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Graduate Studies The Vault: Electronic Theses and Dissertations
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solutions and synovial fluid (Unpublished doctoral thesis).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.
3.6 Acknowledgements
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
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.
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
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.
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.
4.6 Acknowledgements
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
5.6 Acknowledgements
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
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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.
188
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
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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.