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I "‘51" LIBRARY
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This is to certify that the
thesis entitled

BLUNT FORCE INJURY TO CARTILAGE: SOME EFFECTS
OF EXERCISE AND A NUTRACEUTICAL

presented by
LYNN MICHELLE MARTIN

has been accepted towards fulfillment
of the requirements for the

MS. degree in ' Mechanical EngineerinL

 

 

 

mafiy/

flajor Professor’s Signature

00

Date

MSU is an Affirmative Action/Equal Opportunity Institution

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PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2/05 p:lClRC/Date0ue.indd-p.1

 

BLUNT FORCE INJURY TO CARTILAGE:
SOME EFFECTS OF EXERCISE AND A NUTRACEUTICAL

By

Lynn Michelle Martin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Mechanical Engineering

2006

ABSTRACT

BLUNT FORCE INJURY TO CARTILAGE:
SOME EFFECTS OF EXERCISE AND A NUTRACEUTICAL

By
Lynn Michelle Martin

Osteoarthritis is a degenerative joint disease characterized by loss of articular
cartilage and alterations to the underlying subchondral bone. There is evidence of
hereditary defects that may predispose to osteoarthritis, yet other factors such as age,
excessive joint loading, and joint injury increase the risk for development of this disease.
The current study uses an in vivo post-traumatic animal model to investigate blunt
impacts on the patello-femoral joint. Treatment options for the relief of pain due to
chronic joint disease are currently limited. In Chapter 1 the role of two nutraceuticals,
glucosamine and chondroitin sulfate, taken before and after a 6.0 joule blunt impact to the
patello-femoral joint are examined in a regularly exercising animal model. Regular
exercise has been shown to have beneficial effects on preserving articular cartilage in the
knee after a blunt force trauma. Chapter 1 documented that pre-trauma exercise may play
a role in strengthening the cartilage to protect it from severe trauma due to a blunt impact.
Chapter 2 investigates the effects of mechanically stressing cartilage, with intermittent
cyclic compressive loading of chondral explants. This regular loading tends to increase
the mechanical properties of the cartilage. Finally, this investigation of stressed tissue, or
“exercise” versus “no-exercise” is further examined with use of an animal model in
Chapter 3. In this chapter chronic joint degeneration is accelerated in an animal model by

increasing the amount of impact energy to the patello-femoral joint to 10.0 joules.

DEDICATION

I would like to thank my parents for their never ending support and
encouragement throughout my life. Without their support I would never have had the
drive to accomplish all that I have in my life and academic career.

iii

ACKNOWLEDGEMENTS

I would like to acknowledge and thank my professor, mentor, and good friend Dr.
Roger Haut for all he has taught me in the past two years. I am extremely grateful to Dr.
Wright and Dr. Orth for serving on my committee. I would like to extend a sincere thank
you to Clifford Becket, for never hesitating to take the time to what I needed to know and
more, and for always helping to find “the answer” if it wasn’t already known. I would
also like to thank Jane Walsh and Jean Atkinson, for all of their hard work and
dedication. I would like to thank all of the undergraduate students for all of their hard
work and assistance: Zach Kaltz, Austin McPhillamy, and Nurit Golenburg. Last but not
least, I would like to thank my fellow graduate students for their help and friendship:

Eugene Kepich, Mike Sinnott, Eric Meyer, Derek Baars, Steve Rundell.

iv

TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................ vii
LIST or FIGURES .......................................................................................................... vii

BLUNT FORCE INJURY TO CARTILAGE: SOME EFFECTS OF EXERCISE AND A
NUTRACEUTICAL

Introduction ............................................................................................................. 1
References ............................................................................................................... 6
CHAPTER ONE

ADMINISTRATION OF A NUTRACEUTICAL AND EXERCISE TO HELP
PROTECT JOINT CARTILAGE FROM TRAUMA

Abstract ................................................................................................................... 9
Introduction ........................................................................................................... 10
Methods ................................................................................................................. 12
Results ................................................................................................................... 23

Part A: Acute Study ......................................................................................... 23

Part 8: Chronic Study ..................................................................................... 28

Part C: Acute vs. Chronic ............................................................................... 36
Discussion ............................................................................................................. 38
References ............................................................................................................. 44

CHAPTER TWO

MECHANICAL RESPONSE OF CARTILAGE EXPLANTS TO CYCLIC
COMPRESSIVE LOADING

Abstract ............................................................................................................................. 47
Introduction ........................................................................................................... 47
Methods ................................................................................................................. 50
Results ................................................................................................................... 57

Pilot studies ..................................................................................................... 57
Experimental studies ....................................................................................... 65
Two experimental studies combined ............................................................... 71
Discussion ............................................................................................................. 72
References ............................................................................................................. 75

CHAPTER THREE

EFFECTS OF EXERCISE ON JOINT TRAUMA IN A HIGH ENERGY IMPACT

MODEL
Abstract ................................................................................................................. 77
Introduction ........................................................................................................... 77
Methods ................................................................................................................. 79
Results ................................................................................................................... 83

First study: time zero, 1 year exercise (with impact), and 1 year exercise
control (no impact) groups ............................................................................. 83

Second study: 1 year no-exercise and 2 year exercise (control and impact)

groups ............................................................................................................. 88
Histology and comparison between groups and studies ................................. 96
Discussion ........................................................................................................... 101
References ........................................................................................................... 107
CHAPTER FOUR
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK ................... 109
References ........................................................................................................... 1 12
APPENDIX A: Raw data from Chapter 1 ...................................................................... 113
APPENDD( B: Raw data from Chapter 2 ....................................................................... 130
APPENDIX C: Raw data from Chapter 3 ....................................................................... 136
APPENDD( D: Rabbit impact SOP ................................................................................ 150
APPENDD( E: Rabbit indentation SOP ......................................................................... 158
APPENDIX F: Bovine media stock recipe .................................................................... 166
APPENDIX G: Bovine media handling SOP ................................................................ 167

vi

LIST OF TABLES

CHAPTER 1
Table 1 .................................................................................................................. 20
Table 2 .................................................................................................................. 32
Table 3 .................................................................................................................. 37
Table 4 ................................................................................................................. 38

CHAPTER 3
Table 1 .................................................................................................................. 95
Table 2 .................................................................................................................. 98
Table 3 ................................................................................................................ 100

LIST OF FIGURES

INTRODUCTION
Figure 1 ................................................................................................................... 1
Figure 2 ................................................................................................................... 2
Figure 3 ................................................................................................................... 3

CHAPTER 1
Figure 1 ................................................................................................................. 14
Figure 2 ................................................................................................................. 15
Figure 3 ................................................................................................................. 15
Figure 4 ................................................................................................................ 17
Figure 5 ................................................................................................................. 17
Figure 6 ................................................................................................................. 18
Figure 7 ................................................................................................................. 21
Figure 8 ................................................................................................................. 21
Figure 9 ................................................................................................................ 22
Figure 10 ............................................................................................................... 22
Figure 11 ............................................................................................................... 24
Figure 12 ............................................................................................................... 25
Figure 13 ............................................................................................................... 25
Figure 14 .............................................................................................................. 26
Figure 15 ............................................................................................................... 27
Figure 16 ............................................................................................................... 27
Figure 17 ............................................................................................................... 28
Figure 18 ............................................................................................................... 28
Figure 19 .............................................................................................................. 29
Figure 20 ............................................................................................................... 30
Figure 21 ............................................................................................................... 31
Figure 22 ............................................................................................................... 34
Figure 23 ............................................................................................................... 34

vii

Figure 24 .............................................................................................................. 35

Figure 25 ............................................................................................................... 35
Figure 26 ............................................................................................................... 36
CHAPTER 2
Figure 1 ................................................................................................................. 51
Figure 2 ................................................................................................................. 51
Figure 3 ................................................................................................................. 52
Figure 4 ................................................................................................................ 53
Figure 5 ................................................................................................................. 55
Figure 6 ................................................................................................................. 57
Figure 7 ................................................................................................................. 58
Figure 8 ................................................................................................................. 59
Figure 9 ................................................................................................................ 59
Figure 10 ............................................................................................................... 59
Figure 11 ............................................................................................................... 60
Figure 12 ............................................................................................................... 61
Figure 13 ............................................................................................................... 61
Figure 14 .............................................................................................................. 62
Figure 15 ............................................................................................................... 62
Figure 16 ............................................................................................................... 63
Figure 17 ............................................................................................................... 63
Figure 18 ............................................................................................................... 64
Figure 19 .............................................................................................................. 64
Figure 20 ............................................................................................................... 65
Figure 21 ............................................................................................................... 65
Figure 22 ............................................................................................................... 66
Figure 23 ............................................................................................................... 67
Figure 24 .............................................................................................................. 67
Figure 25 ............................................................................................................... 68
Figure 26 ............................................................................................................... 68
Figure 27 ............................................................................................................... 69
Figure 28 ............................................................................................................... 69
Figure 29 .............................................................................................................. 69
Figure 30 ............................................................................................................... 70
Figure 31 ............................................................................................................... 70
Figure 32 ............................................................................................................... 71
Figure 33 ............................................................................................................... 71
Figure 34 ............................................................................................................... 72
CHAPTER 3
Figure 1 ................................................................................................................. 82
Figure 2 ................................................................................................................. 84
Figure 3 ................................................................................................................. 85
Figure 4 ................................................................................................................ 86
Figure 5 ................................................................................................................. 86

viii

Figure 6 ................................................................................................................. 87

Figure 7 ................................................................................................................. 87
Figure 8 ................................................................................................................. 88
Figure 9 ................................................................................................................ 89
Figure 10 ............................................................................................................... 89
Figure 11 ............................................................................................................... 89
Figure 12 ............................................................................................................... 90
Figure 13 ............................................................................................................... 91
Figure 14 .............................................................................................................. 92
Figure 15 ............................................................................................................... 92
Figure 16 ............................................................................................................... 93
Figure 17 ............................................................................................................... 94
Figure 18 ............................................................................................................... 96
Figure 19 .............................................................................................................. 97
Figure 20 ............................................................................................................... 97
Figure 21 ............................................................................................................... 99
Figure 22 ............................................................................................................. 100

ix

INTRODUCTION

Each year in the United States over 775,000 children under the age of 15 are
treated in hospital emergency rooms for sports-related injuries. While apparently .5
percent of these injuries involve broken bones, some of these sports-related injuries can
have more lasting effects to the individual. A single knee injury can put a person at five
times the risk for adulthood osteoarthritis (Arthritis Foundation, 2005). It is estimated that
60% of the population will have symptoms of osteoarthritis by the age of 65 (Green,
2001)

Osteoarthritis is a degenerative joint disease characterized by loss of articular
cartilage and alterations to the underlying subchondral bone. Articular cartilage is a
tough, elastic connective tissue covering the ends of joints. Its purpose is to distribute
load and provide a near frictionless surface for the movement of joint surfaces against
one another. Cartilage is composed of chondrocytes (cells) surrounded by a matrix of
water, collagen (fibrous proteins), and proteoglycans (Figure 1). Proteoglycans are
protein aggregates having polysaccharide side-chain units known as glycosaminoglycans

(GAGS). As the joint is
Collagen subjected to load, the

Cho ndrocyte _ . _
cartilage wrll deform in order

  
   

Proteoglycan to distribute the load, causing

compressive, tensile, and
Interstitial Water

shear stresses throughout the
Figure l. The extracellular matrix of articular cartilage is
composed mainly of collagen fibers, proteoglycans, and water. cartilage (Mow and Setton,

1998). The fimction of the collagen is to provide the cartilage with tensile strength (Mow

and Setton, 1998), whereas the proteoglycans are associated more with the stiffiress
properties of the cartilage in compression (Helminen et al., 1992). The content and
structure of proteoglycans and collagen fibers varies throughout the depth of the
cartilage. The matrix can be divided into three regions: a superficial tangential zone, a
middle zone, and a deep zone (Figure 2).

Ameular surface

Zones

      

. Tide mark
n .,.-. .,: vhhf‘é" 1‘7..." —
Calcrlred cartilage /i$}£&@crcrfiggc°%tr ‘3"? Subchondrai bone
' \ Canoelbus bone

 
 

Figure 2. A sketch showing the cross section of cartilage, illustrating the
collagen network and the three distinct regions of this tissue.

Hereditary defects may predispose to osteoarthritis, yet other risk factors such as
age, excessive joint loading, and joint injury increase the risk for development of this
disease (Buckwalter et al., 2004; Helminen et al., 1992; Gelber et al., 2000; Marsh et al.,
2002). Osteoarthritis is thought to be initiated by fibrillation (the unbinding of collagen
fibrils and surface fraying) and swelling of the cartilage matrix due to the influx of fluid.
This increased hydration leads to a softening of the articular cartilage, which increases
the pressure on the underlying subchondral bone (Radin et al., 1996). These early stages
of osteoarthritis may initiate an increase in the subchondral bone thickness, and lead to
changes such as osteophyte formations (bony outgrowths) and erosion of the articular

cartilage, eventually causing complete loss of this sofi tissue (Figure 3).

 

Figure 3. The progressive stages of 0A. A) Normal articular
cartilage and bone. B) Cartilage surface becomes fibrillated and
the subchondral bone thickens. C) Total loss of cartilage with
bone cyst formation.

