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This is to certify that the

thesis entitled

The effects of a single acute load on an equine
articular cartilage explant system and further studies on
the ability of glucosamine and chondroitin sulfate to
inhibit cytokine-induced cartilage degradation

presented by

Angela Esther Schlueter

has been accepted towards fulfillment
of the requirements for

Masters—degree in AnimaLScience

Major professor

Date vQ////A3

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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6/01 c:/ClRC/Date0ue.p65-p. 15

THE EFFECTS OF A SINGLE ACUTE LOAD ON AN EQUINE ARTICULAR
CARTILAGE EXPLANT SYSTEM AND FURTHER STUDIES ON THE ABILITY
OF GLUCOSAMINE AND CHONDROITIN SULFATE TO INHIBIT CYTOKINE-

INDUCED CARTILAGE DEGRADATION
By
Angela Esther Schlueter

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Animal Science

2003

ABSTRACT
THE EFFECTS OF A SINGLE ACUTE LOAD ON AN EQUINE ARTICULAR
CARTILAGE EXPLANT SYSTEM AND FURTHER STUDIES ON THE ABILITY
OF GLUCOSAMINE AND CHONDROITIN SULFATE TO INHIBIT CYTOKINE-
INDUCED CARTILAGE DEGRADATION
By
Angela Esther Schlueter
Trauma to a joint can initiate of equine osteoarthritis (CA) by damaging

the cartilage and increasing the synthesis of catabolic molecules. Oral treatment
of 0A is also becoming more popular within the equine industry. Glucosamine
(GLN) and chondroitin sulfate (CS) may slow cartilage degeneration associated
with CA and improve lameness in the horse. Equine articular cartilage explants
were used to evaluate the effects of loading (15 or 30 MPa) compared to
cytokine-stimulation (LPS). LPS—treated explants had the highest nitric oxide
(NO) and prostaglandin E2 (PGEz) production over all treatments, while loading
explants at 30 MPa resulted in the highest proteoglycan (PG) release, and
second highest keratan sulfate (KS) degradation and PGE2 production. Explants
loaded at 15 MPa did not differ from the control in NO, PG, or PGE; production,
but had the highest KS loss of any treatment. The same explant system was
used to evaluate if GLN (0.2-0.5 mglml) in combination with CS (0.125 mglml)
are effective in inhibiting cytokine-induced cartilage degradation. NO and PGE2
production and matrix metalloproteinase (MMP) activity were evaluated. NO
production was lowered from 0.3 to 0.5 mglml GLN, while PGE2 production was
decreased at 0.4 and 0.5 mglml. Matrix metalloproteinase-9 activity was

decreased at 0.5 mglml and tended to be decreased at 0.4 mglml.

I would like to dedimte this thesis to my loving family, especially my parents
Randall and Janet Schlueter. Wrthout their strong support, this work would not
have been possible.

ACKNOWLEDGEMENTS

I would like to thank several people for helping and supporting me
throughout my research. I would like to especially thank Raelene Charbeneau
and Pool-See Chan, and my friends back home for all their friendship, help,
humor, and emotional support. Thank you to Doreen Bailey who believed in me
and encouraged me to enter graduate school, not to mention George and Bonnie
Good who supported me through stressful times. In addition, thank you to my
fellow graduate students who have helped to make my transition to Michigan
State more enjoyable.

Thank you to Dr. Orth for trusting in me, giving me the opportunity to
perform research I enjoy, and guiding me through my endeavors. A special
thank you goes out to Karen Waite for all of her mentoring and advice, as well as
Brian Nielsen for his support and guidance. I would also like to thank Dr. Caron,
Dr. Haut, Dr. Nielsen, and Dr. Orth for their guidance and serving on my
committee.

Thank you to all those who were critical in making my research possible:
Tonia Peters for her guidance and training in the lab, the Haut group for helping
impact my explants, all of the people in necropsy at the MSU Veterinary School
who supplied my equine tissue, Bellingar’s Packing for supplying bovine tissue,
and Pooi-See Chan for her knowledge and always being there to assist me in the
lab.

Finally, special thanks to my parents for supporting me throughout my
education, and being there for me during the “best of times and the worst of
times”. Moreover, I would like thank God for the many blessings he has given

me.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xii
INTRODUCTION 1
CHAPTER 1
LITERATURE REVIEW 6
The importance of osteoarthritis in the equine industry 6
Osteoarthritis 8
Significant molecules associated with degradation 11
Causes of OA 17
Trauma 18
Immobilization 22
Conformation 23
Shoeing 24
Age 26
Diagnosis 29
Imaging 31
Arthroscopy 34
'Synovial fluid 36
Therapy 38
Rest 38
Arthroscopic surgery 39
Joint lavage and synovectomy 41
Arthrodesis 42
Conventional medications 44
NSAIDS 44
Corticosteroids 46
Hyaluronic acid 48
Polysulfated GAGs 49
Glucosamine and chondroitin sulfate 51
Focus of research 55
References 56
CHAPTER 2
THE EFFECTS OF A SINGLE ACUTE LOAD ON AN EQUINE ARTICULAR
CARTILAGE EXPLANT SYSTEM
Abstract 76

 

Introduction 77

vi

Materials and Methods 80

 

 

 

Results 85

Discussion 35

References 100
CHAPTER 3

FURTHER STUDIES ON THE ABILITY OF GLUCOSAMINE AND
CHONDROITIN SULFATE TO INHIBIT CYTOKINE-INDUCED CARTILAGE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DEGRADATION
Introduction 105
Materials and Methods 107
Results and Discussion 111
References 1 19

CHAPTER 4

CONCLUSION 123

APPENDIX A

DEVELOPMENT OF A SERUM-FREE MEDIUM FOR AN EQUINE ARTICULAR

CARTILAGE EXPLANT SYSTEM 127
Introduction 128
Experiment 1 129
Materials and Methods 129
Results and Discussion 132
Conclusion 133
Experiment 2 133
Materials and Methods 133
Results and Discussion 135
Conclusion 136

 

References 140

vii

LIST OF TABLES

TABLE 1. Equine events and activities generally associated with OA ,,,,,,,,,,,,,,,,,,, 7
TABLE 2. Classification of osteoarthritis in the horse 30

TABLE 3. Description of treatment groups in equine articular cartilage explant
experiments 108

TABLE 4. Treatment groups for development of serum free culture medium, 130
TABLE 5. Treatment groups for development of serum free culture medium, 134

viii

LIST OF FIGURES

FIGURE 1. Factors involved in articular cartilage degradation in equine OA ,,,,, 18

Figure 2. Mean nitric oxide (N02) (:t SEM) released into the media per well each
day post-treatment for control, loading at 15 MPa (15 MPa), loading at 30 MPa
(30 MPa), and lipopolysaccharide (LPS). N02 concentration was quantified using
an assay employing the Greiss reaction. Different superscripts indicate
significant differences (P<0.05) between treatment groups 92

Figure 3. Mean prostaglandin E2 (P652) (1: SEM) released into the media per
well 24 hours post-treatment for control, loading at 15 MPa (15 MPa), loading at
30 MPa (30 MPa), and lipopolysaccharide (LPS). PGEz concentration was
determined by means of a commercially available competitive enzyme
immunoassay kit Different superscripts indicate significant differences (P<0.05)
between treatment groups 93

Figure 4. Mean proteoglycan (PG) (1: SEM) released into the media per well
each day post-treatment for control, loading at 15 MPa (15 MPa), loading at 30
MPa (30 MPa), and lipopolysaccharide (LPS). Total PG released into the media
was quantified using a dimethymethylene blue (DMB) assay. Different
superscripts indicate significant differences (P<0.05) between treatment

groups 94

Figure 5. Mean keratan sulfate (KS) (:l: SEM) released into the media per well 24
hours post-treatment for control, loading at 15 MPa (15 MPa), loading at 30 MPa
(30 MPa), and lipopolysaccharide (LPS). KS loss in the media was quantified
using an ELISA with a monoclonal antibody specific for KS. Different
superscripts indicate significant differences (P<0.05) between treatment

groups 95

Figure 6. Mean matrix metalloproteinase-2 (:l: SEM) activity measured in the
media per well 24 hours post-treatment for control, loading at 15 MPa (15 MPa),
loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-2 activity was
determined by gel zymography. MMP-2 activity was not significantly different
between groups (P>0.05) 96

Figure 7. Mean matrix metalloproteinase-2 (.1: SEM) activity measured in the
tissue per well 2 days post-treatment for control, loading at 15 MPa (15 MPa),
loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-2 activity was
determined by gel zymography. MMP-2 activity was not significantly different
between groups (P>0.05) 97

Figure 8. Mean matrix metalloproteinase-9 (t SEM) activity measured in the
media per well 24 hours post-treatment for control, loading at 15 MPa ( 15 MPa),
Ioaing at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-9 activity was
determined by gel zymography. MMP—9 activity was not significantly different
between groups (P>0.05) 98

Figure 9. Mean matrix metalloproteinase-9 (1: SEM) activity measured in the
tissue per well 2 days post-treatment for control, loading at 15 MPa (15 MPa),
loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-9 activity was
determined by gel zymography. MMP-9 activity was not significantly different
between groups (P>0.05) 99

Figure 10. Mean nitric oxide (NO) (t SEM) released into the media each day
post-treatment for all 4 horses (36-48 wells per treatment). Values are shown as
log transformed. Treatments: control = no glucosamine (GLN), chondroitin
sulfate (CS), or lipopolysaccharide (LPS); 0. 5= 0. 5 mglml GLN 4- 0. 125 mglml
CS; 0.=4 04mglml GLN+0125mglml CS 0.=3 03mglml GLN+0125mglml
CS, 0. 2= 0. 2 mglml GLN + 0.125 mglml CS; LPS= 10 pg LPS. hcmans not
sharing the same superscript differ (P<0.05) 115

Figure 11. Mean prostaglandin E2 (PGEz) (:t SEM) released into the media each
day post-treatment for all 4 horses (36-48 wells per treatment). Values are
shown as log transformed. Treatments: control = no glucosamine (GLN),
chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0. 5= 0. 5 mglml GLN +
0.125 mglml CS; 0. 4: 0. 4 mglml GLN + 0.125 mglml CS; 0. 3= 0. 3 mglml GLN +
0. 125 “mg/ml CS; 0. 2= 0. 2 mglml GLN + 0.125 mglml CS; LPS= 10 pg LPS.
and“ means not sharing the same superscript differ (P<0. 05) 116

Figure 12. Mean matrix metalloproteinase-2 (MMP—2) activity (:I: SEM) in the
tissue 2 days post-treatment for all 4 horses (36-48 wells per treatment). Values
are shown as log transformed. Treatments: control = no glucosamine (GLN),
chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0.5 = 0.5 mglml GLN +
0.125 mglml CS; 0.4 = 0.4 mglml GLN + 0.125 mglml CS; 0.3 = 0.3 mglml GLN +
0.125 mglml CS; 0.2 = 0.2 mglml GLN + 0.125 mglml CS; LPS = 10 pg LPS.
MMP-2 activity was not significantly different between groups (P>0.05) ,,,,,,,,,,,,, 117
Figure 13. Mean matrix metalloproteinase-9 (MMP-9) activity (:I: SEM) in the
tissue 2 days post-treatment for all 4 horses (36-48 wells per treatment). Values
are shown as log transformed. Treatments: control = no glucosamine (GLN),
chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0. 5= 0. 5 mglml GLN +
0.125 mglml CS; 0. 4= 0. 4 mglml GLN + 0.125 mglml CS; 0. 3= 0. 3 mglml GLN +
0.125 mglml CS; 0. 2= 0. 2 mglml GLN + 0.125 mglml CS; LPS= 10 pg LPS.
means not sharing the same superscript differ (P<0. 05)." indicates a trend
(P<0.08) to differ from LPS 118

FIGURE 14. Mean NO production released into serum-free (SF) or bovine basal
media (BBM) 24 and 48 hours after treatment with ITS, FBS, and LPS.
lTS=insulin-transferrin-sodium-selenite supplement, F BS=fetal bovine serum,
LPS=lipopolysaccharide 137

FIGURE 15. Mean proteoglycan production released into serum-free (SF) or
bovine basal media (BBM) 24 and 48 hours after treatment with ITS, FBS, and
LPS. lTS=insulin-transferrin-sodium-selenite supplement, FBS=fetal bovine
serum, LPS=lipopolysaccharide 138

FIGURE 16. Mean NO production released into serum-free media (SF) 24 and
48 hours prior to treatment with ITS, LLA, T, FBS, and LPS, and 24 hours
following treatment. lTS=insulin-transferrin-sodium—selenite supplement,
LLA=linoleic acid albumen, Tfihyroxine, FBS=fetal bovine serum,
LPS=lipopolysaccharide 139

LIST OF ABBREVIATIONS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Acetylsalicylic Acid ASA
Bovine Basal Media BBM
Bovine Viral Diarrhea Vrrus BVDV
Carprofen CAR
Chondroitin Sulfate CS
Computed Radiology CR
Computed Tomography CT
Cyclooxygenase COX
Cyclooxygenase-2 COX-2
Degenerative Joint Disease DJD
Dexamethasone DEX
Dimethylmethylene Blue DMB
Distal lnterphalangeal DlP
Dulbecco's Modified Eagles Medium DMEM
Extracellular Matrix ECM
Enzyme-Linked Immunosorbent Assay ELISA
Fetal Bovine Serum F BS
Fibronectin Fragments Fn-f
Flunuxin FNX
Glucosamine GLN
Glycosaminoglycan GAG

 

xii

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hyaluronan HA
Insulin Transferrin Sodium Selenite ITS
Insulin-Like Growth Factor 1 lGF-1
Interleukin-1 lL-1
Ketoprofen KET
Keratan Sulfate KS
Leukotriene LTB
Linoleic Acid Albumen LLA
Lipopolysaccharide , LPS
L-Thyroxine T

Magnetic Resonance Imaging MRI

Matrix Metalloproteinases MMP
mega Pascal MPa
Meloxicam MEL
Metacarpalphalangeal MCP
Middle Carpal Joint MCJ

Nitric Oxide Synthase NOS
Nitric Oxide NO

Nonenzymatic Glycation NEG
Non-Steroidal Anti-Inflammatory Drugs NSAle
Nuclear Medicine NM
Osteoarthritis OA

Phenylbutazone PBZ

 

xiii

 

Polysulfated Glycosaminoglycans PSGAGs

 

 

 

 

 

 

 

Prostaglandin E2 ...................................................................................... PGE2
Proteoglycan PG
Proximal Articular Surface PAS
Proximal Phalanx P1
Serum Free SF
Thromboxane TXB
Trssue Inhibitor of Matrix Metalloproteinases TIMP
Ultrasound _ ______ US

 

xiv

INTRODUCTION

Osteoarthritis (OA) is characterized by deterioration of the articular
cartilage, accompanied by changes in the bone and soft tissues of the joint,
including subchondral bone sclerosis and marginal osteophyte formation.1
Considerable attention has been given to OA, because it is one of the most
common causes of lameness in horses. Due to its degenerative effect, OA leads
to significant financial loss in the equine industry.2 Horses most affected by CA
are western performance horses, racehorses, jumping horses, and other sport
horses involved in highly athletic events.“ As athletic events have become
more competitive, so has the breeding and training of the horses, because
exceptional athletes are needed to compete in the industry today.3 The demand
for increased athletic ability and rigorous training predisposes these types of
horses to athletic injury.

Mechanical loading, or trauma to the joint, is a significant cause of GA in
the horse. Athletic horses undergo extensive and intensive training regimens,
which result in repetitive loads being placed on the tissues of the joints.5
Because the tissues of articulating joints are subject to impact loading, cartilage
must be capable of resisting and redistributing the forces arising during joint
movement. Research investigating the in vivo efi‘ects of loading on articular
cartilage has shown that it adapts to load.” In vitm studies of loaded bovine
cartilage explants have indicated changes in the extracellular matrix (ECM),

which include cell death, loss of proteoglycans (PGs), and increased nitric oxide

(NO) production.9 Little research has been done on the effects of mechanical
impact on equine cartilage explants. The biochemical changes that occur
following loading, which predispose the horse to OA, require further investigation.

Increased application of modern technology, including anesthesia and
imaging techniques, has facilitated more accurate and earlier diagnosis of joint
disease.10 However, a lack of information about the basic pathogenesis of OA
has hindered progress in developing innovative treatments. Because 0A is
considered to be a chronic irreversible disease, noninvasive treatments to protect
articular cartilage and prevent further damage are being sought. Glucosamine
(GLN) and chondroitin sulfate (CS) are amino sugars that have a beneficial effect
in the treatment of OA.”‘17 Both of these agents are endogenous to
chondrocytes, which produce the structural components of the ECM, and are
considered chondroprotective.18

My research consists of two objectives. The first objective combines the

“"5 and a method previously designed in our lab

use of an equine explant system
to mechanically load articular cartilage explants,19 and evaluate the effects of
mechanical loading on equine explants. The second objective was to determine
the effectiveness of glucosamine at a lower concentration, in combination with
chondroitin sulfate, in inhibiting degradation of articular cartilage. In my first
experiment, I applied two separate single acute loads (15 and 30 MP3) to equine

articular cartilage explants and compared their biological responses to cytokine

stimulation with LPS. In the second experiment I determined the lower dose at

which GLN HCl, in combination with a fixed dose of CS, is effective in inhibiting in
NO and PGE2 production and MMP activity.

References

1. Mcllwraith CW, Vachon A. Review of pathogenesis and treatment
of degenerative joint disease. Equine Vet J Suppl 1988:3-11.

2. Poole RR. Pathologic manifestations of joint disease in the athletic
horse In: Mcllwraith CW,Trotter GW, eds. Joint Disease in the Horse.
Philadelphia: W.B. Saunders, 1996;87-104.

3. Jackman B, Lewis R, Noble J, et al. Meeting report: lameness in
the western performance horse. J of Equine Vet Sci 2002;22:65-66.

4. Torre F, Ross MW. Veterinary review. lameness in the
Standardbred horse. J Equine Vet Sci 2002;22:429-436.

5. Bird JL, Platt D, Wells T, et al. Exercise-induced changes in
proteoglycan metabolism of equine articular cartilage. Equine Vet J 2000;32:161-
163.

6. Brama PA, Tekoppele JM, Bank RA, et al. Topographical mapping
of biochemical properties of articular cartilage in the equine fetlock joint. Equine
Vet J 2000;32:19-26.

7. Brama PA, Tekoppele JM, Bank RA, et al. Functional adaptation of
equine articular cartilage: the formation of regional biochemical characteristics up
to age one year. Equine Vet J 2000;32:217-221.

8. Brama PA, Te Koppele JM, Bank RA, et al. Development of
biochemical heterogeneity of articular cartilage: influence of age and exercise.
Equine Vet J 2002;34:265-269.

9. Ewers BJ, Dvoracek_Driksna D, Orth MW, at al. The extent of
matrix damage and chondrocyte death in mechanically traumatized articular
cartilage explants depends on rate of loading. J Orthop Res 2001;19:779-784.

10. Trotter GW, Mcllwraith CW. Advances in equine arthroscopy. Vet
Clin North Am Equine Pract 1996;12:261-281.

11. Hanson RR, Smalley LR, Huff HF, et al. Oral treatment with a
glucosamine-chondroitin sulfate compound for degenerative joint disease in
horses: 25 cases. Equine Pract 1997;9:16-22.

