

contributions of PRG4 and HA to friction reduction in the boundary mode, but is not able

to provide insight into wear at the cartilage surface. The test geometry, protocol, and

physiological surfaces allow for friction in a boundary mode of lubrication to be

measured, even in viscous HA solutions - as indicated by the observation that PRG4 is

95

able to reduce friction in a dose dependent manner in high MW HA solutions (3.3

mg/mL). While previous studies have shown that friction and wear are linked at the

articular surface21

, wear prevention and the order in which PRG4 and HA are adsorbed to

the surface have only been studied extensively at model surfaces36

. Model surfaces

provide the advantage of well defined sample surfaces and modes of lubrication, but may

not allow for all operative physiological interactions at a cartilage-cartilage biointerface

to occur. Additionally, the use of conventional Stribeck curve analysis is not able to

account for the macromolecules at non-homogenous cartilage surfaces and in non-

Newtonian lubricant solutions that contribute to friction forces24

. Although the precise

mechanism through which boundary lubrication is provided at these cartilage surfaces

(viscous boundary layer39

, adaptive mechanical control25

) remains to be fully clarified,

the results presented here are in general consistent with PRG4+HA functioning

synergistically to reduce friction at a cartilage surface through thick film boundary

lubrication as proposed by the adaptive multimodal mechanism.

This study used preparations of PRG4 and HA that are representative of their

composition within SF. The PRG4 preparation contained both multimeric and monomeric

PRG4 species typically found in SF59

; future work will be required to determine the

friction reducing ability of each species with HA at a cartilage-cartilage biointerface. A

single high MW HA preparation was used, with 1.5 MDa being within the range of

previously reported HA MW distribution in normal and OA SF81,112

. The results obtained

here with hylan G-F 20 (6 MDa) suggest that higher MW HA also provides similar

reduction of friction with PRG4. Future studies could examine an HA solution composed

of a mixtures of various MW HA at (patho) physiological concentrations to further

96

examine the potential concentration/MW dependence of the PRG4+HA synergism.

Lastly, while a smaller number of replicates has previously been used to assess

differences between lubricants35

, as the lubricating ability of the solutions of interest

become more similar in composition and low-friction function, a higher number of

replicates may help elucidate if the apparent subtle differences observed here (i.e. HA

+R/A PRG4 was higher than but not significantly different from SF (p = 0.07)) are in fact

functionally important.

The coefficients of friction obtained here are consistent with previously measured

values for purified solutions of PRG4 and HA, alone and in combination, at a cartilage-

cartilage biointerface. <μkinetic,Neq> for PRG4 at 4.5 and 45 µg/mL observed in previous

studies was on the order of 0.2, while PRG4 at 450 µg/mL was 0.1034. The <μkinetic,Neq>

obtained here for HA at 0.3 mg/mL with PRG4 at 45 and 450 µg/mL are lower than

previously obtained for PRG4 alone, demonstrating friction reduction compared to PRG4

or HA alone even when low concentrations of high MW HA are added to low

concentrations of PRG4. <μkinetic,Neq> for 1.5 MDa HA alone at 3.3 mg/mL was 0.080

(0.088, 0.072) in this study, and has been observed to be approximately 0.0935

; the values

observed here with PRG4 (even 45 µg/mL) appear to be similar to 1.5 MDa HA alone,

indicating that very low concentrations of PRG4 can limit the boundary lubricating

ability of PRG4+HA solutions. Previous measurements of <μkinetic,Neq> for 450 µg/mL

PRG4 + 3.3 mg/mL 1.5 MDa HA (0.04635

) are consistent with the values observed in this

study.

These results also demonstrate that PRG4 can further contribute to the boundary

lubricating ability of a cross-linked HA clinical product at a cartilage-cartilage

97

biointerface. Indeed, the <μkinetic,Neq> obtained for hylan G-F 20 at 3.3 mg/mL and PRG4

at 450 µg/mL is very close to those discussed above for PRG4 and 1.5 MDa HA. These

results contrast with previous observations using a similar in vitro cartilage boundary

lubrication test, where it was observed that hylan G-F 20 failed to lubricate as well as SF,

and failed to prevent chondrocyte apoptosis compared to SF141

. Subsequent work

demonstrated that addition of purified PRG4 to PRG4-void SF was able to decrease

chondrocyte apoptosis, and lower <μkinetic,Neq> beyond that of PRG4 alone, suggesting

again that the PRG4+HA interaction is critical for normal SF function148

. While the

studies investigating chondrocyte apoptosis and boundary lubrication used a similar in

vitro boundary lubrication test setup as this study, overall values may differ due to test

parameter differences (no annular geometry, less time for stress relaxation, live explants,

12 continuous cycles vs. start and stop). The observation that PRG4+HA friction

reduction is not disrupted by the cross-linking procedure is consistent with previous

evidence suggesting that the PRG4+HA interaction is not a specific site-dependent

binding, but rather a physical interaction35,149

. However, the hylan G-F 20 used in this

study was diluted to 3.3 mg/mL from its clinical concentration of 8 mg/mL, which may

influence interaction with PRG4 in vivo.

The effects of injury and disease on PRG4 structure in SF, including relative

composition of multimers:monomers and fragments of PRG454

, remain to be clarified. It

has been shown that PRG4 can be degraded by enzymes (i.e. neutrophil elastase68

) that

may be up-regulated in inflammatory conditions such as post-ACL tear68

. Despite a slight

reduction in friction, the cartilage boundary lubricating ability of HA alone and HA+R/A

PRG4 were not significantly different, suggesting that degradation of PRG4 structure

98

and/or assembly in SF could potentially impact SF boundary lubricating ability by

altering the PRG4+HA interaction. This suggests that PRG4s disulfide-bonded protein

structure is important in the non-binding interaction with HA. Future studies examining

the role of PRG4 multimer/monomer interaction with HA to reduce friction will help

clarify this issue.

These results demonstrate that both PRG4 and HA are necessary for effective

friction reduction towards the level of whole SF and suggest that deficiency of either or

both may be detrimental to SF cartilage boundary lubricating function. This is also

consistent with observations of cartilage boundary lubrication by SF; when HA MW and

PRG4 content are decreased, lubricating ability is compromised1, but when HA MW is

maintained with low PRG4 concentration, lubricating ability is equivalent to that of

normal SF143

. As cartilage boundary lubrication synergism appears to be lost when both

PRG4 and high MW HA are present in low concentrations, it is possible that a combined

PRG4+HA intra-articular treatment may be able to “rescue” SF deficient in either

lubricant.

This study provides further insight into a fundamental joint lubrication

mechanism and demonstrates the importance of both PRG4 and high MW HA

concentration and PRG4 and HA structure to their synergistic friction-reducing cartilage

boundary lubricating ability. Given that combining PRG4 and HA in an intra-articular

biotherapeutic treatment may be able to impart the benefits of both HA (pain relief,

viscosity) and PRG4 (chondroprotection75,76,123,124

, and potentially viscosity37,150

),

characterizing and understanding the molecular mechanism(s) of the functional

99

synergism could be of great value in optimizing concentrations and/or structural

composition to further improve current intra articular biotherapeutic treatments.


This chapter, in full, is in preparation for submission to the Journal of

Biomechanics. The candidate was the primary author and thanks co-authors Miles Hunter

and Dr. Tannin Schmidt. Study conception and design was performed by TL and TS.

Acquisition of data was performed by MH and TL. Analysis and interpretation of data

was performed by MH, TL, and TS. All authors were involved in revising the article and

approved the final submitted version.

This work was supported by funding from the National Science and Engineering

Research Council of Canada, Canadian Arthritis Network, Alberta Innovates-Technology

Futures, Alberta Innovates Health Solutions OA Team Grant, Faculty of Kinesiology and

Schulich School of Engineering’s Center for Bioengineering Research and Education at

the University of Calgary.

100

Chapter Five: Effects of concentration and structure on proteoglycan 4 rheology and

interaction with hyaluronan

5.1 Abstract

Objective: Determine the viscosity of proteoglycan 4 (PRG4) and recombinant

human PRG4 (rhPRG4) over a range of concentrations, reduced and alkylated (R/A)

PRG4 and rhPRG4, and PRG4 and rhPRG4 with a range of hyaluronan (HA)

concentrations.

Methods: 1.5 MDa HA, PRG4 purified from media condition by bovine articular

cartilage explants, and rhPRG4 were prepared in PBS at varying concentrations of PRG4

(45, 150, 450 µg/mL, R/A 450 µg/mL), rhPRG4 (4.5 45, 150, 450 µg/mL, R/A 450

µg/mL), PRG4+HA, and rhPRG4+HA (HA at 0.3, 1.0, 3.3 mg/mL). Viscosity

measurements were performed on a Nova rotational rheometer with 40 mm parallel plate

fixtures.

Results: PRG4 demonstrated shear thinning behaviour at high concentrations, but

Newtonian behaviour at low concentrations and when R/A. Addition of PRG4 to HA at

0.3 or 1.0 mg/mL increased viscosity compared to HA alone, while addition to HA at 3.3

mg/mL slightly lowered viscosity. Addition of R/A PRG4 had no effect on HA solution

viscosity. rhPRG4 demonstrated Newtonian behaviour over all concentrations tested, as

did R/A rhPRG4. Upon addition of rhPRG4 to HA at 0.3 and 1.0 mg/mL, viscosity was

101

increased compared to HA alone in a concentration-dependent manner. Addition of

rhPRG4 to HA at 3.3 mg/mL decreased viscosity in a concentration-dependent manner.

Discussion: These results provide further support for PRG4 being capable of

influencing solution properties of HA, and that both are important in the rheological

properties in solutions such as synovial fluid. The disulfide-bonded structure of PRG4 is

essential for the observed viscosity effects on HA.

102

5.2 Introduction

The rheological behaviour of synovial fluid (SF) is an important contributor to its

function in articular joints. Healthy SF is able to behave as a viscous fluid at low

frequencies and as an elastic gel at higher frequencies, allowing for storage of mechanical

energy under rapid joint motion, such as running and jumping, to protect cartilage and

surrounding tissues13

. SF demonstrates shear thinning properties, such that it has a higher

viscosity at low shear rates, and viscosity decreases with increasing shear rate151

. With

disease, changes in SF composition and properties alter its functionality. Viscosity of SF

is decreased with osteoarthritis (OA), and some rheumatoid arthritis SF has in fact been

observed to behave as a Newtonian fluid152

. SF from patients with OA also demonstrates

decreased elastic properties13

. These changes in SF behaviour have been attributed to

changes in the hyaluronan (HA) content of SF with aging and disease.

HA is a linear polymer of repeating disaccharides composed of D-glucuronic acid

and D-N-acetlyglucosamine115

, present in SF in a wide range of concentrations (1.8 –

3.33 mg/mL21,102,103,105,106,110

in normal SF) and has been observed to decrease with

arthritic disease102,111,121

. Its molecular weight (MW) distribution, ranging between 27

kDa and 10 MDa in normal human SF and peaking between 6 – 7 MDa81,112-114

, has also

been observed to shift to lower ranges with OA106,111

. Solutions of HA alone demonstrate

shear-thinning behaviour, with increasing concentration and MW increasing the zero-

shear viscosity153,154

; this is attributed to solution non-ideality arising from increased

macromolecular crowding155,156

. HA of varying MW has historically demonstrated

efficacy as a safe intra-articular viscosupplement treatment for pain in osteoarthritis

(OA)92

, though its efficacy has been questioned recently by the American Academy of

103

Orthopaedic Surgeons23

. Although the residence time of injected HA in the joint is short

compared to the duration of pain relief provided88

, suggesting that enhancement of

viscoelastic behaviour is not the only mechanism by which HA injections can relieve

pain91

, viscosupplementation with high MW HA or cross-linked HA products has been

observed to restore SF elasticity in vitro157

. However, HA has not been shown to provide

chondroprotective effects.

Proteoglycan 4 (PRG4, also known as lubricin) is a mucin-like glycoprotein

produced by cells within articular joints including superficial zone chondrocytes6 and

synoviocytes116

. PRG4 works synergistically with HA to reduce friction in the boundary

mode in a cartilage-on-cartilage in vitro friction test34

. While both PRG4 and HA can act

to reduce friction in the boundary mode at a cartilage-cartilage biointerface, PRG4+HA

interaction in solution is also of interest as when PRG4 is depleted from a cartilage

surface it can be replenished by PRG4 from SF158

. In addition to the functional boundary

lubrication synergism, some indirect evidence (single molecule HA tracking149

and

multiple particle tracking micro-rheometry37

) has demonstrated that the PRG4+HA

physical entanglement interaction may also have important effects on solution and SF

rheological behaviour. PRG4+HA viscosity has previously been measured at a single

concentration, and addition of PRG4 purified from SF decreased the viscosity of HA

solutions, potentially by allowing HA molecules to align in the direction of flow37

.

Previous measurements of PRG4-void SF demonstrated an increased zero shear viscosity

compared to normal SF, consistent with the observation above in purified solutions and

suggesting that both PRG4 and HA are important for normal SF rheological function.

104

PRG4 possesses several key properties of mucins including extensive O-linked

glycosylation in the mucin domain60

and disulfide bonded multimerization59

. Previous

studies have shown that viscosity of animal mucins is proportional to mucin

concentration159

, and that small changes in mucin concentration may considerably affect

mucus rheology160

. Disulfide bonded multimerization is an important structural feature of

mucins, and is necessary for gel behaviour 161,162

. It is also important for PRG4 function,

as when inter- and intra-molecular bonds are broken by reduction and alkylation (R/A),

PRG4 fails to bind to cartilage surfaces147

and does not provide boundary lubrication

compared to non-reduced PRG4 145

. Previous boundary lubrication studies have used

PRG4 purified from media conditioned by bovine cartilage explants34

, however full-

length recombinant human PRG4 (rhPRG4) has recently become available46

and has

demonstrated equivalent cartilage boundary lubricating ability to bovine PRG4163

. The

viscosity of this newly available rhPRG4 has not previously been characterized.

Despite thorough characterization of the rheological and viscoelastic properties of

SF and HA solutions, the effects of a range of PRG4 and PRG4+HA concentrations and

PRG4 disulfide-bonded structure (tertiary and quaternary structure) remain to be

determined. The objectives of this study were therefore to determine the viscosity of

PRG4 and rhPRG4 over a range of concentrations, reduced and alkylated (R/A) PRG4

and rhPRG4, and PRG4 and rhPRG4 with a range of HA concentrations. The hypothesis

was that PRG4 and rhPRG4 contribute to the rheological properties of PRG4+HA

solutions through concentration and structurally mediated interactions with itself and HA.