Our laboratory has developed a post-traurnatic animal model to study the
degenerative joint changes in vivo using a Flemish Giant rabbit (I-Iaut et al., 1995).
Methods of evaluating changes in joint tissue include stress—relaxation testing via
mechanical indentation to determine changes in mechanical properties of articular
cartilage (Garcia, 1998; Hayes et al., 1972), histological sectioning to assess structural
and cellular changes throughout the depth of the cartilage, and biochemical testing to
measure alterations in tissue composition, such as in the content of proteoglycans.
Previous studies by our laboratory have shown significant softening of the cartilage,
thickening of the subchondral plate, and histological degradation by 7.5 months post-
irnpact on the patello-femoral joint of rabbits (Newberry et al., 1997; Newberry et al.,
1998; Ewers and Haut, 2000; Ewers et al., 2002). End-stage disease with complete loss of

articular cartilage has eluded various studies by this laboratory.

Treatment options for the relief of osteoarthritis pain are very limited. Mild cases
are commonly treated with non-steroidal anti-inflammatory drugs (N SAIDS).
Unfortunately NSAIDS can have side effects, such as stomach ulcers and kidney damage,
and do nothing to slow the progression of the disease. However, the nutraceuticals
glucosamine and chondroitin sulfate have received considerable attention as a treatment
for relieving pain and delaying the progression of osteoarthritis. (Braham et al., 2003;
Pavelka et al., 2002; Richy et al., 2003; Tiraloche et al., 2005). Little is known about
these nutraceuticals and their effects on the mechanical and biochemical properties of
cartilage in vivo. Chapter 1 evaluates the efficacy of glucosamine and chondroitin sulfate
on enhancing the mechanical and biochemical properties of cartilage in an animal model
prior to and after a blunt impact to its patello-femoral joint. These studies describe the
results of both acute and chronic studies on the tissue after a severe (6.0 J) impact to the
joint.

In vivo experiments on the response of a joint to blunt force trauma using animal
models are very expensive and time consuming (Parkkinen et al., 1989). In vivo models
also create difficulties in controlling the loading situation and the cellular response of
joint tissues following trauma (Parkkinen et al., 1989). Many new mechanical explant
testing systems have been developed to better control loading of the joint tissues by using
chondral or osteochondral cartilage explants (Torzilli et al., 1997; Sah et al., 1989;
Sauerland et al., 2003). Our laboratory has recently created a “cartilage exerciser” to
cyclically load chondral explants. Chapter 2 examines the effects of intermittent cyclic
loading and the differences in mechanical properties and cell death between loaded and

non-loaded chondral explants in a series of pilot studies with this newly developed

device. The role of exercise in the rehabilitation of a joint after trauma has been a subject
of controversy over the years. Numerous studies have shown beneficial effects of regular
exercise (Jurvelin et al., 1986; Ottemess et al., 1998; Weaver and Haut, 2005), however,
evidence has also demonstrated a deleterious effect of excessive (repetitive) joint loading
in normal and injured joints (Buckwalter et al., 2004).

In a previous study by another laboratory using a high intensity 10.0 J blunt
impact animal model, advanced signs of cartilage degeneration were documented as early
as 3 months post-impact (Mazieres et al., 1987). The model showed a thickening of the
subchondral bone at 3 months post-trauma, and exposure of subchondral bone by 6
months. In contrast, in a similar study by our laboratory using Flemish Giant rabbits with
a 10.0 J impact showed there were few signs of advanced disease by 7.5 months post
trauma (Weaver, 2001). However, a major difference noted between the studies by
Mazieres et al. and Weaver was the level of post-trauma exercise. The rabbits in the
Mazieres study were confined to cage activity, whereas the rabbits in the Weaver study
were subjected to a daily exercise regimen. Chapter 3 addresses the issue of regular

exercise versus normal cage activity in a high intensity (10.0 J) impact animal model.

REFERENCES

Braham,R., Dawson,B., and Goodman,C. (2003) The effect of glucosamine
supplementation on people experiencing regular knee pain. Br J Sports Med 37(1), 45-49.

Buckwalter,J.A., Saltzman,C., and Brown,T. (2004) The impact of osteoarthritis:
implications for research. Clinical Orthoaedics and Related Research 427 Suppl, S6-
SIS.

Ewers,B.J. and Haut,R.C. (2000) Polysulphated glycosaminoglycan treatments can
mitigate decreases in stiffness of articular cartilage in a traumatized animal joint. Journal
of Orthopaedic Research 18(5), 756-761.

Ewers,B.J., Weaver,B.T., Sevensma,E.T., and Haut,R.C. (2002) Chronic changes in
rabbit retro-patellar cartilage and subchondral bone after blunt impact loading of the
patellofemoral joint. Journal of Orthopaedic Research 20, 545-550.

Garcia,J.J. (1998) A transversely isotropic hypo-elastic biphasic model of articular
cartilage under impact loading. Michigan State University, East Lansing.

Gelber,A., Hochberg,M., Mead,L., Wang,N., Wigley,F., and Klag,M. (2000) Joint injury
in young adults and risk for subsequent knee and hip osteoarthritis. annals of internal
medicine 133(5), 321-328.

Green,G.A. (2001) Understanding NSAIDs: from aspirin to COX-2. Clinical
Cornerstone 3, 50-60.

Haut,R.C., Ide,T.M., and DeCamp,C.E. (1995) Mechanical responses of the rabbit
patello-femoral joint to blunt impact. Journal of Biomechanical Engineering 117(4), 402-
408.

Hayes,W.C., Keer,I.M., Herrmann,G., and Mockros,I.E. (1972) A mathematical analysis
for indentation tests of articualr cartilage. J Biomechanics 5, 541-551.

Helminen,H.J., Kiviranta,I., Saamanen,A.-M., Jurvelin,J.S., ArokoskiJ., Oettmeier,R.,
Abendroth,K., Roth,A.J., and Tammi,M.I. (1992) Effect of motion and load on articular
cartilage in animal models. In Articular Cartilage and Osteoarthritis (Edited by
Kuettner,K.E., Schleyerbach,R., Peyron,J.G., and Hascall,V.C.) Pp. 501-509. Raven
Press, New York.

Jurvelin,J., Kiviranta,I., Tammi,M., and Hehninen,H. (1986) Effect of physical exercise
on indentation stiffiiess of articular cartilage in the canine knee. Int.J.Sports Med. 7, 106-
110.

Marsh,J.L., Buckwater,J., Gelberrnan,R., Dirschl,D., Olson,S., Brown,T., and Llinias,A.
(2002) Articular fractures: does an anatomic reduction really change the result? Journal
of Bone and Joint Surgery 84-A, 1259-1271.

Mazieres,B., Blanckaert,A., and Thiechart,M. (1987) Experimental post-contusive osteo-
arthritis of the knee: Quantitative microscopic study of the patella and the femoral
condyles. Journal of Rheumatology 14, 1 19-121.

Mow,V.C. and Setton,L.A. (1998) Mechanical properties of normal and osteoarthritic
articular cartilage. In Osteoarthritis (Edited by Brandt,K.D., Doherty,M., and
Lohmander,L.S.) Pp. 108-121. Oxford University Press.

Newberry,W.N., MacKenzie,C., and Haut,R.C. (1998) Blunt impact causes changes in
bone and cartilage in a regularly exercised animal model. Journal of Orthopaedic
Research 16, 348-354.

Newberry,W.N., Zukosky,D.K., and Haut,R.C. (1997) Subfracture insult to a knee joint
causes alterations in the bone and in the fuctional stiffness of overlying cartilage. Journal
of Orthopaedic Research 15, 450-455.

Ottemess,I.G., Eskra,J.D., Bliven,M.L., Shay,A.K., Pelletier,J.-P., and Milici,A.J. (1998)
Exercise protects against articular cartialge degeneration in the hamster. Arthritis Rheum.
41(1 1), 2068-2076.

Parkkinen,J.J., Lammi,M.J., Karjalainen,S., Laakkonen,J., Hyvarinen,E., Tihonen,A.,
Helminen,H.J., and Tammi,M. (1989) A mechanical apparatus with microprocessor
controlled stress profile for cyclic compression of cultured articular cartilage explants.
Journal Biomechanics 22, 1285-1291.

Pavelka,K., Gatterova,J., Olejarova,M., Machacek,S., Giacovelli,G., and Rovati,L.C.
(2002) Glucosamine sulfate use and delay of progression of knee osteoarthritis: a 3-year,
randomized, placebo-controlled, double-blind study. Arch Intern Med 162(18), 2113-
2123.

Radin,E.L., Burr,D.B., Fyhrie,D., Brown,T.D., and Boyd,R.D. (1996) Characterisitcs of
joint loading as it applies to osteoarthritis. In Biomechanics of Diarthrodial Joints
(Edited by Mow,V., Ratcliff,A., and Woo,S.L.-Y.) Pp. 437-451.

Richy,F., Bruyere,O., Ethgen,O., Cucherat,M., Henrotin,Y., and Reginster,J. (2003)
Structural and Symptomatic Efficacy of Glucosamine and Chondroitin Sulfate in Knee
Osteoarthritis. Arch Intern Med 163, 1514-1522.

Sah,R.L., Kim,Y.-J., Doong,J.-Y.H., Grodzinsky,A.J., Plaas,A.H., and Sandy,J.D. (1989)
Biosynthetic response of cartilage explants to dynamic compression. Journal of
Orthopaedic Research 7, 619-636.

Sauerland,K., Raiss,R.X., and steinmeyer,J. (2003) Proteoglycan metabolism and
viability of articular cartilage explants as modulated by the frequency of intermittent
loading. osteoartritis and cartilage 11, 343-350.

Tiraloche,G., Girard,C., Chouinard,L., Sampalis,J., Moquin,L., Ionescu,M., Reiner,A.,
Poole,A.R., and Laverty,S. (2005) Effect of oral glucosamine on cartilage degradation in
a rabbit model of osteoarthritis. Arthritis and Rheumatism 52, 1 118-1 128.

Torzilli,P.A., Grigiene,R., Huang,C., Friedman,S.M., Doty,S.B., Boskey,A.L., and
Lust,G. (1997) Characterization of cartilage metabolic response to static and dynamic
stress using a mechanical explant test system. Journal of Biomechanics 30(1), 1-9.

Weaver,B.T. and Haut,R.C. (2005) Enforced exercise after blunt trauma significantly
affects biomechanical and histological changes in rabbit retro-patellar cartilage. Journal
of Biomechanics In Press.

Weaver, B.T. 2001. Chapter Two: Regular exercise is beneficial in a stable joint after
trauma. The analysis of tissue response following a single rigid blunt impact in an in vivo
animal model: Thesis for the degree of MS Michigan State University, 33-50.

CHAPTER ONE

ADMINISTRATION OF A NUTRACEUTICAL AND EXERCISE TO HELP
PROTECT JOINT CARTILAGE FROM TRAUMA

ABSTRACT

Blunt force trauma to the patello-femoral joint has been shown to cause
degradation of cartilage, often times leading to degenerative disease of the joint such as
osteoarthritis. Treatment options for the relief of pain due to degeneration have been very
limited. New chondroprotective agents have been introduced to help normalize the
cartilage matrix, and possibly strengthen the cartilage by increasing the synthesis of
proteoglycans. Two studies were executed, where we supplemented the daily feed of
Flemish Giant rabbits with 2% Cosamin®DS (containing glucosamine and low molecular
weight chondroitin sulfate) before impacting the patello-femoral joint with a 6.0 Joule
impact. All animals were exercised regularly, and a non-diet supplemented group was
used for a controlled comparison. Mechanical, histological, and biochemical observations
were made in the first study immediately after impact. The second study consisted of a
short-term supplemented group (supplementation only before impact), a long-term
supplemented group (supplementation before and after impact), and a non-supplemented
control group. All specimens underwent an additional 24 weeks of exercise after impact.
Results of the current studies revealed less of a difference in fissuring between impacted
and non-impacted limbs, a slight increase in stiffness, and a trend for a decrease in

permeability in the Cosamin®DS treated groups.

INTRODUCTION

Each year in the US. an estimated 30 million children and adolescents participate
in organized sports (NIH, 1991), and approximately 150 million adults participate in non
work-related physical activities (CDC, 2003). With a recent societal emphasis on a
healthy lifestyle, more people are beginning to exercise and are becoming involved in
sports activities. Yet participation in sports has a risk of injury and has evolved as a cause
of osteoarthritis (OA), especially in hip and knee joints (Gelber et al., 2000).

Severe impact trauma to a joint has been shown to damage the articular cartilage
matrix and kill cells (Lewis et al., 2003), and is also a suspected factor in the initiation of
progressive disease, such as osteoarthritis (Gelber et al., 2000; Marsh et al., 2002). Our
laboratory has developed a model using a single blunt impact to the flexed patello-
femoral (PF) joint of Flemish Giant rabbits (Haut et al., 1995), involving regular
treadmill exercise of the animals (Oyen-Tiesma et al., 1998). This model shows a
significant softening of the retro-patellar cartilage, and an increase in the number of
fissures, average fissure depth, and total fissure length on the impacted limb at 4.5
months post-trauma (Ewers et al., 2002). At 7.5 months post-trauma, a significant
increase in permeability, thickness of the subchondral plate (Ewers et al., 2002), and loss
of proteoglycans has been documented (Ewers and Haut, 2000).

Various interventions have been introduced as possible treatments to mitigate the
development of clinical OA. Polysulfated glycosaminoglycan treatments have been
shown to inhibit the degradation of articular cartilage both clinically (Howell et al., 1986;
May et al., 1988), and also in vivo using a post-trauma animal model (Ewers and Haut,

2000). These treatments have helped to limit softening of the articular cartilage, without

10

changes in underlying bone and fissure depth (Ewers and Haut, 2000). Regular treadmill
exercise of the animal model, as opposed to cage activity, has also shown beneficial
effects by limiting the amount of histological degradation of the cartilage such as
ossification/calcification and erosion (Weaver and Haut, 2005).