12. Hanson RR, Brawner WR, Blaik MA, et al. Oral treatment with a
Nutraceutical (Cosequin®) for ameliorating signs of navicular syndrome in
horses. Vet Ther 2001;2:148-159.

13. . Anderson CC, Cook JL, Kreeger JM, et al. In vitro effects of
glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional
culture. Am J Vet Res 1999;60:1546-1551.

14. Fenton Jl, Chlebek-Brown KA, Peters TL, et al. The effects of
glucosamine derivatives on equine cartilage degradation in explant culture.
Osteoarthritis Cart 2000;8z444-451.

15. Fenton JI, Chlebek-Brown KA, Peters TL, et al. Glucosamine HCI
reduces equine articular cartilage degradation in explant culture. Osteoarthritis
Cart 2000;8z258-265.

16. Fenton Jl, Chlebek-Brown KA, Caron JP, et al. Effect of
glucosamine on interleukin-1-conditioned articular cartilage. Equine Vet J Suppl
2002;34:219-223.

17. Orth MW, Peters TL, Hawkins JN. Inhibition of articular cartilage
degradation by glucosamine-HCI and chondroitin sulfate. Equine Vet J Suppl
2002;34:224-229

18. Lippiello L, Woodward J, Karpman R, et al. In vivo
chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate.
Clin Orthop 2000;381:229-240.

19. Dvoracek-Driksna D. Effects of mechanical impact on a bovine
articular cartilage explant system. Thesis. Animal Science Department. East
Lansing: Michigan State University, 2001;71.

CHAPTER 1
LITERATURE REVIEW

1. The Importance of OA in the Equine Industry

The equine industry is a growing industry that encompasses a diverse
discipline ranging from sport horse to work horse, and has a sizeable share in the
US. economy. The American Horse Council Foundation (Barents Group LLC,
1996) reported that there were 6.9 million horses in the US. in 1996; 725,000
horses were involved in racing, 1,974,000 in showing, 2,970,000 in recreation,
and 1,262,000 in other activities. In addition, the equine industry directly
provided 338,500 full-time jobs. The report also determined that in 1996 the
horse industry produced goods and services valued at $25.3 billion, and the total
contribution to the US. Gross Domestic Product was $112.1 billion. Lameness is
a major cause of wastage in horses and adversely affects the horse industry,
because one of the main factors determining a horse’s value is soundness,
especially in athletic horses."3 Jeffcott1 assessed the wastage in Thoroughbred
race horses from conception to four years of age, and determined that lameness
was the most significant factor responsible for failure to race, outweighing
respiratory problems, colic, or limited racing ability. The study also indicated that
in 140 Thoroughbred 2-year-olds evaluated, only 34 (23%) did not show any
signs of lameness. Similar results were found in a study determining the

wastage of racehorses between 1982 and 1983.2 These authors found that the

greatest number of days lost to training was caused by lameness (68%); among
314 horses examined, 53% were lame at some period during the racing season.
OA is one of the most common causes of lameness, and is of particular
concern in horses because their value is closely tied to their soundness.
Lameness that results from OA is a major cause of poor performance and early
retirement of equine athletes.“6 Table 1 lists equine events and movements

associated with OA.

TABLE 1: EQUINE EVENTS AND ACTIVITIES GENERALLY ASSOCIATED

 

 

WITH OA’
Event Movement Type of OA
Dressage Increased joint fiexion Eariy arthritis of the
tarsal joints
- Collection; extension Fetlock arthritis
(synovitislcapsulitis)
Reining Fast spins Early arthritis of the
tarsal joints
Cutting Lateral driving with Early arthritis of the
forelimbs tarsal joints
Roping Hard stops and abrupt Arthritis of the tarsal
change in direction joints
Western Pleasure Repetitive slow Early arthritis of the
jogging tarsal joints
TB and OH Fatigue Fetlock arthritis
Racing Arthritis of the tarsal
joints
SB racing Extended and fast Fetlock arthritis
pacing and trotting Arthritis of the tarsal
Joints
Barrel racing, Speed and turning Fetlock arthritis
pole bending Torque and twist Arthritis of the inter-
phalangeal joints

 

TB, Thoroughbred; QH, Quarter Horse; 88, Standardbred.

A survey performed at a veterinary school found that 33% of all equine
patients had intra-articular lesions related to OA.” Tew and HooltettB randomly
evaluated 72 equine joints at necropsy and discovered that 35% of the joints had
evidence of grossly visible cartilage damage. Not only is this degenerative
disease found in domestic horses, but it naturally occurs in the joints of wild
horses.10 Despite the huge economic importance of joint disease and DA in
horses, our understanding of the pathophysiological mechanisms involved in joint
degeneration in this species is very poor.11 Whether OA is a single disease or is
caused by several disorders with a similar final common pathway remains

unclear.12

2. Osteoarthritis

Osteoarthritis, often referred to as degenerative joint disease (DJD) in the
horse, is characterized by deterioration of articular cartilage, accompaan by
changes in the bone and soft tissues of the joint. The end stage of OA results in
a net loss of articular cartilage, causing pain, deformity, loss of motion, and
decreased function. Horses have naturally occurring OA, which is similar to that
of humans, and are often used as models to investigate the pathogenesis and
treatment of OA.

Synovial joints are the joints usually associated with lameness in the
horse. These joints have two major functions: 1) to enable movement, and 2) to
transfer load. Synovial joints consist of the articulating surfaces of bone, covered

by articular cartilage, secured by a joint capsule and ligaments, and have a cavity

containing synovial fluid. Articular cartilage is an avascular complex structure,
which serves as a shock absorber for bone and has a frictionless surface bathed
in synovial fluid. This tissue consists of sparsely scattered chondrocytes
(cartilage cells) intenneshed within the ECM. The ECM provides cartilage with
its compressive strength and is primarily composed of type II collagen and P65.
Collagen forms a fibrous network giving cartilage its tensile strength, while large
hydrophilic aggregating PGs (aggrecans) hydrate the collagen network and
provide the tissue with viscoelastic properties and the ability to resist mechanical
compression.13 When the joint capsule is disrupted, proteolytic enzymes are
secreted into the synovial fluid and break down PGs and collagen, the main
components of the ECM.“"15 In an attempt to repair structural changes in the
ECM, chondrocytes proliferate and stimulate synthesis of these components.
However, over time, the metabolic activity of chondrocytes shifts toward a state
where the breakdown of matrix constituents outweighs new matrix synthesis,”16
beginning the gradual process of ECM degeneration and thus the loss of articular
cartilage. Articular cartilage degradation on the joint surface is a common feature
of OA.

Subchondral bone, the bone underlying the articular cartilage, can also be
affected by OA. Because it remodels rapidly, subchondral bone is responsible
for changing the shape and congruity of the joint. Mechanical stimulation of
subchondral bone often leads to microdamage, which may result in 1) normal
remodeling; 2) excessive remodeling, leading to bone sclerosis; or 3)

accumulation of microdamage, leading to gross fracture.° Subchondral bone

thickening is a normal response in exercising horses. However, an increase in
the degree of subchondral bone sclerosis corresponds to greater degrees of
generalized 0A in joints (i.e. fetlock).17 Sclerosis of the subchondral bone can
lead to the development of chip fractures, fowl lesions of traumatic
osteochondritis dissecans, and slab fractures. Articular cartilage covering sites
of subchondral bone sclerosis is predisposed to the development of OA because
the cartilage is no longer supported by healthy subchondral bone.“ Soft tissues
of the joint include the intraarticular ligaments, joint capsule, menisci, and
synovial membrane. Damage to intraarticular ligaments, which provide support
for the joints and distribute normal surface stresses, can stimulate an
inflammatory response and change the loading characteristics of the joint
surface. An example of this phenomenon is shown by mechanical instability of
the joint after transaction of the cranial cruciate ligament. This surgical
procedure produces joint laxity, loss of joint congruency, and abnormal cartilage
weight-bearing forces and trauma that can directly and indirectly induce
abnormal cartilage wear.”22 Degenerative joint disease often develops in
humans following meniscal injury.”0 Increased stress across the knee joint
induced by performing surgical meniscectomies stimulates GA in humans,
rabbits, and guinea pigs."’°""3’25 Chronic disease of the equine joint capsule,
capsulitis, can lead to formation of scar tissue and increased stiffness, leading to
instability of the joint by changing its surface stresses .6 Acute synovitis and
capsulitis may cause significant clinical compromise of the joint, and also

contribute to the degenerative process by the release of enzymes, inflammatory

10

mediators, and cytokines.26 While the cause of acute primary synovitis has never
been determined, the development of an acutely inflamed joint is prevalent in a
racing and training Thoroughbmds and Standardbreds.”

2.1 Pivotal molecules associated with OA

Enzymatic degradation of articular cartilage pmcedes morphologic
breakdown and plays a central role in equine joint disease." Biochemical
degradation is considered to represent an imbalance of the normal homeostatic
processes within the matrix of the articular cartilage. This imbalance causes an
inflammatory cascade, which takes control and is responsible for the majority of
pain in the equine joint. Several inflammatory mediators such as MMPs,
interleukins (lls), prostaglandins, lysozyme, and free radicals have been
incriminated in contributing to this cascade.

Matrix metalloproteinases are the primary class of enzymes responsible
for ECM degradation, and their increased activity plays a crucial role in the
progression of OA. Although all classes of proteinases may be involved in the
degeneration of the ECM, the MMPs are considered to play the most pivotal role
in cartilage destruction.28 These enzymes are characterized by a requirement for
Zn2+ in their active site. Calcium is also required for the expression of full activity
but does not reside in the active site. Overall, the MMPs are capable of
degrading the major cartilage matrix components, such as collagen, aggrecan,
link protein, and cartilage oligomeric protein.28 This growing family of proteolytic
enzymes has been divided into four main classes: collagenases (MMP-1, —8,

and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), and

11

membrane-type (MMP-14, -15, and —17). Matrix metalloproteinases are inhibited
by a group of tissue inhibitors called tissue inhibitors of MMPs (T lMPs).

Relatively few studies have been conducted determining the activity of
MMPs in equine OA. Matrix metalloproteinase activity increases in equine
osteoarthritic joints, and as age increases, MMP activity decreases.29
Specifically, MMPs -2 and -9 have been found in synovial fluid from diseased
equine joints.3032 The activity of both of these MMPs has been found to be
upregulated in normal equine cartilage and synovial fluid following stimulation
with interleukin-1B (IL-1B).” Stimulation of cartilage explants with lL-1 also
induced the synthesis of MMP-3 in young and adult horses.“ Caron35 found that
MMP-13 expression was significantly stimulated by human recombinant lL-1
(rhlL-1), and is produced by equine chondrocytes.

Interleukin-1 is a protein secreted by stimulated cells of macrophages, and
has a number of important biologic activities.”5 This protein has been implicated
in the induction and augmentation of the pathologic processes involved in r
inflammatory joint disease. Morrisa‘6 was the first to identify IL-1 in the equine
osteoarthritic joint, and found that equine lL-1 has many of the characteristics of
lL-1 isolated from other species. Interleukin-1 stimulates chondrocytes and
synovial cells to release enhanced amounts of PGE2, PGs, MMPs, such as
collagenases and stromelysin, and increases NO production.‘7'37'39 Stimulation
by IL-1 creates an inflammatory response that is similar to naturally occurring
OA. As a result, IL-1 is often used as a model in vivo to stimulate an

inflammatory response in chondrocytes.

12

Interleukin-1 has widely been used to study the pathogenesis of OA in
equine articular cartilage. Equine explants stimulated with lL-1 have
demonstrated an increase in the release of GAGs from the ECM.“ Decreased
PG synthesis and increased MMP-3 activity have been reported in equine
explants following stimulation with lL-1,3‘ while recombinant human interleukin-1B
(rhlL-18) induces the expression of MMP-13 in equine chondrocytes in
monolayer culture.“ Interleukin-1 also induces lL-6 synthesis in human cartilage
from normal controls, patients with OA, and patients with rheumatoid arthritis.“
Increased PGE2 and IL-6 concentrations were found in the synovial fluid of
equine joints injected with lL-1(3.42 A subsequent study done by Simmons22
injected rhlL-1l3 into the metacarpalphalangeal (MCP) joints of horses, and also
found an increased concentration of IL-6.

Prostaglandins are widely distributed in the body and mediate or modulate
a variety of physiologic and pathophysiologic processes in many organ systems
and tissues, including the hematopoietic, cardiovascular, and reproductive
systems."6 They are believed to bind to receptors on the sensory nerve endings,
promoting the discharge of impulses and consequently causing an increase in
pain. ‘7

Prostaglandins (primarily E group) are produced in inflamed joints and can
cause a decrease in the PG content of the cartilage matrix.“17 Actions of PGE2
in joints include vasodilation, enhancement of pain perception, degradation of
PGs and inhibition of PG synthesis from cartilage, bone demineralization, and

promotion of plasminogen activator secretion. Cyclooxygenase-2 (COX-2) is one

13

of the enzymes responsible for the production of PGE2 from cell membrane
phospholipids.“3 lL-1 stimulates the synthesis of PGE2, and increased
concentrations of PGE2 in affected joints suggests a causal link of this
inflammatory mediator to the pathophysiologic events of OA.‘°'5° Equine synovial
cells and chondrocytes increased PGE2 production after stimulation with relL-1B
and LPS.5°'5‘ Exposure of equine chondrocytes to relL-lji also resulted in
enhanced expression of COX —2.‘“’ In addition, equine articular cartilage
explants incubated with LPS52 or lL-153'5‘ had an increase of PGE2 released into
the culture medium. In vivo, significantly higher PGE2 production has been
reported in the medium of explants originating from horses with moderate OA
when compared to normal joints.55 These researchers also saw a similar
increased PGE2 content in the synovial fluid of equine osteoarthritic joints when
compared to normal joints.“'57 Prostaglandin E2 may also have an effect on the
expression of MMP activity by inhibiting lL-1 expression through a negative
feedback mechanism in articular cartilage degradation. Exogenous PGE2
significantly reduced relL-1B-induced expression of MMP1, MMP3, MMP13, and
tissue inhibitor of MMP-1 (TIMP-1) in equine chondrocytes.5°

Nitric oxide is another important physiologic mediator that is thought to be
involved in the pathogenesis of OA. This uncharged free radical is released from
various tissues and cells, and is the product of a reaction between L-arginine and
oxygen. Nitric oxide has one unpaired electron and readily reacts with oxygen,
superoxide radicals, or transition metals, which may generate further destructive
species."""59 Stadler°° first showed that articular chondrocytes have the ability to

14

generate large amounts of NO. Nitric oxide is a major component of the
inflammatory response, and may mediate the suppression of cartilage matrix
synthesis occurring in response to intraarticular cytokinesf”;62 Nitric oxide
activates MMPs,‘53 suppresses PG synthesis,“65 and induces apoptosis in
human articular chondrocytes.66 Chondrocyte cell death from N0 occurs under
conditions where other reactive oxygen species are generated.67

An increased interest to determine the extent of the effect of NO
production on equine OA has developed. Although NO is generally thought to be
an important mediator of the inflammatory response, it may have an anabolic
function in inhibiting articular cartilage catabolism. Nitric oxide inhibited aggrecan
degradation in equine explant cultures, suggesting that NO has an anticatabolic
role in PG degradation.62 These researchers suggest that this mechanism may
be mediated by the regulation of aggrecanase activity. Explant cultures of
equine synovial membrane and articular cartilage released significantly higher
amounts of NO in cartilage explants originating from horses with OA.55
Simmons22 injected rhlL-1B intra-articularly into the MCP joints of 6 horses, and
measured nitric oxide synthase (NOS) in the synovial fluid of injected joints 6
hours post treatment. Although intensity and extent of inflammation was
significantly greater in the IL-1B exposed specimens when compared to healthy
specimens, no significant increase in the inducible isoforrn of nitric oxide
synthase (iNOS) was found between the control and the lL-1B exposed joints.
Synovial cell expression of iNOS varies among species, and horses have very

limited iNOS expression. These researchers propose that a longer challenge of

15

lL-1l3 alone, treatment with a combination of cytokines, or a greater challenge
may be necessary to induce an effect from synovium. Increased NO synthesis
occurs in chondrocytes and synoviocytes in response to LPS and lL-1 within a 48
hour incubation period‘s” In addition, LPS or IL-18 dramatically increase NO
synthesis relative to non-stimulated controls in equine explants.“71 In general,
the bulk of information investigating the role of NO on articular cartilage
degradation has demonstrated that it has a negative effect on cartilage
metabolism.

Fibronectin, a noncollagenous protein in articular cartilage that appears to
be important in chondrocyte-matrix interactions and cell adhesion, is another
component of cartilage affected in OA. ln OA, fibronectin content is markedly
increased in the altered matrix because of an increased synthesis by the
chondrocytes and accumulation in the ECM;72 however, the role of fibronectin in
OA has been controversial. Fibronectin localization at sites of cartilage
degeneration and fibrillation was evident in the carpal joints of both gently and
strenuously exercised 2-year-old Thoroughbreds.73 F ibronectin was released
into the matrix by chondrocytes, and distribution between the two exercise
groups was similar. Stimulation of bovine articular cartilage explant cultures with
fibronectin fragments (Fn-f) results in enhanced release of MMP's, rapid cartilage
PG degradation and loss, and decreased PG synthesis.7"73 In addition, general
protein synthesis is suppressed following F n-f incorporation.77 Fibronectin

fragments also stimulated cytokine release in human knee cartilage explants,

16

resulting in an immediate peak release of tumor necrosis factor-alpha (T NF-a)

and IL-18, and an early release of lL-6.7°

3. Causes of OA

Cartilage damage due to trauma, impact injuries, abnormal joint loading,
excessive wear, or as part of an aging process can lead to changes in the
composition, structure, and material properties of the tissue.‘2'7°'°° These
changes can compromise cartilage function in the strenuous mechanical
environment normally found in weight-bearing joints. Regardless of the specific
cause, the initial injury is usually mechanical in nature, with an imbalance
between the load applied and the tissues’ capacity to withstand that load.“6
Trauma to the joint, immobilization of the joint, poor conformation, improper
shoeing, and age are often preliminary factors that contribute to the onset of OA

in the horse. Figure 1 identifies the factors involved in articular OA.17

17

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otPGfrommstnx PGsl-colayen sacorlderytoo/tok'na

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Iorphologlc Breakdown of Articular Cartlhgs

Figure 1. Factors involved in articular cartilage degradation in equine OA."

3.1 Trauma

Trauma to the joint is believed to be the primary cause of OA in the horse.
Mackay-Smith‘ referred to use-trauma, or trauma occurring from normal use of
the joint, as a precurrent factor of OA that had been ignored in previous literature.
Very strenuous exercise injures articular cartilage by increasing fibrillation of the
cartilage and reducing proteoglycan content and quality. Cartilage no longer

responds with improved biomechanical properties, and overload results

18

from such factors as extensive and intensive exercise, fatigue, speed, and poor
conformation or footing.‘6 For example, a racehorse’s pace generates millions of
foot-pounds of force per mile, and the wear and tear produced on the joints
during a race can be severe.4

Most lameness occurs in the forelimbs, because they cany 60 to 65% of
the horse’s weight and are subjected to higher load rates when compared to the
hindlimbs.7'"'82 The hindlimbs propel the horse, while the forelimbs receive the
shock of landing. However, this may vary among breed and performance event.
Different areas of joints and joint surfaces in both the forelimb and hindlimb are
subjected to different types of loading, such as low level constant loading during
weight bearing, intermittent loading during locomotion, and very high and sudden
loading during training or racing.”“‘ The carpal, fetlock, proximal
lnterphalangeal, and distal intertarsalltarsometatarsal joints are most frequently
effected by OA.