105

5.3 Materials and Methods

5.3.1 Materials

Polydisperse sodium hyaluronate (HA, 1.5 MDa) was obtained from Lifecore

Biomedical. HA was dissolved in PBS at 7.0 mg/mL and allowed to dissolve overnight at

room temperature with rocking. PRG4 was purified from media conditioned by bovine

cartilage explants by DEAE-Sepharose anion exchange chromatography as described

previously34

. After buffer exchange into PBS and concentration measurement by

bicinchoninic acid assay (BCA), purity of PRG4 was estimated at 85% by protein stain

after 3 – 8% Tris-Acetate SDS-PAGE (Figure 5-1A). Purified full-length rhPRG446

was

obtained from Lubris. Concentration was measured by BCA, and purity was estimated at

99% by protein stain after 3 – 8% Tris-Acetate SDS-PAGE (Figure 5-1B).

Reduction to break intra- and inter-molecular disulfide bonds was performed by

incubation with dithiothreitol (10mM) in PBS for 2 hours at 60°C. Alkylation was

performed by incubation with iodoacetamide (40mM) for 2 hours at room temperature in

the dark59

. Dithiothreitol and iodoacetamide were removed by dialyzing against PBS (3.5

kDa MW cut off) overnight at 37°C with rocking. R/A of PRG4 and rhPRG4 was

confirmed by protein stain after 3 – 8% Tris-Acetate SDS-PAGE54

(Figure 5-1A and B).

106

Figure 5-1: Characterization of PRG4 (A), reduced and alkylated (R/A)

PRG4 (A), recombinant human (rh) PRG4 (B), and R/A rhPRG4 (B) by

protein stain after 3 – 8% SDS-PAGE. * denotes an ~460 kDa monomeric

species, and ** denotes higher MW species of ~1 MDa and higher MW

aggregates54

107

Solutions of HA alone were prepared at [HA] = 0.3, 1.0, and 3.3 mg/mL. PRG4

was prepared at [PRG4] = 45, 150, and 450 µg/mL, and R/A PRG4 was prepared at 450

µg/mL. Solutions of PRG4+HA were prepared at [HA] = 0.3, 1.0, and 3.3 mg/mL,

[PRG4] = 45, 150, and 450 µg/mL, and [R/A PRG4] = 450 µg/mL. rhPRG4 was prepared

at [rhPRG4] = 4.5, 45, 150, and 450 µg/mL, and [R/A rhPRG4] = 450 µg/mL. Solutions

of rhPRG4+HA were prepared at [HA] = 0.3, 1.0, and 3.3 mg/mL, and [rhPRG4] = 45

and 450 µg/mL (based on observations in PRG4 the 150 µg/mL was not performed with

rhPRG4.) Solutions of (rh)PRG4 and HA were combined and adjusted to final volume

with PBS to achieve the required concentration. Solutions were allowed to equilibrate for

several hours at room temperature with rocking. Samples were then stored at -20°C until

use, and were thawed at room temperature for 2 hours prior to testing.

5.3.2 Viscosity of PRG4+HA Solutions

Steady shear viscosity was measured in stress control mode using a NOVA

Rheometer (ATS RheoSystems) at 25 and 37°C with 40 mm parallel plate geometry and

a 0.3 mm gap. The upper fixture was a lighter disposable aluminum fixture to reduce

inertia effects. Shear stress ranged from 0.5 – 30 Pa depending on the viscosity of the

sample, resulting in shear rates ranging from ~0.01 – 1500 seconds-1

. After reaching the

required shear stress a 20 second delay time was allowed, after which data collected over

30 seconds. Duplicate tests of samples from the same preparations were in excellent

agreement with each other (confirmed visually by overlying duplicate runs on plots).

rhPRG4±HA measurements were conducted at 25°C only.

108

5.4 Results

5.4.1 Viscosity of PRG4+HA Solutions

PRG4 Alone. Solutions of PRG4 alone demonstrated concentration- and disulfide-

bonded structure-dependent shear thinning behaviour. At 25°C, PRG4 at 450 and 150

µg/mL showed strong shear thinning behaviour, but displayed Newtonian behaviour with

a viscosity similar to that of water at 45 µg/mL and when R/A (~0.001 Pa s, Figure

5-2A). Similar trends were observed at 37°C, but measured viscosities were slightly

lower (Figure 5-2B).

109

Figure 5-2: Shear rate dependent viscosity at 25°C (A) and 37°C (B) of

PRG4 alone at 45, 150, 450 µg/mL, and R/A PRG4 at 450 µg/mL.

110

PRG4 + HA. Combination of PRG4 and HA in solution resulted in viscosities

different from HA or PRG4 alone at [HA] = 0.3 and 1.0 mg/mL. At [HA] = 0.3 and 1.0

mg/mL in the absence of PRG4, approximately Newtonian behaviour was observed

(Figure 5-3). Addition of PRG4 increased viscosity, and shifted behaviour of HA

solutions to shear thinning; viscosity enhancement of [HA] = 0.3 and 1.0 mg/mL was

similar for addition of 45 (Figure 5-3A, B), 150 (Figure 5-3D, E), and 450 (Figure

5-3G, H) µg/mL PRG4. Upon addition of R/A PRG4 at 450 µg/mL, viscosity of the HA

at 0.3 or 1.0 mg/mL alone was maintained (Figure 5-4A, B). Trends were similar at 37°C

(not shown).

Combination of HA at 3.3 mg/mL with PRG4 did slightly lowered solution

viscosity. HA at 3.3 mg/mL exhibited strong shear thinning behaviour (Figure 5-3).

Addition of PRG4 at 45 (Figure 5-3C), 150 (Figure 5-3F), and 450 (Figure 5-3I) µg/mL

appeared to lower the viscosity at 25°C slightly. Viscosity of [HA] = 3.3 mg/mL was

maintained with addition of R/A PRG4 at 450 µg/mL (Figure 5-4C). At 37°C addition of

R/A PRG4 appeared to have no effect on viscosity, though addition of R/A PRG4 at 450

µg/mL may have slightly decreased viscosity (not shown).

111

Figure 5-3: Shear rate dependent viscosity at 25°C of HA at 0.3 (A, D,

G), 1.0 (B, E, H), and 3.3 (C, F, I) mg/mL alone and with 45 (A, B C),

150 (D, E, F) and 450 µg/mL (G, H, I) PRG4.

112

Figure 5-4: Shear rate dependent viscosity at 25°C of HA at 0.3 (A), 1.0

(B), and 3.3 (C) mg/mL alone and with R/A PRG4 450 µg/mL

rhPRG4 Alone. Low viscosity Newtonian behaviour was observed for rhPRG4 all

concentrations measured (4.5, 45, 150, and 450 µg/mL, 0.0022 – 0.0029 Pa s), as well as

for R/A PRG4 at 450 µg/mL (0.0027 Pa s). Viscosity of these solutions was slightly

higher than water at 25°C (Figure 5-5).

113

Figure 5-5: Shear rate dependent viscosity at 25°C of rhPRG4 alone at

4.5, 45, 150, 450 µg/mL, and R/A rhPRG4 at 450 µg/mL.

rhPRG4 + HA. Combination of rhPRG4 and HA in solution altered HA solution

viscosity in a dose dependent manner at 25°C. For [HA] = 0.3 and 1.0 mg/mL, addition

of 45 µg/mL rhPRG4 increased viscosity (Figure 5-6A, B), and addition of 450 µg/mL

rhPRG4 increased viscosity slightly more (Figure 5-6D, E). At [HA] = 3.3 mg/mL,

addition of 45 µg/mL rhPRG4 decreased viscosity, and addition of 450 µg/mL decreased

viscosity slightly more (Figure 5-6C, F); addition of rhPRG4 appeared to decrease the

extent of shear thinning in [HA] = 3.3 mg/mL.

114

Figure 5-6: Shear rate dependent viscosity at 25°C of HA at 0.3, 1.0, and

3.3 mg/mL alone and with 45 and 450 µg/mL rhPRG4.

5.5 Discussion

The findings of this study agree with and extend previous viscosity studies on

preparations of PRG4 and HA, and provide insight into the role diminished HA and/or

PRG4 content may play in altered rheological properties of pathological SF. The

observation of shear-thinning behaviour at high concentrations of the PRG4 preparation

used here is consistent with other mucin proteins, albeit at higher concentrations164

. The

highly purified rhPRG4 preparation did not exhibit shear-thinning behaviour at high

115

concentrations. At low HA concentrations, the addition of even a low concentration of

PRG4 or rhPRG4 is able to increase the viscosity of HA solutions, suggesting that PRG4

interacts with HA and contributes to the solution properties. At higher concentrations of

HA (well over the critical concentration of the MW HA preparation used here), addition

of PRG4 or rhPRG4 may limit the viscosity of PRG4+HA solutions by allowing HA to

adopt a more flexible conformation as has previously been suggested37

. These influences

on HA solution behaviour are dependent on the disulfide bonds within and/or between

PRG4. While the mechanism of the PRG4+HA interaction remains to be fully

determined, these results show that it is dependent on PRG4 disulfide-bonded structure

and are consistent with a non-covalent/entanglement mechanism. They suggest that the

combination of PRG4 and HA in normal SF may contribute to its rheological properties

and function, and that PRG4 may play a role in fluid film lubrication in addition to its

contributions to boundary lubrication.

The PRG4 preparation used in this study has previously been used for cartilage

boundary lubrication testing, alone and in combination with HA34,35

, and was purified

using methods that maintain disulfide-bonded structure and are therefore appropriate for

the functional testing performed here. However, estimated purity of the PRG4

preparation is 85% (by densitometric analysis after protein stain on SDS-PAGE), and

other components synthesized and secreted by chondrocytes that may be present, and

potentially contributing to rheological properties, cannot be ruled out. The rhPRG4

preparation used here is a ~99% pure preparation, and previous functional tests using less

pure preparations have confirmed that disulfide bonded multimerization, glycosylation,

and boundary lubricating ability of rhPRG4 are similar to PRG4163

. The HA solutions

116

prepared for this experiment were prepared in several batches, and differences in solution

storage times and preparation may have slightly affected the results; for example the

HA+R/A PRG4 data are very close to but not the same as the HA alone data, but were

prepared from new HA solutions. However, the viscosities of the 1.5 MDa HA solutions

and Newtonian or shear thinning behaviour depending on concentration are in agreement

with previous work153,165

. Repeated testing of different PRG4 preparations at 450 µg/mL

demonstrated that the viscosity measurements are repeatable with careful sample

preparation. Extension of the data to lower shear rates would be useful to confirm these

results (i.e. the reduction of viscosity at 3.3 mg/mL HA with addition of PRG4).

The rheological behaviour of PRG4 alone observed here is consistent with its

classification as a mucin-like glycoprotein. The viscosities of purified porcine gastric

mucin and bovine salivary mucins have been observed to increase with increasing

concentration, however at higher concentrations than those used here159

. It has also been

observed that small changes in mucin concentration can cause large changes in mucus

rheological behaviour160

. Other mucins have demonstrated similar behaviour upon

reduction; porcine gastric mucin fails to demonstrate gelation when reduced, and when

prepared using protease treatment during purification does not gel166

. These results also

are consistent with a previous study of the viscosity of purified synovial lubricating factor

(PSLF). PSLF viscosity has previously been measured as 0.006 Pa s at 35°C167

, however

this preparation was purified by elution from a size exclusion column with a dissociating

buffer containing guanidine hydrochloride, which has been shown to disrupt PRG4

lubricating function168

. This agrees with the viscosity obtained for R/A PRG4 in this

study (0.001 Pa s at 25°C). The speculation that a co-purified species, likely also present

117

in SF, may be contributing to rheological properties is consistent with previous

observations that other proteins (including type VI collagen, tenascin-C, aggrecan,

fibronectin, cartilage oligomeric matrix protein, vitronectin, decorin) are co-purified

during immune-precipitation with anti-PRG4 antibodies in PRG4 preparations extracted

from articular cartilage169

.

These results also agree with previous characterization of PRG4 and PRG4+HA

solutions, where addition of unpurified human umbilical HA appeared to increase the

viscosity of PSLF, while addition of purified human umbilical HA decreased the

viscosity63

. When PRG4 at 300 µg/mL was added to human umbilical cord HA at 3.5

mg/mL, the zero-shear rate viscosity decreased, suggesting that HA is present in a more

rigid conformation in the absence of PRG437

. The observation that R/A PRG4 did not

possess the ability to alter HA solution viscosity is consistent with previous observations

that R/A PRG4 does not significantly enhance cartilage boundary lubricating ability of

HA solutions (see Chapter 4).

The Newtonian behaviour observed for the rhPRG4 alone is consistent with previous

characterization showing it is a very pure preparation. Its ability to contribute to HA

solution viscosity in a dose dependent fashion suggests that the effects observed in the

PRG4+HA solutions are at least in part specific to PRG4 and HA. If there is an additional

species present in the PRG4 preparation, its absence in the rhPRG4 preparation may

explain the Newtonian behaviour observed, and dose dependent effects seen in

rhPRG4+HA solutions. If the PRG4/rhPRG4+HA interaction is provided by molecular

crowding/entanglement, the absence of a contributing species could mean that higher

concentrations of rhPRG4 may be required to observe similar effects, as has been

118

observed for other mucins. Addition of rhPRG4 appears to have decreased the viscosity

of HA at 3.3 mg/mL to a larger extent at both 45 and 450 μg/mL compared to PRG4 at

the same concentrations. Addition of PRG4 lowered viscosity while maintaining the

shear thinning behaviour, but addition of rhPRG4 may have slightly decreased the extent

of shear thinning observed, though measurements at lower shear rates would confirm this.

Both observations are consistent with the presence of another SF component in the PRG4

preparation that may be contributing to PRG4+HA solution viscosity.

While viscosity of PRG4+HA solutions was different than HA or PRG4 alone, this

did not appear to be a purely additive effect; for example, the viscosities of HA at 0.3 and

1.0mg/mL with PRG4 at 45 µg/mL exceeded the sum of the PRG4+HA individual

viscosities, and did not reach the predicted PRG4+HA viscosity when 150 or 450 µg/mL

was added (the predicted sum is not shown here). The viscosities predicted by adding

rhPRG4+HA viscosities were close to those measured for HA at 0.3 and 1.0 mg/ml and

rhPRG4 at 45 and 450 µg/mL, but measured viscosity was significantly lower than

predicted for HA at 3.3 mg/mL. The transition of PRG4s ability to enhance the viscosity

of HA solutions to its tendency to decrease viscosity may be related to the critical

concentration of 1.5 MDa HA, 0.99 mg/mL170,171

. However, the 1.5 MDa HA used is

known to be polydisperse35

, which could influence the actual critical concentration of the

solution used. Differing effects in dilute and concentrated solutions may not be fully

captured here as the 1.0 mg/mL concentration is very close to this critical value. Below

this point it appears that HA may help induce self-aggregation or self-assembly of PRG4

in solution, and increase PRG4+HA solution viscosity. Above this critical concentration,

the HA molecules are already completely entangled, and self-aggregation of PRG4 has

119

less of an effect. Previous work has suggested that addition of PRG4 enables HA

molecules to align in the direction of flow, reducing the viscosity of high concentration

HA solutions37

. This is consistent with the observation (especially in rhPRG4) here that

viscosity of 3.3 mg/mL HA is decreased with addition of 45 or 450 μg/mL rhPRG4,

however the mechanism remains to be confirmed. It is also possible that in a crowded HA

solution, PRG4 molecules can no longer self-aggregate or self-assemble. The observation

in PRG4+HA that 45, 150, and 450 µg/mL PRG4 enhance viscosity to the same extent is

consistent with boundary lubrication observations, where addition of small amounts of

the same PRG4 preparation to increase boundary lubricating ability to a point, beyond

which PRG4 can no longer make any further contributions to solution cartilage boundary

lubricating ability172

.