Chondroprotective therapeutic agents have been introduced as a method of
slowing cartilage degeneration, normalizing the cartilage matrix, and possibly stimulating
the synthesis of glycosarninoglycans (Lippiello et al., 2000). Two of these agents,
glucosamine and chondroitin sulfate, have shown some degree of efficacy in the relief of
joint pain and reduction of joint space narrowing in patients with clinically diagnosed
osteoarthritis (Richy et al., 2003). In vitro and in vivo studies have shown that this
nutraceutical enhances the synthesis of cartilage matrix proteoglycans (PG’s); (Oegema
et al., 2002; Tiraloche et al., 2005), especially in mechanically stressed tissue (Lippiello,
2003). However, little is known about this nutraceutical and its effects on the mechanical
properties in the in vivo setting.

Two separate studies will be discussed in this chapter. In the first study, the acute
study, the hypothesis was that 2 months administration of a commercial nutraceutical,
Cosamin®DS (containing glucosamine and low molecular weight chondroitin sulfate), to
an exercising animal model would be effective in enhancing the tolerance of joint tissue
cartilage to acute blunt impact, by increasing the level of tissue proteoglycans (PG’s). In
theory, this increase in PG’S would stiffen the retro-patellar cartilage and reduce the
extent of acute damage under a defined impact load.

After the results of the acute study, it was hypothesized that more time for

degradation may be necessary for the supplement to have a significant effect, so a chronic

11

study was conducted with another group of animals. The hypothesis of the chronic study
was that continued diet supplementation after blunt impact trauma to the joint would limit
the degree of chronic degeneration in the joint cartilage, based on its biomechanical

properties and histological appearance after 6 months post blunt impact trauma.

METHODS

A total of sixty-one mature Flemish Giant rabbits were used in two separate
studies. Twenty-five of the rabbits (5.7 i 0.5 kg, 6-8 months of age) were used in an
acute study. Another group of animals, thirty-six rabbits (5.7 i 0.6 kg, 6-8 months of age)
were purchased from the same breeder at the end of the acute study, and used for a
chronic study. Animal experiments were conducted with the approval of the All-
University Committee on Animal Use and Care. For the acute study the rabbits were
randomly split into two groups: a control group with no dietary supplementation (n=12),
and a group that had their 200g of daily feed supplemented with 2% Cosamin®DS
(Nutramax Laboratories, Inc., Edgewood, MD) (n=l3) (Figure 1a). For a two-month pre-
impact period, all animals were exercised 10 minutes a day, 5 days a week at 0.3 mph on
a treadmill (Oyen-Tiesma et al., 1998). When not exercising, all animals were housed
individually in cages (122 cm x 61 cm x 49 cm). At the end of the two-month exercising
period, both diet-supplemented and normal diet animals were euthanized with a lethal
injection of Pentabarbitol (85.9 g/kg) and within 5 minutes, received a single blunt
impact to the right patello-femoral joint (discussed later).

For the chronic study the rabbits were first randomly divided into two groups: a

control group with no dietary supplementation (n=12); and a group that had their 200g of

12

daily feed supplemented with 2% Cosamin®DS (Nutramax Laboratories, Inc., Edgewood,
MD) (n=24). For the two-month pre-impact period all animals were exercised 10 minutes
a day, 5 days a week at 0.3 mph on a treadmill. When not exercising, all animals were
housed individually in cages (122 cm x 61 cm x 49 cm). After the 8 weeks of exercise, all
animals were anesthetized (2% Isoflurane and oxygen) and received a single blunt impact
to the right patello-femoral joint. The animals receiving dietary supplementation were
then split into two groups of 12. One group continued to receive daily dietary
supplementation for 24 weeks, while the other group had a normal diet. Post trauma, all
animals were allowed a 5-day period of rest before continuing their daily exercise
regimen for 24 weeks (Figure 1b). At this time all animals were euthanized with a lethal

injection of Pentabarbitol (85.9 g/kg) (Weaver, 2001).

13

 

Treatment 8 Weeks 6 J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N=13 ercrse Impact
L .

25 2 Weeks Mechanical,
Animals Conditfining Histological.
Biochemical
Evaluations

~=12 W Impact

(a)

 

 

 

 

 

 

Treatment ‘ 5 um: I 6 J
=24

Exercise lrnpact

 

 

 

 

 

 

     
 

Mechanical, ‘

 

 

 

 

36 2 Weeks Exercise Histological.
Animals CBnHitr'onr'ng Biochemical
‘——‘ Evaluations

 

 

 

 

 

No Treatment 8 Weeks 6 J
#
N=12 Exercise Impact Treatment

 

 

 

 

 

 

Exercise N=12

 

 

(b)

Figure 1. (a) Acute study schedule; (b) chronic study schedule.

Impacts were administered with a gravity drop fixture, which has been used in
previous studies by this laboratory. Blunt impact was administered to the right hind
patello-femoral joint (Newberry et al., 1998) (Figure 2) (see Appendix D for standard
operating procedure). Each animal was placed in a specially designed chair that held the
right hind limb rigid, while flexed at 120° with the animal supine and the femur aligned
vertically. A strap was placed across the left hind limb, which prevented the pelvis from

rotating during impact. Six joules of impact energy was administered by dropping a 1.33

14

kg mass from a height of 0.46 m
with a rigid impact interface.

(b)
The impact did not result in

bone fracture. The dropped mass
(A was arrested electronically after
\ \l“
|
|\H/ l the first impact to prevent

multiple impactions. A load

 

 

 

  

" i ‘ _ transducer (model 31/1432:
Figure 2. (21) Photograph of the impact set-up; (b) Sketch
'llustratin the ‘ act load directed onto the t 11a.

1 g “up pa 6 Sensotec, Columbus, OH,

USA.) with a 2.2 kN capacity was attached behind the impact head to record the impact
loads. Experimental data were collected at 10 kHz by a personal computer equipped with
an analog-to-digital board. The peak load and time to peak were recorded from the load

versus time curves (Figure 3).

 

800 Jn‘

 

 

Peak load
600 - '

E400 ,

200 -

 

     

 

 

o 0.002 from 0.006 0.008 0.01

True (sec)

 

 

 

 

Time to peak

Figure 3. Typical plot of load-time data collected during impact.

15

Immediately after sacrifice, impacted and un-impacted patellae were excised and
matrix damage was assessed. Retro-patellar surfaces were wiped with India ink,
photographed at 25X under a dissection microscope (Wild M5A, Wild Heerbrugg Ltd.,
Switzerland), and evaluated in terms of total surface fissure length using digital image
software (SigmaScan, SPSS Inc., Chicago, IL) (Ewers et al., 2002). The patellae were
then immersed in room-temperature, phosphate buffered saline (pH 7.2) for mechanical
indentation tests on the retro-patellar cartilage (see Appendix E for standard operating
procedure). Briefly, each patella was placed in a clamp attached to a camera mount
(Bogen, Ramsey, NJ) which was secured to the base of a custom made mounting frame,
which allowed three degrees of movement for precision placement of the patella under
the indenter tip (Figure 4). The camera mount allowed rotation of the patella, while the
mounting plate allowed translation. The mounting insured indentation tests were
performed perpendicular to flat locations on the patella. The tests were performed using a
computer controlled stepper motor (Physik Instruments, Waldbrom, Germany: model M-
16830), at two different sites on the lateral retro-patellar facet (Figure 5). A 1.0 mm
diameter flat, non-porous probe was pressed 0.1 mm into the cartilage in 30 ms and held
for 150 seconds while resistive loads of relaxation were measured (Data Instruments,
Acton, MA: model JP-25, 25 lb capacity), amplified, and collected at 1000 Hz for the
first second and 20 Hz thereafter. The cartilage was then allowed to recover for 5
minutes, and the test was repeated with a 1.5 mm diameter flat, non-porous probe. After
another 5-minute recovery, the thickness of the indentation site was determined by

depressing a needle probe into the cartilage.

l6

Specimen clamp

Stepper motor

  
 

Z plate l
Load cell
lndenter

Camera mount

 

X-Y mounting
plate

Figure 4. Photograph of the indentation test fixture. The X-Y mounting plate allows for
left/right or forward/backward placement, and the Z plate allows for up.down placement.
The camera mount allowed for rotation of the sample to set the surface perpendicular to
the indenter.

Retro-patellar surface

Lateral Facet

Central Ridge

 

Figure 5. Photograph of excised rabbit patella.
(Original photograph = 25X). 0 Indicates two
sites where mechanical indentation tests are
performed.

Cartilage has two phases (solid and liquid) with a superficial zone formed by
sheets of tightly woven collagen fibrils (Figure 2, pg 2), which suggests a model with a
Young’s modulus in the plane (E 1 1) different than that in the direction perpendicular to

the surface (E33) (Garcia et al., 1998)

3

(Figure 6). Therefore, mechanical data
were analyzed using a biphasic
(poroelastic) model having a transversely

isotropic (TI) solid structure. Four elastic

 

parameters (E11. E33, 013. and V31) and

Figure 6. Illustration of coordinate system
designated to cartilage. Isotropy is assumed in
the plane 1-2.

two permeability measures (k; and k3)
were computed using a curve-fitting
algorithm (Garcia et al., 1998). Poisson’s ratio V]; was assumed equal to:

V12 =1 ‘ 0-5(E11/E33) (1)

After completion of the mechanical indentation tests, the patellae were split in
half for evaluation of tissue proteoglycan content using the DMB assay (Farndale et al.,
1982) and histological sectioning using standard methods (Atkinson et al., 1998). All
patellae were histologically processed by placing them in 10% buffered formalin for
seven days, followed by decalcification in 20% formic acid for another seven days.
Tissue blocks were cut transversely across the patella in areas of high contact pressure
(Haut et al., 1995). Histological sections (six) were cut 8 microns thick and stained with
Safi'anin O-Fast Green and examined in light microscopy at 12—100X. Each histologic
section was analyzed to determine the area most affected by impact. A histopathalogic

scoring system was developed based on the literature (Colombo et al., 1983; Mazieres et

al., 1987). This system was used to quantify the progression of degenerative changes in
the cartilage (examples shown in Figures 7-9). Each aspect was graded from 0 (normal or
absent) to +4 using the guidelines in Table 1. One independent blinded reader (JW)
examined each of six slides for each sample to find the most representative slide. This
slide was then assessed for each parameter at three locations on the patella: medial,
central, and lateral, and index scores were recorded. The scores of each parameter were
then summed across these locations. The mean and range of each parameter were
documented for the impacted and non-impacted limb of both impact groups. The
thickness of the subchondral bone plate underlying the retro-patellar cartilage was
measured at 25X for all six histology sections with a calibrated eye-piece at the center of
each facet (medial, central, and lateral) by a single investigator (J .W.) using established

protocols (Newberry et al., 1998) (Figure 10).

19

 

 

 

 

 

 

 

 

 

 

 

 

0 +1 +2 +3 +4
. . . Moderately .
Surface Integrity Regular Slightly irregular irregular Focally severe Extensrvely severe
Proteoglycan Focally severe
staining Normal Slight loss ”9‘1”“ '°55 (‘° (loss beyond Total loss
. mid zone) .
(Figure 8) mid zone)
- - 5 or more (small), 3
l... .. 24:32.12: 1 2:22:25” .. m...
or 1 (full thickness)
1 2 5-6 (small) or 7 or more ,
Clones ' a 3-4 (small) orb 3,4 (medium) or (small) or 5-6 7 or more (medium)
(small) 1-2 (medium) 1 c (medium) or 3- or 5 or more (large)
1'2 ( arge) 4 (large)
Disruptions Absent --....---..--.. Compression .. Horizontal or
(Figure 9) ridges Vertical splits
Ossification Absent ------------- ---------- Present ----------
Exposure of
Subchondral Absent -------------- -------------- Present - ----------
Bone
5'9”" Absent Detectable Moderate Focally severe Extensively severe
(Figure 8)
Zone of C alcified
“Image 0-10 units 11-13 units 1447 units 18-23 units 24 or more units
Thickness (at
100x)
Z0"? “C“C‘fie" Normal Slight Moderate Fm"? Excessive
Cartilage Cells excessrve
Subchondral Less than
Bone Thickness . 20-29 units 30-39 units 40-49 units 50 or more units
(at 40x) 20 units

 

b
aSmall = 2-4 cells Medium = 5-8 cells cLarge = 9 or more cells

1 unit = 0.02 mm at 40x = 0.008 mm at 100x

Table l. Histopathologic scoring system.

20

 

Figure 7. Section from a chronic non-supplemented animal showing
local erosion and loss of proteoglycans in the vicinity of irnpact-
induced lesions. (Original photograph= 40X).

'Bu40 Riht

 

Figure 8. Section from a chronic short-term supplemented animal
showing horizontal disruption. (Original photograph = 40X).

21

Deep-zone fissure Mid-zone fissure

81153 Left

 

Figure 9. (a) Section from an acute supplemented animal showing surface and
deep-zone fissuring. (b) Section from a chronic control animal showing mid-
zone fissuring. (Original photographs = 40X).

    

_ A ‘ -
Figure 10. Histologic section of patella: arrows indicate the
subchondral bone thiclcness measurements.

A two way (limb, group) repeated factor analysis of variance (ANOVA) with a
Student-Newman—Keuls post hoc test was used to test for statistical differences between

impacted and non-impacted limbs within a group, and to test differences between groups.

22

The Wilcoxon-Signed Rank test was used to test for differences between impacted and
non-impacted limbs in all groups based on the histopathologic index scores, while the
Mann-Whitney Rank Sum test was used to test for differences between groups. The
Pearson Product Moment Correlation test was used to study statistical correlations
between mechanical parameters and proteoglycan content of the tissue. Statistical

significance was set at p < 0.05.