The fetlock joint of the foreleg has the largest number of unique
degenerative and traumatic lesions of any limb joint in racing horses.85 Brama86
topographically mapped contact areas and pressure distributions on the proximal
articular surface (PAS) of the proximal phalanx (P1) under various clinically
relevant loading conditions in the forelimbs of 13 horses. These authors found
that certain areas of the PAS of the P1 are permanently loaded in the standing
horse, and as the load was increased to mimic the walk or trot, the contact area
enlarged in the dorsal, dorsolateral and dorsomedial direction. The joint

pressures in the continuously loaded central area of the equine fetlock joint are

19

relatively low in the standing horse, but may increase up to 6-fold when loads are
applbd that can be expected during athletic performance.

Articular cartilage degeneration of the dorsal joint margins of the carpal
bones in race horses may be the direct result of trauma.” Repetitive exercise
may induce the replacement of normal subchondral bone by sclerotic bone,
therefore contributing to the pathogenesis of OA. Research into the effects of
exercise on PG metabolism in the carpal joints has produced conflicting results.
Palmer"7 assessed the relevance of site and the influence of exercise on articular
cartilage PG synthesis and metabolism on third carpal articular cartilage in 16
horses. PG synthesis was increased in exercised horses relative to
nonexercised horses at the end of a 6-week period. However, the increase in
newly synthesized PG was not reflected in endogenous PG within the matrix at
different sites on the third carpal bone. A significant correlation of site on
endogenous PG was evident, with a greater concentration of PG located in the
palmar aspect of the radial facet compared to the sites located on the dorsal
aspect of the radial facet or all sites on the intermediate facet. Total PG content
on sites of the middle carpal joint (MCJ) increased in untrained Thoroughbreds
with short-term exercise."8 Proteoglycan content was greater at palmar sites
overall, and dorsal sites of the high-intensity trained group had 12% higher PG
when compared to those of the low-intensity trained group. A contradictory study
to those previously described evaluated the effect of strenuous versus moderate
exercise on the metabolism of PGs in the articular cartilage from different weight-

bearing regions in the equine third carpal bone.11 PG synthesis was reduced in

20

both exercise groups, and greater PG loss was found in the different joint regions
of the strenuously trained animals. No change in PG size or ability to aggregate
in different regions of any articular cartilage site was found in this study.

Low-motion joints such as the proximal interphalangeal, distal intertarsal,
and tarsometatarsal joints are vulnerable to the development of OA, because
they have a relatively smaller area of joint surface that must sustain the same
weight-bearing load for a relatively longer period of time during joint movement.”
Both ring-bone and bone spavin can produce crippling lameness in horses.
Although the etiopathogenesis of ring-bone and bone spavin is undetermined,
the cause is probably trauma to the periarticular soft tissues including the joint
capsule insertions and periosteum.”

Ring-bone is a term used to describe DJD of the proximal and distal
interphalangeal joint. This disorder most commonly occurs in horses forced to
make quick turns and abrupt stops, such as western performance horses, polo
ponies, and jumpers.90 Ellis and Greenwood91 evaluated six cases of ring-bone
in young Thoroughbreds ranging from the age of three months to four years. All
cases except one had other pre-existing or concurrent bone disease, which could
have consequently placed abnormal weight on the interphalangeal joint resulting
in DJD. Ring-bone has been the most serious cause of wasting in Norwegian
Dble horses.92

Bone spavin is the most common cause of hindlimb lameness of athletic
horses, and involves the distal intertarsal, tarsometatarsal, and occasionally the

proximal intertarsal joints.7'93'95 This degenerative disorder has been found in a

21

variety of breeds including Quarter Horses, Thoroughbreds, Standardbreds, and
Icelandic horses. Wyn-Jones and May” treated 30 horses and ponies for
lameness due to bone spavin, and found that 25 of the 30 horses were lame in
both hindlegs and lameness varied from slight to severe. Twenty-three percent of
Icelandic horses radiographically evaluated (379 total) had signs of bone spavin,
suggesting a predispostion to the disease.96

3.2 Immobilization

Reduced loading or immobilization, due to lack of exercise, can lead to
atrophy or degeneration of articular cartilage. While excessive forces may lead
to articular cartilage loss, removal of all mechanical stimulation leads to atrophy.
When cartilage is subjected to high-pressure loads, PGs are compressed and
water is expressed from the cartilage.“ Cartilage then expands as it is
rehydrated upon alleviation of pressure. Physiologic loading and motion are
therefore essential to maintain the normal nutrition and metabolism of articular
cartilage provided by exchange with synovial fluid.

Although several immobilization studies have been conducted, few have
been done on horses. An early study investigating changes in the metabolism of
proteoglycans in immobilized limbs of sheep, showed a decrease in
glycosaminoglycan (GAG) content of the non-loadbearing joint.97 Proteoglycans
isolated from the immobilized limb were smaller than those isolated from load-
bearing joints. Instability of the MCP joint was surgically performed in six horses
by transacting the collateral and lateral sesamoidean ligaments.22 This

procedure induced CA in all horses, which resulted in lameness, increased joint

22

circumference, decreased joint range of motion, and increased new synthesis of
PG production. Horses immobilized with fiberglass casts from the proximal
portion of the metacarpus down to the hoof tended to have lower hexosamine
concentrations in articular cartilage biopsied from their casted joints.” The
contralateral limbs of each horse served as a mobilized control and the control
articular cartilage tended to gain hexosamine during the 30-day trial. These
researchers saw little change in the GAG synthesis of the casted joints, while the
largest significant change occurred in the control. Similar results have been
found in the rabbit.” Thus, contlalateral limbs are unsuitable for controls in
immobilization studies because of their biological response to increased weight
bearing. Palmer87 found a lower concentration of newly synthesized PGs in
nonexercised horses when compared to exercised horses. Exercised horses
had a noticeable increase in the early PG peak of newly synthesized PGs, while
this did not occur in the sites of the non-exercised group. Immobilization studies
performed with canine and rabbit limbs have indicated a depletion of PGs,
defective aggregation of PGs, and accumulation of water in the tissue.""“°1
These problems may be reversed after remobilization.
3.3 Conformation

Conformation is defined by the physical appearance and outline of a
horse, which is dictated primarily by bone and muscle structures. Certain
conformational traits can predispose the horse to lameness. Conformation
defects, such as “calf knees”, “knocked knees” (carpus valgus), “bowed knees”

(carpus varus), or “bench knees”, cause the animal to load its carpus abnormally,

23

and 0A can result.102 In the rear legs, horses that are extremely straight in
angulation of the stifle and hock or are obviously sickle- or cow-hooked are
predisposed to conforrnationally induced lameness.103 Certain breeds’
characteristic conformation magnifies their risk of developing OA. Icelandic
horses with sickle hooks had a prevalence of radiographic signs of bone spavin
of 42%, which was significantly higher than that of horses with straight (20%) or
normal (19%) conformation.96 In addition, the prevalence of bone spavin was
found in 19% of horses with a light skeletal type, whereas lesions were identified
in 23% of those with intermediate and in 24% of those with heavy skeletal type.
A more recent study confirmed this finding, and indicated that the prevalence of
radiographic signs of DJD in the distal tarsus of Icelandic horses increased in
horses with a smaller tarsal angle.‘°‘ Upright pastems, base narrow front limbs,
and a rectangular shaped P1 in the Nonlvegian Ddle horse are conformation
defects that contribute to the development of ring-bone.92 Quarter Horses are
often more prone to OA because they have a relatively large body mass, poor
carpal conformation, small feet, and short upright pastems.‘°5
3.4 Shoeing

Since the hoof capsule is malleable, the manner in which it is trimmed and
shod can have a marked effect on performance and soundness of the equine
athlete. The hoof of the horse must be balanced to absorb high impact vibrations
when it is exposed to the repetitive trauma incurred during performance events

and normal use. Maximum energy dissipation depends on proper hoof

24

preparation and shoeing.“ Good shoeing Is an art and maintenance of the
natural angle and balance of the hoof is critical.

Improper shoeing can change the limb configuration of the horse, resulting
in a modification of the forces placed on the joint surfaces.""5 Increased
abnormal wear and loading on the joint surface due to improper shoeing can
contribute to degeneration of articular cartilage. The typical long toe/low heel
conformation commonly seen in Thoroughbred racehorses can accentuate
hyperextension type injuries in the fetlock and carpus and cause direct injury to
the foot in the form of 0A in the distal interphalangeal (DIP) joint.107

Corrective trimming and shoeing alters the hoof shape or angle to affect
stance or stride and breakover in order to help the horse achieve a more normal
movement. Altered foot orientation, which could result from trimming and
shoeing, influences intra-articular pressure in the articular contact area of the
DIP.‘°‘3 When a hoof is being actively re-fonned, the change in shape during one
trimming may be dramatic. Types of shoes and shoeing devices can alter the
traction of the hoof. For instance, sliding plates and wide web shoes are often
used on reining horses. These types of shoes provide inadequate traction for the
horse, and can result in strained tendons or sprained ligaments. Traction
devices, such as toe grabs, heel calks, and borium can provide too much
traction. Excess torque on the limb and joints resulting from using these devices
can lead to strain or sprain and may contribute to the development of OA.
Horses shod with hoof caulks had altered joint angles, which could change the

forces placed on the joint surfaces, or the soft tissue structures in the lower

25

limb.106 A study evaluating the effects of shoeing horses with mdgec (angle 3.7
and 5') showed that an increased elevation of the heel delayed unloading of the
heel and an increased elevation of the toe advanced unloading.“ These results
suggested that the horse is unable to compensate for an acute foot imbalance by
redistributing the load under the foot Increased joint pressure has been
implicated in the progression of OA."° An in vitro study evaluating the intra-
articular pressure in the DIP showed that elevating the heels by 5' significantly
increased DIP pressure.""3
3.5 Age

Advancing age is the most significant risk factor for 0A in humans.111 In
horses, however, OA is known to develop in animals as young as two years of
age. Young performance horses are most likely to develop OA early in life,
because of the emphasis on racing and showing young horses in futurities and
other events. Training horses at a young age may precipitate damage to joints
unable to withstand the extreme forces they are subjected to during training and
competition.102 Racing and training may accelerate the naturally occurring age-
related changes. In addition, some horses may be genetically predisposed to
developing OA due to either age or training, while other horses may never be
prone to the disease.10

Pathologic and arthroscopic examinations have shown that OA is
commme observed in the joints of older horse.7"°'°5'"2 Naturally occurring OA
also becomes more severe with age in untrained wild horses.10 Increased
severity of lesions is correlated with subchondral bone sclerosis and ossicles with

26

increasing age. Age is also a significant cause for the prevalence of 0A in
Icelandic Horses.°6'"3 Many studies have described surgical treatment of horses
diagnosed with OA ranging from the age of one year up to the age of 21.

Similar to humans, as the horse ages, the biochemical properties of
articular cartilage change. Several recent studies have investigated the effect of
age on the biochemical characteristics of equine articular cartilage. Variations in
biochemical characteristics of cartilage in relation to site and age showed no
significant change in cartilage collagen between horses ranging from age 4 to 30
years old, but indicated that nonenzymatic crosslinking was higher in older
horses and was linearly related to age.” A steady increase in pentosidine
crosslinking increased with age from 5 years onward resulting in a 10-fold
increase up to the age of 30 years. Crosslinklng of articular cartilage by
nonenzymatic glycation (NEG) is expected to result in stiffer, more brittle tissue
that is more vulnerable-to damage by mechanical loading. Nonenzymatic
crosslinking during aging may predispose older horses to development of OA.

The biochemical characteristics of articular cartilage in mature cartilage
differ from those of immature cartilage at different sites on the joint surface. No
significant differences in water content and hydroxylysylpyrodinoline crosslinks
were found at two different sites of the MCP joint in neonatal, 5-month-old, and 1-
year old horses.83 However, differences in DNA, GAG, collagen and
hydroxylysine content between sites paralleled those shown in the mature
horse.114 In a more recent study, the same researchers investigated the

influences of age and exercise on the biochemical characteristics of articular

27

cartilage."5 Neonatal foals showed no site specific biochemical heterogeneity in
contrast to mature horses. The process of formation of site differences was
almost comNeted in exercised foals at age 5 months, but was delayed in those
deprived of exercise. These researchers concluded that the functional
adaptation of articular cartilage to mechanical loading occurs during the first 5
months postpartum, and that a certain amount of exercise is required to sustain
this adaptation. Joints of horses less than two years of age had significantly
higher cell numbers, total collagen, and DNA content, and lower PG content
relative to mature horses ranging in age from 2 to 20 years old."° No significant
difference in these measurements was found within the mature age groups.
Another study has reported no significant difference in collagen or GAG content
in mrtilage derived from horses 2 to 5 years of age.117

Two specific GAGs, which are the main GAGs found in aggrecan, have
been linked to age—related changes in equine articular cartilage. Chondroitin
sulfate, the most abundant GAG in aggrecan, and keratan sulfate (KS), the most
widely distributed GAG in aggrecan, have both been reported to change with
age.

The sulfation patterns in CS chains affect specific properties and functions
of these molecules. Cartilage degeneration in the MCP joints of racehorses was
accompanied by deposition of CS chains with altered sulfation pattems."° Six-
sulfation of internal and terminal CS residues increased with age. The same

phenomenon has also been reported in human studies.119

28

High KS concentrations were reported in foals from 1 week after birth to 3
months of age.‘2° These values decreased rapidly from 3 to 5 months, and
gradually reached adult values between the ages of 5 to 18 months. This pattern
also has been reported in children121 and puppies.122 Todhunter123 had a similar
finding, and reported a significant relationship between age of foals and plasma
KS concentration. Mean plasma KS concentration peaked when foals were 10
weeks old. Age affected KS concentration in the synovial fuid of 32 clinically
normal horses. However, no significant effect of age on plasma KS
concentration was seen in normal adult horses with a mean age of 65 months.
An earlier study also reported no age-related changes in synovial fluid KS

concentrations in mature horses ranging in age from 8 to 30 years old.124

4. Diagnosis

The clinical signs of 0A in the horse vary with the type and degree of OA
as well as with the amount of acute inflammation.125 The ultimate goal in
properly treating horses with joint disease is to make an accurate and early
diagnosis, institute appropriate treatment, and prevent ongoing deterioration of
joint tissues.17

Clinically, CA is characterized by pain and disfunction of the joint. There
have been various interpretations of OA in the horse; as a result, it has been
found necessary to divide equine OA into 3 different classifications.17 Table 2
describes the classifications of OA in the horse. Originally, Mcllwraith125

classified OA into 5 different types; however, this author later found it more

29

appropriate to combine 2 of the original types while eliminating another. Type 1
OA typically affects young racehorses in highly mobile joints (carpal or MCP),
and also involves high-load-low-motion joints such as the interphalangeal and
intertarsal joints in both mature and young horses (ring-bone and bone spavin).
Cases that develop secondarily to primary joint problems have been classified
under Type 2 OA.

 

TABLE 2: CLASSIFICATION OF OSTEOARTHRITIS IN THE I'IORSE17

 

Type 1 Associated with synovitis and capsulitis (common in carpus,
fetlock, and distal tarsal and distal interphalangeal joints)

Type 2 Associated with (and usually secondary to) other identified
injuries or problems including intra-articular fractures,
traumatic articular cartilage ligamentous injuries,
osteochondritis, subchondral bone injury and disease, sub-
chondral cystic lesions, septic arthritis, and fragmentation
of distal patella

Type 3 Incidental or nonprogressive articular cartilage erosion

 

This includes intraarticular fractures, unresolved osteochondrosis, tarsal bone
collapse, distal palmar metacarpal lesions, and septic arthritis.17 Type 1 and 2
can overlap. Type 3 OA may be recognized during routine necropsies, and
includes a series of changes in the cartilage that are of questionable clinical

significance. These changes do not always correlate with clinical problems, and

30

can be associated with degeneration of the articular cartilage with age or trauma
to the joint, which results from continued use.

Clinical signs of OA in high-motion joints with acute inflammation include
lameness, heat, swelling of the joint, pain on flexion, and decreased motion.
Pain is detected either as a painful withdrawal response on joint palpatation or
joint flexion, or as lameness when the horse is jogged in-hand or exercised.
Horses affected with OA usually show greater lameness when worked on a hard
surface or in circles, and often elicit pain upon flexion and rotation of the digits.
However, even in clinically affected joints, a relationship between the amount of
lameness and the cartilage degeneration is not easily established.126 Joint
enlargement, which is associated with fibrous tissue deposition or bone
enlargement, also occurs in more chronic cases of OA, but acute inflammatory
signs may persist to various degrees. The most prominent signs of OA in low
motion joints are joint enlargement and exacerbation of the lameness with
flexion.7
4.1 Imaging

An increased understanding of clinical findings, appreciation of diagnostic
local anaesthesia and improvements in technology have facilitated more
accurate and earlier diagnosis of OA in the horse. Imaging plays a major role in
the diagnosis, and is a critical diagnostic tool for identifying the cause, location,
extent, and severity of lesions associated with OA. Radiography was the earliest
and is currently the most widely used diagnostic imaging modality for detection of

equine OA.127 There have been several reports on the incidence of degenerative

31

i 93,128,129 I 130.131 l“5132-134

joint disease of the tarsa carpa and pastem joi as
assessed radiographically. Radiologic signs of OA include a narrowed joint
space, widened joint space (when there is destruction of the subchondral bone
plate), periarticular osteophytes, and soft tissue swelling. For most lamenesses,
the traditional methods of careful examination, selective local anesthesia, and
radiography provide sufficient diagnostic information for prognostic and treatment
purposes.”8 Unfortunately, a significant group of horses develop an obscure,
mild to moderate impairment of locomotory performance for which the traditional
methods of examination are unreliable. A poor correlation between the
radiographic and arthroscopic assessment of degenerative joint disease has
been reported.‘3°"3"135 Therefore, combining a number of newer imaging
methods with conventional radiography may greatly improve diagnostic accuracy
and provide considerable insight into the pathogenesis of OA.‘2°"35"3°

Ultrasound (US), nuclear medicine (NM), therrnography, computed
radiology (CR) and tomography (CT), and magnetic resonance imaging (MRI)
offer the potential for a more accurate and detailed diagnosis of OA. Choosing a
particular imaging procedure requires not only an understanding of the relative
strengths of available techniques, but also an appreciation of the clinical criteria
needed to chose the appropriate imaging technique for a particular case most
efficiently.126

Ultrasound is a useful noninvasive imaging modality that provides images

of periarticular soft tissue structures, such as the joint capsule and articular

surface. It has been used extensively in the evaluation of equine lameness. A

32

major advantage that US has over other imaging procedures is its real time
dynamic capability.137 Comparison views can be taken immediately, and, if
necessary, treatment decisions can be made without delay.

The field of equine NM is developing rapidly.138 This technique involves
administering a radioactively labeled substance to a patient, and quantifying the
emitted radiation outside of the body by a scintillation (or gamma) camera. The
resulting image is a record of the differential uptake of labeled substance in
various parts of the skeleton including soft tissue.