Future work will be centered on clarifying the mechanism of this physical PRG4+HA

functional interaction, and investigating how it differs in solution and at cartilage

surfaces. Substitution of HA for another aggregating polymer and evaluation of

PRG4+polymer viscosity, as well as repeating these experiments with smaller HA, would

help clarify if HA can influence PRG4 aggregation in solution via molecular crowding.

Lipid content may also affect rheology of other mucins160

; surface active phospholipids

are present in SF32

and while they have not been observed to contribute to cartilage

boundary lubricating ability in vitro, their contributions to SF viscosity are currently

unknown. The contributions of PRG4 multimeric/monomeric structure and glycosylation

patterns65,160

to solution viscosity also remain to be elucidated. Other rheological

techniques may be useful in evaluation of PRG4, PRG4+HA, and SF rheology including

dynamic oscillatory testing (to evaluate viscoelasticity) and micro-rheology160

. Multiple

120

particle tracking microrheology has been observed to be well correlated with bulk

rheology37

, and given the limited volumes of normal human SF available, micro-rheology

techniques may be valuable.

The observation that addition of (rh)PRG4 can alter solution properties of HA

suggests that both HA and PRG4 are key contributors to the rheological properties of SF,

as has been observed for boundary lubricating function. The ability of (rh)PRG4 to both

increase and decrease the viscosity of HA solutions, depending on HA concentration,

suggests that SF composition may be “designed” for optimal rheological performance.

While the mechanisms of the PRG4+HA interaction observed here and in SF cartilage

boundary lubricating ability remain to be clarified and may be different, these results

agree with observations in lubrication that the disulfide bonded structure of PRG4 is

essential for function. As intra-articular treatment with PRG4 has been shown to protect

against cartilage degeneration in animal models of OA75,76,123,173

, it will be valuable to

understand the rheological effects of combining PRG4±HA in biotherapeutic treatments

to restore and maintain SF function, as a combined treatment may be able to provide both

chondroprotective and viscosupplement effects in SF where either PRG4 and/or HA

composition is altered.

121


This chapter, in full, is in preparation for submission to Biomacromolecules. The

candidate is the primary author and thanks co-authors Dr. Mary K. Cowman, Dr. Gregory

Jay, and Dr. Tannin A. Schmidt. Study conception and design was performed by TL,

MK, and TS. Acquisition of data was performed by TL. Analysis and interpretation of

data was performed by TL, MK, GD, and TS.

This work was supported by funding from the National Science and Engineering

Research Council of Canada, Canadian Arthritis Network, Alberta Innovates-Technology

Futures, Alberta Innovates Health Solutions OA Team Grant, Faculty of Kinesiology and

Schulich School of Engineering’s Center for Bioengineering Research and Education at

the University of Calgary.

122

Chapter Six: Conclusions

6.1 Summary of Findings

The overall goals of this thesis work were to investigate the relationship between

boundary lubricant composition and cartilage boundary lubricating function in normal

and diseased SF, and to investigate how PRG4 contributes to the boundary lubricating

and rheological properties of SF through concentration and interactions with itself and

HA in solution. The major findings were:

1. PRG4 and HA composition can be altered in chronic OA SF, post intra-articular

injection flare SF, and repeat donor OA human SF compared to normal human SF.

PRG4 concentrations spanned a wide range in both OA and normal SF samples.

PRG4 concentrations are not reduced in all chronic OA or flare SF samples.

2. Some chronic OA SF was deficient in PRG4, had low HA MW, and failed to

lubricate as well as normal SF. Some flare-SF was deficient in PRG4, had

approximately normal HA MW distribution, and lubricated as well as normal SF.

3. The diminished human cartilage boundary lubricating ability of PRG4-deficient

chronic OA SF with low HA MW could be restored with supplementation with

PRG4; subsequent addition of HA had no further effect. Cartilage boundary

lubricating ability of flare-SF deficient in PRG4 with normal HA MW distribution

was not altered by addition of PRG4 or PRG4+HA.

123

4. Low concentrations of PRG4 or decreased concentration of high MW HA can limit

the cartilage boundary lubricating ability of PRG4+HA solutions; this is exacerbated

when both PRG4 and high MW HA are decreased.

5. PRG4 is able to contribute to the cartilage boundary lubricating ability of cross-linked

HA in solution. R/A PRG4 is not able to contribute to the cartilage boundary

lubricating ability of HA solutions, suggesting that PRG4 disulfide-bonded structure

is important for the PRG4+HA boundary lubricating functional synergism.

6. PRG4 alone demonstrates strong shear thinning behaviour at high concentrations,

while rhPRG4 is Newtonian even at high concentrations. Addition of even small

amounts of PRG4 or rhPRG4 to low concentrations of HA increases solution

viscosity. Addition of PRG4 or rhPRG4 to high concentrations of HA appears to

decrease solution viscosity. Both effects are dependent upon the disulfide-bonded

structure of PRG4.

This thesis work has contributed to the understanding of the functional synergism

between PRG4 and HA and demonstrated that changes in PRG4 and/or HA content in SF

may have deleterious effects on SF cartilage boundary lubricating ability and rheological

function. They suggest that maintaining PRG4 and HA content in SF during injury and

disease may be able to retain function, and that the development of new PRG4±HA

biotherapeutic treatments may be able to provide both chondroprotective and

viscosupplementation effects in vivo.

124

6.2 Discussion

6.2.1 Measurement of PRG4 Concentration in SF

PRG4 concentration in human and animal SF can be measured by semi-quantitative

western blot87,103

, and by sandwich ELISAs using other antibodies20,68,104,127,174

. The

western blot technique is time consuming and quantification is difficult. The long term

availability of proprietary antibodies may also limit long-term use of some ELISA

techniques.

The ELISA developed here uses commercially available antibodies/reagents and has

been used to measure PRG4 concentration in human1 and ovine

137 SF. The capture

antibody LPN identifies the C-terminal of full length PRG459

, and detection with PNA

identifies glycosylations important for cartilage boundary lubricating function65

.

Blocking and dilution reagents were selected based on the highest signal to noise ratios

obtained during assay development, and such that linearity of dilution was achieved in

the samples and controls. In this ELISA, PRG4 controls and SF samples are pre-treated

with hyaluronidase, to eliminate the possibility of a PRG4+HA interaction interfering

with antibody recognition of PRG4, and Sialidase A-66 to remove sialic acid caps prior

to quantification using PNA-HRP. This may result in differences from previously

measured values, although the average value in normal SF is consistent with previously

reported normal values20

. While this assay has contributed improvements to measurement

of PRG4 in SF, and data from PRG4 measurement in clinical samples was valuable in

guiding experiment planning for the purified solution experiments, it is somewhat costly

due to the LPN, and cannot distinguish between PRG4 multimers and monomers. Other

125

techniques that could potentially quantify the PRG4 multimer:monomer distribution in

SF are discussed in the Future Work section below.

There appears to be some differences in the changes reported in PRG4 concentrations

after acute injury in post-traumatic SF within human and animal studies. PRG4

concentrations in SF after acute injury in animal models have been observed to both

increase (equine87

, rabbit68

, ovine study in preparation for publication175

) and decrease

(rat176

). Furthermore, some studies showed that PRG4 returned to normal in these models

(at 20 weeks post injury in ovine SF137

, at 2 – 3 weeks after injury in rabbit SF68

) while

others remained decreased (rat176

). In longer term models, PRG4 concentration was

observed to decrease in guinea pig SF 9 months after ACL transection117,118

, and down-

regulation of PRG4 mRNA expression was observed 3 months after meniscectomy in an

ovine model119

.

Reported changes are also varied in human SF. PRG4 concentrations have been

observed to both decrease20

and increase174

after ACL tear, and both recover to normal

over 1 year20

and decrease from acute injury to follow up ~30 days later174

. PRG4

concentrations have also been observed to increase after tibial plateau fracture103

and in

end stage OA104

, and some chronic OA patients can demonstrate decreased

concentrations1. Furthermore, PRG4 expression has been used to sub-classify RA patients

as high- or low-expressors109

, and SF composition and properties can differ between the

right and left knees of one patient (Appendix C177

).

The underlying cause(s) of these differences in PRG4 concentration in both animal

and human SF remain to be determined, and are compounded by potential differences due

126

to varying methodologies, animal models, time points of analysis after injury discussed

above, and several other factors:

1. Average normal values of PRG4 concentration appear to be quite variable1

2. PRG4 expression is regulated by both mechanical stimulation71

and cytokines

that may be dis-regulated in injury or disease77

3. PRG4 concentration can change quickly with loading of the joint73

4. Increases in degradative enzymes in post-injury or inflammatory situations

may increase PRG4 degradation20

.

These factors suggest that the timing of SF aspiration/PRG4 measurement may be

important to consider in future analysis, and that changes in an individual’s SF

composition may be of more value than comparison to the average normal.

6.2.2 PRG4+HA Functional Synergism

While not directly designed to probe the molecular mechanism of PRG4 interaction,

the results discussed in this thesis provide some insight into the mechanism of PRG4+HA

interaction. It should be noted that the mechanism of interaction of PRG4+HA may be

different in solution and at cartilage surfaces.

The cartilage boundary lubricating ability experiments in SF and purified solutions

performed in this work are summarized in Table 6-1 below. They suggest that both

PRG4 and high MW HA content can be limiting in achieving reduction of friction in the

boundary mode at cartilage surfaces. They also demonstrate that PRG4 tertiary and

quaternary structure, via disulfide bond formation, is important in PRG4+HA synergistic

127

boundary lubricating ability. Furthermore, these results are consistent with previous

observations of a transient, entanglement mechanism of interaction for PRG4+HA35

.

128

Table 6-1: Summary of boundary lubricant composition and boundary

lubrication function of SF and purified solutions tested.

[PRG4] [HA] HA MW

Boundary

Lubricating Ability

OA-LO SF* ↓ ≅ ↓ ↓

Flare-SF* ↓ ≅ ≅ ≅

PRG4+HA Low

(4.5, 45 μg/mL)

High 1.5 MDa ↓

PRG4+HA ~Normal

(150, 450

μg/mL)

Low 1.5 MDa ↓

PRG4+HA Low

(45 μg/mL)

Low 1.5 MDa ↓↓

PRG4+HA R/A

(450 µg/mL)

High 1.5 MDa ↓

PRG4+HA ~Normal

(450 μg/mL)

High Cross-linked ≅

*For normal SF, average PRG4 concentration measured in this thesis work

was 287 μg/mL. Average PRG4 concentration in OA-LO SF was 147

μg/mL, and flare-SF 103 – 231 μg/mL.

129

In the HA and PRG4 dose responses presented in Chapter 4, it was observed that

equilibrium loads experienced by the cartilage samples at 18% deformation seemed to

decrease with increasing boundary lubricant concentration, suggesting that perhaps

PRG4±HA aggregates or a viscous boundary layer39

at the surface may be increasing in

thickness and bearing load.

In both the cartilage boundary lubrication and viscosity experiments, there seems to

be a concentration plateau past which PRG4 can no longer additionally contribute to the

friction reduction or viscosity changes of PRG4+HA solutions. rhPRG4 viscosity data is

not consistent with this, where there was a concentration dependent effect on viscosity of

addition of 45 vs. 450 μg/mL; this discrepancy could potentially be accounted for by a

species co-purifying with the PRG4 preparation. Both sets of experiments do suggest that

adding a small amount of PRG4 (or rhPRG4) is able to somewhat affect the behaviour of

low concentrations of HA.

PRG4 and/or high MW HA concentration may be limiting in the cartilage boundary

lubricating ability or rheological properties of purified solutions, and potentially SF.

These results reinforce the notion that both HA and PRG4 are critical contributors to the

normal function of SF, and suggest that a combined biotherapeutic treatment may be

useful to “rescue” SF when either of these species is diminished.

The work presented here also suggested that perhaps HA (or possibly another SF

constituent that is co-purified with the PRG4 preparation) helps induce assembly of

PRG4 multimers/aggregates in solution. Preliminary atomic force microscopy images of

PRG4 have shown it may exist as aggregated globules on hydrophobic surfaces, can

entangle with other PRG4 molecules, and interact along the length of HA chains. (This

130

preliminary unpublished data was collected by an undergraduate student in the lab of

collaborator Dr. Mary Cowman, NYU.)

6.3 Future work

6.3.1 Measurement of PRG4 Concentration in SF

Multiplex assays allow simultaneous detection of many analytes in small sample

volumes. In this technology, a target-specific antibody is coated onto a unique fluorescent

dye color-coded microparticle. The particles are used in microtiter plate format, and

biotinylated antibodies are used to detect each analyte of interest. The first step in

quantification is to identify the analyte associated with the color of the microparticle,

followed by quantification of the signal provided by the detection antibody178

. This

technology has shown promise in discriminating between normal, mild/moderate, and

severe OA SF cytokine profiles using as little as 20 μL of human SF179

. Customizing a

multiplex assay for PRG4 may enable identification of PRG4 fragments in SF and

potentially multimer/monomer distribution of PRG4 in SF. PRG4, HA, and other

cytokines of interest could be quantified simultaneously, which would reduce the amount

of SF required.

The distribution of PRG4 multimers/monomers in SF is of interest because of the

effects this could have on SF boundary lubricating function146

. The SDS-PAGE/Western

blot protocol used here does not adequately separate very high MW PRG4 species

(~1MDa). Agarose gel electrophoresis has shown separation of PRG4 species in SF59

, but

may be complicated by varying glycosylation patterns in SF samples65

. High performance

131

liquid chromatography using size exclusion columns may also be useful to quantify the

multimer/monomer distribution in SF.