RESULTS
Part A: Acute Study

In the acute study no significant differences were noted by the veterinary
technician (J .A.) in the gait or health of rabbits on the supplemented or non-supplemented
diets. The blunt impact forces on the patello-femoral joints and the times to peak were not
different between the non-supplemented (654 :1: 154 N; 5.92 i 0.48 ms) and
supplemented (649 i 204 N; 5.55 i 0.42 ms) groups.

Gross photographs of the retro-patellar cartilage surface were studied and
revealed significant differences in the total length of surface fissures between the non-
impacted (4.93 d: 6.57 mm) and the impacted (11.33 :t 11.53 mm) limbs of the non-
supplemented group (p=0.044, Figure 11). The same trend was present between the non-
impacted (5.65 :l: 8.28 mm) and the impacted (11.20 i 6.04 mm) limbs of the

supplemented group (p=0.074), yet the difference was not significant (Figure 11).

23

L3 Non-Impacted Limb

 

I Impacted Limb

Total Fissure Length (mm)

 

 

 

Acute Control Acute Supplemented

'Significantly different from contralateral non-impacted limb

Figure 11. Bar graph of total surface fissure lengths of impacted and non-
impacted contralateral limbs of each group in the acute study. Lengths were
measured using digital imaging software.

Analyses of the indentation relaxation data revealed no significant differences
between contralateral limbs for En (p=0.93), E33 @=0.53), k, (p=0.56), and k3 (p=0.38)
of the non-supplemented group, or for E11 (p=0.26), E33 Q3=O.18), or k3 (p=0.23) in the
supplemented group. There was however, a significant difference between limbs for the
k, permeability in the supplemented group (p=0.04). No differences between groups were
found for E11 (p=0.93), E33 (p=0.69), and k3 (p=0.63), yet there was a significant
difference between groups in the k, permeability in the impacted limbs (p=0.04) (Table 3,

Figures 12-13).

24

 

 

E11 (MPa)

   

 

 

Acute Control Acute Acute Control Acute
Supplemented Supplemented
El Non-impacted Limb I Impacted Lime la Non-impacted Limb I Impacted Limbj
(a) (b)

Figure 12. (a) Bar chart of the E 1 1 modulus for impacted and non-inrpacted limbs of each group in
the acute study. (b) Bar chart of the E 33 modulus for irrrpacted and non-impacted limbs.

 

 

 

   

in
‘7
<
O
1-
3!.
tn
Z
st
<
E
1')
x
Acute Control Acute
Supplemented Acute Control Acute

Supplemented

 

 

ENOn-impacted Limb I impacted Limb]

 

IENon-impacted Limb I Impacted Limb

 

'StatisticaI/y different from
contralateral non-impacted limb

(a) (b)

Figure 13. (a) Bar chart of the k, permeability for impacted and non-impacted limbs of each group in
the acute study. (b) Bar chart of the k3 permeability for impacted and non-impacted limbs.

The tissue proteoglycan (PG) content was significantly greater in the impacted
limb of the supplemented group (21.8 d: 5.5 rig/mg W.W.) compared to the non-impacted
limb (18.3 :1: 6.1 rig/mg W.W., p=0.023). Conversely, PG content was not different

between the impacted (22.0 i 7.7 rig/mg W.W.) and non-impacted (20.9 i 7.7 rig/mg

25

W.W.) limbs of the non-supplemented group (p=0.364, Figure 14). There was also no
difference found between non-supplemented (21.5 :l: 7.5 pig/mg W.W.) and supplemented
(20.4 :t 5.7 rig/mg W.W.) groups (p=0.672, Figure 14). The correlation analyses revealed
a significant, negative correlation between the proteoglycan content of retro-patellar
cartilage and k, in the non-supplemented group (p=0.037, Figure 15). No other
significant correlations were documented between proteoglycan content and any of the
other mechanical parameters for either group (Figures 16-18), however, there did seem to
be a positive trend in the non-supplemented group between proteoglycan content and

both the E l 1 (p=0.168, Figure 16) and the E33 moduli (p=0.072, Figure 17).

 

t3 Non-impacted Limb
I Impacted Limb

 

 

 

 

 

 

 

 

 

 

 

A 35
at
g 30+ ....................................
a
3- 251 ------------------------ 3 ----------
E 203-": --------- I
a 15----— ——————— /
3
e 10..." ....... -..-
8 5.
2 _--- _______ _--
0 .
Acute Control Acute Supplemented

‘Significantl y different from contralateral impacted limb.

Figure 14. Bar chart of proteoglycan contents, measured in wet
weight, for contralateral limbs in each group of the acute study.

26

 

 

 

 

 

 

R = 0.606

8.0
R = 0.272
2": 0 Controls
3
S.
g I Supplemented
3
<
E .
:1 —Linear
3 (Controls)
, Linear
0.0 i i . (Supplemented)
0 1O 20 30 40
PG Content (pg/mg WW)

Figure 15. Correlation plot of the k, permeability vs. the proteoglycan content for the
supplemented vs. non-supplemented groups of the acute study. Right and left limbs were

 

 

 

 

 

 

averaged for each group.
14-0 R = 0.425
R = 0.057
0 Controls
1?
n.
E I Supplemented
E
4.0 - —————————————————————————————————————————————— —— Linear
(Supplemented)
2.0 - _______________________________________________ .
-—Linear
0.0 i i . (Controls)
0 10 20 30 40

PG Content (pg/mg WW)

Figure 16. Correlation plot of the E 1 , modulus vs. the proteoglycan content for the
supplemented vs. non-supplemented groups of the acute study. Right and left limbs were

averaged for each group.

27

 

3.5 R = 0.538

 

 

 

 

3.0 - ------------------------------------------------ R = 0.340
O

2.5 4 0 Controls
2’
g 2'0 q I Supplemented
8 1.5 -
m -——Linear

1'0 l --------------------------------------- (Supplemented)

0'5 ' """""""""""""""""""""""""""""""""" —Linear

(Controls)
0.0 i . .
0 10 20 30 40
PG Content (pg/mg WW)

Figure 17. Correlation plot of the E 3 3 modulus vs. the proteoglycan content for the supplemented
vs. non-supplemented groups of the acute study. Right and left limbs were averaged for each group.

 

 

 

 

 

3.0
R = 0.355
A 25 . ________________________ ‘ ................................
g R = 0.320
<
E . Controls
0
E - Slpplemented
5" —Linear
(Supplemented)
0.0 I . r — Linear (Controls)
0 10 20 30 40
PG Content (pglmg WW)

Figure 18. Correlation plot of the k3 permeability vs. the proteoglycan content for the supplemented
vs. non-supplemented groups of the acute study. Right and left limbs were averaged for each group.

Part B: Chronic Study
In the chronic study no significant differences were noted by the veterinary
technician (J .A.) in the gait or health of rabbits on the supplemented or non-supplemented

diets. The blunt impact forces on the patello-femoral joints and the times to peak were not

28

different between the non-supplemented (633 a: 114 N; 4.4 a: 1.0 ms), short-term
supplemented (576 i 118 N; 3.5 i 1.9 ms), and long-term supplemented (593 i 96 N; 4.6
:1: 1.1 ms) groups.

Gross photographs of the retro-patellar cartilage surface were studied and
revealed significant increases in the total length of surface fissures in the non-impacted
limb (11.09 :i: 8.00 mm) versus the impacted limb (17.56 d: 13.34 mm) of the non-
supplemented group (p=0.047) (Figure 19). Conversely, no differences were documented
between impacted (18.11 :t 13.09 mm) and non-irnpacted (21.97 i 16.30 mm) limbs of
the short-term supplemented group (p=0.295), or between impacted (22.72 i 11.95 mm)

and non-impacted (20.38 i 12.26 mm) limbs of the long-terrn supplemented group

 

 

 

 

(p=0.517).
D Non-Impacted Limb

E 45 l Impacted Limb
E 40-.. 77777777777777
.r:
‘63
c
til
.i
2
3
(It
.2
u.
.72
o
.—

Chronic Control Chronic Short-term Chronic Long-terrn

Supplemented Supplemented

*Signiflcantly different from contralateral non-impacted limb

Figure 19. Bar graph of total surface fissure lengths of impacted and non-irnpacted contralateral
limbs of each group in the chronic study. Lengths were measured using digital imaging software.

Analyses of the indentation relaxation data revealed no significant differences
between contralateral limbs for E11 (p=0.668), E33 (p=0.680), k) (p=0.846), and k3

(p=0.507) of the non—supplemented group, for k, (p=0.640), or k3 (p=0.141) in the short-

29

term supplemented group, or for E11 (p=0.446), E33 (p=0.123), k, (p=0.069), or k3
(p=0.284) in the long-term supplemented group (Figures 20-21). There was however, a
significant increase in the E“ (p=0.050) and E33 (p=0.020) modulii in the impacted limb
of the short-term supplemented group. No differences between groups were found for any
of the mechanical parameters, with p values of 0.817, 0.634, 0.457, and 0.834, for E11,

E33, k), and k3, respectively (Table 2, Figures 20-21).

 

 

0.5 --

é ‘/ ' 0.0-

l

 

 

 

 

 

1:. ............. ............ Ziil
Edw/ ._ £220:
3::6"/ __ $1.5
E4.._/ 1.0--

2 g y

o
l

 

Control Short-tenn Long-term

Control Short-terrn Long-term Supplement Supplement

Supplement Supplement

 

 

|n Non-impacted Limb I impacted Limb

 

la Non-impacted Limb I Impacted Limb)
(a) (b)

Figure 20. (a) Bar chart of the E“ modulus for impacted and non-impacted limbs of each group
in the chronic study. (b) Bar chart of the E 3 3 modulus for impacted and non-irnpacted limbs.

 

30

 

 

 

 

 

 

 

 

 

 

 

 

2.0
‘n . In ..
‘r 3.0 71-5
< <
o o
E ‘ 31.2 --
3 2"
3 3 0.8 »
e ' a
E ' 9 0.4 -
0.0 '
Control Short-temi Long-term Control 3110114917“ Long-term
Supplement Supplement Supplement Supplement
|izi Non-impacted Limb I impacted Limb] [E Non-impacted Limb l Impacted Limbl
(‘0 (b)

Figure 21. (a) Bar chart of the k, permeability for impacted and non-impacted limbs of each group
in the chronic study. (b) Bar chart of the k, permeability for impacted and non-impacted limbs.

31

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32

The tissue proteoglycan (PG) content was not different between the impacted
(39.7 i 5.4 rig/mg W.W.) and non-impacted (39.8 i 6.1 jig/mg W.W.) limbs of the non-
supplemented group (p=0.964, Figure 22). There was a slight trend for an increase in PG
content in the impacted limb (37.6 :b 7.4 rig/mg W.W.) compared to the non-impacted
limb (35.5 at 7.4 rig/mg W.W.) of the short-term supplemented group (p=0.470), however
the difference was not significant (Figure 22). No difference was found between
impacted (35.5 i 6.2 rig/mg W.W.) and non-impacted (36.5 :L 8.8 rig/mg W.W.) limbs of
the long-term supplemented group (p=0.577, Figure 22). PG content was nearly the same
between the non-supplemented (39.7 i 5.4 rig/mg W.W.), short-term supplemented (36.5
d: 6.5 rig/mg W.W.) and long-term supplemented (36.0 i 7.0 pg/mg W.W.) groups. The
correlation analyses revealed no significant correlations between proteoglycan content
and any of the mechanical parameters for the non-supplemented group (Figures 23-26).
However, in the long-term supplemented group there was a significant, positive
correlation documented between proteoglycan content and each of the following
parameters: in-plane modulus (E11) (p=0.026); thickness direction modulus (E33)

(p=0.002); and a negative correlation for in-plane permeability (kt) (p=0.005).

33

a Non-impacted Limb
I Impacted Limb

 

PG Content (pg/mg)

 

 

 

Chronic Control Chronic Short-terrn Chronic Long-term
Supplemented Supplemented

Figure 22. Bar chart of proteoglycan contents, measured in wet
weight, for contralateral limbs in each group of the chronic study.

 

    

 

 

 

 

 

 

R=0.636
14.0 R:0.171
R 0.179
12.0 ' “““““““““““““““ 1- """ .— """""""
“ 0 Long-term
10.0 - ------------------------ n - 4— 1°- ---------
? I Control
CL 8.0 - ---------------------------------------
E u
: 6 0 I A Short-terrn
. ___________________ l """""""""""""""
E . .
4 0 _______________________________________________ Linear
' (Long-term)
2_o ——————————————————————————————————————————————— Linear
(Control)
0.0 I I I I I Linear
0.0 10.0 20.0 30.0 40.0 50.0 60.0 (Short-term)
PG content (mg/mg W.W.)

Figure 23. Correlation plot of the E, , modulus vs. the proteoglycan content
for the supplemented vs. non-supplemented groups of the chronic study. Right

and left limbs were averaged for each group.

34

 

4.0

3.5 - ------------------------------------------------

3.0 -
2.5 -
2.0 -
1.5 1
1.0 -
0.5 -

E33 (M Pa)

 

 

,._.._-__..._..___.________.__________________...._-..___.

=0.798

R

O

A

 

 

0.0

0.0

10.0

20.0

30.0

40.0
PG content (pg/rm W.W.)

 

 

R=0.440

41389
Long-term

Control

Short-terrn

— Linear (Long-

term)

Linear
(Control)
Linear (Short-
term)

Figure 24. Correlation plot of the E 33 modulus vs. the proteoglycan content for the

 

 

 

 

 

supplemented vs. non-supplemented groups of the chronic study. Right and left limbs
were averaged for each group.