Thennography is an imaging modality that uses infrared radiation emitted
from the skin to produce a visual image. The skin surface temperature reflects
changes in circulation and deeper tissues such as those resulting from synovitis.
Joint inflammation produces characteristic thermal patterns and studies are
underway to attempt to correlate the inflammatory response with the gross
damage to the joint.139 Thennography may be able to locate inflammation before
clinical signs are evident, and as a result, training programs can be changed to
reduce stress on the inflamed area.

Computed radiography produces a radiographic image from digital
information similar to that of conventional radiography, while CT uses digital
information to provide an image in the third dimension. An advantage of CR is
the ability to alter the imaging algorithm and produced improved edge
enhancement and wide contrast latitude.“0 Computed tomography scanning has

excellent contrast resolution and demonstrates subtle differences in bone or soft-

33

tissue density displayed in cross-section, which cannot be attained by
radiography.‘2""“1

Magnetic resonance imaging is a noninvasive effective technique that
provides very good soft tissue and intraarticular information, information which is
presently only available using arthroscopy.”mm“1 Magnetic resonance
images are proton images, mainly of hydrogen nuclei, with high contrast and
spatial resolution. This technique provides exceptionally good anatomic,
pathoanatomic, and pathophysiologic information of intraarticular and
periarticular structures. Both bone and soft tissues can be imaged with high
contrast and sections can be made in any plane.141 The development of this
technique for use in equine limbs is required in order to validate its cost and use
in providing a beneficial technique for clinically defining OA.7"2°"3°"‘°"‘2"‘3
4.2 Arthroscopy

Development of equine arthroscopy in the late 1970s was one of
the major revolutions in equine surgery, and has been referred to as the ‘gold
standard’ for detecting articular derangement‘35"“ Arthroscopy is a useful
diagnostic technique that, when used in conjunction with traditional diagnostic
methods enables the examiner to make a more accurate and detailed diagnosis
of OA.

The acceptance of arthroscopy as a primary method of treatment of
articular disease in the horse has resulted from its use in removing osteochondral

fragments and the treatment of articular lesions via minimally invasive

arthroscopic portals. This technique enables evaluation of the nonosseous

tissues of the joint, including synovial membrane and associated villi, articular
cartilage, intraarticular ligaments, and menisci. In addition, the use of the
arthroscope in the horse is used to make an assessment of articular cartilage
when radiographic signs are equivocal or nonexistentm'146 The principal
advantages of arthroscopy as a diagnostic surgical tool are: 1) the greatly
increased accuracy with which a joint can be evaluated, and 2) all types of
surgical manipulations can be performed through the small stab incisions with
decreased postoperative morbidity, decreamd convalescence time and improved
performance.“7

Diagnostic arthroscopy Is a relatively atraumatic technique and can be
used on multiple joints during a single period of anesthesia. Initially, arthroscopy
was restricted to the carpus and fetlock joints, but in later years, it has been used
to investigate and treat the shoulder, elbow, DIP (coffin joint), hock, and stifle
jointsm'm

The arthroscopic procedure consists of introducing an arthroscope into the
fibrous joint capsule through a cannula placed in a 5 mm skin incision.“9 Interior
examination of the joint is performed using the arthroscope and the aid of light
source, lens, and video technology. Introducing the arthroscope into the proper
site is critical, because if it is inserted in the wrong area, its maneuverability can
become limited and the arthroscope or the instruments may be placed through

sites or structures of undesirable penetration.

35

4.3 Synovial Fluid

Synovial fluid analysis in combination with clinical and radiologic
examinations is a valuable tool in determining the cause and probable duration of
OA in the horse.’"""’“”2 Although analysis of the synovial fluid alone will not
render a specific diagnosis, it can give you an indication of the degree of
synovitis and metabolic derangement within the joint.

Synovial fluid has an essential role in the nutrition and lubrication of
articular cartilage.153 This protein-containing dialysate of blood plasma has
hyaluronan (HA) widely distributed throughout it, which acts as a boundary
lubricant of the synovial membrane and gives synovial fluid its viscosity.‘25"5"‘5‘
Alterations in the concentration of HA in the synovia may indicate a functional
abnormality of the synovial membrane. Hyaluronan content in horses with OA is
similar to values found in normal synovial fluid; however, animals with septic or
infectious arthritis have a reduced hyaluronan concentration.‘5“""""“"5

Routine examination of synovial fluid should also include determination of
appearance, volume, clot formation, sugar content, microscopic character of the
sediment, and cytologic properties.151 Normal synovial fluid in the horse is pale
yellow, clear, and free of flocculent material. Horses with OA have clear to pale
yellow and opaque colored fluid in conjunction with signs of flocculent material in
the fluid sample. Synovitis leads to increased formation and decreased
absorption of synovial fluid, resulting in increased synovial fluid volume and

distention of the joint capsule.”6 Total volume of aspirated synovial fluid from

joints with synovitis is generally increased. Fluid volume will vary in direct

36

proportion to the size of the joint, and younger horses often contain a

'5‘ Although normal

proportionally greater volume of fluid than mature horses.
equine synovial fluid does not clot, joints moted with acute to subacute
traumatic arthritis clot rapidly. Synovial fluid sugar content in horses with OA
decreases with increased inflammation. In addition, cartilaginous fragments
increase in fluid from joints affected with 0A or traumatic arthritis.°'“""156 A
marked increase in protein is shown in acute synovitis, but this increase may only
be slight in chronic OA. Increased synovial membrane permeability and altemd
joint metabolism are reflected by an increase in serum enzymes in the synovial
fluid.‘5° Enzymes generally affected are alkaline phosphatase, lactate
dehydrogenase, aspartate aminotransferase, and alkaline dehydrogenase.
Additional analysis of synovial fluid includes the use of synovial fluid to
assess GAG concentration, synovial fluid cytokines, and eicosanoids as markers
of joint disease of the horse. Horses with OA have increased synovial fluid GAG
levels when compared to normal horses.”157 High levels of GAGS in the
synovial fluid of horses with OA presumably reflects the rate of PG loss from the
articular cartilage during the disease process.‘57"5° In addition, a large increase
of specific GAGS such as CS may reflect alterations in the synthetic or
degradative process of both the articular cartilage and synovial membrane since
CS is a ground substance GAG common to both tissues.87 Furthermore, high
levels of KS indicate the rate of aggrecan degradation in articular cartilage within

the joint.1259 Bertone160 evaluated the value of various synovial fluid cytokines

and eicosanoids in the diagnosis of joint disease. The results of this study

37

indicated that IL-6 was the most sensitive and specific for joint disease when
compared other factors such as tumor necrosis factor-a (T NF-ot), IL-1B, PGE2,
thromboxane2 (TXB2), prostaglandin F1-a(PGF1-a), and leukotriene B4 (LTB4).
Prostaglandin E2 levels were also found to be useful as a functional screening

test for the presence of joint disease.

5. Therapy

In recent years, significant advances have been made in the ability to
diagnose arthropathies in the horse. Although successful treatment of
established equine OA has not progressed to the same extent, a rapid
advancement in knowbdge of articular structure, physiology and pathology has
facilitated development of effective OA therapies. Osteoarthritis is the result of a
number of different pathologic processes and the choice of treatment and its
effectiveness depends on the stage of the disease and the degree of active
inflammation present.112 The goals of any treatment for DA include: 1) decrease
pain; 2) improve function and/or range of motion; and 3) minimize or reverse the
progression of the OA process.161 Several of the primary methods of treatment
of equine OA will be discussed below.
5.1 Rest

It is well recognized that rest, limited motion and physical therapy are the
most obvious and perhaps most essential contributors to the normal return to
function without further cartilaginous damage to the equine joint.27 Rest, either

alone or in conjunction with other forms of therapy, has long been proposed as a

38

treatment for OA.27"’5°"""'“‘3 However, the economic limitations of the racing and
showing industries often prevent or limit this option.

Complete joint immobilization has been shown to promote articular
cartilage degeneration.2"°°"°°"°‘ However, discontinuance of hard work is
essential in the management of OA. Richardson and Clark” demonstrated that
normal equine joints can tolerate at least 4 meks of cast immobilization without
significant irreversible change to the articular cartilage.

The extent to which rest has on the resolution of cartilage erosion or bony
proliferation is uncertain.7 Stall rest is the most common form of rest
recommended in horses. The amount of stall rest depends on the degree of
articular cartilage damage; however, 60 days is the minimum amount of recovery
time for the repair of soft tissues and the return of a horse to training.‘°5 Hand
walking is often recommended in conjunction with stall rest, and paddock rest
following stall rest is the traditional method used before an arthritic horse is
returned to active training.

5.2 Arthroscopy Surgery

Arthroscopic surgery has revolutionized equine joint surgery and has
become a routine method of joint surgery for nearly all conditions, including
OA.“° Surgery consists of the removal of bone chips and debri, deep curettage
of the cartilaginous defect into subchondral bone in order to encourage a good
healing response from the specific region, production of a defect with vertical
rather than sloping edges, and conservative smoothing and removal of new bone

from the dorsal surface of the bone.‘6 Although arthroscopy originally started as

39

a diagnostic tool, techniques for doing surgery under arthroscopic visualization
developed and virtually all joint surgery is now done ralrthroscopically.131

Several studies have documented arthroscopic removal of chip fractures
and cartilage fragments. Debris from osteochondral or cartilage fragments can
cause mechanical damage to the articular surface in addition to initiating an
inflammatory response in the affected joint."""""3 Severe compromise of the
articular surface leads to instability, as does tearing of fibrous joint capsule and
ligaments.17 Surgical removal of these fragments is a means of preserving the
economic value of the horse, and may allow a rapid and successful return to
performance.

Osteochondral chip fractures of the carpus and the proximal phalanx are
primarily a problem in racehorses.‘:"'"“"172 Arthroscopic removal of chip
fractures decreases lameness and increases the likelihood of racehorses to
resume racing. Chip fractures are considemd to cause pain through tugging on
synovial membrane attachment, inducing synovitis due to release of debris, and
by causing damage to the apposing articular surface (kissing lesions).173 All of
these factors lead to the cyclic process of OA.

After arthroscopic surgery, 63% percent of 87 Standardbred racehorses
returned to preoperative racing levels after surgical removal of osteochondral
fragments of the proximal phalanx,170 while 74% of 176 returned to racing
following carpal chip fragments.172 Houttu‘" operated on 45 horses of which 42
were Standardbred trotters. This researcher reported that 51% of the horses

returned to speed training in 3 months and 91% retumed to speed training in 6

40

months. Additionally, a study demonstrating removal of chip fractures in
Standardbreds and Thoroughbmds resulted in 80% of treated horses returning to
racing, and 68% of these horses raced at a level of competition equal to or better
than the pre-injury level.167

Studies in Quarter Horses and Thoroughbreds have also shown promising
results. Eighty-nine percent of Thoroughbreds raced after arthroscopic removal
of fractures of the proximal phalanx and 82% did so at the same or higher
class.168 An Australian study reported that 76% of Thoroughbreds returned to
racing following surgery, however, 48% failed to earn in more than $1,000 in
post-operative races."1 Carpal fracture removal in Quarter and Thoroughbred
horses resulted in a 68% return to racing that was at a level equal to or better
than their pre-injury level.131 The severity of articular cartilage damage also had
a detrimental effect on horses’ return to racing. This study also evaluated
degradation of the articular cartilage and separated damage into 4 grades,
classifying grade-1 as the least severe OA and grade-4 the most severe; 71.1%
of grade-1 damage, 75% of grade-2 damage, 53% of grade-3 damage, and 54%
of grade-4 damage horses returned to racing at a level equal to or better than
their pre-injury level.
5.3 Joint lavage and synovectomy

Two treatments associated with synovitis and septic arthritis are joint

lavage and synovectomy. As noted above, cartilage breakdown products can

promote or induce synovitis. Both of these treatments have potential benefits of

41

lowering the level of deleterious factors such as MMPs, PGE2, cytokines, and
free radicals.112

The pathologic changes associated with many arthroses and the
accompanying mechanical destruction of the articular surfaces produce
significant debris within the joint capsule.9 Joint lavage was initially proposed to
remove cartilaginous debris that caused synovitis.“ This technique is often
used in conjunction with arthroscopic surgery to remove debris from the joint
following surgery.

The rational for synovectomy is to remove the inflamed, hypertrophic
synovial membrane in an attempt to decrease the production and stimulation of
cartilage destructive enzymes.” Removing the synovium is advantageous in
severe inflammation, but removal of synoviocytes may be detrimental to synovial
physiology and may alter and induce changes in articular chondrocyte
metabolism."5'"°
5.4 Arthrodesis

Arthrodesis is a technique that was first described for the treatment of
bone spavin by Adams,128 although May and Larsen had used a similar
technique with success prior to this point.”7 At this time, numerous types of
therapy to treat bone spavin had failed, and horses affected with bone spavin
remained lame and resistant to all methods of therapy. Bone spavin may later
result in ankylosing arthritis of the involved joint, which is a slow progress and

may not establish complete immobility of the joint.128 Treatment by surgical

arthrodesis is an internal fixation of the joint that is aimed at eliminating any

42

motion of the joint thereby decreasing pain and lameness. The arthrodesis
procedure involves removal of the articular cartilage, internal fixation with screws,
or with a bone plate, followed by external immobilization with a cast for 3 to 5
weeks. Although this procedure is most commonly used to treat bone
spavin,°""""‘7"""9 it also is effective in the fixation of the pastem joint,‘32"3‘ coffin
joint‘°°'133 and carpus.173

Arthrodesis of the joint is often accompanied by a prolonged convalescent
period that can require up to 1 year before soundness can be evaluated.“
Success rate for horses returning to performance following treatment with
surgical arthrodesis has varied. Caronm showed that the success rate for return
to serviceability following arthrodesis of the proximal interphalangeal joint was
approximately 46% for the forelimbs and 83% for the hind limbs. A more recent
study of the proximal interphalangeal joint in 34 horses indicated a successful
outcome in 85% of forelimbs and 89% of hindlimbs."31 Surgical arthrodesis in so
horses for the treatment of OA of the proximal intertarsal, distal intertarsal and
tartsometatarsal joints revealed that 78% of horses treated in the distal intertarsal
and tarsometatarsal and 55% in the proximal intertarsal became sound following
surgery.93 Surgery of the distal intertarsal and tarsometatarsal joints in 17 racing
Standardbreds and Thoroughbreds resulted in a 71% success rate as
determined by clinical examination or communication with clients.“ All
Standardbreds and 67% of Thoroughbreds in this study had successful

outcomes.

43

Complications of arthrodesis as reviewed by Mcllwraith” include implant
breakage or loosening, bent screws, fracture of the proximal phalanx, infection,
laminitis, toe elevation, DJD of the distal interphalangeal joint, and navicular
abnormalities. The definition of successful outcomes also vary from study to
study, and depending on the opinion of the author, certain degmes of
improvement may or may not be classified as a successful outcome.

5.5 Conventional Medications

Medications used to treat joint disease can provide relief by decreasing
pain, acting as anti-inflammatory agents, and contributing chondroprotection.
The term chondroprotection implies the maintenance, or restoration of normal
homeostatic mechanisms to protect cartilage from injury.” Many drugs have
been termed chondroprotective by their function of either enhancing cartilage
repair or retarding cartilage degradation. Non-steroidal anti-inflammatory drugs
(NSAIDS), corticosteroids, HA, polysulfated glycosaminoglycans (PSGAGs), and
glucosamine and chondroitin sulfate have all been used in the treatment of OA.
These drugs have been extensively reviewed by Mcllwraith and Trotter in Joint
Disease in the Horse,183 and recent investigations of the effects of these
chondroprotective drugs will briefly be described here.

NSAIDS

Nonsteroidal anti-inflammatory drugs provide anti-inflammatory and

analgesic effects to acutely inflamed joints. The mechanism by which NSAIDS

exhibit their anti-inflammatory effect is through inhibition of cox.“"'-"’5

Cyclooxygenase is the first of a series of enzymes that convert arachidonic acid
to prostaglandins, and all NSAIDS inhibit COX activity to some degree.

The most common NSAIDS approved for use in the horse include
phenylbutazone (PBZ), flunixin melamine (FNX), and ketoprofen men.“ Other
NSAIDS that have been evaluated for their anti-inflammatory properties in the
horse include carprofen (CAR), meloxicam (MEL), diclofenac, acetylsalicylic acid ‘
(ASA), naproxen, and indomethacin. Phenylbutazone has been shown to
decrease PGE2 concentration in equine articular cartilage explants and
chondrocytes,”49 suppress PG loss,183 and function as a more potent inhibitor of
cox-1 than cox-2.180 Carprofen was the weakest inhibitor of cox-2 when
compared to PBZ, FNX, KET, diclofenac, indomethacin, and MEL in the whole
blood of horses, dogs and cats, while PBZ and FLX were more potent overall in
inhibiting cox-1 than cox-2.18° This is in agreement with Tung‘9 who
demonstrated that PBZ had no effect on the expression of COX-2 in equine
chondrocytes; however, PBZ did inhibit COX-2 activation. Phenylbutazone had
no effect on inhibiting the expression of iNOS or influencing nitrite concentrations
in cytokine-stimulated equine Chondrocyte cultures.”7 Diclofenac, ASA,
indomethacin, MEL, and naproxen profoundly differ in their ability to modulate
proteolytic activities by articular chondrocytes.37 All of these NSAIDS were
effective in inhibiting collagenase activity in lL-1 stimulated bovine chondrocytes;
however, only indomethacin, MEL, and naproxen reduced transcript levels of
MMP-1.23 In addition, indomethacin and MEL specifically reduced
proteoglycanase activity; however, no NSAID had an effect on TIMP activity or

45

TIMP-1 biosynthesis. Neither PBZ or FLX have been shown to regulate MMP-2
or -9.31
Corticosteroids

Synthetic analogs of natural corticosteroids possess potent anti-
inflammatory activity and are commonly injected intra-articularly for local relief of
inflammatory lesions in performance horses?“ However, they are also anti-
catabolic, which can delay healing of injured tissue. Corticosteroids prevent
inflammation through an interaction in which they bind to steroid-specific
receptors in the cellular cytoplasm of steroid-responsive tissues. The
corticosteroid binds to the receptor, resulting in a change in the allosteric nature
of the receptor-steroid complex, and ultimately, directing the synthesis of new
proteins."35 Similar to NSAIDS, corticosteroids predominantly function by
inhibiting prostaglandin production.”"°"° Additionally, their beneficial effects
may be mediated by inhibiting cartilage MMPs, cytokines such as IL-1 and TNF—
or, and by enhancing synthesis and release of TlMPs.3"‘85"°°"°°

Although corticosteroids seem beneficial in preventing the inflammatory
response involved in OA, the deleterious effects of these drugs on articular
cartilage have been questioned. Detrimental effects of infra-articular
corticosteroids on articular cartilage include: 1) decreased cartilage elasticity and
GAG content with progressive degeneration of the cartilage; 2) formation of
calcium deposits on the hyaline surface; 3) thinning and fissuring of the cartilage,
and 4) decreased viscosity and HA content of synovial fluid.‘°‘ The doses of

corticosteroids achieved in a joint after intra-articular administration are often