Several other characteristics of PRG4 remain to be quantified in SF to fully

understand the SF composition/structure/cartilage boundary lubricating function

relationship. The candidate has sent normal SF and age and sex matched OA samples,

OA SF deficient in PRG4, and flare-SF deficient in PRG4 to collaborator Dr. Niclas

Karlsson (University of Gothenburg, Gothenburg, Sweden, sent February 2013), for

glycosylation analysis. Previous work has suggested that OA-specific glyco-epitopes or

fragments of PRG4 may exist66

, and that glycosylation of PRG4 may be altered in RA65

.

Taken together with the fact that glycosylations on PRG4 (including sialic acid caps)

have been shown to be important for boundary lubricating function62

, and could also

affect viscosity of PRG4 solutions160

, alterations in glycosylations are of interest for their

effects on SF function.

The candidate has also performed PRG4 measurement in normal, early OA, late OA,

and RA SF provided by collaborator Dr. Juergen Steinmeyer (Justus-Liebig-University of

Giessen, Giessen, Germany, analysis completed March 2014). Previous work by Dr.

Steinmeyers group has observed differences in relative composition and abundance of

phospholipid species between normal, early OA, and late OA SF32

. These results are

being correlated with HA and surface active phosopholipid composition of these samples

and will provide a complete picture of putative boundary lubricants contributing to SF

function.

While not directly related to PRG4, the candidate also was also involved with a study

investigating whether metabolomic methods (nuclear magnetic resonance (NMR)

132

spectroscopy and gas chromatography-mass spectrometry (GC-MS)) could be used to

identify metabolic patterns in SF samples of symptomatic chronic knee OA patients (Dr.

Hans Vogel, University of Calgary, Calgary, draft manuscript completed December 2013,

Submitted to Osteoarthritis and Cartilage April 2014). Using these techniques and

multivariate statistical analysis, OA patients could be distinguished from the normal

cadaveric controls, and the 23 metabolites important for identifying OA SF may be useful

for future diagnosis.

6.3.2 PRG4+HA Functional Synergism

In future studies, it would be interesting and valuable to test the synergistic effect

both at a cartilage-cartilage interface and viscosity with another molecule that may be

able to push PRG4 into a more entangled confirmation or increase its functional

concentration by crowding. Evaluating the boundary lubricating ability and rheology of

PRG4 combined with a lower MW HA may also provide insight into the mechanism of

interaction. The failure of the dot blot (Appendix A) and ITC (Appendix B) experiments

performed by the candidate to capture the interaction further support a non-

covalent/entanglement mechanism. However, given the availability of rhPRG4 it may be

feasible to repeat the ITC experiments with higher concentrations, as discussed in

Appendix B.

Previous evidence has demonstrated that mucin-alginate gel rheology can be

disrupted by addition of low MW alginate oligomers180

. While this was porcine gastric

mucin and alginate, it is conceivable that low MW HA fragments could have a similar

effect in the PRG4+HA interaction. A linear correlation between [HA < 0.5 MDa] and

133

<μkinetic,Neq> was observed for the flare-SF presented in Chapter 3 (increasing COF with

increasing [HA < 0.5 MDa, p < 0.05, data not shown); however this SF retained normal

boundary lubricating ability (possibly due to retention of enough HMW HA), and the

PRG4 deficient OA SF that demonstrated decreased boundary lubricating ability (Chapter

2) did not demonstrate such a correlation. It would be interesting to assess this in less

complicated purified solution in both cartilage-cartilage boundary lubricating and

rheology experiments; very small HA (20 kDa) could be added to PRG4 (450 μg/mL)

and 1.5 MDa HA (3.3 mg/mL) to evaluate disruption of the synergism.

While the in vitro cartilage-cartilage boundary mode lubrication test29

used here has

been used to evaluate lubricating ability of purified PRG4+HA solutions34,35

, ovine

SF137,175

, and human SF, the effect of the order of lubricants was not explicitly tested

here. Previous work has suggested that the order of the lubricants does not affect the

calculated coefficient of friction34

. In the work presented here an order effect may have

obscured contributions by HA alone, for example, in the human SF boundary lubricating

ability tests. The samples used in this study were also frozen at -80°C prior to testing, and

while coefficients of friction obtained were similar to those obtained using fresh

samples35

, this may have affected mechanical properties of the tissues. The equilibrium

stresses obtained for the frozen bovine samples used here (0.09 MPa) were similar to

those previously obtained for fresh samples (0.11 MPa34

, 0.10 MPa35

); the equilibrium

stress achieved in the human studies was slightly higher (0.21, 0.17 MPa), consistent with

previous observations that human patellar groove cartilage is stiffer than bovine5.

Future work on PRG4+HA interaction influence on rheological properties could use

micro-rheology to reduce the volume required, monitor higher frequencies in low

134

viscosity solutions, and connect bulk rheological properties to solution micro-

structure181,182

; this technique may allow extension of these measurements to normal and

diseased human SF samples. It may also be possible to extend current confocal-

fluorescence recovery after photobleaching techniques183

to allow calculation of solution

viscosity160

. While this work has demonstrated that the potential use of PRG4±HA as a

biotherapeutic treatment may be able to contribute to both boundary lubricating ability

and viscosity of SF, future work will require animal models to ensure these effects can

also persist in vivo in the more complicated environment of the intact joint. Biological

factors such as degradation and clearance of exogenous lubricant molecules will

influence the biomechanical effects observed in this study.

135

Bibliography

1. Ludwig TE, McAllister JR, Lun V, Wiley JP, Schmidt TA. Diminished cartilage

lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan

4: Restoration through proteoglycan 4 supplementation. Arthritis Rheum.

2012;64(12):3963-3971.

2. Chubinskaya S, Malfait AM, Wimmer MA. Form and Function of Articular

Cartilage. In: O'Keefe RJ, Jacobs JJ, Chu CC, Einhorn TA, eds. Orthopaedic

basic science: foundations of clinical practice American Academy of Orthopaedic

Surgeons; 2013:183-198.

3. Ateshian GA, Mow VC. Friction, lubrication, and wear of articular cartilage and

diarthrodial joints. In: Mow VC, Huiskes R, eds. Basic Orthopaedic

Biomechanics and Mechano-Biology. 3rd ed. Philadelphia: Lippincott Williams &

Wilkins; 2005:447-494.

4. Koopman WJ, Moreland LW, eds. Arthritis and allied conditions : a textbook of

rheumatology 15th ed: Lippincott Williams & Wilkins; 2005.

5. Mow VC, Gu WY, Chen FH. Structure and Function of Articular Cartilage and

Meniscus. In: Mow VC, Huiskes R, eds. Basic Orthopaedic Biomechanics and

Mechano-Biology. Third Edition ed. Philadelphia, PA: Lippincott Williams &

Wilkins; 2005:181-258.

6. Schumacher BL, Block JA, Schmid TM, Aydelotte MB, Kuettner KE. A novel

proteoglycan synthesized and secreted by chondrocytes of the superficial zone of

articular cartilage. Arch Biochem Biophys. 1994;311:144-152.

136

7. Schmidt TA, Schumacher BL, Klein TJ, Voegtline MS, Sah RL. Synthesis of

proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer

cultures, and resurfaced cartilage cultures. Arthritis Rheum. 2004;50:2849-2857.

8. Goldring MB. Cartilage and Chondrocytes. In: Firestein GS, Budd RC, Gabriel

SE, McInnes IB, O'Dell JR, eds. Kelley's Textbook of Rheumatology. Ninth

Edition ed. Philadephia: Elsevier Saunders; 2013:33-60.

9. Simkin PA. Synovial physiology. In: Koopman WJ, ed. Arthritis and Allied

Conditions. Vol 1. 15 ed. Baltimore: Williams & Wilkins; 2005:193-205.

10. Goldring SR, Goldring M. Biology of the Normal Joint. In: Firestein GS, Budd

RC, Gabriel SE, McInnes IB, O'Dell JR, eds. Kelley's Textbook of Rheumatology.

Ninth Edition ed. Philadelphia: Elsevier Saunders; 2013:1-19.

11. Pan J, Zhou X, Li W, Novotny JE, Doty SB, Wang L. In Situ Measurement of

Transport between Subchondral Bone and Articular Cartilage. J Orthop Res.

2009;27:1347-1352.

12. Rubenstein D, Yin W, Frame MD. Lubrication of Joints. In: Rubenstein D, Yin

W, Frame MD, eds. Biofluid Mechanics: An Introduction to Fluid Mechanics,

Macrocirculation, and Microcirculation: Elsevier; 2012.

13. Balazs EA. Viscoelastic Properties of Hyaluronan and Its Therapeutic Use. In:

Garg HG, Hales C, eds. Chemistry and Biology of Hyaluronan: Elsevier Ltd;

2004:415-474.

14. Levick JR. Blood flow and mass transport in synovial joints. In: Renkin E, ed.

Handbook of Physiology, Section 2 The Cardiovascular System. Vol IV

Microcirculation Part 2: American Physiological Soc; 1984:917-947.

137

15. McCarty WJ, Cheng JC, Hansen BC, et al. The Biophysical Mechanisms of

Altered Hyaluronan Concentration in Synovial Fluid After Anterior Cruciate

Ligament Transection. Arthritis Rheum. 2012;64(12):3993-4003.

16. Bresnihan B, Flanagan AM, Firestein GS. Synovium. In: Firestein GS, Budd RC,

Gabriel SE, McInnes IB, O'Dell JR, eds. Kelley's Textbook of Rheumatology.


17. Vos T, Flaxman AD, Naghavi M, Lozano R, Michaud C, Ezzati Mea. Years lived

with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990–2010:

a systematic analysis for the Global Burden of Disease Study 2010. The Lancet.

2012;380:2163-2196.

18. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: A Disease

of the Joint as an Organ. Arthritis Rheum. 2012;64(6):1697-1707.

19. Yelin E. Economic Burden of Rheumatic Diseases. In: Firestein GS, Budd RC,

Gabriel SE, McInnes IB, O'Dell JR, eds. Kelley's Textbook of Rheumatology.


20. Elsaid KA, Fleming BC, Oksendahl HL, et al. Decreased lubricin concentrations

and markers of joint inflammation in the synovial fluid of patients with anterior

cruciate ligament injury. Arthritis Rheum. Jun 2008;58(6):1707-1715.

21. Jay GD, Torres JR, Rhee DK, et al. Association between friction and wear in

diarthrodial joints lacking lubricin. Arthritis Rheum. Nov 2007;56(11):3662-3669.

22. Gallagher B, Tjoumakaris FP, Harwood MI, Good RP, Ciccotti MG, Freedman

KB. Chondroprotection and the Prevention of Osteoarthritis Progression of the

138

Knee: A Systematic Review of Treatment Agents. Am J Sports Med. 2014;DOI:

10.1177/0363546514533777.

23. AAOS. Treatment of Osteoarthritis of the Knee: Evidence-Based Guideline 2nd

Edition. American Academy of Orthopaedic Surgeons. 2013.

24. Greene GW, Lee DW, Yu J, Das S, Banquy X, Israelachvili JN. Lubrication and

Wear Protection of Natural (Bio)Systems. In: Zeng H, ed. Polymer Adhesion,

Friction, and Lubrication. Hoboken, New Jersey: John Wiley and Sons, Inc;

2013:83-133.

25. Greene GW, Banquy X, Lee DW, Lowrey DD, Yu J, Israelachvili J. Adaptive

mechanically controlled lubrication mechanism found in articular joints. Proc

Natl Acad Sci USA. 2011;108(13):5255-5299.

26. Chan SMT, Neu CP, DuRaine G, Komvopoulos K, Reddi AH. Tribological

altruism: A sacrificial layer mechanism of synovial joint lubrication in articular

cartilage. J Biomech. 2012;45:2425-2431.

27. Gleghorn JP, Bonassar LJ. Lubrication mode analysis of articular cartilage using

Stribeck surfaces. J Biomech. 2008;41(9):1910-1918.

28. Morrell KC, Hodge WA, Krebs DE, Mann RW. Corroboration of in vivo cartilage

pressures with implications for synovial joint tribology and osteoarthritis

causation. Proc Natl Acad Sci U S A. Oct 11 2005;102(41):14819-14824.

29. Schmidt TA, Sah RL. Effect of synovial fluid on boundary lubrication of articular

cartilage. Osteoarthritis Cartilage. Jan 2007;15(1):35-47.

139

30. Eckstein F, Lemberger B, Gratzke C, et al. In vivo cartilage deformation after

different types of activity and its dependence on physical training status. Ann

Rheum Dis. Feb 2005;64(2):291-295.

31. Barker MK, Seedhom BB. The relationship of the compressive modulus of

articular cartilage with its deformation response to cyclic loading: does cartilage

optimize its modulus so as to minimize the strains arising in it due to prevalent

loading regime? Rheumatology. 2001;40:274-284.

32. Kosinska MK, Liebisch G, Lochnit G, et al. A Lipidomic Study of Phospholipid

Classes and Species in Human Synovial Fluid. Arthritis Rheum. 2013;65(9):2323-

2333.

33. Barat B. Boundary Lubrication and Lubricants. In: Barat B, ed. Tribology in

Practice Series: Principles and Applications to Tribology (2nd Edition): Wiley;

2013:655-716.

34. Schmidt TA, Gastelum NS, Nguyen QT, Schumacher BL, Sah RL. Boundary

lubrication of articular cartilage: role of synovial fluid constituents. Arthritis

Rheum. Mar 2007;56(3):882-891.

35. Kwiecinski JJ, Dorosz SG, Ludwig TE, Abubacker S, Cowman MK, Schmidt

TA. The effect of molecular weight on hyaluronan’s cartilage boundary

lubricating ability - alone and in combination with proteoglycan 4. Osteoarthritis

Cartilage. 2011;19(11):1356-1362.

36. Das S, Banquy X, Zappone B, Greene GW, Jay GD, Israelachvili J. Synergistic

Interactions between Grafted Hyaluronic Acid and Lubricin Provide Enhanced

Wear Protection and Lubrication. Biomacromolecules. 2013;14:1669-1677.

140

37. Jay GD, Torres JR, Warman ML, Laderer MC, Breuer KS. The role of lubricin in

the mechanical behavior of synovial fluid. Proc Natl Acad Sci U S A. Apr 10

2007;104(15):6194-6199.

38. Hsu SM. Boundary lubrication: current understanding. Tribology Letters.

1997;3:1-11.

39. Yakubov GE, McColl J, Bongaerts JHH, Ramsden JJ. Viscous Boundary

Lubrication of Hydrophobic Surfaces by Mucin. Langmuir. 2009;25:2313-2321.

40. Chang DP, Abu-Lail NI, Guilak F, Jay GD, Zauscher S. Conformational

mechanics, adsorption, and normal force interactions of lubricin and hyaluronic

acid on model surfaces. Langmuir. Feb 19 2008;24(4):1183-1193.