=0.745
6.0 R=0.281
RZOZSS
i? 5.0 d 0 Long-term
:9 4.0 - I Control
TIT
§ 3 0 d A Short-temi
<
‘E’Z'O ' ——Linear
: (Long-term)
x 1.0 " ---------------------------------- _. _____________ __,. Linear
(Control)
0.0 . . , - ' —Linear
0.0 10.0 20.0 30.0 40.0 50.0 60.0 (sm't‘te'm)
PG content (pg/mg W.W.)

Figure 25. Correlation plot of the k, permeability vs. the proteoglycan content for
the supplemented vs. non-supplemented groups of the chronic study. Right and left
limbs were averaged for each group.

k3 ((m‘4INs)10"-15)

 

3.0 R=O.480

 

 

 

 

R=0.069
2.5 - R=().032
2 0 Long-term
1 I Control
A Short-term
1.
—Linear
-
05 . __________________________ u -- - f. -_.’ .............. (Long-term)
-——Linear
0.0 . . . . I (Control)
—— Linear
0.0 10.0 20.0 30.0 40.0 50 .0 60.0 (Short-term)

PG content (pg/mg W.W.)

Figure 26. Correlation plot of the k3 permeability vs. the proteoglycan content for the
supplemented vs. non-supplemented groups of the chronic study. Right and left limbs were
averaged for each group.

Part C: Acute vs. Chronic

A significantly greater amount of surface fissuring was documented in the short-

term supplemented group in the chronic study (20.04 :1: 14.69 mm) compared to the acute

supplemented group (8.43 i 8.86 mm, p=0.02). There was also a significant difference in

fissuring in the chronic long-term supplemented group (21.55 1 11.90 mm) compared to

the acute supplemented group (8.43 i 8.86 mm, p=0.03).

Analyses of the indentation relaxation data revealed a significant decrease in k, in

the chronic control group (2.23 i 1.13 (m4/Ns)*10"5) compared to the acute control

group (3.86 :t 1.89 (m4/Ns)*10'15, p=0.005). No differences were found, however,

between these groups for the k3 permeability. In comparing the overall effects of each

study (combining groups within a study), a 16% increase in the E 3 3 modulus (p=0.004), a

36

35% decrease in the k, permeability (p<0.001), and a 45% increase in proteoglycan
content (p<0.001) were documented in the chronic study versus the acute study. No

differences between groups were found for E11 (p = 0.779) or k3 (p=0.499), (Table 3).

 

 

 

Group Eli/523:? (Siva) (151131) (m‘m131045 (m4/hli::)l 0'”
Acutea 20.69 :h 6.29 8.76 .4: 2.10 1.73 a: 0.42 3.35 a: 1.51 0.86 :t 0.73
Chronicb 37.42 i: 695* 8.91 a: 1.93 2.06 :h 049* 2.17 :t 091* 0.94 i 0.48
a N=25
b N=36

*Statistically difl’erent from Acute group
Table 3. Average proteoglycan content, stiffness, and permeability for all groups combined in the
acute and chronic studies.

Histologically, the chronic control group had a trend for a greater loss of
proteoglycans than the acute control group, yet the difference was not significant
(p=0.068). The acute control group had a significantly greater loss of tidemark than the
chronic control group (p=0.021). Neither the articular cartilage thickness nor the
subchondral bone thicknesses were different between impacted and non-impacted
patellae, or between groups within a study (Table 4). However, a significant decrease in
subchondral bone thickness fi'om the acute to the chronic study was documented. The
acute non-supplemented group was significantly different from the chronic non-
supplemented group at both the central (p=0.001) and lateral (p=0.014) locations of the
impacted limb. The acute supplemented group was significantly different from the
chronic short-term supplemented group at the central (p<0.001) and lateral (p=0.02)
locations, and also from the chronic long-term supplemented group at the central

(p<0.001) and lateral (p=0.001) locations.

37

 

Subchondral bone

 

 

 

Group Limb Cartilage
Medial Central Lateral
Non-impacteda 0.52 :t 0.12 0.86 :l: 0.33 0.68 :t 0.14 0.61 i 0.09
Acute Controls
lmpacteda 0.59 i 0.22 1.02 :l: 0.38* 0.70 :t 0.24* 0.57 :t 0.06
Acme Non-impactedb 0.54 i 0.11 0.97 1: 0.18+’x 0.69 i 0.15" 0.58 i 0.09
Supplemented
lmpactedb 0.59 i 0.16 0.95 i 0.27“x 0.76 i 0.21” 0.62 i: 0.08
Non-impacteda 0.51 i 0.10 0.65 i 0.26 0.55 i 0.12 0.60 :t 0.06
Chronic Controls
lmpacteda 0.52 i: 0.09 0.56 i 0.18 0.54 i 0.10 0.59 i 0.07
, a
Chronic Short-term Non-Impacted 0.53 i 0.14 0.65 i 0.26 0.57 :l: 0.25 0.59 It 0.06
Supplemented
lmpacteda 0.53 i 0.14 0.63 :t 0.20 0.57 i 0.17 0.61 i 0.07
Non-impacteda 0.49 i 0.10 0.54 i 0.18 0.52 at 0.1218 0.61 i 0.06
Chronic Long-term
Supplemented a
Impacted 0.54 :l: 0.13 0.62 i 0.26 0.55 :1: 0.1618 0.64 i 0.08
“ N=12
b N=13

* Significantly different from impacted limb of the chronic control group.
+ Significantly different from impacted limb of the chronic short-term supplemented group.
x Significantly different from impacted limb of the chronic long-term supplemented group.

Table 4: Subchondral bone thickness (mm) was measured at 40X with a calibrated eye-piece at the central
region of the patella and midline of the lateral and medial facets (average i S.D.); the cartilage thickness
was measured at the sites of biomechanical testing on the lateral facet (average i S.D.).

DISCUSSION

One objective of the acute study was to test the hypothesis that administration of
Cosamin®DS over a period of two months prior to a blunt impact would increase the
level of tissue proteoglycans. The study did show a significant increase in the amount of

proteoglycans in the impacted limb compared to the non-impacted limb in the diet

38

supplemented animals. However, the supplement was not able to increase the level of
PG’s in comparison with the non-supplemented group. While the non-supplemented
group, and others from the literature, have shown positive correlations between tissue
proteoglycan content and aggregate modulus (Mizrahi et al., 1986), and negative
correlations between proteoglycan content and permeability (Mow and Hayes, 1991), the
acute study did not show similar correlations in the diet supplemented group. Analyses of
the correlation plots suggest that while some diet supplemented animals have relatively
low PG contents, the mechanical properties of retro-patellar cartilage were relatively
high. Whereas subjects in the normal diet group with similarly low PG contents showed
rather poor mechanical properties. These data showing an increase in mechanical
properties in supplemented animals with low PG contents may also agree with recent
clinical data suggesting that those patients with early stages of disease also seem to
benefit the most fiom this nutraceutical (Das and Hammad, 2000).

Another objective of the acute study was to test the hypothesis that two months of
Cosamin®DS would stiffen the cartilage and reduce the extent of acute damage under
impact. It was expected that an increase in tissue proteoglycan content would result in an
increase in the stiffness of the cartilage (Mizrahi et al., 1986, Helminen et al., 1992). The
supplement was able to significantly decrease the k, permeability in the impacted limb
compared to both the contralateral non-impacted limb, and the non-impacted limb of the
non-supplemented group. There was also a trend for an increase in the E 3 3 modulus in the
impacted limb of the supplemented group, however it was not significant. While previous
studies show that resultant degenerative effects cannot be expected immediately after

impact (Ewers et al., 2002; Newberry et al., 1998), these changes in k, and E 3 3 suggested

39

that the Cosamin®DS was beginning to show positive effects, but possibly needed more
time for cartilage degradation to have a more significant effect.

One more objective of the acute study was to test the hypothesis that two months
of Cosamin®DS would reduce the extent of acute damage under impact. In comparison
with the non-supplemented group, the acute study showed reduced differences in the
amount of surface fissuring between limbs in those animals treated with Cosamin®DS.
One possible explanation could be that the nutraceutical had a slight effect on minimizing
the amount of impact-induced fissuring. However, since the p-value for the difference
between limbs of the non-supplemented group was 0.044, and the p-value for the
difference between limbs of the supplemented group was only 0.074, it is possible that
with the small number of samples, the low power and the increased variance in the
supplemented group caused the difference to be insignificant. Also, the difference
between supplemented and non-supplemented groups was not different, agreeing with a
recent study which demonstrated that a two month administration of glucosamine after
induced injury in a rabbit ACL transaction model did not prevent fibrillation of the
articular cartilage (Tiraloche et al., 2005). However, the chronic animals had more
surface damage, even in the non-impacted limbs compared to the acute study. It is
believed that it is difficult to create further extensive surface damage on animals that
already have a considerable amount of baseline damage (Silyn-Roberts and Broom,
1990), which could be an alternative explanation for the reduced difference in fissuring
between limbs in the chronic animals. The results for the control groups of each study
compare well with those of an earlier study using this model, such that there were

significant differences between limbs both at time 0 and at 7.5 months post-trauma

40

(Ewers et al., 2002). However, in the previous study no significant time-dependent
changes were found in the total length of surface fissures, which gives reason to believe
that the significant increase in the amount of fissuring over time in the current study
could be due simply to more initial baseline damage in these chronic animals which came
from a different population than the acute animals.

The mechanical data from the transversely isotropic biphasic analysis of the
traumatized retro-patellar cartilage revealed a significant decrease in the k, permeability,
and a subtle trend for an increase in the E 3 3 modulus in the acute study after just two
months of feeding Cosamin®DS. In the chronic short-term supplemented group, where
the animals also received two months of pre-impact treatment (before waiting six more
months to be sacrificed), similar effects were seen. This time there was a significant
increase in both the E I 1 and the E 3 3 moduli, and a trend for a decrease in both the k; and
k3 permeabilities. These effects however, were not significant in the long-term
supplemented group. Previous studies have shown that high doses of glucosamine over
long periods of time can have a toxic effect (De Mattei et al., 2002). The recommended
daily dosage for humans is 20 mg/kg a day. The rabbits in the current study were fed 2 %
of their daily 200 g feed, which is equal to 4 g/day. With an average weight of 5.7 kg, this
is approximately equal to 700 mg/kg a day. This is approximately 35 times the
recommended daily dosage per bodyweight for humans. The long-term supplemented
animals received this diet supplementation for a total of 8 months. Lippiello et al. (2000)
used the same 2% concentration in a rabbit ACL transaction model, and saw positive
effects of the Cosamin®DS. However, those animals only received supplementation for 4

months. The long-term supplemented animals in the current chronic study may have

41

received too high of a dose for too long of a period of time. This may have begun to have
a slightly negative effect that was reversing any positive effects shown by the 2 months
of supplementation.

The current study produced some results different from those of other
investigations. A study by Ewers et al. (2002) showed that at 7.5 months post-impact
there was a significant reduction in stiffness and an increase in cartilage permeability in
the impacted limb versus the non-impacted limb (although not significant compared to
time 0). Other studies by our laboratory also documented a softening of the cartilage in
the impacted limb after 6 months and 7.5 months, respectively, following a blunt force
impact at 6.0 J (Newberry etal., 1998; Ewers and Haut, 2000). Yet in the current study, 6
months after impact there were no significant changes in the impacted limb of the non-
supplemented and long-term supplemented groups, and in the short-term supplemented
group the cartilage actually appeared stiffer and less permeable in the impacted than non-
impacted limbs. Also, the chronic groups overall seemed stiffer and less permeable than
the acute results (Ewers et al., 2002). One aspect of the current model that has not been
investigated by our laboratory was the exercise regimen that included 8 weeks of exercise
prior to the impact on the knee. This regimen may have had a protective effect on the
cartilage, defending it from extensive damage in the impact situation, helping to explain
why the severe degenerative effects normally seen with this model did not occur in the
current study. Other researchers believe that glucosamine has no effect on normal tissue
homeostasis (Oegema et al., 2002; Deniz et al., 2003). If we were unable to cause severe
damage to the cartilage in the current model due to the pre-impact exercise, this would

explain why the supplement appeared to have no major effects on the tissue.

42

ACKNOWLEDGEMENT

This research was supported by the CDC (R49/ CCR503607). Nutramax
Laboratories, Inc., Edgewood, MD provided the supplement. The authors wish to
acknowledge Steven Rundell for his assistance in the experimental impact protocol,
Clifford Beckett for his technical assistance, Jane Walsh (J.W.) for preparation of the
histological sections and reading of the subchondral bone thickness, and Jean Atkinson

(J .A.) for care and regular exercise of the animals during this study.

43

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Deniz,M., Oegema,T.R., Schiffman,E.L., and Look,J.O. (2003) The effect of exogenous
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Ewers,B.J. and Haut,R.C. (2000) Polysulphated glycosaminoglycan treatments can
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Ewers,B.J., Weaver,B.T., Sevensma,E.T., and Haut,R.C. (2002) Chronic changes in
rabbit retro-patellar cartilage and subchondral bone after blunt impact loading of the
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F amdale,R.W., Sayers,C., and Barrett,A.J. (1982) A direct spectrophotometric

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Gelber,A., Hochberg,M., Mead,L., Wang,N., Wigley,F., and Klag,M. (2000) Joint injury
in young adults and risk for subsequent knee and hip osteoarthritis. annals of internal
medicine 133(5), 321-328.