46

many orders of magnitude greater than required to produce a cellular effect in
vitro.‘°° This leads to the question whether inappropriately high doses of these
drugs are used in joints and if previously reported deleterious articular effects of
corticosteroids may, in part, be due to incorrect closing.“""'“’°'"’o
The best corticosteroid drug and the appropriate dose rate remain to be
elucidabd. Corticosteroids approved for intra-articular use in the horse include
cortisol, methylprednisolone, triamcinolone acetonide, betamethisone,
isoflupredone, and flumethasone.‘“ Recent in vivo studies on corticosteroids in
equine articular cartilage have focused on their effects on PGE2 synthesis and
gene expression. Betamethasone, dexamethasone, and methylprednisolone
have been evaluated for their possible inhibitory effect on the activity of MMP-2
and -9 in equine synovial fibroblasts and peripheral blood neutrophils.31
Betamethasone had no inhibitory activity on either MMP, while higher
concentrations of dexamethasone and methylprednisolone significantly inhibited
MMP-2 activity. In spite of this, the high doses used in this study are unlikely to
be achieved in vivo for any length of time. High concentrations of
methyprednisolone and betamethasone inhibited PG synthesis; however,
synthesis was increased with a lower concentration of methylprednisolone, while
it remained unchanged with a lower concentration of betamethasone.”"°9
Methylprednisolone has been shown to have similar effects on PGE2 synthesis.
Prostaglandin E2 production was suppressed by low concentrations of
methylprednisolone, while higher concentrations had no effect.48 In addition,

methyprednisolone acetate decreases type-ll procollagen synthesis."38

47

Dexamethasone (DEX), another potential anti—inflammatory corticosteroid,
significantly reduces PGE2 production in equine chondrocytes.‘9 It is also
involved in pre-translational regulation of COX-2 and iNOS gene in equine
chondrocytes.“""’7 Lower concentrations of dexamethasone have also been
found to reduce transcript levels of MMP-1, decrease the expression of MMP-3,
and inhibit collagenase and proteoglycanase activity."‘7
Hyaluronic Acid

Hyaluronic acid belongs to the category of local anti-inflammatory drugs.
It is a weak acid, nonsulfated polysaccharide that is synthesized by special cells
of the synovial layer of the joint capsule and is responsible for the high viscosity
of the synovial fluid. Hyaluronate was first isolated in 1939 from bovine synovial
fluid.156 Large quantities of HA are obtained for experimental and commercial
use from umbilical cords, rooster combs, and bacteria. Administration of purified
HA restores the permeability barrier restricting the flow of leukocytes and active
plasma components into the joint, and mduces the effusive component of the
inflammation process.191

Intra-articular injection of HA (HYVISC®) resulted in the highest clinical
improvement (87%) in horses diagnosed with OA compared to arthritic horses
treated with rest, intra-articular corticosteroid injections, NSAIDS, systemic anti-

‘“ Lameness was

inflammatory agents, physical therapy, or counterirritation.
greatly improved showing 54% of HA treated horses having a lameness score of
zero post-treatment, while only 1% of these horses had been diagnosed with a

lameness score of zero pretreatment. A study using amphotericin B to induce

48

synovitis had conflicting results to the previous study, and indicated that intra-
articular Hylan® had no effect on lameness grade, total nucleated cell count,
total protein, or polymorphonuclear cell count in the synovial fluid.192 However,
horses injected with HA following surgically induced Chip fractures to produce
lameness, had a highly significant increase in weight bearing on the treated
limb.‘°3"°‘ Horses with unilateral front limb lameness also showed improved
bilateral weight bearing.‘°‘ In vitro, IL-1 stimulated canine explants had reduced
PG degradation following treatment with HA compared to untreated lL-1
explants.195 A more recent study showed that HA had no effect on the
expression of iNOS or nitrite content in cultured equine chondrocytes.187 In
addition, HA treated equine chondrocytes showed no significant effect in the
reduction of the gene expression of COX-2.49 However, high concentrations of
HA inhibited PGE2 synthesis in LPS stimulated equine synoviocytes.196

Hyaluronic acid has also proved to be effective in rabbit models in vivo.
Rabbit knees pre-treated with HA followed by F n-f injection had significantly less
decrease in PG content compared to Fn-f injection alone.197 Similar results were
found in bovine articular cartilage explants.“ lntraarticular hyaluronan down
regulated MMP-3 and lL-1B,‘°9 and suppressed NO production2°° in New
Zealand rabbits.
Polysulfated GAGs

Polysulfated GAG agents are a group of biosynthetic compounds
composed of repeating units of hexosamine and hexuronic acid designed to re-

establish normal joint conditions via their inhibition of lysosomal and MMP

49

enzymes, stimulation of HA synthesis, and proposed chondroprotective effect of
diffusing into the articular cartilage superficial matrix and acting as a replacement
GAG.201 The routes by which PSGAGs are administered include intraarticular,
intramuscular, and intravenous injection. Studies investigating the effects of
PSGAGs on inhibiting articular cartilage degradation have been controversial,
and their effect on Chondrocyte metabolism may be explained by the lack of
consistency between different experimental designs.“"2°2'203
A survey completed by equine practitioners to evaluate the perceived
efficacy of PSGAG, discovered that practitioners found PSGAG to be more
effective than sodium hyaluronate for the treatment of subacute degenerative
joint disease?“ In addition, practitioners found the efficacy of PSGAG for
incipient and chronic forms of degenerative disease compamd favorably to
sodium hyaluronate. Polysulfated GAG was not proven to be chondroprotective
in equine joints of ponies injected with methylprednisolone201 or equine explants
in culture obtained from mildly osteoarthritic joints?05 However, a previous study
indicated increased rates of cartilage-specific type-ll collagen and CS-rich GAG
synthesis following treatment with PSGAGs.2°‘5 The varying outcomes of these
studies may have been due to the use of different experimental designs to
determine the efficacy of PSGAG. Prostaglandin E2 synthesis was decreased in
LPS stimulated equine synoviocytes by concentrations of PSGAG similar to
those estimated to be obtainable by intra-articular injection.196 Polysulfated GAG

also reversed the concentration-related suppression of PG synthesis induced by

lL-1B in equine chondrocytes.” In addition, keratan sulfate concentration was

50

found to be decreased in synovial fluid from non-exercised ponies with
Chymopapain induced arthritis following treatment with PSGAG.” This indicated
that, in nonexercised joints, medication with PSGAG may have decreased either
release of KS from the articular cartilage into the synovial fluid or inhibited
synthesis of KS. Although intra-articular PSGAG combined with exercise in
ponies with OA induwd a more fibrous repair tissue than tissue from
nonmedicated joints,205 PSGAG ameliorated the soft tissue swelling and bony
Changes of OA in the exercised joints of ponies.209 It also significantly reduced
iNOS gene expression and nitrite concentrations in cytokine-stimulated
cultures.187 Polysulfated GAG did not, however, have any effect on inhibiting the
activity of MMP-2 or -9.2‘°
Glucosamine and chondroitin sulfate

The combined use of the nutraceuticals GLN and CS in the treatment of
OA has become an extremely popular supplementation protocol in arthritic
conditions of joints. Glucosamine is an amino-monosaccharide that is
biosynthesized endogenously by animals and humans from glucose and
glutamine.211 It plays an important role in the biochemistry of cartilage as it is a
fundamental molecule for the synthesis of GAGs and HA which make up the
foundation of aggrecan.212 Absorption of GLN is very high (about 90% in
humans), and articular cartilage concentrates glucosamine to a greater extent
than any other structural tissue.213 Chondroitin sulfate is a long chain polymer of
a repeating disaccharide unit, and is the predominant GAG found in articular

cartilage. The metabolic fate of CS is complex due to the variability in molecular

51

weight, Chain length, electrical charge distribution, location of sulfate groups, and
percentage of similar disaccharide residues.213 However, disaccharides formed
specifically from the breakdown of CS have been found in plasma samples of
horses following oral dosing of CS.""“'215

Oral GLN decreases pain and improves mobility in humans with OA;2“"218
however, the use of GLN as an alternative treatment for OA requires more
attention in the scientific community. Glucosamine sulfate is commonly used in
the treatment of or».219 and recently has been found to inhibit IL-1B-induced NFch
activation in human osteoarthritic chondrocytes in vine.”0 Small-animal
practitioners evaluated a GLN/CS product and rated it good or excellent for the
treatment of OA in small animals and found the product to be safe with minimal
side effects.221 Both GLN and CS were chondroprotective in a rabbit knee
instability model.222 In addition, prior treatment with GLN and CS reduwd
lameness in dogs with induced synovitis.1o

Studies performed in our laboratory have evaluated the effects of GLN
HCL, GLN sulfate, n-acetylglucosamine, and CS in inhibiting cytokine induced
cartilage degradation in equine explants. Glucosamine HCL and glucosamine
sulfate, but not N-acetylglucosamine, prevented LPS and rhIL-1 induced cartilage
degradation in equine explantsm'”4 The addition of GLN HCL prevented
increased NO production, PG release, and MMP activity induced by LPS or rhlL-
1 in equine explants?” A similar study using equine lL-18-stimulated equine
explants demonstrated that GLN HCL inhibited cartilage catabolic responses in a

dose-dependent manner. Stromelysin activity was suppressed at a dose of 0.25

52

mglml, while collagenase/gelatinase activity was inhibited at 2.5 mglml!39 In
addition, a GLN HCL concentration of 25 mglml prevented lL-18-induced
increases in NO production, PGE2 production, and PG release into the media.
Glucosamine inhibited aggrecanase activity in vitro in rat and bovine cartilage ‘
explants?25 An evaluation of the combined effects of GLN HCL and CS on LPS
stimulated equine cartilage explants determined that a low concentration of GLN
(1 mglml) alone was capable of decreasing NO production relative to LPS
stimulated cartilage, while the addition of CS at either .25 or .5 mglml in
combination with GLN did not further inhibit NO production.“ Furthermore, GLN
HCL inhibited PGE2 production, whereas CS had no effect on PGE2. The
combination of GLN HCUCS decreased MMP-9 gelatinolytic activity, but had no
effect on MMP-2. Most recently, it was found that the response of bovine
explants from aged animals to GLN/CS under simulated conditions of stress was
significantly greater than that seen in nonstressed or young tissue.226 In vivo,
lameness in horses with OA was reversed after supplementation with the
GLN/CS product Cosequin®.227 Twenty-five horses with natural OA induced
lameness showed significant improvement in lameness grade, flexion test, and
stride length within 2 weeks following oral supplementation with Cosequin®.
The mechanism by which GIN and CS inhibit mediators of articular
cartilage degradation remains unclear. It has yet to be determined whether the
combination of these two molecules provides an additive or synergistic effect.
Our research suggests that GLN specifically could be regulating cell signaling

molecules, such as NO and PGE2, while the combination suppresses proteolytic

53

activityfit£59 It has been hypothesized from results of in vitro research that GLN
and CS may work by stimulating the synthesis of matrix macromolecules,
specifically type II collagen, aggrecan and HA; decreasing the activity of MMPs
and aggrecanases; and decreasing the production of cell signaling molecules
such as NO and PGE2. However, the mechanism of action of GLN and CS in
vivo is unknown. Furthermore, the in vivo pharmacological concentration and
dosage of the combination of GLN and CS most effective in preventing articular
cartilage degradation and maintaining cartilage metabolism remains to be
elucidated.

Because GLN and CS are considered nutritional supplements, they are
not subject to the same stringent requirements for quality manufacturing as are
pharmaceutical products. An analysis of the GLN and CS content in oral joint
supplement products revealed that only 5 out of the 11 products claiming to
contain either GLN, CS, or a combination of both contained GLN only, while 6 out
of 11 contained CS only.228 The amount of GLN found in the product was
different from that suggested by the label, and ranged from as low as 62.6% to
112.2% of label Claims. The amount of CS was also differed from the content
suggested on the product label, and ranged from 22.5% to over 155.7% of label
Claims. Similar results have been produced in a study analyzing 32 products for
their GLN and CS content.229 In general, both of these studies found that the
least expensive products were most likely to be seriously deficient in meeting the

label Claim.

6. Focus of my research

The use of equine joint tissues provides a valuable in vitro model for
investigation of OA in all species, and tissue from horses of all ages, levels of
joint disease, and athletic performance are available for investigation.

Mechanical loading or trauma to a joint can initiate osteoarthritis (OA) in
horses. The biochemical Changes that occur following loading, which predispose
the horse to OA, require further investigation. Therefore, my first objective is to
determine the effect of a single acute load in vitro on an equine articular cartilage
explant model, and compare its biological response to that of a cytokine (LPS)
stimulated explant model. I will measure the following parameters to identify
whether loading of equine explants results in distinct biochemical changes to
those resulting from LPS stimulation: NO production, PGE2 production, PG
release, MMP-2 and -9 activity, and KS content. The lower dose at which GLN,
in combination with CS, inhibits LPS-induced NO production, PGE2 production,
and MMP activity in equine explant culture is yet to be elucidated. Hence, my
second objective is to determine the lower dose at which GLN and CS combined
are effective in inhibiting cytokine induced OA in equine explants. I will
investigate this relationship by measuring NO and PGE2 production in
conditioned media surrounding the explants, and analyzing explants for MMP-2

and -9 activity.

55

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202. Todhunter RJ, Lust G. Polysulfated glycosaminoglycan in the
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206. Glade MJ. Polysulfated glycosaminoglycan accelerates net
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207. Todhunter RJ, Yeager AE, Freeman KP, et al. Keratan sulfate as a
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208. Todhunter RJ, Minor RR, Wootton JA, et al. Effects of exercise and
polysulfated glycosaminoglycan on repair of articular cartilage defects in the
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209. Todhunter RJ, Freeman KP, Yeager AE, et al. Effects of exercise
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211. Setnikar I, Pacini MA, Revel L. Antiarthritic effects of glucosamine
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213. Kelly GS. The role of glucosamine sulfate and chondroitin sulfates
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214. Eddington ND, Du J, White N. Evidence of the oral absorption of
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217. McCarty MP. The neglect of glucosamine as a treatment for
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225. Sandy JD, Gamett D, Thompson V, et al. Chondrocyte-mediated
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229. Adebowale AO, Cox DS, Liang Z, et al. Analysis of glucosamine
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permeability of chondroitin sulfate raw materials. JANA 2000;3z37-44.

75

CHAPTER 2
THE EFFECTS OF A SINGLE ACUTE LOAD ON AN EQUINE ARTICULAR
CARTILAGE EXPLANT SYSTEM

Abstract

Trauma to a joint is a major initiator of OA Research studying the effects
of trauma on equine cartilage in vitro is limited. Thus, our aim was to evaluate
the effects of mechanical loading on equine articular cartilage explants. Six mm
diameter cartilage discs were extracted from the knee joints of horses. Explants
were equilibrated for 2 days. At the start of the experiment, 2 groups of explants
were subjected to a single load of either 15 or 30 MPa within 50 ms, while a third
group was treated with LPS (10 jug/ml) to induce cartilage degradation. An
additional untreated group of explants served as a control. Nitric oxide and total
PG released into the media were measured on days 1 and 2, while the
concentrations of PGE2 and KS in the media and MMP activity in tissue and
media were measured on day 1. Experiments were replicated 4 times with
cartilage from 4 horses. LPS significantly increased NO production and PG
release similar to the 30 MPa loaded treatment on day 1, had the highest NO on
day 2, and increased PGE2 production the highest over all treatments except the
30 MPa treatment. The 15 MPa treatment was lower in NO and total PG release
on day 2, had lower PGE2 production on day 1 than both the LPS and 30 MPa
treatment, but had the highest KS loss of any treatment. The 30 MPa loaded

treatment had the highest NO and PG release among treatments on day 1, PGE2

76

production similar to LPS, and higher KS loss relative to the LPS and control
treatments. Minimal changes were seen in MMP activity. We have confirmed
that acute trauma does initiate a catabolic response in equine cartilage explants,

and differs to some degree fiom cytokine stress.

Introduction

Wastage due to lameness results in a significant loss of performance and
productivity in the horse industry.” Osteoarthritis is a major cause of lameness
in the horse. Lameness resulting from DA is an principal cause of poor
performance and early retirement of equine athletes."'5 The deteriorating effects
of OA are frequently seen in western performance horses, racehorses, jumping
horses, and other sport horses involved in highly athletic events."6'7 Not only is
this disease prevalent in domestic horses, but it also occurs naturally in the joints
of wild horses.“ Although OA significantly contributes to financial loss in the
equine industry and remains a problem in the horse, our understanding of the
pathophysiological mechanisms involved in this disease is poor.9

Osteoarthritis is characterized by deterioration of articular cartilage
accompanied by changes in the bone and soft tissues of the joint. The end stage
of OA results in a net loss of articular cartilage, causing pain, deformity, loss of
motion, and decreased function. Enzymatic degradation of articular cartilage

precedes morphologic breakdown and plays a central role in OA. Several

77

inflammatory mediators such as lL-l,‘°-11 PGE2,‘2"3 no,“15 and matrix
MMPS‘B'" may contribute to the cascade of events leading to DA in the horse.

Although factors such as increasing age, poor conformation, and improper
shoeing have been shown to contribute to the onset of OA in the horse, trauma
to the joint is believed to be the primary source of equine OA.‘°"° Proteoglycans
are one of the main components of the ECM of articular cartilage, and provide
cartilage with its ability to withstand the compressive deformation associabd with
loading. The PG molecule has GAG highly sulfated disaccharide chains that
attract water into the tissue for cushion and support. Very strenuous exercise
injures articular cartilage by increasing fibrillation of the cartilage and reducing
PG content and quality. The properties of the cartilage become altered and
overload results, which may lead to the development of OA.

Wrthin equine joints, cartilage adapts to load by altering the biochemical
composition of cartilage early in life.”22 Topographical and exercise-related
differences in fibronectin distribution,23 GAG, collagen and DNA content, and
chondrocyte density exist in the composition of equine carpal articular
cartilage?” Changes in these parameters in a load bearing region may
predispose this site to clinical injury such as OA. Decreased PG synthesis has
been observed in the articular cartilage of strenuously exercised horses 2
compared to that of moderately exercised horses?" Severe loading due to
overload trauma may occur in horses that are subjected to extensive and
intensive exercise or speed, experience fatigue, or have poor conformation or

footing. For example, joint pressures in the continuously loaded area of the

78

fetlock joint are relatively low in the standing horse, but may increase up to 6-fold
when loads are applied that can be expected during athletic performance.”

Synovitis is often present following trauma to the joint, and may be the
initial sign of injury in situations of ‘wear and tear".27 A cytokine-induced in vivo
model, using lL-1 or LPS, targeted at stimulating an inflammatory response
similar to that of synovitis in the joint, has frequently been used to study the
pathogenesis of OA in the horse. Cytokine stimulation induces changes in the
ECM of cartilage similar to that of OA, including PG loss, increased NO and
PGE2 release, and synthesis of MMPs. Several experimental in vitro studies
employing an articular cartilage explant model to investigate the effect of trauma
on articular cartilage have been conducted in various species; however, little
research has studied the effects of trauma on equine cartilage. Mechanically
loaded articular cartilage explants studies have indicated that loaded explants
have altered biochemical and physical Characteristics including increased cell
death and matrix damage,”3 PG Ioss,29'30 decreased PG synthesis, and
increased NO production and swelling of the tissue.31

The objectives of this study were to (1) determine the effect of trauma on
equine explants, and (2) compare these catabolic effects to selected indices of
cartilage degradation induced by cytokine stimulation. A recent study,
researching the combined effects of injurious compression and cytokine
stimulation on articular cartilage explants, suggests that these two processes
may induce cartilage catabolism through distinct pathways?1 Therefore, we

hypothesize that the biologic response of equine articular cartilage explants

79

subjected to a single acute load will differ from that of explants stimulated with

LPS.