41. Chang DP, Guilak F, Jay GD, Zauscher S. Interaction of lubricin with type II

collagen surfaces: Adsorption, friction, and normal forces. J Biomech.

2013;47(3):659-666.

42. Lakin B, Wathier M, Grinstaff M, Snyder B. Novel Synthetic Biolubricant

Reduces Friction in Previously-Worn Cartilage Evaluated by Long-Duration

Torsional Friction Test (Abstract). Trans Orthop Res. 2014;39:0096.

43. Samaroo KJ, Tan M, Barrett AR, et al. Characterization of binding and lubricating

properties of biomimetic boundary lubricants for articular cartilage (Abstract).

Trans Orthop Res. 2013;38:0392.

44. Samaroo KJ, Tan M, Demange MK, et al. Prevention of Cartilage Damage by

Intraarticular Treatment with Lubricin-Mimetics in the Rat Following Anterior

Cruciate Ligament Transection (Abstract). Trans Orthop Res. 2014;39:1210.

141

45. Unterman SA, Gibson M, Lee JH, et al. Hyaluronic Acid-Binding Scaffold for

Articular Cartilage Repair. Tissue Eng Part A. 2012;18(23-24):2497-2506.

46. Samsom ML, Morrison S, Masala N, et al. Characterization of Full-Length

Recombinant Human Proteoglycan 4 as an Ocular Surface Boundary Lubricant.

Exp Eye Res. 2014;Submitted.

47. Swann DA, Silver FH, Slayter HS, Stafford W, Shore E. The molecular structure

and lubricating activity of lubricin isolated from bovine and human synovial

fluids. Biochem J. 1985;225:195-201.

48. Schumacher BL, Hughes CE, Kuettner KE, Caterson B, Aydelotte MB.

Immunodetection and partial cDNA sequence of the proteoglycan, superficial

zone protein, synthesized by cells lining synovial joints. J Orthop Res.

1999;17:110-120.

49. Swann DA, Slayter HS, Silver FH. The molecular structure of lubricating

glycoprotein-I, the boundary lubricant for articular cartilage. J Biol Chem.

1981;256:5921-5925.

50. Swann DA, Radin EL. The molecular basis of articular lubrication. J Biol Chem.

1972;247:8069-8073.

51. Flannery CR, Hughes CE, Schumacher BL, et al. Articular cartilage superficial

zone protein (SZP) is homologous to megakaryocyte stimulating factor precursor

and is a multifunctional proteoglycan with potential growth-promoting,

cytoprotective, and lubricating properties in cartilage metabolism. Biochem

Biophys Res Commun. 1999;254(3):535-541.

142

52. Schumacher BL, Schmidt TA, Voegtline MS, Chen AC, Sah RL. Proteoglycan 4

(PRG4) synthesis and immunolocalization in bovine meniscus. J Orthop Res.

2005;23:562-568.

53. Ikegawa S, Sano M, Koshizuka Y, Nakamura Y. Isolation, characterization and

mapping of the mouse and human PRG4 (proteoglycan 4) genes. Cytogenet Cell

Genet. 2000;90:291-297.

54. Steele BLS, Alvarez-Veronesi C, Schmidt TA. Molecular weight characterization

of PRG4 proteins using multi-angle laser light scattering (MALLS).

Osteoarthritis Cartilage. 2013;21:498-504.

55. Jay GD, Tantravahi U, Britt DE, Barrach HJ, Cha CJ. Homology of lubricin and

superficial zone protein (SZP): products of megakaryocyte stimulating factor

(MSF) gene expression by human synovial fibroblasts and articular chondrocytes

localized to chromosome 1q25. J Orthop Res. 2001;19:677-687.

56. Lord MS, Estrella RP, Chuang CY, et al. Not All Lubricin Isoforms Are

Substituted with a Glycosaminoglycan Chain. Connect Tissue Res. 2011.

57. Rhee DK, Marcelino J, Baker M, et al. The secreted glycoprotein lubricin protects

cartilage surfaces and inhibits synovial cell overgrowth. J Clin Invest.

2005;115(3):622-631.

58. Ruan MZC, Erez A, Guse K, et al. Proteoglycan 4 Expression Protects Against

the Development of Osteoarthritis. Science Transl Med. 2013;5(176):34.

59. Schmidt TA, Plaas AH, Sandy JD. Disulfide-bonded multimers of proteoglycan 4

(PRG4) are present in normal synovial fluids. Biochim Biophys Acta. May

2009;1790(5):375-384.

143

60. Swann DA, Sotman S, Dixon M, Brooks C. The isolation and partial

characterization of the major glycoprotein (LGP-I) from the articular lubricating

fraction of synovial fluid. Biochem J. 1977;161:473-485.

61. Garg HG, Swann D. The structure of the O-glycosylically-linked oligosaccharide

chains of LPG-I, a glycoprotein presnt in articular lubricating fration of bovine

synovial fluid. Carbohydrate Res. 1979;78:79-88.

62. Jay GD, Harris DA, Cha C-J. Boundary lubrication by lubricin is mediated by O-

linked ß(1-3)Gal-GalNAc oligosaccharides. Glycoconj J. 2001;18:807-815.

63. Jay GD, Lane BP, Sokoloff L. Characterization of a bovine synovial fluid

lubricating factor III. The interaction with hyaluronic acid. Connect Tissue Res.

1992;28:245-255.

64. Benz M, Chen N, Israelachvili J. Lubrication and wear properties of grafted

polyelectrolytes, hyaluronan and hylan, measured in the surface forces apparatus.

J Biomed Mater Res A. Oct 1 2004;71A(1):6-15.

65. Estrella RP, Whitelock JM, Packer NH, Karlsson NG. The glycosylation of

human synovial lubrcin: implications for its role in inflammation. Biochem J.

2010;429:359-367.

66. Ali L, Jin C, Karlsson NG. Glycoproteomics of Lubricin-Implication of Important

Biological Glyco- and Peptide-Epitopes in Synovial Fluid. In: Lemmey AB, ed.

Rheumatoid Arthritis - Etiology, Consequences and Co-Morbidities2012:131-152.

67. Coles JM, Chang DP, Zauscher S. Molecular mechanisms of aqueous boundary

lubrication by mucinous glycoproteins. Curr Opin Colloid Interface Sci.

2010;15(6):406-416.

144

68. Elsaid KA, Jay GD, Warman ML, Rhee DK, Chichester CO. Association of

articular cartilage degradation and loss of boundary-lubricating ability of synovial

fluid following injury and inflammatory arthritis. Arthritis Rheum. Jun

2005;52(6):1746-1755.

69. Lotz M. Osteoarthritis year 2011 in review: biology. Osteoarthritis Cartilage.

2012;20(3):192-196.

70. Nugent GE, Schmidt TA, Schumacher BL, et al. Static and dynamic compression

regulate cartilage metabolism of proteoglycan 4 (PRG4). Biorheology.

2006;43:191-200.

71. Nugent GE, Aneloski NA, Schmidt TA, Schumacher BL, Voegtline MS, Sah RL.

Dynamic shear stimulation of bovine cartilage biosynthesis of proteoglycan 4.

Arthritis Rheum. 2006;54:1888-1896.

72. Nugent-Derfus GE, Takara T, O'Neill JK, et al. Continuous passive motion

applied to whole joints stimulates chondrocyte biosynthesis of PRG4.

Osteoarthritis Cartilage. 2007;25:566-573.

73. Abusara Z, Krawetz R, Steele BLS, Schmidt TA, Herzog W. Muscular loading of

joints triggers cellular secretion of PRG4 into the joint fluid. J Biomech.

2013;46(7):1255-1230.

74. Ni G, Lei L, Zhou Y. Intensity-dependent effect of treadmill running on lubricin

metabolism of rat articular cartilage. Arthritis Res Ther. 2012;14(6):R256.

75. Jay GD, Fleming BC, Watkins BA, et al. Prevention of Cartilage Degeneration

and Restoration of Chondroprotection by Lubricin Tribosupplementation in the

145

Rat Following Anterior Cruciate Ligament Transection. Arthritis Rheum.

2010;62(8):2382-2391.

76. Elsaid KA, Zhang L, Waller K, et al. The impact of forced joint exercise on

lubricin biosynthesis from articular cartilage following ACL transection and intra-

articular lubricin’s effect in exercised joints following ACL transection.

Osteoarthritis Cartilage. 2012;20(8):940-948.

77. Schmidt TA, Gastelum NS, Han EH, Nugent-Derfus GE, Schumacher BL, Sah

RL. Differential regulation of proteoglycan 4 metabolism in cartilage by IL-

1alpha, IGF-I, and TGF-beta1. Osteoarthritis Cartilage. Jan 2008;16(1):90-97.

78. Neu CP, Khalafi A, Komvopoulos K, Schmid TM, Reddi AH.

Mechanotransduction of Bovine Articular Cartilage Superficial Zone Protein by

Transforming Growth Factorβ Signaling. Arthritis Rheum. 2007;56(11):3706-

3714.

79. Kupcsik L, Stoddart MJ, Li Z, Benneker LM, Alini M. Improving

Chondrogenesis: Potential and Limitations of SOX9 Gene Transfer and

Mechanical Stimulation for Cartilage Tissue Engineering. Tissue Eng Part A.

2010;16(6):1845-1855.

80. Cowman MK, Spagnoli C, Kudasheva D, et al. Extended, Relaxed, and

Condensed Conformations of Hyaluronan Observed by Atomic Force

Microscopy. Biophys J. 2005;88:590-602.

81. Dunn S, Kolomytkin OV, Marino AA. Pathophysiology of Osteoarthritis:

Evidence against the Viscoelastic Theory. Pathobiology. 2009;76:322-328.

146

82. Miyaguchi M, Kobayashi A, Kadoya Y, Ohashi H, Yamano Y, Takaoka K.

Biochemical change in joint fluid after isometric quadriceps exercise for patients

with osteoarthritis of the knee. Osteoarthritis Cartilage. 2003;22:252-259.

83. Kim YJ, Grodzinsky AJ, Plaas AHK. Compression of cartilage results in

differential effects on biosynthetic pathways for aggrecan, link protein, and

hyaluronan. Arch Biochem Biophys. 1996;328:331-340.

84. Recklies AD, White C, Melching L, Roughley PJ. Differential regulation and

expression of hyaluronan synthases in human articular chondrocytes, synovial

cells and osteosarcoma cells. Biochem J. 2001;354:17-24.

85. Asari A, Miyauchi S, Kuriyama S, Machida A, Kohno K, Uchiyama Y.

Localization of hyaluronic acid in human articular cartilage. J Histochem

Cytochem. Apr 1994;42(4):513-522.

86. Sigma-Aldrich. Glycobiology Analysis Manual - Glycosaminoglycans and

Proteoglycans. 2nd Edition: http://www.sigmaaldrich.com/technical-

documents/articles/biology/glycobiology/glycosaminoglycans-and-

proteoglycans.html. Accessed 03/19/2014, 2014.

87. Antonacci JM, Schmidt TA, Serventi LA, et al. Effects of Equine Joint Injury on

Boundary Lubrication of Articular Cartilage by Synovial Fluid: Role of

Hyaluronan. Arthritis Rheum. 2012;64(9):2917-2926.

88. Waddell DD. Viscosupplementation with Hyaluronans for Osteoarthritis of the

Knee Clinical Efficacy and Economic Implications. Drugs and Aging.

2007;24(8):629-642.

147

89. Balazs EA, Band PA. Therapeutic Use of Hyaluronan-Based Products. In: Garg

HG, Cowman MK, Hales C, eds. Carbohydrate Chemistry, Biology, and Medical

Applications: Elsevier 2008:311-349.

90. Moreland LW. Intra-articular hyaluronan (hyaluronic acid) and hylans for the

treatment of osteoarthritis: mechanisms of action. Arthritis Res Ther.

2003;5(2):54-67.

91. Masuko K, Murata M, Yudoh K, Kato T, Nakamura H. Anti-inflammatory effects

of hyaluronan in arthritis-therapy: Not just for viscosity. International J Gen Med.

2009;2:77-81.

92. Conrozier T, Chevalier X. Long-term experience with hylan GF-20 in the

treatment of knee osteoarthritis. Expert Opin Pharmacother. 2008;9(10):1797-

1804.

93. Balazs EA, Denlinger JL. Viscosupplementation: a new concept in the treatment

of OA. J Rheumatol. 1993;20 S39:3-9.

94. Bagga H, Burkhardt D, Sambrook P, March L. Long term effects of intraarticular

hyaluronan on synovial fluid in osteoarthritis of the knee. J Rheumatol.

2006;33:946-950.

95. Wang CT, Lin YT, Chiang BL, Lin YH, Hou SM. High molecular weight

hyaluronic acid down-regulates the gene expression of osteoarthritis-associated

cytokines and enzymes in fibroblast-like synoviocytes from patients with early

osteoarthritis. Osteoarthritis Cartilage. 2006;14:1237-1247.

148

96. Smith MM, Ghosh P. The synthesis of hyaluronic acid by human synovial

fibroblasts is influenced by the nature of the hyaluronate in the extracellular

environment. Rheumatol Int. 1987;7(3):113-122.

97. Hochberg MC, Altman RD, April KT, et al. American College of Rheumatology

2012 Recommendations for the Use of Nonpharmacologic and Pharmacologic

Therapies in Osteoarthritis of the Hand, Hip, and Knee. Arthritis Care Res.

2012;64(4):465-474.

98. Balazs EA. Therapeutic uses of hyaluronan. Struct Chem. 2009;20:341-349.

99. Frobell RB, Le Graverand M-P, Buck R, et al. The acutely ACL injured knee

assessed by MRI: changes in joint fluid, bone marrow lesions, and cartilage

during the first year. Osteoarthritis Cartilage. 2009;17(2):161-167.

100. Offiah AC, Woo P, Prieur A-M, Hasson N, Hall CM. Camptodactyly-

Arthropathy-Coxa Vara-Pericarditis Syndrome Versus Juvenile Idiopathic

Arthropathy. Am J Roentgenol. 2005;185:522-529.

101. Hui AH, McCarty WJ, Masuda K, Firestein GS, Sah R. A systems biology

approach to synovial joint lubrication in health, injury, and disease. WIREs Syst

Biol Med. 2012;4:15-37.

102. Mazzucco D, Scott R, Spector M. Composition of joint fluid in patients

undergoing total knee replacement and revision arthroplasty: correlation with

flow properties. Biomaterials. Aug 2004;25(18):4433-4445.

103. Ballard BL, Antonacci JM, Temple-Wong MM, et al. Effect of Tibial Plateau

Fracture on Lubrication Function and Composition of Synovial Fluid. J Bone

Joint Surg Am. 2012;94(e64):1-9.