Haut,R.C., Ide,T.M., and DeCamp,C.E. (1995) Mechanical responses of the rabbit
patello-femoral joint to blunt impact. Journal of Biomechanical Engineering 1 17(4), 402-
408.

Helminen,H.J., Kiviranta,I., Saamanen,A.-M., Jurvelin,J.S., ArokoskiJ., Oettrneier,R.,
Abendroth,K., Roth,A.J., and Tammi,M.I. (1992) Effect of motion and load on articular
cartilage in animal models. In Articular Cartilage and Osteoarthritis (Edited by
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Lewis,J.L., Deloria,L.B., Oyen-Tiesma,M., Thompson,R.C., Ericson,M., and
Oegema,T.R. (2003) Cell death after cartilage impact occurs around matrix cracks.
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Lippiello,L. (2003) Glucosamine and chondroitin sulfate: biological response modifiers
of chondrycytes under simulated conditions of joint stress. Osteoarthritis and Cartilage
1 1, 335-342.

Lippiello,L., Woodward,J., Karpman,R., and Hammad,T. (2000) In vivo
chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate.
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Mow,V.C. and Hayes,W.C. (1991) Basic orthopaedic biomechanics. Raven Press, New
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45

Newberry,W.N., MacKenzie,C., and Haut,R.C. (1998) Blunt impact causes changes in
bone and cartilage in a regularly exercised animal model. Journal of Orthopaedic
Research 16, 348-354.

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Oegema,T.R., Deloria,L.B., Sandy,J.D., and Hart,D.A. (2002) Effect of oral glucosamine
on cartilage and meniscus in normal and chyrnopapain-injected knees of young rabbits.
Arthritis and Rheumatism 46(9), 2495-2503.

Oyen-Tiesma,M., Atkinson,J., and Haut,R.C. (1998) A method for promoting regular
exercise in rabbits involved in orthopedics research. Contemporary Topics in Laboratory
Animal Science 37(6), 77-80.

Quinn,T.M., Grodzinsky,A.J., Buschmann,M.D., Kim,Y.-J., and Hunziker,E.B. (1998)
Mechanical compression alters proteoglycan deposition and matrix deformation around
individual cells in cartilage explants. Journal of Cell Science 111(5), 573-583.

Richy,F., Bruyere,O., Ethgen,O., Cucherat,M., Henrotin,Y., and Reginster,J. (2003)
Structural and Symptomatic Efficacy of Glucosamine and Chondroitin Sulfate in Knee
Osteoarthritis. Arch Intern Med 163, 1514-1522.

Silyn-Roberts,H. and Broom,N.D. (1990) Fracture behavior of cartilage-on-bone in
response to repeated impact loading. Connective Tissue Research 24, 143-156.

Tiraloche,G., Girard,C., Chouinard,L., Sampalis,J., Moquin,L., Ionescu,M., Reiner,A.,
Poole,A.R., and Laverty,S. (2005) Effect of oral glucosamine on cartilage degradation in
a rabbit model of osteoarthritis. Arthritis and Rheumatism 52, 1118-1128.

Weaver,B.T. and Haut,R.C. (2005) Enforced exercise after blunt trauma significantly
affects biomechanical and histological changes in rabbit retro-patellar cartilage. Journal
of Biomechanics In Press.

Weaver, B.T. 2001. Chapter Two: Regular exercise is beneficial in a stable joint after
trauma. The analysis of tissue response following a single rigid blunt impact in an in vivo
animal model: Thesis for the degree of M. S. Michigan State University, 33-50.

46

CHAPTER TWO
MECHANICAL RESPONSE OF CARTILAGE EXPLANT S TO CYCLIC
COMPRESSIVE LOADING

ABSTRACT

As a way to examine more controlled loads an articular cartilage in a more cost
effective manner than our in vivo animal models, our laboratory has been developing a
mechanical testing apparatus to load cartilage explants in vitro. The objective of the
current study was to determine whether, and to what extent, regular cyclic compressive
loading alters the biomechanical properties of articular cartilage. Chondral explants were
taken from the bovine metacarpophalangeal joint and exposed to 10 cycles of loading
every hour at a magnitude of 0.5 MP3 for 0, l, 2, 3, or 6 days. Stiffness of the cartilage
was measured by mechanical indentation, and the tissue was evaluated for cell viability.
Results indicated a general trend for a stiffening of cartilage explants when exposed to
regular cyclic loading at low intensities, along with a decrease in fluid gain and an

increase in deep zone cell death.

INTRODUCTION

Numerous in vivo animal models have been utilized to examine changes in joint
tissue, along with effects of regular exercise in contrast with normal cage activity or joint
immobilization (Newberry et al., 1997; Ewers et al., 2002; Weaver and Haut, 2005;
Helminen et al., 1992; Jurvelin et al., 1986). While immobilization has been shown to
soften the articular cartilage (Helminen et al., 1992), regular exercise has been shown to

have a stiffening effect (Jurvelin et al., 1986). Mechanical stiffness of articular cartilage

47

has been associated with proteoglycan content (Helminen et al., 1992; Mizrahi et al.,
1986), and in vivo models have shown that increased weight-bearing and regular exercise
have an upregulating effect on tissue proteoglycans (PGs) in articular cartilage in the
knee (Kiviranta et al., 1987; Helminen et al., 1992). However, in vivo animal models
limit the degree of control on factors such as amplitude and distribution of physical
forces, and create difficulties in directly relating the cellular response to various loading
situations. As a result, in vitro mechanical explant test systems have been developed to
address these issues (Torzilli et al., 1997; Sah et al., 1989; Sauerland et al., 2003).

Various studies have attempted to quantify the effects of static (constant) or
dynamic (cyclic) compressive stresses on cartilage explants in vitro (Quinn et al., 1998);
(Sah et al., 1989; Torzilli et al., 1997). Although some researchers were not able to show
an increase in proteoglycan biosynthesis (Torzilli et al., 1997), the general consensus is
that static stress tends to decrease proteoglycan synthesis while cyclic stresses tend to
increase the synthesis of tissue PGs (Palmoski and Brandt, 1984; Parkkinen et al., 1989;
Quinn et al., 1998).

Our laboratory has developed a “cartilage exerciser” to cyclically load chondral
explants in vitro, simulating normal loading or exercise on the joint. The cartilage
exerciser was designed to be load controlled, representing the physical conditions
cartilage might experience in a synovial joint. A low level of stress (5 0.5 MPa) and a
higher frequency of compression (0.01 - 1 Hz) were chosen based on a study by Sah
showing a threshold of biosynthesis at these levels (Sah et al., 1989).

Other studies have investigated the in vitro mechanical response on cartilage

explants to injurious compression (Loening et al., 2000; Morel et al., 2005; Rundell and

48

Haut, 2005; Thibault et al., 2002). These researchers have found that an increase in fluid
gain corresponds to a decrease in cartilage stiffness (Jurvelin et al., 1986) and an increase
in surface fissuring when subjected to high compressive loads (Loening et al., 2000;
Morel et al., 2005; Rundell and Haut, 2005; Thibault et al., 2002). Other laboratories
have used mechanical explant testing systems to focus primarily on the response of
proteoglycan synthesis to applied static compressive stresses or cyclical strains (Torzilli
et al., 1997; Sah et al., 1989; Sauerland et al., 2003). Unfortunately, few studies have
investigated the direct correlation between regular cyclic loading of chondral or
osteochondral explants and mechanical stiffness in non-injurious compression. The
hypothesis of the current study was that regular, intermittent in vitro cyclic loading of
cartilage explants would cause an increase in the mechanical stiffiress of the cartilage.
Since the cartilage exerciser was a newly developed device in our laboratory, pilot
studies were executed in order to define the desired loading parameters for the apparatus.
This chapter will discuss both these initial results from pilot studies, as well as results

obtained from experimental studies with a regular intermittent loading regimen.

49

METHODS

Six pairs of skeletally mature (12-24 months) bovine forelegs were obtained from
a local abattoir within 3 hours of slaughter. The legs were cut proximal to the
metacarpophalangeal surface leaving the joint intact. The legs were rinsed with distilled
water, skinned, and rinsed again prior to opening the joint under a laminar flow hood. A
6-mm biopsy punch was used to make 154 cartilage plugs. These plugs were removed
from the underlying bone with a scalpel, thickness measurements were taken (described
later), and the explants were divided into two groups, exercise and no-exercise.

All explants were washed three times in bovine media with Dulbecco’s Modified
Eagle’s Medium (Gibco, USA #12500-039) (see Appendix F for stock recipe), and then
placed in the medium supplemented with 10% fetal bovine serum and antibiotics
(penicillin 100 units/ml, streptomycin lug/ml, amphotericin B 0.25 jug/ml) in a 24 well
plate (see Appendix G for media handling standard operating procedure). The bovine
media was replaced with fresh media every 2 days throughout a study. The well plate was
placed in a mechanical loading device (the “cartilage exerciser”, Figure 1) inside of a
humidity-controlled incubator (37° C, 5% C02, NuAire, Plymouth, MN). The cartilage
exerciser consists of 12 loading chambers simultaneously powered by air. Pneumatic
cylinders force the pistons downward to apply a compressive load to the specimens
through 14.60 mm diameter nonporous Teflon® platens (Figure 2). The cartilage
exerciser was designed to hold a 24 well culture plate, so that 12 cartilage samples would
be mechanically loaded and 12 unloaded control explants would be subjected to an

identical culture environment (Figure 3).

50

 

Figure 1. The “cartilage exerciser” mechanical loading device with 12 cylinders
to apply compressive loads to the cartilage explants.

cyclic load

   
 
   

stainless
steel piston

Teflon tip
cartilage cell culture
explant media

Figure 2. Each piston of the cartilage exerciser is
powered by air to simultaneously load the explant
with a nonporous TeflonO platen.

51

   

pistons

cartilage
explants in
media

24—well cell culture plate

Figure 3. The cartilage exerciser consists of 12 pistons and is
designed to simultaneously load 12 explants in a 24 well plate,
leaving 12 explants as non-loaded controls.

Pilot studies involved various loading conditions in order to determine the most
optimal parameters. In each of the three pilot studies, intermittent uniaxial cyclic loads
were administered using a sinusoidal waveform of 1 Hz at a peak stress of 0.05 MPa. Ten
cycles of load were applied once every hour starting at day 0 (right off the joint) and
ending at 1, 2, or 7 days. Between loading cycles the pistons rise above the cartilage so
that zero load is applied to the samples. A total of twenty-seven samples were used for
the first pilot study (6/14/05), where explants were separated into 5 groups: time zero
(n=4), 2 day exercise (n=6), 2 day no-exercise (n=6), 7 day exercise (n=5), and 7 day no-
exercise (n=6). Thirty explants were used for the following two pilot studies (6/28/05)
and (7/12/05), with 6 samples in each of the 5 groups: time zero, 2 day exercise, 2 day
no-exercise, 7 day exercise, and 7 day no-exercise. In the final pilot study (1 1/1/05) the
peak load was raised from 0.05 MPa to 0.5 MPa, at a frequency of 0.2 Hz, and the

samples were again loaded with 10 cycles every hour. This study included the following

52

five groups: time zero (n=9), 2 day exercise (n=4), 2 day no-exercise (n=4), 3 day
exercise (n=4), and 3 day no-exercise (n=4).

Based on the results of the initial pilot studies, a set loading pattern was
determined. The cyclic loads were to be administered at 0.2 Hz with a peak stress of 0.5
MPa for ten cycles applied once every hour. These parameters remained constant for each
of the two final experimental studies: (11/8/05) and (11/ 16/05). These two studies each
consisted of time 0 (n=8), 1 day exercise (n=4) and no-exercise (n=4), 3 day exercise
(n=4) and no-exercise (n=4), and 6 day exercise (n=4) and no-exercise (n=4) groups.
After a desired loading time of 1, 3, or 6 days the specimens were then evaluated in terms
of mechanical stiffness, cell viability, and proteoglycan content.

Structural integrity of the metacarpophalangeal cartilage was determined using
indentation stress relaxation tests (see Appendix F for standard operating procedures).
After allowing a minimum of 30 minutes in media for the explants to swell, mechanical
indentation tests were performed at one of six times: 0, l, 2, 3, 6, or 7 days of exercise
after removal fiom the joint. Cartilage explant thickness was measured immediately off
the joint, and again before mechanical indentation testing. The percentage in thickness
change due to swelling was found by dividing the change in thickness (final minus initial)
by the initial (off the bone)
thickness. Thickness was

measured at two perpendicular

   

t2 sites across the center of the

explant using a digital Vernier
Figure 4. Explant thickness taken at two perpendicular
sites across the explant using digital calipers.

caliper (Mitutoyo Corp:

53

Abosolute Digimatic, Model No. CD-6” CS) with a resolution of 0.01 mm (Figure 4).
The two thickness values were then averaged together and recorded. The explants were
placed on a flat level surface so that the face of the explant was perpendicular to the
indenter tip. A magnet with a 4.5 mm diameter hole was placed on top of the explant to
secure the edges to help resist curling of the explants (Figure 5). The explant and fixture
were then submerged in a room temperature phosphate buffered solution (pH 7.2) (Figure
5). A 1.0 mm diameter flat, non-porous probe was lowered into the cartilage until a
preload of 0.03 N was attained and held for 60s. After the 60s preload, the indenter was
pressed into the cartilage 15% of the total thickness in 2s and maintained for 360s while
resistive loads of relaxation were measured (Data Instruments, Acton, MA: model JP-25,
25 lb capacity), amplified, and collected at 1000 Hz for the first second and 20 Hz for the
remainder. Note that this protocol differs slightly from that in Chapter 1 due to the nature
of the specimens being tested. In Chapter 1, a 0.02 N preload was applied and
immediately the indenter was pressed 0.01 mm into the cartilage. Due to the curling
nature of the cartilage explants in the current study, a slightly larger (0.03 N) preload was
used to ensure that the entire tip of the indenter was in contact with the explant. Since a
larger preload was applied, the cartilage was allowed 60 seconds to relax in order to
verify that the cartilage was not in a stressed state at the time of indentation. Also, it was
decided to indent 15 % of the cartilage thickness, rather than a prescribed distance, due to

the variability of the explant thicknesses.