Materials and Methods

Experimental design

Articular cartilage was obtained from the antebrachio-carpal and middle
carpal joints of 4 horses (1-8 years old) euthanized for reasons other than
lameness. Four separate experiments were conducted using tissue from each of
the 4 horses. Cartilage discs (6.0 mm in diameter) were obtained from the load
bearing region of the joint and were washed for a total of 3 washes in Dulbecco’s
Modified Eagles Medium (DMEM): nutrient mixture F-12 (Ham) (1:1).' Two
randomly-selected discs (approximately 40 mg of cartilage) were placed in each
well of a 24 well Falcon culture plate.b Each well consisted of 1 ml of DMEM/F-
12 (Ham) (1:1) serum free media supplemented with Iinoleic acid (5 pglml),° L-
thyroxine (40 ng/ml),° insulin-transferrin-sodium selenite supplement (ITS) (1
pl/ml)," 50 pg ascorbic acid, 100 units/ml penicillin/streptomycin,‘ and all 20
amino acids.° The explants were maintained in culture in a humidified incubator
with 7% CO2 at 37°C.

Explants were equilibrated in media 2 days prior to the first of 2 treatment
days. There were 6-8 wells per treatment, depending on the amount of tissue
obtained from each horse, and each experiment had an equal number of wells
randomly assigned to 1 of 4 treatment groups: (1) control- no impact, no LPS,

(2) impact- single acute load of 15 MPa within 50 mseC, (3) impact- single acute

80

load of 30 MPa within 50 msec, (4) LPS. An Instron (model 1331, Canton MA)
was used to deliver a peak pressure of either 15 or 30 MPa to impacted explants.
Briefly, specimens were placed between two highly polished stainless steel
plates of the servo-hydraulic testing machine in an unconfined compression,
where peak load, time to peak, and maximum displacement were recorded for
each experiment. After loading, explants were washed 3 times in fresh media
(10 min/wash) and were placed in pro-assigned wells of 24 well culture plates.
Lipopolysaccharide-stimulated explants received 10 pglml of LPS on days 1 and
2 of treatment. Conditioned media were collected and replaced with new media
daily and stored at 4°C until analysis.
Nitric oxide analysis- Nitric oxide (NO) was measured indirectly in the
conditioned media on days 1 and 2 as described previously.32 Nitrite (NO2), a
stable end-product of NO metabolism, was quantified using the Greiss reaction
and sodium nitrite as a standard. Briefly, 75 pl conditioned medium was
incubated with 75 pl 1.0% sulfanilamide, 0.1% N-1-naphthylethylenediamide
dihydrochloride in 25% phosphoric acid at room temperature for 5 min.
Absorbance at 540 nm was determined using the Spectromax 300 plate reader.‘9
Results are expressed as nmol NO2Iwell.
Proteoglycan analysis

Proteoglycan (PG) release into conditioned media was measured on days
1 and 2 as previously described33 using a dimethylmethylene blue assay (DMB).

Briefly, PG content was determined by measuring sulfated GAG content

81

compared to a chondroitin sulfate standard. Results are expressed as pg
PGIwelI.
Keratan sulfate analysis

The DMB assay measures both degraded and newly synthesized PG.
Thus, we also measured KS levels in the conditioned media as a specific
indicator of proteoglycan degradation. KS released into conditioned media was
measured on day 1 using a previously described enzyme-linked immunosorbent
assay (ELISA) with an inhibition step using a monoclonal antibody“ specific for
KS.“ Media was incubated with an anti-KS monoclonal antibody to bind the
antigen to the antibody. A secondary antibody, goat anti-mouse lgG HRP-
conjugated,f was then added to the antigen-coated plate. Color development
was initiated using O-phenylenediaminec and stopped with 2 M sulfuric acid and
read at 490 nm on a Spectromax 300 plate reader. Intensity is inversely
proportional to the amount of KS antigen present in the sample. Samples are
compared to a KS standard (kindly provided by the laboratory of Dr. Eugene
Thonar, Rush University,Chicago, Illinois) and are expressed as pglml.
Cartilage extraction

Matrix metalloproteinases were extracted from articular cartilage using a
modified protocol.35 Following treatment on day 2, explants from each well were
placed in a cold stainless steel mortar apparatus and snap froze with liquid
nitrogen. They were then powdered immediately using a stainless steel pestle
and hammer. Powdered explants were placed in microcentrifuge tubes with stir

bars and 1 ml extraction buffer (50 mmolll Tris HCI, 10 mmolll CaCI2, 2 mol/l

82

guanidine HCI and 0.05% Brij-35: pH 7.5) was added to 2 powdered explants
from each well. Samples were stirred overnight at 4°C and then centrifuged at

18,000 g for 30 min at 4°C. Supematant was dialysed (24 h) against assay
buffer (50 mmolll Tris HCI, 10 mmolll CaCl2, 0.2 mol/l NaCl, 0.05% Brij-35: pH
7.5) using Spectrapor 2 dialysis tubing with a 12-kd cutoff.‘I Dialysis was
continued for 48 h with distilled water. The amount of protein in the extracts was
determined using the Pierce Micro BCA Protein Assayh with bovine serum
albumen as the standard. Extractions were stored at 4°C and immediately

analyzed for MMP activity.

Gel zymography
Matrix metalloproteinase activity in conditioned media and tissue was

detected by gel zymography. Extracts of articular cartilage samples containing 7
to 8 pg of protein (each gel received the same amount of protein) were applied
without reduction to an 8% polyacrylamide gel with 1 mglml gelatin incorporated
as the substrate. Samples were diluted with 4x sample buffer and were
electrophoresed at room temperature. Conditioned media samples from day 1 of
treatment were prepared for gel zymography by diluting samples 1:4 with 4x
sample buffer. A gelatinase A (MMP-2) molecular weight standard served as the
gelatinase control. Following electrophoresis, gels were then incubated in 2.5%
(vlv) Triton X—100 for 1h and then overnight at 37°C in 50 mmolll Tris (pH 7.5)
containing 200 mmolll NaCI, 10 mmolll CaCl2, 10 pmol/l ZnCI2 and 0.02% Brij-35.

The gels were stained with Coomassie Blue R250 for 1 h at room temperature

83

and enzyme activity was measured by scanning densitometry (Gel Doc 2000),i
using Quantity One 4.0.1 software.
Prostaglandin E2 immunoassay

Prostaglandin E2 (PGE2) was measured using a commercially available
competitive enzyme immunoassay kit.j lndomethacin (10 pglml) was added to
conditioned media samples after 1 day of treatment and samples were then
stored at -20°C until analysis. Samples were diluted 10-fold in provided assay
buffer and run in duplicate. Multiple washes were performed to remove excess
conjugate and unbound sample from the plate. Substrate solution was then
added to determine bound enzyme activity and the absorbance was read at 405
nm with a wavelength correction set at 590 nm. A four parameter logistic curve
ranging from 39-5,000 pglml PGE2 was used to determine sample
concentrations. All samples from each PGE2 microplate had acceptable total
activity, nonspecific binding, maximum binding and substrate blanks.
Statistical analysis

Data for indicators of degradation were analyzed using the repeated
measure option of the SAS statistical software PROC MIXED (2001)." Data was
combined by pooling wells from all 4 horses according to treatment. Each group
of treatments using tissue from one animal was considered a replicate (n=4).
Experimental effects of treatment, treatment‘day, and treatrnent*horse were
assessed for the pooled treatments. Differences between effects were compared

using difference of least square means and Tukey's multiple comparison

procedure. All data were presented as mean :I: SEM, and statistical significance

was considered at P<0.05 unless noted.

Results

Loaded and LPS-treated explants had higher media content of NO2 than
untreated controls on day 1, however, this difference was statistically significant
for only the 30 MPa loaded specimens (P<0.05). In contrast, NO2 content on day
2 was higher in the LPS and 30 MPa treatment groups than the control and 15
MPa group, but this difference was only significant for LPS treated explants
(P<.05). The 30 MPa loaded and LPS-treated groups had higher PGE2
production than the control and 15 MPa loaded explants (P<0.05) (Figure 3),
whereas loading at 15 MPa did not differ from the control. Specimens from the
LPS-treated group produced two fold greater PGE2 than the 30 MPa loaded
group.

30 MPa loaded specimens had the highest total PG release among
treatments on day 1 (P<0.05) (Figure 4). In contrast, on day 2 of culture, the 30
MPa loaded and LPS-treated explants had higher PG release than the control
and 15 MPa loaded groups (P<0.05). Loaded explants had the highest KS
concentrations 24 hours post-treatment relative to the control and LPS groups
(P<0.05) (Figure 5). Lipopolysaccharide-treated specimens did not differ in KS

loss from the control group.

85

No differences between treatment groups were detected in MMP-2
activity in the media (Figure 6) or tissue (Figure 7). Similarly, there was no
difference in MMP-9 activity in the media (Figure 8) or tissue (Figure 9).

Discussion

Under normal physiological circumstances, articular cartilage is exposed
to a complex and diverse array of mechanical stresses and strains due to loading
of the joint.36 The results in this study confirm that a single acute load on equine
articular cartilage explants induces catabolic effects as it had in other in vitro
models. We applied two separate loads, a lower load of 15 MPa, and a higher
load of 30 MPa to equine articular cartilage explants. Previous work in our
laboratory has shown that loading at 15 MPa causes slight fissuring and cell
death in the superficial tangential zone of articular cartilage.37 The macroscopic
damage seen following loading at 15 MPa is similar to the damage observed
over time in in vivo trauma models. Loading at 30 MPa causes more extensive
fissuring in the superficial layer and cell death up to 40% throughout the
superficial tangential and middle zone of cartilage.38 Stimulating equine cartilage
explants with 10 pglml of LPS induces a catabolic response in cartilage that
includes increased NO and PGE2 production, increased KS loss, and an up-
regulation of MMPs?“0

Nitric oxide is a major component of the inflammatory response, possibly
mediating pro—inflammatory cytokines.15 Nitric oxide activates MMPs,‘1

42,43

suppresses PG synthesis, and induces apoptosis in human articular

86

chondrocytes. In vitro, equine articular cartilage chondrocytes‘4 and explants39"°
produce significant amounts of NO following stimulation of LPS. In several
studies, NO production was also significantly increased in chondrocytes following
mechanically loading. Bovine chondrocytes exposed to dynamic compressive
strain had decreased N02 production in response to increasing load, while LPS
stimulated chondrocytes had increased production 2.5-fold greater than the
lowest compressive strain.45 Bovine chondrocytes subjected to fluid-induced
shear force also had a five-fold increase in NO release relative to a non-treated
control.‘6 This study demonstrated a decrease in GAG synthesis in response to
shear stress was blocked by NO synthase inhibitors. A similar study shomd a
fourfold increase in NO release in human chondrocytes subjected to the same
type of force.47 Both static and intermittent compression indumd an up- ,
regulation of NO production, while only intermittent compression increased nitric
oxide synthase (NOS), the enzyme that catalyzes the reaction producing NO."8
Mechanical compression of articular cartilage increases NO synthesis in a
manner dependent on the magnitude of stress.“""“'49 This proved to be true both
days post-impact, 15 MPa specimens consistently had less production of NO
compared to the 30 MPa treatment group. Loading at 30 MPa was not different
from LPS on day 1; however, LPS was greater than both loading groups by
almost two-fold on day 2. Our results agree with previous studies that suggest
that mechanical loading influences and increases NO production. Different types
of loading, the duration of loading, and imposed strain likely elicit different levels

of NO production. In this study, a single acute load of 15 MPa was not different

87

from LPS on day 1, but was distinctly different from cytokinestimulated explants
on day 2. In general, LPS-stimulated NO response peaks on day 2 in our equine
explant cultures, thus the response elicited in LPS on day 1 may have not been
great enough to differ from loading at 15 MPa. On the other hand, loading at 30
MPa had the highest NO response on day 1, and continued to be elevated
similar to LPS on day 2. The trauma incurred by loading at higher loads such as
30 MPa, which includes increased cell death, does induce a higher level of NO
production. Thus, a single acute traumatic load may cause an increase in NO
production that is similar to cytokine induction, whereas loading at a lower load
may not induce the cell signaling necessary to stimulate significant NO
production.

Lipopolysaccharide and 30 MPa treated explants had the highest PGE2
production overall. Dramatically increased production of PGE2 has been

125° and explant cultures39"'°'51 following

demonstrated in equine chondrocytes
stimulation with LPS. This trend was demonstrated in 3 out of the 4 horses used
in our study; however, due primarily to an increased response of PGE2
production in the 30 MPa loaded group in one horse, this group was not different
from LPS. Similar to NO production on day 1, PGE2 production was greater in
the 30 MPa and LPS groups compared to the control and 15 MPa groups. Nitric
oxide can inhibit or stimulate PGE2 production due to interactions between NO
and prostanoid production.52 Mechanical compression of articular cartilage does

increase PGE2 production through a NO-dependent pathway.36 Nitric oxide may

have a role in intracellular signaling induced PGE2 production in chondrocytes.53

88

The similarity in our NO and PGE2 data supports this proposed mechanism.

Little work has investigated the effect of loading on PGE2 production; however,
fluid-induced shear force was reported to induce a 10-20-fold increase in PGE2
production relative to a non-loaded control in bovine“ and human"
chondrocytes. Our PGE2 data could suggest that similar to the NO response,
PGE2 may be strain dependent The up-regulation of PGE2 in acute loaded
explants in this study indicates that mechanically induced PGE2 production
potentially may play a role in the physiological or pathophysiological regulation of
chondrocyte metabolism.

Glycosaminoglycan (GAG) release?"31 PG synthesis are increased and
decreased respectively in loaded bovine explants.“5 Our results indicating
increased PG release from loaded explants, measured by two different methods,
is consistent with results from leading bovine explants similarly.55 The slight
differences between the results of these two methods could be due to the fact
that the DMB assay measures total PG release, both synthesized and degraded,
while KS is a measurement of PG degradation or synthesis without aggregation.
D’Lima"O found that after 48 hours of culture, explants receiving a load of 14 or
23 MPa had increased PG loss relative to unloaded explants or those receiving a
load of 7 MPa. Additionally, these loads showed higher cellular apoptosis
relative to all treatments. Cell viability experiments conducted in our lab (results
not published) have detected very little cell death in LPS-stimulated explants. A
single acute load on explants caused increased PG turnover, as indicated by an

up-regulation of total PG and KS loss, which may be caused by mechanical

89

deformation of the matrix versus the regulation of catabolic mediators as
commonly seen in cytokine degradation. This study agrees with others
demonstrating that mechanical loading affects PG turnover, and further indicates
that loading equine explants induces greater total PG loss and degradation
relative to LPS.

Matrix metalloproteinase activity measured in the tissue or media did not
differ among treatments. Equine explants stimulated with LPS have increased
MMP-2 and -9 activityf”9 however, LPS stimulation did not up-regulate either of
the MMPs in this study. Little work has reported the effects of loading on MMP
expression in cartilage explants; however, both MMP-2 and —9 have increased
expression following cyclical loading at 01-05 MPa for a period of 1-16 h in
bovine explants.56 Shear stress of 1.6 MPa for 30 min to 24 h also induced
MMP-9 expression in rabbit chondrocytes.57 One study has indicated that
porcine explants subjected to cyclic dynamic or static loading over a time period
of 10 min did not Change the expression of MMP -1,-3, -13, or 14, or the tissue
inhibitors TIMP 1 and -3.58 The few studies showing increased MMP-2 or -9 in
loaded explants used different loading models than that used in our study and
our method used to measure MMP activity was semi-quantitative and this may
have had a direct effect on our results. An acute load may increase proteoch
activity through other enzymes such as aggrecanase, or MMP-3 or -13. Further
investigations are needed to determine whether the effects of loading on matrix
loss are mechanical via loosening or destabilizing the matrix, or if loading actually

induces an enzymatic response through up-regulating MMPs or aggrecanase.

90

Elucidating the mechanism by which trauma to the joint contributes to OA
in the horse is important in defining the pathogenesis of this degenerative
disease. This study has provided biochemical evidence confirming that acute
trauma does initiate a catabolic response in equine cartilage explants; however,
the response (especially at 15 MPa) differs somewhat from LPS-stimulated
explants, suggesting at least some unique cell signaling pathways. In agreement
with this, a recent study reported that lL-1 caused a synergistic loss of PG from
mechanically injured bovine and human cartilage.31 During a traumatic injury in
vivo, the synovial tissue, a significant producer of IL-1, will also likely be affected.
Thus, an in vitro model employing the use of both mechanical loading and
cytokine stimulation may be more beneficial in understanding how trauma to a
joint induces a catabolic response in articular cartilage in the horse, so that more

effective treatment protocols can be established.

a'Gibco, Grand Island, New York, USA.

bFisher Scientific, Pittsburgh, Pennsylvania, USA.

cSigma Chemical, St Louis, Missouri, USA.
dRocheDiagnostics Corporation, Indianapolis, Indiana, USA.
°Molecular Devices, Sunnyvale, California, USA.

'lCN Pharmaceuticals Inc, Costa Mesa, California, USA.
9Spectrum Medical industries, Los Angeles, California, USA.
f‘Pierce, Rockford, Illinois, USA.

fBioRad, Hercules, California, USA.

’R&D Systems, Minneapolis, Minnesota, USA.

kSAS Institute, Inc., Cary, North Carolina, USA.

91

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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(30 MPa), and lipopolysaccharide (LPS). NO2 concentration was quantified using
an assay employing the Greiss reaction. Different superscripts indicate

significant differences (P<0.05) between treatment groups.

92

 

 

 

 

 

 

 

 

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Figure 3. Mean prostaglandin E2 (PGE2) (:t SEM) released into the media per

loading at 15 MPa (15 MPa), loading at

well 24 hours post-treatment for control,

30 MPa (30 MPa), and lipopolysaccharide (LPS). PGE2 concentration was

determined by means of a commercially available competitive enzyme

immunoassay kit. Different superscripts indicate significant differences (P<0.05)

between treatment groups.

93

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4. Mean proteoglycan (PG) (1 SEM) released into the media per well
each day post-treatment for control, loading at 15 MPa (15 MPa), loading at 30
MPa (30 MPa), and lipopolysaccharide (LPS). Total PG released into the media
was quantified using a dimethymethylene blue (DMB) assay. Different

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Figure 5. Mean keratan sulfate (KS) (:l: SEM) released into the media per well 24
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(30 MPa), and lipopolysaccharide (LPS). KS loss in the media was quantified
using an ELISA with a monoclonal antibody specific for KS. Different

superscripts indicate significant differences (P<0.05) between treatment groups.

95

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 6. Mean matrix metalloproteinase-2 (1: SEM) activity measumd in the
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loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-2 activity was
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Figure 7. Mean matrix metalloproteinase-2 (1 SEM) activity measured in the
tissue per well 2 days post-treatment for control, loading at 15 MPa (15 MPa),
loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-2 activity was

determined by gel zymography. MMP-2 activity was not significantly different
between groups (P>0.05).

97

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Figure 8. Mean matrix metalloproteinase-9 (1 SEM) activity measured in the
media per well 24 hours post-treatment for control, loading at 15 MPa (15 MPa),
loading at 30 MPa (30 MPa), and lipopolysaccharide (LPS). MMP-9 activity was
determined by gel zymography. MMP-9 activity was not significantly different
between groups (P>0.05).

98

 

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Figure 9. Mean matrix metalloproteinase-9 (:1: SEM) activity measured in the
tissue per well 2 days post-treatment for control, loading at 15 MPa (15 MPa),
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determined by gel zymography. MMP-9 activity was not significantly different
between groups (P>0.05).