149

104. Neu CP, Reddi AH, Komvopoulos K, Schmid TM, Di Cesare PE. Increased

Friction Coefficient and Superficial Zone Protein Expression in Patients with

Advanced Osteoarthritis. Arthritis Rheum. 2010;62(9):2680-2687.

105. Hansen BC, Temple-Wong MM, Antonacci JM, et al. Internal Derangement of

the Knee is Associated with Impaired Synovial Fluid Lubricant Function and

Composition (Abstract). Trans Orthop Res. 2010;35:1986.

106. Temple-Wong MM, Hansen BC, Grissom MJ, et al. Effect of Knee Osteoarthritis

on the Boundary Lubricating Molecules and Function of Human Synovial Fluid

(Abstract). Trans Orthop Res. 2010;56:340.

107. Schmid T, Lindley K, Su J, et al. Superficial zone protein (SZP) is an abundant

glycoprotein in human synovial fluid and serum (Abstract). Trans Orthop Res

Soc. 2001;26:82.

108. Rhee DK, Marcelino J, Al-Mayouf S, et al. Consequences of disease-causing

mutations on lubricin protein synthesis, secretion, and post-translational

processing. J Biol Chem. 2005;280(35):31325-31332.

109. Ungethuem U, Haeupl T, Witt H, et al. Molecular Signatures and new candidates

to target the pathogenesis of rheumatoid arthritis. Physiol Genomics.

2010;42A(4):267-282.

110. Jay GD, Elsaid KA, Zack J, et al. Lubricating ability of aspirated synovial fluid

from emergency department patients with knee joint synovitis. J Rheumatol.

2004;31:557-564.

150

111. Dahl LB, Dahl IM, Engstrom-Laurent A, Granath K. Concentration and molecular

weight of sodium hyaluronate in synovial fluid from patients with rheumatoid

arthritis and other arthropathies. Ann Rheum Dis. 1985;44(12):817-822.

112. Lee HG, Cowman MK. An agarose gel electrophoretic method for analysis of

hyaluronan molecular weight distribution. Anal Biochem. Jun 1994;219(2):278-

287.

113. Laurent TC, Laurent UB, Fraser JRE. Structure and function of hyaluronan: an

overview. Immunol Cell Biol. 1996;74:A1-7.

114. Ghosh P, Guidolin D. Potential mechanism of action of intra-articular hyaluronan

therapy in osteoarthritis: are the effects molecular weight dependent? Semin

Arthritis Rheum. Aug 2002;32(1):10-37.

115. Fam H, Bryant JT, Kontopoulou M. Rheological properties of synovial fluids.

Biorheology. 2007;44(2):59-74.

116. Jay GD, Britt DE, Cha D-J. Lubricin is a product of megakaryocyte stimulating

factor gene expression by human synovial fibroblasts. J Rheumatol. 2000;27:594-

600.

117. Wei L, Fleming BC, Sun X, et al. Comparison of Differential Biomarkers of

Osteoarthritis with and without Posttraumatic Injury in the Hartley Guinea Pig

Model. J Orthop Res. 2010;28(7):900-906.

118. Teeple E, Elsaid KA, Fleming BC, et al. Coefficients of friction, lubricin, and

cartilage damage in the anterior cruciate ligament-deficient guinea pig knee. J

Orthop Res. Feb 2008;26(2):231-237.

151

119. Young AA, McLennan S, Smith MM, et al. Proteoglycan 4 downregulation in a

sheep meniscectomy model of early osteoarthritis. Arthritis Res Ther. Jan 31

2006;8(2):R41.

120. Marcelino J, Carpten JD, Suwairi WM, et al. CACP, encoding a secreted

proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis

syndrome. Nat Genet. 1999;23(3):319-322.

121. Asari A, Miyauchi S, Sekiguchi T, et al. Hyaluronan, cartilage destruction and

hydrarthrosis in traumatic arthritis. Osteoarthritis Cartilage. Jun 1994;2(2):79-89.

122. Watterson JR, Esdaile JM. Viscosupplementation: therapeutic mechanisms and

clinical potential in osteoarthritis of the knee. J Am Acad Orthop Surg. Sep-Oct

2000;8(5):277-284.

123. Flannery CR, Zollner R, Corcoran C, et al. Prevention of cartilage degeneration in

a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin.

Arthritis Rheum. Mar 2009;60(3):840-847.

124. Jay GD, Elsaid KA, Kelley KA, et al. Prevention of Cartilage Degeneration and

Gait Asymmetry by Lubricin Tribosupplementation in the Rat Following Anterior

Cruciate Ligament Transection. Arthritis Rheum. 2012;64(4):1162-1171.

125. Estrella RP WJ, Packer NH et al. The glycosylation of human synovial lubricin:

implications for its role in inflammation. Biochem J. 2010;429:359-367.

126. McDonald J. Handbook of Biologial Statistics (2nd ed). Baltimore, Maryland:

Sparky House Publishing; 2009.

152

127. Su J-L, Schumacher BL, Lindley KM, et al. Detection of superficial zone protein

in human and animal body fluids by cross-species monoclonal antibodies specific

to superficial zone protein. Hybridoma. 2001;20(3):149-157.

128. Ballard BL, Antonacci JM, Temple-Wong MM, et al. Tibial plateau and tibial

pilon fractures disrupt the lubrication function and composition of synovial fluid

(Abstract). Trans Orthop Res Soc. 2009;34:1539.

129. McCutchen CW. Boundary lubrication by synovial fluid: demonstration and

possible osmotic explanation. Fed Proceedings. 1966;25:1061-1068.

130. Brandt KD, Smith GNJ, Simon LS. Intraarticular injection of hyaluronan as

treatment for knee osteoarthritis: what is the evidence. Arthritis Rheum.

2000;43(6):1192-1203.

131. Owen DS, Jr. Diagnostic Tests and Procedures: Aspiration and Injection of Joints

and Soft Tissues. In: Ruddy S, Harris ED, Sledge CB, eds. Kelley's Textbook of

Rheumatology. Vol 1. Sixth Edition ed. Philadelphia: WB Saunders; 2001:583-

603.

132. Chevalier X, Jerosch J, Goupille P, et al. Single, intra-articular treatment with 6

ml hylan G-F 20 in patients with symptomatic primary osteoarthritis of the knee:

a randomised, multicentre, double-blind, placebo controlled trial. Ann Rheum Dis.

2010;69(113-119).

133. Goldberg V, Coutts R. Pseudoseptic Reactions to Hylan Viscosupplementation:

Diagnosis and Treatment. Clin Orthop Rel Res. 2004;419:130-137.

153

134. Rutjes AWS, Juni P, da Costa BR, Trelle S, Nuesch E, Reichenbach S.

Viscosupplementation for Osteoarthritis of the Knee: A Systematic Review and

Meta-analysis. Ann Intern Med. 2012;157:180-191.

135. Marino AA, Waddell DD, Kolomytkin OV, Pruett S, Sadasivan KK, Albright JA.

Assessment of Immunologic Mechanisms for Flare Reactions to Synvisc(R). Clin

Orthop Rel Res. 2006;442:187-194.

136. Conrozier T, Mathieu P, Vignon E, Piperno M, Rinaudo M. Differences in the

osteoarthritic synovial fluid composition and rheology between patients with or

without flare-up: a pilot study. Clin Exp Rheumatol. 2012;30:729-734.

137. Barton KI, Ludwig TE, Achari Y, Shrive NG, Frank CB, Schmidt TA.

Characterization of proteoglycan 4 and hyaluronan composition and lubrication

function of ovine synovial fluid following knee surgery. J Orthop Res.

2013;31(10):1549-1554.

138. Blewis ME, Nugent-Derfus GE, Schmidt TA, Schumacher BL, Sah RL. A Model

of Synovial Fluid Lubricant Composition in Normal and Injured Joints. Eur Cell

Mater. 2007;13:26-39.

139. Zappone B, Ruths M, Greene GW, Jay GD, Israelachvili JN. Adsorption,

lubrication, and wear of lubricin on model surfaces: polymer brush-like behavior

of a glycoprotein. Biophys J. Mar 1 2007;92(5):1693-1708.

140. Abubacker S, Alvarez MC, Messersmith PB, Schmidt TA. Cartilage Boundary

Lubricating Ability of PRG4 Monomers versus Multimers (Abstract). Trans

Orthop Res. 2013;38:0394.

154

141. Waller K, Zhang L, Fleming BC, Jay GD. Preventing Friction-induced

Chondrocyte Apoptosis: Comparison of Human Synovial Fluid and Hylan G-F

20. J Rheumatol. 2012;39(7):1473-1480.

142. Ateshian GA. The role of interstitial fluid pressurization in articular cartilage

lubrication. J Biomech. 2009;42:1163-1176.

143. Ludwig TE, McAllister JR, Lun V, Wiley JP, Schmidt TA. Effect of Flare

Reaction to Intra-Articular Injection on Cartilage Lubricating Ability of Human

Synovial Fluid (Abstract). Clin J Sport Med. 2013;23(2):e20.

144. Milas M, Rinaudo M, Roure I, Al-Assaf S, Phillips GO, Williams PA.

Comparative Rheological Behavior of Hyaluronan from Bacterial and Animal

Sources with Cross-Linked Hyaluronan (Hylan) in Aqueous Solution.

Biopolymers. 2001;59:191-204.

145. Schmidt TA, Schumacher BL, Gastelum NS, Plaas AHK, Sah RL. PRG4

boundary lubrication of articular cartilage: role of disulfide bonding and

chondroitin sulfate (Abstract). Trans Orthop Res Soc. 2006;52:84.

146. Abubacker S, Alvarez-Veronesi C, Messersmith PB, Schmidt TA. Cartilage

Boundary Lubricating Ability of PRG4 Monomers versus Multimers: Effect of

Inter- and Intra-Molecular Bonds (Abstract). Trans CCTC. 2013:P42.

147. Swann DA, Hendren RB, Radin EL, Sotman SL, Duda EA. The lubricating

activity of synovial fluid glycoproteins. Arthritis Rheum. 1981;24:22-30.

148. Waller K, Zhang L, Elsaid KA, Fleming BC, Warman M, Jay GD. Role of

lubricin and boundary lubrication in the prevention of chondrocyte apoptosis.

Proc Natl Acade Sci USA. 2013;110(15):5852-5857.

155

149. Kappler J, Kaminski TP, Gieselmann V, Kubitscheck U, Jerosch J. Single-

molecule imaging of hyaluronan in human synovial fluid. J Biomed Opt.

2010;15(6).

150. Ludwig TE, Cowman MK, Schmidt TA. Effects of Concentration and Structure

on PRG4 Viscosity and Interaction with Hyaluronan (Abstract). Trans Orthop

Res. 2014;39:0372.

151. Seller PC, Dowson D, Wright V. The rheology of synovial fluid. Rheol Acta.

1971;10:2-7.

152. Fam H, Bryant JT, Kontopoulou M. Rheological properties of synovial fluids.

Biorheology. 2009;44:59-74.

153. Fouissac E, Milas M, Rinaudo M. Shear-Rate, Concentration, Molecular Weight

and Temperature Viscosity Dependences of Hyaluronate, a Wormlike

Polyelectrolyte. Macromolecules. 1993;26:6945-6951.

154. Morris ER, Rees DA, Welch EJ. Conformation and Dynamic Interactions in

Hyaluronate Solutions. J Mol Biol. 1980;138:383-400.

155. Cowman MK, Matsuoka S. Experimental approaches to hyaluronan structure.

Carbohydr Res. Apr 11 2005;340(5):791-809.

156. Cowman MK, Hernandez M, Kim JR, Yuan H, Hu Y. Macromolecular Crowding

in the Biomatrix. In: Balazs EA, ed. Structure and Function of Biomatrix: Control

of Cell Behaviour and Gene Expression. Edgewater, NJ: Matrix Biology Institute;

2012:45-66.

156

157. Mathieu P, Conrozier T, Vignon E, Rozand Y, Rinaudo M. Rheologic Behavior

of Osteoarthritic Synovial Fluid after Addition of Hyaluronic Acid. Clin Orthop

Rel Res. 2009;467:3002-3009.

158. Nugent-Derfus GE, Chan AH, Schumacher BL, Sah RL. PRG4 exchange between

the articular cartilage surface and synovial fluid. J Orthop Res. 2007;25(10):1269-

1276.

159. Park M-S, J-W C, Kim Y-K, Chung S-C, Kho H-S. Viscosity and wettability of

animal mucin solutions and human saliva. Oral Diseases. 2007;3:81-86.

160. Lai SK, Wang Y-Y, Wirtz D, Hanes J. Micro- and macrorheology of mucus. Adv

Drug Deliv Rev. 2009;61:86-100.

161. Bell AE, Sellers LA, Allen A, Cunliffe WJ, Morris ER, Ross-Murphy SB.

Properties of Gastric and Duodenal Mucus: Effect of Proteolysis, Disulfide

Reduction, Bile, Acid, Ethanol, and Hypertonicity on Mucus Gel Structure.

Gastroenterology. 1985;88:269-280.

162. McCullagh CM, Gupta R, Jamieson AM, Blackwell J. Gelation of fractionated

canine submaxillary mucin in a chaotropic solvent. Int J Biol Macromol.

1996;18:247-253.

163. Dorosz SG, Abubacker S, Masala N, Jay GD, Schmidt TA. Cartilage Boundary

Lubricating Ability of Full-Length Human Recombinant PRG4: Alone and in

Combination with Hyaluronan (Abstract). Trans Orthop Res Soc. 2013;38:1276.

164. Pearson PJ, Allen A, Hutton DA. Rheology of Mucin. In: Corfield A, ed.

Glycoprotein Methods and Protocols - The Mucins. Vol 125. Totawa, NJ:

Humana Press Inc; 2000:99-109.

157

165. Krause WE, Bellomo EG, Colby RH. Rheology of Sodium Hyaluronate under

Physiological Conditions. Biomacromolecules. 2001;2:65-69.

166. Bansil R, Turner BS. Mucin structure, aggregation, physiological functions and

biomedical applications. Curr Opin Colloid Interface Sci. 2006;11:164-170.

167. Jay GD. Characterization of a bovine synovial fluid lubricating factor. I.

Chemical, surface activity and lubricating properties. Connect Tissue Res.

1992;28:71-88.

168. Kooyman J, Alvarez MC, Schmidt TA. Cartilage Boundary Lubricating

Properties of Native Proteoglycan 4 Purified from Normal Bovine Synovial Fluid

(Abstract). Trans Orthop Res. 2010;56:255.

169. Flannery CR, Yang Z, Zeng W, et al. Proteomic Identification of Novel Lubricin-

Binding Ligands at Cartilage Surfaces (Abstract). Trans Orthop Res Soc.

2010;35:164.