54

\

Explant

 

Figure 5. (A) Explant indentation test system and fixture. (B) First, explant is placed in hole of bottom
magnet on flat steel surface. (C) Second, a top magnet is lowered over the top of the explant to hold
down edges. (D) Finally, the indenter tip is lowered to a preload of 0.03 N.

The stiffness of the cartilage was determined from the results of the indentation
and thickness test by a calculation of the shear modulus from an assumed elastic layer on
a rigid half space (unbonded) (Lebedev and Ufliand, 1958). The instantaneous shear
modulus (Gu) and the relaxed shear modulus (6,) were calculated based on the peak load
at 62 s (60 s of preload, and 2 s for indentation application) and the relaxed load at 360 s,

respectively, using equation 1.

55

P(1—v)
a (1)
4*a*K[—)*w
h

Where P = the measured load
v = Poisson’s ratio (Assumed to be 0.5 for GU and 0.4 GR)
a = Indenter radius
h = layer thickness
(0 = penetration depth
G = Elastic shear modulus
K = scaling factor.

G:

 

For cell viability, three 0.5-mm slices were taken through the thickness at the
center of each explant using a customized cutting tool (Ewers et al., 2001). The sections
were stained with a kit containing calcein and ethidiurn bromide homodimer (Live/Dead
&Viability/Cytotoxity, Molecular Probes, Oregon). Each section was viewed and
photographed under a fluorescence microscope at lOOX (Lecia DM LB (frequency: 50-60
Hz), Lecia Mikroskopie and Systeme GmgH, Germany). Full thickness, digital images
were taken of a 2.5-mm length at the center of each explant. The percentage of dead cells
(red) to viable cells (green) were manually determined by taking a measurement of the
thickness of the dead zone and dividing it by the full thickness of the sample using digital
imaging software (Sigma Scan, SPSS Inc., Chicago, IL). This was done at three sites on
each of three slices from every explant (Figure 6), and the percentages were averaged

together and recorded.

56

 

Figure 6. Gross photographs were used to determine
percentage of viable cells in the cartilage explants. The
thickness of the death zone and the total thickness of the
explant were measured at three sites.

After completion of the mechanical indentation and cell viability testing, the
remaining end pieces of the explant samples were evaluated for tissue proteoglycan
content using the DMB assay (Famdale et al., 1982).

A one way ANOVA with Student-Newman-Keuls post hoc tests was used to
compare the mechanical stiffness, proteoglycan contents, and percentage of cell death
between exercised and non-exercised groups. All data are reported as mean i standard

deviation. Statistical significance was indicated at p<0.05.

RESULTS
Pilot Studies

Mechanical stiffness results from the first pilot study (6/14/05) revealed a
significant increase in both the instantaneous shear modulus G" (p=0.005) and the relaxed

shear modulus G, (p=0.009) in the exercise group compared to the non-exercise control

57

group at 2 days (Figure 7). There was also a slight trend for an increase in G" and G, after

7 days of exercise compared to the non-exercise group, yet neither was statistical (Figure

 

 

   

7).
0.12 .
0.10
A A 0.08
a (I
n. n.
g g 0.06
5 I-
" 0 0.04
0.02
0.00
Time 2Day ZDay 7Day 7Day Time 2Day ZDay 7Day 7Day
Zero No Ex Ex No Ex Ex Zero No Ex Ex No Ex Ex
(net) (HS) (n=6) (n=5) (n=6) (n=4) (n=6) (n=6) (n=5) (n=6)
(8) (b)

Figure 7. (a) Bar chart of the instantaneous modulus (Gu) and (b) the relaxed shear modulus (G,)
for no-exercise and exercise samples from each group in the 6/14/05 study.

Staining for cell viability showed a band of death in the deep zone of the cartilage,
as well as a smaller band of death in the superficial zone in both non—exercise (7.7 d: 7.8
% cell death) and exercise (26.1 i 11.1 % cell death) groups after 2 days (Figure 8). The
same effect, only more extensive, was seen in the exercise (44.4 at 9.7 % cell death) and
no-exercise (26.6 d: 21.5 % cell death) groups after 7 days (Figure 9). These increases in
cell death in the exercised explants in comparison with the non-exercised controls were
found to be significant at both 2 days (p=0.032) and 7 days (p=0.047). Over time there
was also a trend for an increase in cell death from 2 days to 7 days in non-loaded controls
(p=0.069), and from 2 days of exercise to 7 days of exercise (p=0.098), although these

results were not quite significant (Figure 10).

58

5L2:

. ‘_ 5 “- h
" it”. ‘~"\5‘.).~l\:‘- a.
a T ‘

 

OSSCC .. 0.1 mm OZsEb ~ 0.1 mm
(a) (b)

Figure 8. Representative cell viability photographs for (a) the 2 day no-exercise group and (b) the 2 day
exercise group in the 6/14/05 study. Green cells are viable and red cells are dead.

 

 

(b)

Figure 9. Representative cell viability photographs for (a) the 7 day no-exercise group and (b) the 7 day
exercise group in the 6/14/05 study. Green cells are viable and red cells are dead.

% Cell Death

60%

 

50% i
40% -
30% -
20% .

10% '

 

 

 

 

0%-

2 Day No Ex 2 Day Ex 7 Day No Ex 7 Day Ex

Figure 10. Percentage of cell death in each of the no-exercise and
exercise groups in the 6/14/05 study.

59

The second set of experiments (6/28/05) revealed a trend for an increase in G“ in
the exercise group (0.52 :h 0.35 NEPa) versus the non-exercise group (0.45 i 0.42 MPa)
after 2 days (Figure 11). There was also a trend for an increase in G, in the exercise (0.09
i 0.06 MPa) group compared to the non-exercise (0.08 i: 0.04 MPa) group after 2 days,
however, the differences in G“ and G, were not statistically significant. The 7 day groups
showed no change in G“ and a slight decrease in G, in the exercise group (0.11 :l: 0.05

MPa) compared to the non-exercise (0.13 i 0.05 MPa) group (Figure 11).

 

 

0.21 .
0.18
0.15
0.12
0.09
0.06
0.03

0.00
Time 2 Day 2 Day 7 Day 7 Day Tlme 2 Day 2 Day 7 Day 7 Day
Zero No Ex Ex No Ex Ex Zero No Ex Ex No Ex Ex

(n=6) (n=6) (n=6) 0%) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6)
(3) (b)
Figure 11. (a) Bar chart of the instantaneous modulus (Gu) and (b) the relaxed shear modulus (0,)
for no—exercise and exercise samples from each group in the 6/28/05 study.

Gu (MPa)
Gr (MPa)

   

Cell viability staining revealed complete death in all exercise specimens after 2
days (Figure 12) and 7 days (Figure 13) of loading. Minimal death occurred in the deep
zone of the control samples after 2 days (Figure 12). However, the band of death was

increased in the 7 day no-exercise group (Figure 13).

60

OQSFD

   

(b)

Figure 12. Representative cell viability photographs for (a) the 2 day no-exercise group and (b) the 2
day exercise group in the 6/28/05 study. Green cells are viable and red cells are dead.

 

Figure 13. Representative cell viability photographs for (a) the 7 day no-exercise group and (b) the 7
day exercise group in the 6/28/05 study. Green cells are viable and red cells are dead.

The (7/12/05) pilot study indicated a significant increase in G“ (p=0.020) and G,
(p=0.014) in the no-exercise group compared to exercise group after 2 days of loading
(Figure 14). The same result was found in G, after 7 days of loading in comparison with
non-loaded controls (p=0.02) (Figure 14). There was also a trend for 7 days of loading to
increase 6,, yet the result was not quite significant (p=0.064) (Figure 14). However, cell
viability revealed 100% death in all exercised specimens, and no death in the non-

exercised controls.

61

 

 

Gu (MPa)
Gr (MPa)

   

Time 2 Day 2 Day 7 Day 7 Day Time 2 Day 2 Day 7 Day 7 Day
Zero No Ex Ex No Ex Ex Zero No Ex Ex No Ex Ex
(n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6)

(2) (b)

Figure 14. (a) Bar chart of the instantaneous modulus (Gu) and (b) the relaxed shear modulus (6,)
for no—exercise and exercise samples from each group in the 7/12/05 study.

Proteoglycan analysis showed a trend for an increase in PG content after 2 days of
exercise compared to the non-loaded control group, however the difference was not
significant (Figure 15). An overall decrease was seen fiom time zero to 2 day no-exercise
and both 7 day groups (Figure 15). The 2 day and 7 day exercise groups showed an
increase in percent thickness change in comparison to the no-exercise group from initial

reading off the joint to 2 or 7 days in media (Figure 16).

 

PG Content (“g/mg W.W.)

 

 

 

 

Time Zero 2 Day No 2 Day Ex 7 Day No 7 Day Ex
Ex Ex

Figure 15. Proteoglycan contents for no-exercise and exercise
samples from each group in the 7/12/05 study.

62

 

% Thickness Change
8
a?

 

 

 

Time Zero

2 DayNo Ex 2 DayEx 7 Day No Ex 7 DayEx

Figure 16. Increases in thickness due to swelling in the 7/12/05 study
are plotted here. Explant thickness was measured directly off the joint,
and again after 30 min, 2 days, or 7 days of equilibration.

In the final pilot study (11/1/05) a slight decrease in 0,, was found in the 2 day
exercise group (1.07 :t 0.56 MPa) compared to the 2 day no—exercise group (1.43 i 0.52
MPa), as well as a slight decrease in G, in the exercise (0.14 :t 0.03 MPa) vs. no-exercise
(0.19 i 0.07 MPa) group (Figure 17). A very subtle decrease in 0,, and G, of the
exercised explants was also found after 3 days of loading compared to the non-exercised

control group, although the differences were not significant (Figure 17 ).

 

 

3.5
3.0- ----------- - ~ -
2.5- ----------
{£2.0- ; - ~ -
E
31.5- . eeeeeeee ‘
O

 

 

 

 

 

 

Time 2031' 203)! 3Day 3Day Time 2Day 2Day 3Day 3Day
Zero NoEx Ex NoEx Ex

(n=9) (n=4) (n=4) (n=4) (n=4)
0‘) 0))

Zero NoEx Ex NoEx Ex
(n=9) (n=4) (n=4) (n=4) (n=4)

Figure 17. (a) Bar chart of the instantaneous modulus (0,) and (b) the relaxed shear modulus (0,) for
no-exercise and exercise samples from each group in the 1 1/1/05 study.

63

Cell viability showed a slight increase in cell death in the exercise compared to
non-exercise group after 2 days (Figure 18). This effect was increased after 3 days of
loading, and a larger band of death was seen in the exercised explants (Figure 18). Death
in the control specimens mainly occurred in the superficial zone, whereas death in the
exercised samples occurred mostly in the deep zone with a small amount of death in the

superficial zone (Figures 19-20).

 

35%-L ,, -------

% Cell Death
to
a:
33

 

 

 

2DayNoEx ZDayEx 30ayNoEx 3DayEx

Figure 18. Percentage of cell death in each of the no-exercise
and exercise groups in the 11/ 1/05 study.

 

(ill—.1.)

(‘0 (b)
Figure 19. Representative cell viability photographs for (a) the 2 day no—exercise group and (b) the 2
day exercise group in the l l/ l/05 study. Green cells are viable and red cells are dead.

64

(b)

Figure 20. Representative cell viability photographs for (a) the 7 day no-exercise group and (b) the 7
day exercise group in the 11/1/05 study. Green cells are viable and red cells are dead.

 

Experimental Studies

In the first of the experimental studies, there was a trend for an increase in 0.,
after 1 day of exercise, compared to the 1-day control group (Figure 21). The opposite
trend was seen for the 0, values after 1 day (Figure 21). Differences between exercise
and no-exercise were very minimal at 3 days, yet after 6 days an increase in both 0., and
0, was found in the no-exercise group compared to the exercised specimens (Figure 21).

No differences between limbs or between groups were statistically significant.

 

 

   

E
E
h
0
Time 1Day 1Day 3Day 3Day 6Day 6Day Time 1Day1Day 3Day3Day GDay SDay
ZeroNoExExNoExExNoExEx ZeroNoExExNoExExNoExEx
(a) (b)

Figure 21. (a) Bar chart of the instantaneous modulus (0,) and (b) the relaxed shear modulus (0,) for
no—exercise and exercise samples from each group in the l 1/8/05 study. In the time zero group n=8. in all
other groups (no-exercise and exercise) n=4.

65

After 1 day of loading there was a decrease in percent thickness change compared
to the non-loaded group (Figure 22). The trend alter 6 days was similar, yet less evident,
however at 3 days the trend was reversed, with a slight increase in percent thickness
change in the exercised samples (Figure 22). A decrease in percent thickness change

seemed to correspond with an increase in 0., at 1 and 6 days (Figure 22).

 

25%

20%

15%

10%

% Thickness Change

5%

 

0%

30min 1Day 1Day 3Day 30ay 6Day 6Day
NoEx Ex NoEx Ex NoEx Ex

Figure 22. increases in thickness due to swelling in the 1 1/8/05
study are plotted here. Explant thickness was measured directly
off the joint, and again after 30 min, 1 day, 3 days, or 6 days of
equilibration.