99

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50. May SA, Hooke RE, Lees P. Equine chondrocyte activation by a
variety of stimuli. Br Vet J 1992;148:389-397.

51. MacDonald MH, Stover SM, erlits NH, et al. Effect of bacterial
lipopolysaccharides on sulfated glycosaminoglycan metabolism and
prostaglandin E2 synthesis in equine cartilage explant cultures. Am J Vet Res
1994;55:1127-1138.

52. Weinberg JB. Nitric oxide synthase 2 and cyclooxygenase 2
interactions in inflammation. lmmun Res 2001;22:319-341.

53. Notoya K, Jovanovic DV, Reboul P, et al. The induction of cell
death in human osteoarthritis chondrocytes by nitric oxide is related to the
production of prostaglandin E2 via the induction of cyclooxygenase-2. J Immunol
2000;165:3402-3410.

54. Smith RL, Donlon BS, Gupta MK, et al. Effects of fluid-induced
shear on articular Chondrocyte morphology and metabolism in vitro. J Orthop Res
1995;13:824-831.

55. D'Lima DD, Hashimoto S, Chen PC, at al. Impact of mechanical
trauma on matrix and cells. Clin Orthop 2001:890-99.

56. Blain EJ, Gilbert SJ, Wardale RJ, et al. Up-regulation of matrix
metalloproteinase expression and activation following cyclical compressive
loading of articular cartilage in vitro. Arch Biochem Biophys 2001;396:49-55.

57. Jin G, Sah RL, Li YS, et al. Biomechanical regulation of matrix
metalloproteinase-9 in cultured chondrocytes. J Orthop Res 2000;18:899-908.

58. Fehrenbacher A, Steck E, Rickert M, et al. Rapid regulation of
collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of
metalloproteinase 1, 2, 3 expression in response to mechanical loading of
cartilage explants in vitro. Arch Biochem Biophys 2003;410:39-47.

104

CHAPTER 3
FURTHER STUDIES ON THE ABILITY OF GLUCOSAMINE AND
CHONDROITIN SULFATE TO INHIBIT CYTOKINE-INDUCED CARTILAGE
DEGRADATION

Introduction

Osteoarthritis, also known as DJD, is a permanent condition that can
cause lameness in horses at all levels of performance. Increased athletic ability
and rigorous training predispose many horses to the degenerative effects of OA.
Preliminary factors often involved in contributing to OA in the horse include
trauma to the joint, instability of the joint, fatigue, and increased age. As a rule,
the earlier the disease is diagnosed and properly treated, the greater the
likelihood of improvement in the affected joint.

The outcome of OA generally results in decreased motion, pain, and
dysfunction of the joint. The inflammatory response caused by this degenerative
disease up regulates inflammatory mediators such as NO, PGE2, and MMPs. In
humans, NO activates MMPs, suppresses PG synthesis, and induces apoptosis
in articular chondrocytes."2 Increased production of NO may mediate the
suppression of cartilage matrix synthesis occurring in response to intra-articular
cytokines. Explants of equine synovial membrane and articular cartilage
released significantly higher amounts of NO in tissue originating from horses with
OA.3 Prostaglandin E2 is produced in inflamed joints, and can regulate PG
degradation and inhibit PG synthesis.4 Increased PGE2 production occurred
after cytokine stimulation in equine synovial cells and chondrocytes in vitrof"6

and was upregulated in cartilage explants from horses with moderate OA.3

105

 

Exogenous PGE2 significantly reduced relL-1B induced expression of MMP-1, -3,
-13, and TIMP 1 in equine chondrocytes.5 Matrix metalloproteinases are the
primary class of enzymes that are considered to play the most pivotal role in
destruction of the cartilage ECM. Specifically, MMPs —2 and -9 have been found
in synovial fluid from diseased equine joints, and are up-regulated in equine
cartilage and synovial fluid following stimulation with lL-1I3.7'8

Oral non-invasive treatment of this disease with alternative therapeutic
agents is becoming more popular within the equine industry. Two nutraceuticals
(nutritional compounds added to the diet that have pharmacological-like effects)
shown to slow the progression of OA are GLN and CS. In combination, these
compounds suppress NO production, PGE2 release, and MMP-2 activity in
equine cartilage explants.N1 Horses diagnosed with OA and supplemented with
GLN and CS orally had significantly improved lameness scores.“13 The same
effects have been seen in humans and other animals. A combination of GLN

I,“ and in patients

and CS relieved symptoms of knee OA in military personne
with radiographically diagnosed mild to moderate OA.15 Joint improvement after
intercarpal injection with the combination has been seen in dogs with chemically
induced OA.16 Furthermore, GLN and CS, in combination with manganese
ascorbate, was effective in reducing OA-induced lesions in rabbits.17

The lowest doses at which GLN and CS are most effective in inhibiting
the degenerative effects of 0A in vitro remain to be elucidated. High
concentrations of GLN (6.5, 25 mglml) have been found to be toxic to bovine

articular chondrocytes in vitro; however, these doses are extremely high, and the

106

actual dose in vivo is considered significantly less.“ We have recently reported
that the combination of GLN (1 mglml) and CS (0.25 mglml) inhibited the
synthesis of several mediators of cartilage degradation in equine articular
cartilage explants.11 These results were at least 2.5-fold lower than
concentrations in our previously reported work.‘9 This research project was
designed to study even lower concentrations of GLN HCI, in combination with a
fixed concentration of CS, to determine if they would also inhibit NO production,
PGE2 release, and MMP activity in equine explants in vitro.

Materials and Methods

Experimental design

Articular cartilage was obtained from the antebrachio-carpal and middle
carpal joints of 4 horses (2-14 years old) euthanized for reasons other than
lameness. Four separate experiments were conducted using tissue from each of
the 4 horses. Cartilage discs (3.5 mm in diameter) were biopsied from the load
bearing region of the joint and were washed for a total of 3 washes in Dulbecco’s
Modified Eagles Medium (DMEM): nutrient mixture F-12 (Ham) (1:1).' Three
randomly selected discs (approximately 40 mg of cartilage) were placed in each
well of a 24 well Falcon culture plate.” Each well consisted of 1 ml of DMEM:
F12 media supplemented with 10% fetal bovine serum,‘ 10 pllml ascorbic acid,
100 units/ml penicillin/streptomycin,‘I and all 20 amino acids°.2° The explants

were maintained in culture in a humidified incubator with 7% CO2 at 37°C.

107

 

 

Explants were equilibrated in media for 2 days. Lipolysaccharidec (10
pglml) was added to induce cartilage degradation in the presence or absence of
GLN-HCI (FCHG49)d in combination with CS (T RH122)‘I (see Table 3) both days
of treatment Conditioned media were collected and replaced with new media
daily and stored at 4°C until analysis. Treatments and controls of each

experiment were identical and treatment groups are summarized in Table 3.

 

Table 3: Description of tr'eatr'nent groups In equine articular cartilage explant

 

 

experiments
Treatment F BS LPS GLN CS
FBS-Control 1 10%
LPS-Control 2 10% 10 pglml
Treatment 1 10% 10 pglml 0.50 mglml 0.125 mglml
Treatment 2 10% 10 pglml 0.40 mglml 0.125 mglml
Treatment 3 10% 10 pglml 0.30 mglml 0.125 mglml
Treatment 4 10% 10 pglml 0.20 mglml 0.125 mglml

 

’FBS = fetal bovine serum; LPS = lipopolysaccharide; GLN = glucosamine HCI; CS =
Chondroitin sulfate

Nitric oxide analysis

Nitric oxide (NO) was measured indirectly in the conditioned media as
described previously.‘ Nitrite (NO2), a stable end-product of NO metabolism,
was quantified using the Greiss reaction and sodium nitrite as a standard.
Briefly, 75 pl conditioned medium was incubated with 75 pl 1.0% sulfanilamide,
0.1% N-1-naphthylethylenediamide dihydrochloride in 25% phosphoric acid at
room temperature for 5 min. Due to some precipitation of reagents with CS,

plates were spun at 950 g at a temperature of 4°C. Following centrifugation, the

108

remaining supernatant was transferred to a new 96-well plate, and absorbance at
540 nm was determined using a Spectromax 300 plate reader.’ Results are
expressed as nmol NO2/Vlrell.
Cartilage extraction

Matrix metalloproteinases were extracted from articular cartilage using a
modified protocol.21 Explants from each well were placed in a cold stainless steel
mortar apparatus and snap frozen with liquid nitrogen. They were then
powdered immediately using a stainless steel pestle and hammer. Powdered
explants were placed in microcentrifuge tubes with stir bars and 600 pl extraction
buffer (50 mmolll Tris HCI, 10 mmolll CaCl2, 2 molll guanidine HCI and 0.05%
Brij-35: pH 7.5) was added to 3 powdered explants from each well. Samples
were stirred overnight at 4°C and then centrifuged at 18,000 g for 30 min at 4°C.
Supematant was dialysed (24 h) against assay buffer (50 mmolll Tris HCI, 10
mmolll CaCl2, 0.2 molll NaCI, 0.05% Brij-35: pH 7.5) using Spectrapor 2 dialysis
tubing with a 12-kd cutoff.’ Dialysis was continued for 48 h with distilled water.
The amount of protein in the extracts was determined using the Pierce Micro
BCA Protein Assay° with bovine serum albumen as the standard. Extractions
were stored at 4°C and immediately analyzed for MMP activity.
Gel zymography

Gelatinase activity was detected by gel zymography. Extracts of

articular cartilage samples containing 7 or 8 pg of protein (each gel received the
same amount of protein) were applied without reduction to an 8% polyacrylamide

gel with 1 mglml gelatin incorporated as the substrate. Samples were diluted

109

with 4x sample buffer and samples were electrophoresed at room temperature.
A gelatinase A (MMP-2) molecular weight standard served as the gelatinase
control. Following electrophoresis, gels were then incubated in 2.5% (vlv) Triton
X-100 for 1h and then overnight at 37°C in 50 mmolll Tris (pH7.5) containing 200
mmolll NaCl, 10 mmolll CaCl2, 10 pmolll ZnCI2 and 0.02% Brij-35. The gels were
stained with Coomassie Blue R250 for 1 h at room temperature and enzyme
activity was measured by scanning densitometry (Gel Doc 2000)," using Quantity
One 4.0.1 software.
Prostaglandin E2 immunoassay

Prostaglandin E2 (PGE2) was measured using a commercially available
competitive enzyme immunoassay kit.‘ lndomethacin (10 pglml) was added to
conditioned media samples after 1 day of treatment and samples were then
stored at -20°C until analysis. Samples were diluted 10-fold and run in duplicate.
Briefly, the sample competes with a fixed amount of alkaline phosphatase-
labeled PGE2 for sites on a mouse monoclonal antibody. The antibody becomes
bound to the goat anti-mouse antibody coating the microplate. Excess conjugate
and unbound sample were removed from the plate through multiple washes.
Absorbance was read at 405 nm with a. wavelength correction set at 590 nm. A
four parameter logistic curve ranging from 39-5,000 pglml PGE2 was used to
determine sample concentrations.
Statistical analysis

Data for indicators of degradation were analyzed using the repeated

measure option of the SAS statistical software PROC MIXED (2001).i Data were

110

combined by pooling wells from all 4 horses according to treatment. Each group
of treatments using tissue from one animal was considered a replicate (n=4).
Data were normalized using log transformation due to variation in the level of
response between horses. Experimental effects of treatment, and treatment by
day effect were assessed for the pooled treatments. Differences between effects
were compared using difference of least square means and Tukey’s multiple
comparison procedure. Statistical significance was considered at P-<0.05 unless

otherwise noted.

Results and Discussion

The concentrations of GLN and CS used in this study were developed
from a previously published observation11 indicating that concentrations of GLN
as low as 0.50 mglml and CS as low as 0.125 mglml were beneficial in inhibiting
some catabolic mediators of cytokine-induced cartilage degradation.

Equine cartilage explants demonstrated decreased NO production (Figure
10) with supplementation of GLN between the range of 0.30 and 0.50 mglml
(P<0.05). Previously, we have reported that concentrations of GLN as low as
0.50 mglml inhibited NO production in equine explants.11 Nitric oxide is a major
component of the inflammatory-like response generally seen in OA, and has
been implicated as a mediator of some of the effects of the pro-inflammatory
cytokines LPS and lL-1 in equine articular chondrocytes.22'23 Blocking NO

production can prevent cartilage degradation in animal models.24

111

We have previously shown that GLN alone,“19 and in combination with CS11 is
effective in inhibiting the NO response.

Prostaglandin E2 production (Figure 11) also tended to decrease with
increased GLN concentration, and the minimum amount of GLN required to
significantly inhibit production was 0.40 mglml (P<0.05). Concentrations of GLN
that proved to be effective in this study were similar to the efficacy of GLN at 0.50
mglml in our previous studies.“"11 Prostaglandin E2 is an important cellular signal
in both normal and pathological joint metabolism. Significantly greater
concentrations of PGE2 were detected in synovial fluid from equine joints
affected with OA.”27 Cytokine stimulated explants and chondrocytes have
enhanced levels PGE2 production.5'°2° Blocking PGE2 production is an important
strategy to decrease symptoms of GA in humans as evidenced by the
development of COX-2 inhibitors.

In agreement with our previous study,11 GLN and CS had no effect on
MMP-2 activity (Figure 12). In addition, LPS-stimulated explants did not display
an up-regulation of MMP-2 as demonstrated in the prior study. Matrix
metalloproteinase-9 activity was suppressed, however, at a concentration of 0.50
mglml GLN (P<0.05) and tended to be decreased at 0.40 mglml GLN (P=0.08)
(Figure 13). This is in agreement with our previous study demonstrating that the
combination of GLN and CS down-regulate MMP-9 activity.11 However, this
study used a concentration of 1.0 mglml GLN and 0.25 mglml CS. We have
shown that concentrations even lower than this may be effective in suppressing

MMP-9 activity. The differences between the two MMPs could be that in articular

112

cartilage, MMP-2 is constitutively expressed while MMP-9 is induced by pro-
inflemmatory cytokines such as lL-1.29 MMP activity has been reported to be
nearly twice as high in equine joints with OA compared to normal joints, and most
likely reflects matrix destruction.3° Thus, if GLN and cs could inhibit at least
some proteolytic activity, this could prove beneficial to horses with OA.

Our results have provided further evidence that GLN and CS in
combination are chondroprotective and may sews as an effective treatment for
inhibiting the catabolic response of articular cartilage degradation found in OA.
The combination may suppress cartilage catabolism through regulating cell
signaling molecules, such as NO and PGE2, which regulate MMPs. Most
recently, GLN alone appeamd capable of pre-trensletional, and possibly also
translational, regulation of MMP expression." Both NO and PGE2 are up-
regulated in equine synovial membrane and cartilage obtained from osteoarthritic
joints.3 Weinberg” has suggested that inhibiting the production of NO and PGE2
simultaneously would provide potent anti-inflammatory effects. The
concentrations of GLN and CS used in this study are still somewhat higher than
values measured in blood, although the concentration of CS (0.125 mglml) is
closer to the higher end of plasma concentrations seen in dogs33 and horses.34
Further studies investigating even more physiological concentrations of GLN and
CS in combination are ongoing. Elucidating the mechanism of action of these
compounds should further increase our understanding of how to maintain and

prevent CA in athletic and performance horses.

113

'Gibco, Grand Island, New York, USA.

"Fisher Scientific, Pittsburgh, Pennsylvania, USA.

cSigma Chemical, St Louis, Missouri, USA

‘Nutremax Laboratories, Edgewood, Maryland, USA.
‘Molecular Devices, Sunnyvale, California, USA.
'Spectrum Medical Industries, Los Angeles, Celifomie, USA
“Pierce, Rockford, Illinois, USA.

f'BioRad, Hercules, California, USA.

'R&D Systems, Minneapolis, Minnesota, USA.

jSAS Institute, Inc., Cary, North Carolina, USA.

114

(log scale) nmol NOIWeII

 

 

 

l EICorlrol cos no.4 ao.3 002 llrLPs J

 

Figure 10. Mean nitric oxide (NO) (1 SEM) released into the media each day
post-treatment for all 4 horses (36-48 wells per treatment). Values are shown as
log transformed. Treatments: control = no glucosamine (GLN), chondroitin
sulfate (CS), or lipopolysaccharide (LPS); 0.5 = 0.5 mglml GLN + 0.125 mglml
CS; 0.4 = 0.4 mglml GLN + 0.125 mglml CS; 0.3 = 0.3 mglml GLN + 0.125 mglml
CS; 0.2 = 0.2 mglml GLN + 0.125 mglml CS; LPS = 10 pg LPS. °° means not

sharing the same superscript differ (P<0.05).

115

 

(log scale) PGE2 (pglml)

 

 

 

 

I nCortrol no.5 no.4 ao.3 c102 aLPs |

Figure 11. Mean prostaglandin E2 (PGE2) (:t SEM) released into the media each
day post-treatment for all 4 horses (36-48 wells per treatment). Values are
shown as log transformed. Treatments: control = no glucosamine (GLN),
chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0.5 = 0.5 mglml GLN +
0.125 mglml CS; 0.4 = 0.4 mglml GLN + 0.125 mglml CS; 0.3 = 0.3 mglml GLN +
0.125 mglml CS; 0.2 = 0.2 mglml GLN + 0.125 mglml CS; LPS = 10 pg LPS. "’

and "° means not sharing the same superscript differ (P<0.05).

116

2.82 -

2.80 1

2.78 I

2.76 -

(log scale) INT/mmz

2.74 .

 

 

2.72

 

 

 

| o Comol 0.5 a 0.4 a 0.3 o 02 a LPS ]

Figure 12. Mean matrix metalloproteinase-2 (MMP-2) activity (:t SEM) in the
tissue 2 days post-treatment for all 4 horses (36-48 wells per treatment). Values
are shown as log transformed. Treatments: control = no glucosamine (GLN),
Chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0.5 = 0.5 mglml GLN +
0.125 mglml CS; 0.4 = 0.4 mglml GLN + 0.125 mglml CS; 0.3 = 0.3 mglml GLN +
0.125 mglml CS; 0.2 = 0.2 mglml GLN + 0.125 mglml CS; LPS = 10 pg LPS.

MMP-2 activity was not significantly different between groups (P>0.05).

117

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(log scale) lrlrlmm2

 

 

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.................... IlilMlllM/Illll ————-—-—~

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. ' . ‘ . ............. Illfllllmfllllll/ ————-

.................. ”WW/”III ——————

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'... .......... ”warranty/””11 .

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2 7O _..._:_-..‘._. ...... A I’llfllfllllllllll --——-——-
I

 

 

3w

 

 

El Corlml 0.5 B 0.4 B 0.3 D 0.2 B LPS

 

 

Figure 13. Mean matrix metalloproteinase-9 (MMP-9) activity (1 SEM) in the
tissue 2 days post-treatment for all 4 horses (36-48 wells per treatment). Values
are shown as log transformed. Treatments: control = no glucosamine (GLN),
Chondroitin sulfate (CS), or lipopolysaccharide (LPS); 0.5 = 0.5 mglml GLN +
0.125 mglml CS; 0.4 = 0.4 mglml GLN + 0.125 mglml CS; 0.3 = 0.3 mglml GLN 1-
0.125 mglml CS; 0.2 = 0.2 mglml GLN + 0.125 mglml CS; LPS = 10 pg LPS. "’
means not sharing the same superscript differ (P<0.05). * indicates a trend

(P<0.08) to differ from LPS.