170. Cowman MK, Chen CC, Pandya M, et al. Improved agarose gel electrophoresis

method and molecular mass calculation for high molecular mass hyaluronan. Anal

Biochem. 2011;417:50-56.

171. Coleman PJ, Scott D, Mason RM, Levick JR. Characterization of the effect of

high molecular weight hyaluronan on trans-synovial flow in rabbit knees. J

Physiol. 1999;514(1):265-282.

172. Ludwig TE, Hunter MM, Schmidt TA. Effects of Concentration on Synergistic

Hyaluronan-PRG4 Cartilage Boundary Lubrication (Abstract). Trans Orthop Res.

2013;38:1275.

158

173. Teeple E, Elsaid KA, Jay GD, et al. Effects of Supplemental Intra-articular

Lubricin and Hyaluronic Acid on the Progression of Posttraumatic Arthritis in the

Anterior Cruciate Ligament–Deficient Rat Knee. The American Journal of Sports

Medicine. 2011;39(1):164-172.

174. Catterall JB, Stabler TV, Flannery CR, Kraus VB. Changes in serum and synovial

fluid biomarkers after acute injury (NCT00332254). Arthritis Res Ther.

2010(12):R229.

175. Atarod MA, Ludwig TE, Schmidt TA, Frank CB, Shrive NG. Diminished

Cartilage Lubrication Early After ACL Injury and Increased Meniscal Loads

Later After an ACL Injury May Both Contribute To OA Development In an

Ovine Model (abstract). Trans Orthop Res Soc. 2014;39:0086.

176. Elsaid KA, Machan JT, Waller K, Fleming BC, Jay GD. The impact of anterior

cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor

necrosis factor alpha on chondroprotection in an animal model. Arthritis Rheum.

Oct 2009;60(10):2997-3006.

177. Bhuanantanondh P, Grecov D, Kwok E. Rheological Study of Viscosupplements

and Synovial Fluid in Patients with Osteoarthritis. J Med Biol Eng.

2010;21(1):12-16.

178. Houser B. Bio-Rad’s Bio-Plex® suspension array system, xMAP technology

overview. Arch Physiol Biochem. 2012;118(4):192-196.

179. Heard BJ, Fritzler MJ, Wiley PJ, et al. Intraarticular and systemic inflammatory

profiles may identify patients with osteoarthritis. J Rheumatol. 2013 40(8):1379-

1387.

159

180. Nordgard CT, Draget KI. Oligosaccharides As Modulators of Rheology in

Complex Mucous Systems. Biomacromolecules. 2011;12:3084-3090.

181. Instruments M. An Introduction to DLS Microrheology. Version 7:

http://www.malvern.com/en/support/resource-

center/Whitepapers/WP120917IntroDLSMicro.aspx, 2013.

182. Miri T. Viscosity and Oscillatory Rheology. In: Norton IT, Spyropoulos F, Cox P,

eds. Practical Food Rheology: An Interpretive Approach: Wiley-Blackwell;

2011:7-28.

183. Bloom AK, Samsom ML, Steele BL, Schmidt TA. Investigating the Effect of

Proteoglycan 4 on Hyaluronan Solution Properties using Confocal Fluorescence

Recovery after PhotoBleaching (Abstract). Trans Orthop Res Soc. 2013;39:1240.

184. Perozzo R, Folkers G, Scapozza L. Thermodynamics of Protein–Ligand

Interactions: History, Presence, and Future Aspects. J Recept Signal Transduct

Res. 2004;24(1):1-52.

185. Yamniuk AP, Burling H, Vogel HJ. Thermodynamic characterization of the

interactions between the immunoregulatory proteins osteopontin and lactoferrin.

Mol Immunol. 2009;46:2395-2402.

186. Ball V, Maechling C. Isothermal Microcalorimetry to Investigate Non Specific

Interactions in Biophysical Chemistry. Int J Mol Sci. 2009;10:3283-3315.

187. Velázquez-Campoy A, Ohtaka H, Nezami A, Salman Muzammil S, Freire E.

Isothermal Titration Calorimetry. Curr Protoc Cell Biol. 2004;Supplement

23:17.18.11-17.18.24.

160

188. Tolg C, Hamilton SR, Zalinska E, et al. A RHAMM Mimetic Peptide Blocks

Hyaluronan Signaling and Reduces Inflammation and Fibrogenesis in Excisional

Skin Wounds. Am J Pathol. 2012;181(4):1250-1270.

189. Edmondson EG, Dent SYR. Identification of Protein Interactions by Far Western

Analysis. In: Coligan JE, Dunn BM, Speicher DW, Wingfield PT, eds. Current

Protocols in Protein Science: John Wiley & Sons, Inc.; 2001.

190. Hall RA. Studying Protein-Protein Interactions via Blot Overlay or Far Western

Blot. In: Fu H, ed. Protein-Protein Interactions Methods and Applications. Vol

261. Totowa, New Jersey: Humana Press; 2004:167-174.

191. Yuan H, Tank M, Alsofyani A, et al. Molecular mass dependence of hyaluronan

detection by sandwich ELISA-like assay and membrane blotting using

biotinylated hyaluronan binding protein. Glycobiology. 2013;23(11):1270-1280.

192. Sankar C. Binding Interactions Between Hyaluronan and Lubricin: Potential Role

in Osteoarthritis [Bachelors]: Chemical and Biomeolecular Engineering,

Polytechnic Institute of New York University; 2011.

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Appendix A: Probing the PRG4+HA Interaction: Isothermal Titration Calorimetry

A.1 Introduction

While indirect biophysical evidence of PRG4+HA interaction has been observed

(as discussed in Chapter 5), direct biophysical evidence is lacking and the mechanism of

interaction remains to be determined. Isothermal titration calorimetry (ITC) is a

biophysical technique that measures the heat generated or absorbed when molecules bind

together, and can provide information regarding the stoichiometry and dissociation

constants of the interaction184

. ITC allows binding characterization of species without

attachment of probes, which could interfere with the interaction of interest185

. ITC can

also provide information about non-specific interactions186

.

An ITC apparatus is a heat-flux calorimeter that measures the amount of power

input required to maintain a constant temperature difference between a reference cell and

a sample cell; the sample cell contains the first species of interest and the other species is

titrated into the solution using an injection syringe. Formation of a complex between the

2 species is accompanied by either a release or absorption of heat, causing a temperature

difference from the reference cell. Once the temperature balance is restored the area

under the peak of the heat flux versus molar ratio graph provides the amount of heat

associated with each injection187

. ITC has previously been used to evaluate the binding of

an HA receptor mimicking peptide to HA oligosaccharides and 240 and 500 kDa HA188

.

The objective of this preliminary study was to use ITC to further characterize the

PRG4+HA interaction.

162

A.2 Materials and Methods

In order to optimize identification and interpretation of an interaction, the most pure

PRG4 and HA preparations available were used. The same phosphate buffered saline

(PBS) buffer was used for all sample preparation and experiments, to ensure that

equilibration of buffers did not contribute to signal.

PRG4 was purified from media conditioned by bovine explants as described

previously34

, followed by further purification with size exclusion chromatography (SEC,

Superose 6, 10/300 GL – GE Healthcare) in PBS buffer. Fractions identified as PRG4 on

SDS-PAGE were pooled and concentrated in a 30 kDa molecular weight cut-off filter

unit, purity was confirmed on SDS-PAGE, and concentration was measured using the

BCA assay. For ITC experiments, 3 SEC runs were pooled and concentrated, resulting in

PRG4* in PBS at 1050 µg/mL (Figure A-1). Based on work performed to characterize

the MW of PRG454

and densitometry on the sample prepared for ITC, it was estimated

that approximately 50% of the PRG4* preparation had a MW of 1 MDa (Figure A-1, top

arrow), and 50% of 460 kDa (Figure A-1, bottom arrow). An average molecular weight

of 730 kDa was used to calculate the approximate molarity of the PRG4* solution

(1.4×10-6

M) such that the appropriate HA concentration could be prepared in order to

perform 8 – 10 injections before a 1:1 molar ratio was reached, and 8 – 10 injections after

the 1:1 ratio. Based on these calculations, a monodisperse Select HA of 150 kDa size

(Lifecore Biomedical) was prepared in PBS at 2.94 mg/mL (1.96×10-5

M). ITC

experiments were performed on a Microcal VP ITC.

163

Figure A-1: SDS-PAGE of non-reduced PRG4* used for ITC

experiments (right lane) showing ~1 MDa (top arrow) and 460 kDa

species (bottom arrow), and reduced PRG4* (Red PRG4* - left lane)

showing lower MW species for comparison.

All ITC injections were performed at 37°C. A baseline injection of de-gassed PBS

into de-gassed PBS was performed to ensure there were no temperature differences

arising from buffer equilibration. HA was then injected into PBS buffer, again to ensure

no temperature changes were observed. Finally, HA was injected into PRG4; HA was

diluted to 1.94 mg/mL, in order to have sufficient loading volume, and the number of

injections was increased to 30.

164

A.3 Results

Injection of PBS into PBS showed negligible heat flux with each injection (not

shown), indicating that the buffers were well matched. Injection of HA into PBS (Figure

A-2A) showed one initial peak, but otherwise showed relatively uniform heat flux

throughout injection. A similar initial peak was seen when HA was injected into PRG4

(Figure A-2B), followed again by uniform peaks at higher molar ratios.

Figure A-2: Power required to maintain temperature in sample cell (top

panel) and integrated heat plot (bottom panel) for (A) HA injected into

PBS and (B) HA injected into PRG4. Note the values on the y-axes are

very small.

165

A.4 Discussion

No evidence for a PRG4+HA interaction was observed with the ITC method, and

thus it failed to provide additional information on the nature of the PRG4+HA

interaction. The scale of any changes observed here are well below the recommended

signal to accurately determine any change of heat involved in the interaction; the

sensitivity of this instrument is 0.1 µcal, so a minimum of 1 µcal is recommended187

. The

initial HA peak observed upon injection of HA into PBS could have been HA

aggregating in the syringe and breaking up upon injection. The small peaks observed

throughout injections may be due to unspecific phenomena such as effect of dilution of

the reactants or friction of the injected liquid.

Micro-molar concentrations are typically used for ITC experiments, and the

concentrations of PRG4 and HA used here were theoretically within that range (1.4×10-6

M and 1.96×10-5

M respectively). However, previous work has shown that the PRG4+HA

interaction may not be a strong interaction, but perhaps a reversible interaction or

entanglement35

, as the lack of binding seen in this data suggests. In order to observe the

interaction in solution using ITC it may be necessary to use more concentrated PRG4 and

HA (10 – 100 times more concentrated). Using purer species, such as PRG4 multimers or

monomers alone140

(both of which were present in the preparation used here) may help

isolate an interaction. Furthermore, using a higher MW HA, or a concentration of HA

closer to its critical concentration, may allow an interaction to be observed by ITC.

166

A.5 Acknowledgements

Thank you to Dr. Evan Haney from Dr. Hans Vogel’s lab for training on the

Microcal VP ITC, assistance with experiments, and interpretation of results.

167

Appendix B: Probing the PRG4+HA Interaction: Slot Blot Far-Western

B.1 Introduction

As discussed throughout this thesis, direct observation of the PRG4+HA

interaction and information regarding its mechanism is lacking. Furthermore, the

interplay between PRG4+HA interactions in solution and at surfaces is not well

understood and may differ in their mechanisms. In order to function as a boundary

lubricant a molecule must be attached to the articular cartilage surface. However, both

surface and solution interactions are of interest in the context of joint lubrication, as HA

and PRG4 are present both in the SF and at the articular surface. When PRG4 is removed

from the articular surface, PRG4 can be replenished by native SF, indicating an exchange

between the surface and SF under certain conditions, although this is not an equilibrium

between surface and solution PRG4158

. Understanding molecular interactions in solution

and how interactions affect adsorption to the surface will be important in future

development of biotherapeutic lubricant treatments and basic understanding of

lubrication.

A far-western blot (or overlay blot)189,190

captures a “bait” molecule on a

membrane surface, which can then be probed with “prey” molecules to identify

interactions. In this study, this is done after vacuum slot blotting, where samples are

immobilized on a membrane by vacuum filtration, rather than electro-blotted after gel

electrophoresis (as previously described in this thesis). Vacuum blotting has previously

been used to investigate the MW-dependent specific detection of HA191

. The purpose of

168

this study was to further investigate the PRG4+HA interaction at membrane surfaces, and

elucidate the effects of concentration and HA MW on PRG4+HA interaction.

B.2 Methods

B.2.1 Materials

Polyvinylidene fluoride (PVDF) and Hybond-N+ membranes were obtained from

GE Healthcare. HA (132 kDa and 1.5 MDa) were from Lifecore Biomedical, and PRG4

was prepared from media conditioned by bovine cartilage explant culture as described

previously34

. Biotinylated recombinant human HA binding protein (HABP) was from

CosmoBio, and hyaluronidase (HA’se) was from Seikagaku. The secondary detection

species for the biotinylated HABP, streptavidin conjugated horse radish peroxidise

(streptavidin-HRP) was from Sigma Aldrich. Primary anti-PRG4 antibody H140 (against

the C-terminus) was from Santa Cruz Biotechnology Inc, and anti-PRG4 primary

antibody 9G3124

was a generous gift from Dr. Gregory Jay. Secondary antibody for H140

(goat anti rabbit IgG-HRP) was from Fisher Scientific, and secondary for 9G3 (goat anti

mouse IgG-HRP) was from Sigma Aldrich. PBST was prepared by adding 0.05% Tween

20 to phosphate buffered saline (PBS) solution, TAE buffer consisted of 40 mM Tris

Base, 5 mM sodium acetate, 0.09 mM ethylenediaminetetraacetic acid, pH 7.9. TBS

buffer was 20 mM Tris Base, and 137 mM sodium chloride, pH 7.6, and TBST was TBS

with 0.1% Tween 20. Blocking was performed with non-fat milk solutions (BioRad) or

Protein-Free Blocking Buffer (PBS based, Thermo Scientific). Membranes were

169

developed using SuperSignal West Femto (Thermo Scientific) or ECL Prime (GE

Healthcare Life Sciences).