Cell death after 1 day (exercise and no-exercise samples) was negligible (Figure
23). Although cell death was still minimal after 3 days, there was a slight increase in
death in the exercised specimens (3.4 i 1.1 % cell death) compared to the non-exercised
group (1.5 i 0.6 % cell death) (Figure 23). However, after 6 days there was a significant
increase in cell death in the no—exercise group Gigure 23). Unfortunately, two of the
control samples were contaminated (Figure 24), so it is unknown if exercise would have

caused more death otherwise.

66

100%
900/0. .4. . . _ L .....

 

% Cell Death

 

 

 

1Day 1Day 3Day 30ay 6Day 6Day
NoEx Ex NoEx Ex NoEx Ex

Figure 23. Percentage of cell death in each of the non-
exercised and exercised groups in the 11/8/05 study.

 

Figure 24. Representative cell viability photographs for (a) the 6 day no-exercise group and (b) the 6
day exercise group in the 11/8/05 study. Green cells are viable and red cells are dead.

Results of the final study (11/16/05) revealed a consistent trend for an increase in
0,, after 1, 3, and 6 days of loading in comparison with the non-loaded controls (Figure
25). This trend remained the same for G, at 3 and 6 days, however at 1 day the exercise
group actually saw a slight decrease in 0, compared to the no-exercise group (Figure 25).
Percent thickness change was consistent throughout the study, with a decrease in the

exercise groups at l, 3, and 6 days (Figure 26), corresponding to the increases in 0...

67

 

 

Gu (MPa)

 

 

 

 

   

Time 1Day1Day 3Day3DayBDay6 Day Time 1031/ 1Day 3931! 3081! GDay6Day
ZeroNoExExNoExExNoExEx Z°'°N°EXEXN°ExExNoF-X£x
(a) (b)

Figure 25. (a) Bar chart of the instantaneous modulus (0“) and (b) the relaxed shear modulus (0,)
for exercise and no-exercise samples of each group in the 1 1/16/05 study. In the time zero group n=8.
In all other groups (no-exercise and exercise) n=4.

 

% Thickness Change

 

 

30min 1Day 1Day 3Day 3Day 6Day 6Day
NoEx Ex NoEx Ex NoEx Ex

Figure 26. Increases in thickness due to swelling in the 1 1/16/05
study are plotted here. Explant thickness was measured directly
off the joint, and again after 30 rrrin, 1 day, 3 days, or 6 days of
equilibration.

No significant differences in cell death were found between groups (Figure 27).
There was a slight trend for an increase in cell death in the no-exercise group after 1 day.
However, after 3 and 6 days, more cell death was found in the exercise samples (Figures
28-30). Cell death in the control specimens was mostly confined to the superficial zones,

whereas death in the exercised specimens was in both the superficial and deep zones.

68

14%
12%
10%
8%
6%
4%

 

% Cell Death

2 %

 

 

0 %

 

1 Day 1 Day 3 Day 3 Day 6 Day 6 Day
No Ex Ex No Ex Ex No Ex Ex

Figure 27. Percentage of cell death in each of the no-
exercise and exercise groups in the 1 1/16/05 study.

MH‘IK

,111
‘

   

(a)

Figure 28. Representative cell viability photographs for (a) the 1 day no—exercise group and (b) the 1
day exercise group in the 1 1/16/05 study. Green cells are viable and red cells are dead.

 

Figure 29. Representative cell viability photographs for (a) the 3 day no—exercise group and (b) the 3
day exercise group in the 1 1/16/05 study. Green cells are viable and red cells are dead.

69

   

(b)

Figure 30. Representative cell viability photographs for (a) the 6 day no—exercise group and (b) the 6
day exercise group in the 11/16/05 study. Green cells are viable and red cells are dead.

Proteoglycan analysis showed a trend for an increase in PG content after 3 and 6
days of exercise compared to the non-loaded control group, however the difference was
not significant (Figure 31). A significant correlation was found between the relaxed shear

modulus (0,) and the tissue proteoglycan content (p=0.036, Figure 31).

 

PG Content (pg/mg W.W.)

 

 

 

Time 1Day 1Day 3Day 3Day SDay 6Day
Zero No Ex Ex No Ex Ex No Ex Ex

Figure 31. Proteoglycan contents for no-exercise and
exercise samples from each group in the 11/16/05 study.

70

 

0.35
0.30 ..
0.25
0.20
0.15 (
0.10 -
0.05 ~
0.00 . . .

Gr (MPa)

 

 

 

 

PG Content (rig/mg W.W.)

Figure 32. Correlation between relaxed shear modulus 0, and
proteoglycan contents for no-exercise and exercise samples
from each group in the 11/16/05 study.

Two experimental studies combined

When combining the two final studies, there was a trend for increased 0., at 1 and
3 days, and an increased 0, at 3 days in the exercise groups (Figure 33). However, the
no-exercise groups demonstrated an increase in G, at 1 and 6 days (Figure 33). There was

also a trend for an increase in thickness change in the no-exercise samples at 1, 3, and 6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

days (Figure 34).
3.5 0.35
3.0-~- ------ 0.30-
2.5- ------- ,,,,,,,,. 0.25-
E 2.0- ------ -- ------- E 0.20 -
a a
8 1.5 -» , - > - a 0.15-
1.0-- - -- *7 —- ~ — 0.10--
05-» — ~ —- -- - o.05--«
0.00
Time 1 Day1 Day 3 Day3 Day 6 Days Day Time 1 Day 1 Day 3 Day 6 Day 6 Day
Zero No Ex Ex No Ex Ex NoEx Ex Zero NoEx Ex Ex No Ex Ex
(8) (b)

Figure 33. (a) Bar chart of the instantaneous modulus (0.) and (b) the relaxed shear modulus (0,)
for no—exercise and exercise samples for the 11/8/05 and ll/l6/05 studies combined.

71

 

% Thickness Change
8
o\°

 

 

 

30min 1DayNo1DayEx SDayNo SDayEx 6DayNo 6DayEx
Ex Ex Ex
Figure 34. Increases in thickness due to swelling in the 11/8/05 and 1 1/16/05
studies combined are plotted here. Explant thickness was measured directly
off the joint, and again after 30 rrrin, 1 day, 3 days, or 6 days of equilibration.

DISCUSSION

In a previous study by our laboratory the mechanical response to injurious
compression was examined in equilibrated and non-equilibrated chondral explants
(Rundell and Haut, 2005). The results of the previous study demonstrated that the
stiffness of the equilibrated specimens was less than that of the non-equilibrated
specimens. Unfortunately, few studies investigate the mechanical response to regularly
loaded explants at non-injurious levels. The hypothesis of the current study was that
regular cyclic loading of chondral explants would cause an increase in mechanical
stiffness of the cartilage.

The initial three pilot studies (6/14/05; 6/28/05; 7/12/05) showed a significant loss
of viable cells, with 26 — 100 % cell death occurring in the exercised samples, and 8 — 27
% in the non-loaded controls. These large amounts of cell death can be explained by
contamination in the system. Twelve diaphragrns are used inside the cartilage exerciser
chambers to assist in the vertical plunging action of the pistons. After eliminating

numerous factors, it was determined that the diaphragm material (neoprene) was

72

biologically unsafe. This material had more direct contact with the exercised wells, and
was causing complete death in these specimens. It is also possible that this material was
contarrrinating the air, causing some death in the control specimens. To eliminate this
problem, the diaphragrns were replaced with new biologically safe (silicone) diaphragrns.
In these pilot studies, major increases in mechanical stiffness were seen in exercised
specimens compared to non-exercised controls. With minimal amounts of viable cells
remaining in the exercised specimens, it can be concluded that the stiffening effect seen
in these initial pilot studies was strictly due to a mechanical response rather than a
cellular response.

In the final pilot study and the following two experimental studies, slightly more
cell death occurred in the exercised samples compared to the non-exercised controls.
When cell death did exist in the control specimens, it was typically located in the
superficial zone. Conversely, there was a consistent band of death in the deep zone of the
exercised samples. These studies also showed a trend for an increase in fluid gain in the
non-loaded explants compared to the exercised samples. These data agree with Rundell’s
study, where he documented an increase in superficial zone cell death, and a decrease in
deep zone cell death, with an increase in water gain (Rundell and Haut, 2005).

One limitation of the current study was that the cartilage explants were not
weighed before and after equilibration in media. The percentage change in thickness was
attributed to fluid gain, and this was used as a comparison to previous equilibration
studies (Rundell and Haut, 2005). However, a better measure of fluid gain would be
increase in wet weight of the tissue. Another limitation of the current study was the

method of measuring the percentage of cell death. The method used assumed a

73

uniformity in cell density throughout the depth of the explant. If more time were
available, a better measure of cell death would be to individually count live and dead
cells in each explant.

A consistent association between an increase in fluid content and a decrease in the
instantaneous shear modulus 0., was shown in the current study. These results agree with
other researchers, who have also shown a decrease in cartilage stiffness with an increase
in fluid gain (Armstrong and Mow, 1982; Morel et al., 2005; Rundell and Haut, 2005). In
Chapter 1, both diet supplemented and non-supplemented animals showed no softening
of the articular cartilage in impacted limbs (short-term or long-term) versus non-impacted
limbs. These data were not characteristic of earlier studies by this laboratory (Newberry
et al., 1997; Newberry et al., 1998; Ewers and Haut, 2000; Ewers et al., 2002). Recall
that a major difference between the Chapter 1 study and previous studies by our
laboratory was that the Chapter 1 animals involved a two-month period of pre-impact
exercising. Morel believes that increased matrix swelling may cause the cartilage to be
more susceptible to injury (Morel et al., 2005). Therefore, if exercise can help reduce
fluid content in joint cartilage, as shown in the current study, the cartilage may be stiffer
and better able to withstand a blunt impact with less damage than a non-exercising

specimen.

ACKNOWLEDGEMENT

The authors wish to acknowledge Bellingar Packing (Ashley, M1) for providing
the bovine limbs for the study, Nurit Golenberg for her assistance in the mechanical
indentation and cell viability testing, and Clifford Beckett for his design of the “cartilage

exerciser” and technical assistance.

74

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Ewers,B.J. and Haut,R.C. (2000) Polysulphated glycosaminoglycan treatments can
mitigate decreases in stiffness of articular cartilage in a traumatized animal joint. Journal
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Ewers,B.J., Weaver,B.T., Sevensma,E.T., and Haut,R.C. (2002) Chronic changes in
rabbit retro-patellar cartilage and subchondral bone after blunt impact loading of the
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Famdale,R.W., Sayers,C., and Barrett,A.J. (1982) A direct spectrophotometric
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mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.
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Morel,V., Mercay,A., and Quinn,T.M. (2005) Prestrain decreases cartilage susceptibility
to injury by ramp compression in vitro. Osteoarthritis and Cartilage 13, 964-970.

Newberry,W.N., MacKenzie,C., and Haut,R.C. (1998) Blunt impact causes changes in
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CHAPTER THREE

EFFECTS OF EXERCISE ON JOINT TRAUMA IN A HIGH ENERGY IMPACT
MODEL

ABSTRACT

High intensity impacts (10.0 J) have been shown to accelerate the degradation of
articular cartilage in the patello-femoral joint of rabbits in both exercising and non-
exercising models. However, no studies have directly investigated the effects of exercise
versus no-exercise in an impact model at this high intensity. In this study we impacted the
rabbit patello-femoral joint with a severe impact (10.0 J) and compared a group of
regularly exercised animals to animals restricted to cage activity only. The rabbits were
sacrificed at one of three times: 0, 12, and 24 months post-impact, and the retro-patellar
cartilage was biomechanically and histologically examined. Stiffiress and permeability of
the cartilage were measured by indentation, pathology was scored histologically, and the
thickness of the zone of calcified cartilage and subchondral bone were measured. The
data indicated that a restriction to cage activity accelerated the histological degeneration
of the cartilage in comparison with the regularly exercised animals, yet no loss of

articular cartilage was found in any of the groups.

INTRODUCTION

Osteoarthritis (0A) is a chronic joint disease which can be characterized by loss
of articular cartilage. OA affects over 20 million Americans, and costs the United States
economy more that $60 billion per year (Buckwalter et al., 2004). The incidence of 0A is

known to rise with age, yet age is not the only factor contributing to OA. Impact trauma

77

to the joint can cause severe articular deterioration by damaging the cells and disrupting
the cartilage matrix (Marsh et al., 2002).

Our laboratory has previously developed a post-traumatic animal model using the
rabbit (Flemish Giant) to examine the development of osteoarthritis (Haut et al., 1995). It
has been shown that a high severity (6.0 J) impact with a rigid interface causes a
softening of the retropatellar cartilage adjacent to superficial lesions, and thickening of
underlying subchondral bone at 12 months post-impact in an exercising rabbit model
(Ewers et al., 2002). In another study thickening of the subchondral bone was
documented at 3 months post impact in a non-exercising rabbit model (New Zealand
White), using a higher intensity 10.0 J impact (Mazieres et al., 1987). Histologically this
study showed significantly higher scores for degradation of articular cartilage in the
contusive knees compared to the opposite control knees, with increasing scores over time
in the impacted knees. Softening of the cartilage on the first day post-contusion was also
documented, persisting for the first month yet disappearing by 3 months. Based on the
Mazieres study, it appears that a higher-intensity impact (> 6.0 J) may be able to
accelerate the onset of post-traumatic osteoarthritis. However, few studies explore these
effects in an exercising animal model, to examine whether or not exercise will he