118

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2. Hauselmann HJ, Oppliger L, Michel BA, et al. Nitric oxide and
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3. von Rechenberg B, Mcllwraith CW, Akens MK, et al. Spontaneous
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122

CONCLUSION

The previous chapters have demonstrated the differences between
applying a single acute load or cytokine stimulation to equine articular cartilage
explants, and have further investigated whether reduced concentrations of GLN
(in combination with CS) are effective in inhibiting cytokine-induced cartilage
degradation. Several catabolic measurements of articular cartilage were used to
assess degradation, which included NO and PGE2 production, PG release, KS
degradation, and MMP activity. These factors are up-regulated in OA, and can
contribute to ineversible cartilage damage in a joint.

Evidence is accumulating that direct damage to the cartilage is not the
only route by which trauma contributes to OA, and that fibrillation and fissuring of
the cartilage surface is not necessarily the primary indicator of cartilage
degradation. Our study employing two different levels of loading on cartilage
explants agrees with similar mechanical loading studies demonstrating increased
NO and PGE2 production, and increased PG release. We have shown that acute
trauma causes distinct biochemical differences in cartilage catabolism compared
to cytokine-induced degradation. This evidence is supported by gene expression
studies performed in our lab indicating that loading explants significantly
increased MMP-3 expression, but had little effect on other MMP expression;
whereas lL-1 stimulated explants had an increase in MMP-3, -13, and -14.

Biochemical analysis revealed that loaded explants peaked in nitric oxide

production on day 1; whereas cytokine treated explants peaked on day 2.

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Proteoglycan release was significantly higher overall in the 30 MPa loaded
explants, and both loaded groups demonstrated the greatest KS degradation.
Although MMP-2 and -9 activity were not significantly different across any
treatment, LPS treated explants has the highest MMP-9 activity among
treatments. Other studies have indicated up-regulation of MMP-9 following LPS
stimulation, and increasing the number of animals and replication in this study
may have resulted in a significant up—regulation in LPS treabd explants.
Contrary to cytokine treated explants, loaded explants showed little difference, if
any, from the control explants. Results from previous studies employing different
models of mechanical loading to study proteolytic activity have had conflicting
results. Further investigations to determine whether the loss of PCs in models of
injury to cartilage is due to mechanical disruption or dislodgement of the matrix
rather than cell-mediated enzymatic degradation would be helpful. Induction of
cytokines following an in vitro injury to articular cartilage synergistically increases
the loss of GAGs from the tissue. This finding, combined with the work of our
study, suggests that factors external to cartilage, such as cytokines in the
synovial fluid, could play an important role in the development of cartilage
degradation after acute joint trauma. Future studies observing the effects of
cytokine stimulation following loading could further aid in explaining the trauma-
induced OA commonly seen in horses.

Glucosamine and CS, in combination, are proposed to have
chondroprotective properties, and have become a popular supplementation

protocol in arthritic conditions of joints. The hypothesis that the combination of

124

these two molecules provides an additive or synergistic chondroprotective effect
is not firmly established. Furthermore, the physiological concentrations of GLN
and CS in vivo that are most effective in inhibiting cartilage degradation remain to
be elucidated. Using indicators of cartilage metabolism, we have shown that
GLN-HCL, in combination with CS (0.125 mglml), at concentrations ranging from
0.30 to 0.50 mglml is effective in inhibiting several inflammatory mediators of
cytokine-induced cartilage degradation. The combination of GLN and CS
decreased NO and PGE2 production along with MMP activity. These molecules
may suppress cartilage catabolism through regulating cell signaling molecules
such as NO and PGE2, which regulate MMPs. Although the concentrations of
GLN and CS used in this study are still somewhat higher than values in the blood,
the concentration of CS is close to higher values found in the plasma of dogs.
One drawback to this study verses our previous loading study is that the horses
used in this study were from a broad age group (4 to 14 years). Therefore,
increased variation was observed between animals. However, this variable
response may be helpful in understanding the pattern in efficacy of GLN and CS
in inhibiting the inflammatory-like response of cartilage degradation in animals of
different age groups. The biochemical evidence resulting from this study
supports the limited in vivo research indicating that GLN and CS may prevent
equine articular degradation.

An in vitro articular explant model may not be an ideal system to study the
effects of articular degradation in vivo; however, it enables us to study possible

pathways and mechanisms of action of contributing factors involved in OA. In

125

turn, this information can lead to a greater understanding of the physiological
adaptations taking place during cartilage degeneration, without sacrificing the

animal.

126

APPENDIX

127

 

APPENDIX A

DEVELOPMENT OF A SERUM-FREE MEDIUM FOR AN EQUINE ARTICULAR
CARTILAGE EXPLANT SYSTEM

Various growth factors, hormones and proteins present in serum are
presumed to be responsible for its growth-stimulating activity in culture media.1
However, serum is a complex mixture, and many serum components are poorly
characterized or have not been completely studied.2 In addition, the
concentrations of some of the components of serum vary drastically among
several different serum batches. Fetal bovine serum is commonly used in
articular cartilage tissue culture. Infection with viruses such as bovine viral
diarrhea virus (BVDV) is frequent in the bovine population.3 In utero infection
leads to virus and antibody contamination of fetal and other serum used in cell
culture production.

Serum substitute media eliminates the complex and variable effects of
serum on cell growth. These substitutes can be mixtures of salts, amino acids,
vitamins, glucose and various compounds such as nucleic acid and lipid
precursors or antioxidative substances.‘ They are supplemented with hormones
(insulin, growth factors, steroids), binding proteins (transferring, albumin) and
trace elements, which replace the growth stimulating activities of serum with the
appropriate serum substituents at the right concentrations.

Currently, the serum supplemented medium used in our laboratory limits
our ability to detect certain known mediators of articular cartilage degradation in

cultured media. Eliminating serum from our culture medium allows the simple

128

design and interpretation of experiments which would be difficult or impossible to
carry out in serum-containing medium. Thus, my objective in this study is to
develop a serum-free medium that sustains articular cartilage chondrocytes in

cartilage explant culture.

Experiment 1

Materials and Methods

Experimental design

Articular cartilage was obtained from 1 pair of bovine forelegs (18 to 24
months of age) obtained from a local abattoir within three hours of slaughter.
Cartilage discs (3.5 mm) were biopsied from the load-bearing region of the joint
and were washed for a total of 3 washes in Dulbecco’s Modified Eagles Medium
(DMEM): nutrient mixture F-12 (Ham) (1:1).‘I Three randomly selected discs
(approximately 40 mg of cartilage) were placed in each well of a 24 well Falcon
culture plateb (42 wells total). Six wells from each plate were randomly assigned

to one of 7 treatment groups shown in Table 4.

129

 

Table 4: Treatment ggupe for development of serum free culture medium

 

Treatment group Treatments"
1 Serum-free media + 1 ullml ITS
2 Serum-free media + 10% FBS
3 Serum-free media + 1 ule ITS + 10 pglml LPS
4 Serum-free media + 10% FBS + 10 pglml LPS
5 Bovine basal media + 1 ule ITS
6 Bovine basal media + 1 ullml ITS + 10 pglml LPS
7 Bovine basal media + 10% FBS + 10 pglml LPS

 

*ITS = Insulin Transferrin Sodium Selenite Supplement, ° FBS = Fetal bovine
serum, ' LPS = Lipopolysaccharide.“

The serum-free medium used in treatments 1 through 4 consisted of
DMEMIF-12 (Ham) (1:1) serum free media supplemented with all 20 amino
acidsd (Gln 2.19 g/L, Gly 5.63 mglL, His 5.26 mglL, 25.17 mglL, Leu 22.88 mglL,
Lys 27.63 mglL, Met 6.38 mglL, Phe 15.26 mglL, Pro 8.64 mglL, Ser 7.88 mglL,
Thr 20.88 mglL, Trp 3.49 mglL, Tyr 24.00 mglL, Val 20.38 mglL, 2.23 mglL, 31.6
mglL, 3.76 mglL, 3.33 mglL), ascorbic acid (50 pllml),“ sodium bicarbonate (3.89
gIL)," Iactalbumin hydrolysate (2 mglL),° dexamethasone (100 pglml),d
magnesium sulfate (16.9 mglml),‘I and penicillin/streptomycin (100 units/ml).‘I
The bovine basal medium used in treatments 5 through 7 was identical to the
recipe for the serum free medium listed prior, except it had twice the amounts of
amino acids and 1 pglml sodium selenited added to the medium.

The explants were maintained in culture in a humidified incubator with 7%
C02 at 37°C. Explants from treatments 1 through 4 were equilibrated in serum-

free media + ITS 2 days prior to the first of 4 treatment days, while explants from

130

 

treatments 5 through 7 were equilibrated in bovine basal media + 10% FBS prior
to treatment. On day 1 of treatment, explants in groups 3, 4, 6, and 7 were
incubated with 10 pglml LPS. Conditioned media were collected daily and stored
at 4°C until analysis. In addition, one explant from one well of each treatment
was saved to study cell viability of the explants.
Nitric oxide analysis

Nitric oxide (NO) was measured indirectly in the conditioned media as
described previously (Blanco et al., 1995). Nitrite (N02), a stable end-product of
NO metabolism, was quantified using the Greiss reaction and sodium nitrite as a
standard. Briefly, 75 pl conditioned medium was incubated with 75 pl 1 .0%
sulphanilamide, 0.1% N-1-naphthylethylenediamide dihydrochloride in 25%
phosphoric acid at room temperature for 5 min. Absorbance at 540 nm was
determined using the Spectromax 300 plate reader.“ Results are expressed as
nmol Nozlwell.
Proteoglycan analysis

Proteoglycan (PG) release into conditioned media was measured as
previously described (Chandrasekhar, 1987) using a dimethylmethylene blue
(DMB) assay. Briefly, PG content was determined by measuring sulfated
glycosaminoglycan (GAG) content compared to a chondroitin sulphate standard.
Results are expressed as pg PGIwell.
Cell viability

On the final day of tissue culture, one explant from each group was

randomly chosen for a cell viability study. Explants were removed from their

131

assigned wells and sliwd into approximately 0.5 mm sections using a scalpel
blade. The sections were stained using a kit containing calcein and ethidium
bromide homodimer (Live/Dead and \frabilitleytotoxicity).f All specimens were
viewed in a florescence microscope (Leica DM LB) 9 (frequency: 50-60 Hz). Cell
viability was determined by visual detection of dead (red) and viable (green) cells.

Results and Discussion

The serum-free medium + ITS treated control (treatment 1) and the bovine
basil medium + ITS (treatment 5) were very similar in NO production on both
days 1 and 2 (Figure 14). In addition, the senrm-free LPS treated positive
controls (treatments 4 and 7) for each of these mediums were very comparable
in NO production on these two days. Proteoglycan release showed similar results
to that of NO production for the different mediums (Figure 15). Treatments 1 and
2 comparing the ITS supplement to F BS in serum-free media had almost
identical PG release, as did treatments 3 and 7 in both mediums with added LPS.
Although there was a huge spike in PG release in treatment 4 on day 1, no visual
differences are seen in the figure on day 2 when comparing the serum-free
medium with FBS and LPS and the bovine basal media with FBS and LPS. This
spike was also seen 1 day prior to treatment (data not shown), and thus was a
result of factors other than addition of LPS. Cell viability was normal under

serum-free culture conditions. Explants showed a small amount of cell death

132

around biopsied edges, but no abnormal cell death was observed upon detection

with florescence.

Conclusion

The ITS supplement used in this experiment has been used with success
in equine cartilage explant cultures."5 I have also proved this to be true in our
equine articular cartilage explant system in the current experiment. Insulin,
transferrin, and selenium are important in cellular maintenance and are
constituents that are often added to media.2 Insulin regulates cell metabolism
and promotes growth, transferrin works as an iron transporter, and selenium
works as a component of the free-radical scavenger glutathione peroxidase, and
as a nutrient for cell proliferation.5 The results of this experiment indicate that
incubating bovine articular cartilage in serum-free media containing 9‘.- of the
normally added amino acids in conjunction with the ITS supplement nourishes
and helps chondrocytes to flourish similar to that of incubation with full amino
acids and FBS. Although FBS treatments tended to have higher NO production
and PG release, the ITS treated explants showed similar trends and had parallel

results.

Experiment 2

Materials and Methods

133

Experimental design

Articular cartilage was obtained from 1 pair of bovine forelegs ( 18 to 24
mo of age) obtained from a local abattoir within three hours of slaughter.
Cartilage discs (3.5 mm) were biopsied from the load-bearing region of the joint
and were washed for a total of 3 washes in Dulbecco’s Modified Eagles Medium
(DMEM): nutrient mixture F-12 (Ham) (1:1). Four randomly selected discs
(approximately 50 mg of cartilage) were placed in each well of a 24 well Falcon
culture plate (42 wells total). Six wells from each plate were randomly assigned

to one of 7 treatment groups shown in Table 5.

 

 

Table 5: Treatment groups for development of serum free culture medium

 

 

Treatment group Treatments“

1 Serum-free media + 1 pllml ITS

2 Serum-free media + 1 pllml ITS + 10 pglml LPS

3 Serum-free media + 1 pllml ITS + 5 pglml LLA +10 pglml
LPS

4 Serum-free media + 1 pllml ITS + 40 ng/ml Thy +10 pglml
LPS

5 Serum-free media + 1 pllml ITS + 5 pglml LLA + 40 nglml
Thy + 10 pglml LPS

6 Serum-free media + 10% FBS + 10 pglml LPS

7 Serum-free media + 10% FBS

 

*ITS = Insulin Transferrin Sodium Selenite Supplement, FBS = Fetal bovine
serum, LPS = Lipopolysaccharide, LLA = Linoleic acid albumen,“ T= Thyroxine.d

The serum-free medium used in treatments 1 through 7 was identical to
that described in experiment 1 of this appendix (APPENDIX A). The explants

were maintained in culture in a humidified incubator with 7% 002 at 37°C.

134

Explants from treatments 2 through 6 were equilibrated in serum-media + ITS
and either LLA, T, or both (as indicated in Table 5) 2 days prior to the first of 1
treatment day. Explants in treatments 1 and 7 did not receive additional
treatments except their maintenance supplement of ITS or F BS. On day 1 of
treatment, explants in groups 2 through 6 were incubated with 10 pglml LPS.
Conditioned media were collected daily and stored at 4°C until analysis.
Nitric oxide

Nitric oxide (NO) was measured indirectly in the conditioned media as
described previously (Blanco et al., 1995) and in experiment 1 of this appendix
(APPENDIX A).

Results and Discussion

Figure 16 shows the NO release for explants in experiment 2 for two days
prior to treatment and the day of treatment. The addition of LLA by itself in
treatment 3 shows a marked increase in NO production compared to treatment
without LPS (treatment 1), with ITS + T + LPS (treatment 4), with FBS + LPS
(treatment 5), and with FBS alone (treatment 6). Thyroxine alone with ITS and
LPS (treatment 4) did not have the same effect; however, the combination of
both T and LLA (treatment 5) showed the highest NO production over all
treatments. The NO release seen in this treatment was also closer to the range
of release typically seen in other experiments conducted in our lab using LPS

treated bovine explants.

135

 

Conclusion

Linoleic acid, a prostaglandin precursor, has previously been used in
equine articular cartilage explant cultures."5 Thyroxine is a growth hormone that
supports matrix assembly and hypertrophic expression in growth plate
chondrocytes.6 This experiment indicates that the combination of supplements,
in conjunction with LPS, that causes chondrocytes to release NO in a manner
most similar to culturing with FBS in serum-free medium is ITS + LLA + T. In the
previous experiment, I confirmed the beneficial effect of supplementing ITS to
cartilage explants in serum-free media. In the present experiment, I have shown
that two addition supplements, LLA and T, in combination with ITS, have a

positive effect on chondrocyte metabolism.

aGibco, Grand Island, New York, USA.

I’Fisher Scientific, Pittsburgh, Pennsylvania, USA.

“Sigma Chemical, St Louis, Missouri, USA.

°Roche Diagnostics Corporation, Indianapolis, Indiana, USA.
°Molecular Devices, Sunnyvale, California, USA.
'Molecular Probes, Oregon, USA

9Lecia Mikroskopie und Systeme GmgH, Wetlzar, Germany

136

 

 

40.00 i

 

 

nmol NOzIWelI

 

 

 

 

 

 

 

BSFM'S ISFFBS BSFLPSM'S DSFFBSLPS IBBIMTS IBBMLPSFBS IBBMLPSMS

 

 

 

Figure 14. Mean nitrite (N02) released into serum-free (SF) or bovine basal
media (BBM) 24 and 48 hours after treatment with ITS, FBS, and LPS. N02
concentration was quantified using an assay employing the Greiss reaction.
ITS=insulin-transferrin-sodium-selenite supplement, FBS=fetal bovine serum,

LPS=lipopolysaccharide.

137

2504
200-
E
2 150-
a
:-
100<

 

 

 

 

 

IE SF/ITS III SFIFBS E3 SFILPS +H'S El SF/FBS +LPS I BBM/ITS I BBM/LPS+FBS I BBM/LPSHTS

Figure 15. Mean proteoglycan (PG) released into serum-free (SF) or bovine
basal media (BBM) 24 and 48 hours after treatment with ITS, FBS, and LPS.
Total PG released into the media was quantified using a dimethymethylene blue
(DMB) assay. |TS=insuIin-transferrin-sodium-selenite supplement, FBS=fetaI

bovine serum, LPS=|ipopolysaccharide.

138

8.000 -

7.000 t

3???

nmol NOIWeII

é

 

 

 

Dav

 

 

E ITS I ITS+LPS I3 IIS+LLA+LPS D ITS+T+LPS I IIS+LLA+T+LPS I LPS I FBS l

 

Figure 16. Mean nitrite (N02) released into serum-free (SF) media 24 and 48
hours prior to treatment with ITS, LLA, T, FBS, and LPS, and 24 hours following
treatment. N02 concentration was quantified using an assay employing the
Greiss reaction. ITS=insulin-transferrin-sodium-selenite supplement, LLA=Iinoleic

acid albumen, T=thyroxine, FBS=fetal bovine serum, LPS=lipoponsaccharide.

139

 

References

1. Mariani E, Mariani AR, Monaco MC, et al. Commercial serum-free
media: hybridoma growth and monoclonal antibody production. J Immunol
Methods 1991;145:175—183.

2. Barnes D, Sato G. Methods forgrovvth of cultured cells in serum-
free medium. Anal Biochem 1980;102:255-270.

3. Wessman SJ, Levings RL. Benefits and risks due to animal serum
used in cell culture production. Dev Biol Stand 1999;99:3-8.

4. Loredo GA, MacDonald MH, Benton HP. Regulation of
glycosaminoglycan metabolism by bone morphogenetic protein-2 in equine
cartilage explant cultures. Am J Vet Res 1996;57:554-559.

5. Kawcak CE, Trotter GW, Frisbie DD, et al. Maintenance of equine
articular cartilage explants in serum-free and serum-supplemented media,
compared with that in a commercial supplemented medium. Am J Vet Res
1996;57:1261-1265.

6. Alini M, Kofsky Y, Wu W, et al. In serum-free culture thyroid

hormones can induce full expression of chondrocyte hypertrophy leading to
matrix calcification. J Bone Miner Res 1996;1 1 :105—1 13.

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