B.2.2 PRG4 Bait on PVDF Membrane

The PVDF membrane was prepared by soaking in 100% methanol for 10 seconds,

followed by PBS for 10 minutes. The membrane was loaded onto the apparatus, tightened

and re-tightened under vacuum pressure. PRG4 was prepared in PBS at 45, 150 and 450

μg/mL, and was digested with HA’se for 3 hours at 37°C (preliminary experiments

suggested that the HABP used to detect HA may also detect PRG4192

). PRG4 samples

(30 μL or PBS for negative controls) were sucked through the membrane in the slot blot

apparatus, followed by vacuum rinse with 200 μL PBS. The membrane was left in the

apparatus while incubated with 30 μL HA at 0.3, 1.0, or 3.3 mg/mL (prepared in PBS) for

2 hours at room temperature with rocking. HA was also sucked through one lane of the

membrane as a positive control. The HA solutions were discarded from the apparatus,

and the membrane was rinsed in TBS twice for 5 minutes and then blocked with 5% milk

in TBST for 1 hour at room temperature with rocking. After 3 rinses of 5 minutes in

PBST the membrane was soaked in HABP as a primary detection antibody overnight at

4°C. After 3 rinses in PBST for 10 minutes, the membrane was soaked in secondary

antibody streptavidin-HRP for 1 hour at room temperature, rinsed 3 times 10 minutes in

PBST, and developed with Femto.

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B.2.3 HA Bait on Hybond-N+ Membrane

The Hybond-N+ membrane was prepared by rehydrating in TAE for 10 minutes,

loading into the slot blot apparatus and tightening, followed by tightening again under

vacuum and rehydration with 100 µL TAE sucked through the membrane. As per

previous successful blotting of HA to Hybond-N+ membranes, HA was prepared in

TAE191

. HA of 132 kDa and 1.5 MDa size was prepared at a range of concentrations

from 0.0003 to 3.3 mg/mL and 200 µL was vacuum blotted (sucked through) to the

membrane alone in order to select a range of concentrations that demonstrated a dose-

dependent binding response. The membrane was then blocked in 10% milk, incubated in

the primary antibody HABP in PBST for 1 hour at room temperature, incubated in the

secondary antibody streptavidin-HRP in PBST for 1 hour at room temperature, and

developed with ECL. Based on this concentration analysis, concentrations of 0.0003,

0.003, and 0.03 mg/mL were chosen for subsequent experiments (Figure B-1).

171

Figure B-1: Determination of concentrations for HA on PVDF slot blot

using HA alone. Detection with biotinylated HABP and streptavidin-HRP.

Concentrations outlined in green box (0.0003, 0.003, 0.03 mg/mL)

selected for subsequent experiments.

For the far-western assay, HA was immobilized to the membrane as above. One

hundred μL of TAE was sucked through the membrane as a rinse, and the membrane was

blocked overnight at room temperature in 10% milk, Pierce Protein-Free Blocker, or

172

without blocking (in TAE). Membranes were then rinsed with PBS 3 times for 10

minutes (PBS for the protein free block), and incubated in PRG4 prepared in PBS at 0 or

150 µg/mL for 2 hours at room temperature. The membranes were rinsed in TBS 3 times

for 10 minutes, and blocked again in the same block as above for 1 hour at room

temperature. Following another 3 x 10 minute TBS wash, membranes were probed with

primary anti-PRG4 Ab 9G3 and secondary antibody goat anti-mouse HRP and developed

with ECL.

B.3 Results

B.3.1 PRG4 Bait on PVDF Membrane

Sucking the HA through the membrane (left-most lane of Figure B-2) increased

HABP signal compared to soaking in HA (next lane to the right), likely because the HA

was also immobilized on the membrane. While not quantified, it appears there may be

dependence of signal on both PRG4 and HA concentration. However, as the HABP

attached to the PRG4 that was immobilized to the membrane when HA was not present

(top 2 rows in the PRG4 45, 150, 450 μg/mL lanes), it is not possible to say that this

dose-dependent signal is from HA interaction with PRG4.

173

Figure B-2: Far-western blot of HA on PRG4-blotted PVDF membrane.

Detection with biotinylated HABP and streptavidin-HRP.

B.3.2 HA Bait on Hybond-N+ Membrane

When blotted with HA and then incubated with PRG4, some specific PRG4 signal

was observed. On membranes incubated in PRG4 that were not blocked, there was non-

specific background signal throughout the membrane (right-most lanes of Figure B-3A)

detected by the primary antibody H140. When blocked with milk, practically all signal

was abolished (left-most lanes of Figure B-3A), and the protein free block appeared to

174

diminish non-specific signal. The membrane shown in Figure B-3A was re-probed with

primary antibody 9G3, which is more sensitive to PRG4. In this situation all membranes

incubated in PRG4 showed a higher background signal (Figure B-3B); it is unknown if

this is 9G3 non-specific signal due to the protein binding capacity of the membrane, or

more sensitive detection of PRG4 on the entire membrane. The milk block again reduced

all signal, and the protein free block reduced non-specific signal.

175

Figure B-3: Far-western blot of PRG4 onto HA-blotted Hybond-N+

membrane. (A) Detection with PRG4 antibody H140. (B) Detection with

PRG4 antibody 9G3, reprobe.

176

B.4 Discussion

Despite the fact that the HABP sticks to PRG4, the PVDF assay provided

encouraging suggestion that the PRG4+HA interaction is preserved at membrane

surfaces. HABP identification of PRG4 was not abolished with HA’se digestion of the

PRG4, suggesting this is not due to the small amount of HA present in the PRG4

preparation. There is some preliminary evidence (not shown), that HABP does not stick

to recombinant human PRG4; this may be useful in further investigating the PRG4+HA

interaction. If successful in finding an HA detection species that does not stick to PRG4,

it will be important to optimize the amounts of captured PRG4 so that a dose response

can be observed, and to use a higher loading volume to ensure more even signal in each

slot.

As expected due to the high protein binding capacity of the Hybond-N+

membrane (as stated in the product manual), PRG4 is immobilized to it when sucked

through (not shown), even under the small amount of gravity filtration occurring during

incubation periods in the apparatus; for this reason the membranes were removed for

incubation with PRG4. The binding capacity also makes blocking important but difficult,

and previous work has shown that bovine serum albumin, casein, and Tween do not work

well as blocking reagents with Hybond-N+191

. Finally the binding capacity made it

difficult to reduce background signal and identify specific vs. non-specific signal; these

membranes are not recommended for western blotting and may not be ideal for this far-

western slot blot assay.

In future work, it would be ideal to immobilize PRG4 on the PVDF membrane,

incubate in HA, and detect with a molecule that identifies HA but not PRG4. Once the

177

immobilization of PRG4 or HA to a membrane is finalized, HA and PRG4 can also be

combined in solution as it has been shown that the presence of PRG4 in solution can alter

HA conformation and affect friction and wear properties at model surfaces36

. It will be

important to optimize the buffers used in this assay as, for example, PBS appears to

interfere with HA binding to positively charged membranes191

.

B.5 Acknowledgements

Thanks to Rachel Malone for beginning the slot blot/far-western experiments with

PRG4 “bait” on PVDF membranes in Summer 2013, to Mary Cowman for her HA

expertise and to Curt Sankar (undergraduate student with Mary Cowman) for sharing his

thesis work on slot-blotting PRG4+HA, as well as evidence that HABP may bind to

PRG4.

178

Appendix C: Temporal Effects of Intra-Articular HA and/or Corticosteroids on OA

Synovial Fluid Boundary Lubricant Composition: A Case Series

C.1 Purpose

Proteoglycan 4 (PRG4) and hyaluronan (HA) are critical boundary lubricants

present in synovial fluid (SF) and at the surface of articular cartilage. Deficiency of

PRG4 or HA concentration and/or molecular weight (MW) in SF may lead to

compromised boundary lubrication, which can be restored in vitro by lubricant

supplementation1,87

. Intra-articular (IA) corticosteroids (CST) can provide short-term

pain relief for patients with osteoarthritis (OA), and IA HA can provide pain relief for up

to 6 months despite its comparatively short residence time in the joint (hours to days).

While IA CST can decrease local inflammation and IA HA may stimulate endogenous

HA production, the effects of IA treatment on SF boundary lubricant composition over

time remain unclear. The purpose of this study was to measure PRG4 and HA content in

SF aspirated from the same OA patient knee joint over time during the course of

treatment with IA CST and/or HA.

C.2 Methods

In an ongoing study, knee SF was aspirated from chronic OA patients prior to IA

treatment. Patients were included in this case series if 3 or more SF aspirations were

available for analysis. SF was stored at -80°C with protease inhibitors (PI’s) until use,

and without PI’s for HA MW analysis when enough volume was available. In total, 4

179

knees (3 patients) with 3-5 aspirations were available for analysis (age 49 – 54, aspirated

SF volume range 2 – 60 mL). Two patients (3 knees) had 1 HA and 2 CST injections

over 6 – 9 months, and 1 patient had 1 HA and 4 CST injections over 34 months. IA HA

received was hylan G-F 20, CST received was Depo-Medrol. PRG4 and HA

concentration was measured by sandwich enzyme linked immunosorbent assay. HA MW

was measured by 1% agarose gel electrophoresis. SF boundary lubricant composition

data from 29 normal cadaveric SF samples are included for comparison (average value ±

95% confidence interval (CI)).

C.3 Results

No consistent trends in SF lubricant composition after IA HA or CST treatment

were observed over time. PRG4 concentration in 3 of the 4 knees appeared to be lower

than the normal range. PRG4 concentration increased in 1 knee over time with IA CST,

decreased in 1 knee with IA HA and CST, and fluctuated over time in 2 knees with IA

HA and CST (Figure C-1).

180

Figure C-1: PRG4 concentration in OA SF over time during treatment

with IA HA or corticosteroid. Each line represents 1 knee, and circular

markers denote knee SF from the left (filled circles) and right (open

circles) knee of 1 patient. SF was aspirated prior to therapeutic injection.

Red markers denote an IA injection was received after aspiration, all other

markers are corticosteroid injections. Grey shaded area shows average

[PRG4] in normal SF ± 95% confidence interval.

181

HA concentration in 2 knees appeared to be higher than the normal range; HA

concentration decreased over time in 1 knee with IA CST, and fluctuated in 3 knees with

IA HA and CST (Figure C-2).

Figure C-2: HA concentration in OA SF over time during treatment with

IA HA or corticosteroid. Each line represents 1 knee, and circular markers

denote knee SF from the left (filled circles) and right (open circles) knee

of 1 patient. SF was aspirated prior to therapeutic injection. Red markers

denote an IA injection was received after aspiration, all other markers are

corticosteroid injections. Grey shaded area shows average [HA] in normal

SF ± 95% confidence interval.

182

Three of 4 knees appeared to have lower than normal high MW (3–6 MDa) HA

content. High MW HA fluctuated over time in 1 knee with IA HA and CST and 1 knee

with IA CST, decreased over time in 1 knee with IA CST and remained stable over time

in 1 knee with IA HA and CST (Figure C-3, top). Three of 4 knees appeared to have

higher than normal low MW (<0.5 MDa) HA content. Low MW HA fluctuated over time

in 1 knee with IA HA and CST and 1 knee with IA CST, remained stable over time in 1

knee with IA HA and CST and increased over time in 1 knee with IA CST (Figure C-3,

bottom).

183

Figure C-3: HA MW distribution (high MW 3 – 6 MDa and low MW <

0.5 MDa) in OA SF over time during treatment with IA HA or

corticosteroid. Each line represents 1 knee, and circular markers denote

knee SF from the left (filled circles) and right (open circles) knee of 1

patient. SF was aspirated prior to therapeutic injection. Red markers

denote an IA injection was received after aspiration, all other markers are

corticosteroid injections. Grey shaded area shows average HA MW in

normal SF ± 95% confidence interval.

184

C.4 Conclusions

IA HA did not appear to result in an increased abundance of high MW HA in

these patients, nor in consistent changes in HA or PRG4 content. The times between

aspirations in this study were above the predicted times for PRG4 and HA to reach

steady-state following joint lavage, so aspirate composition likely reflects disease state

and/or response to IA treatments. Other factors including joint loading, activity level, and

inflammation may also influence SF lubricant composition over time. As such, the

present study suggests that boundary lubricant composition of SF in OA joints can

change over time with repeated IA treatment, and that this response to IA CST or HA

treatment appears to vary between individuals. IA PRG4 has been shown to stimulate

endogenous production of PRG4 in preclinical models75

, and PRG4 can affect boundary

lubricating and rheological properties of PRG4+HA solutions and SF in vitro.

Furthermore, PRG4 concentrations appear to be decreased in some chronic OA patients,

suggesting that future study of IA PRG4±HA is warranted and could provide further

insight into the mechanism of action of IA PRG4±HA biotherapeutic treatments. The

outcome of such future analysis of SF lubricant composition, boundary lubricating

function, and pain relief provided by IA PRG4±HA might ultimately be beneficial for

chronic, symptomatic OA patients with compromised SF boundary lubricant

composition.

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Appendix D: Description of Cartilage-Cartilage Boundary Lubrication test and

Lubricant Sequences

D.1 Introduction

This previously characterized in vitro cartilage-cartilage boundary lubricating

ability test29

is used in Chapter 2, Chapter 3, and Chapter 4 to evaluate boundary

lubricating ability of various human SF and purified PRG4+HA solutions. Figure D-1

below shows schematics of sample acquisition and the test sequence. It should be noted

that for the tests in human SF performed in Chapters 2 and 3, normal human

osteochondral samples were used and the 1200 second pre-spin duration was not

performed. For tests of purified solutions of PRG4 and HA, bovine SF served as the

positive control lubricant, and osteochondral samples were from bovine stifle joints. The

lubricant sequences used in each test in each chapter are summarized in the schematics in

Figure D-2 and Figure D-3.

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Figure D-1: Schematic depicting location osteochondral samples are

harvested from (A), annulus and core shaped samples (B), sample

immersion overnight in lubricant bath (C), sample orientation, applied

load, and rotation during testing (D), and test sequence schematic showing

compression, stress relaxation, and order of pres-spin durations (Tps) over

the duration of the tests (E).

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Figure D-2: Lubricant sequences used in Chapters 2 and 3 to evaluate

boundary lubricating ability of various human SF.

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Figure D-3: Lubricant sequences used in Chapter 4 to evaluate boundary

lubricating ability of various PRG4+HA solutions.

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Appendix E: PRG4 Concentration in all SF Samples Measured

Figure E-1: PRG4 concentration measured in all SF samples that were

measured in this thesis work. Grey bars indicate that those samples were

identified as having low PRG4 and were selected for friction testing. The

average normal value in N = 29 cadaveric SF samples (±95% confidence

interval) is shown in the black horizontal lines. (Please note the average

normal changed between Chapters 2, 3, and Appendix C, as more normal

samples were acquired and measured.)

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Appendix F: Figure Reprint Permissions

F.1 Reprint Permissions for Chapter 2, Published in Arthritis & Rheumatism

Figure F-1: Reprint permissions for Chapter 2, published in Arthritis &

Rheumatism, 2012; 64 (12): 3963-3971

191

F.2 Reprint Permissions for Appendix D, Published in Osteoarthritis and Cartilage

Figure F-2: Reprint permission for Appendix C, published in

Osteoarthritis and Cartilage 2014; Supplement 22: S481-S482.

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