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Thisistccertifythatthe
thesisentitled

RESPONSE OF ARTICULAR CARTILAGE TO A BLUNT
ACUTE OVERLOAD CAN BE AFFECTED BY
INTERMITTENT CYCLIC PRELOAD AND ALTERATION OF
PROTEOGLYCAN CONTENTS

presented by

FENG WEI

has been accepted towards fulfillment
of the requirements for the

MS. degree in Mechanical Engineering

 

 

 

 

 

' @jor Professor's Signature

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Date

MSUbanaflinnafiw-ecflcn,cqud—cpportunityemployer

 

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/07 p:/CIRC/DaIeDue.indd-p.1

 

RESPONSE OF ARTICULAR CARTILAGE TO A BLUNT ACUTE OVERLOAD
CAN BE AFFECTED BY INTERMITTENT CYCLIC PRELOAD AND ALTERATION
OF PROTEOGLYCAN CONTENTS

By

Feng Wei

A THESIS

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

MASTER OF SCIENCE

Department of Mechanical Engineering

2007

ABSTRACT
RESPONSE OF ARTICULAR CARTILAGE TO A BLUNT ACUTE OVERLOAD

CAN BE AFFECTED BY INT ERMITTENT CYCLIC PRELOAD AND ALTERATION
OF PROTEOGLYCAN CONTENTS

By
F eng Wei
Mechanical loading of articular cartilage can influence chondrocyte metabolism and lead
to alterations in cartilage matrix composition. Most previous studies have focused on the
effect of cyclic loading on cartilage mechanical properties and proteoglycan (PG)
synthesis. However, the role of PGs synthesized from cyclic loaded cartilage in response
to an acute overloading has not been elucidated. We have therefore conducted studies
where low intensity, intermittent cyclic loading was applied to chondral explants prior to
an acute unconfined compression on the tissue. Chapter One documented a study by our
laboratory showing that 14 days of intermittent cyclic loading on chondral explants has a
positive effect on the tissue, by causing mechanical stiffening prior to a blunt force
overloading, to limit acute tissue damage. And yet, longer term loading to 21 days results
in tissue degradation prior to the acute traumatic event. In Chapter Two a supplement of
glucosamine-chondroitin sulfate was used in an experimental setting to alter PG synthesis
during cyclic loading of explants and its effect on the response of cartilage to an acute
overload was documented. The results showed that experimentally increased tissue PGs
during cyclic loading of cartilage help strengthen the cartilage, making them inhibit the
degradation of the tissue after long-terrn cyclic compression and reduce the susceptibility
of cartilage to a severe level of mechanical injury. In the long term these types of studies
may help understand the role of biologic-based pre-conditioning of articular cartilage for

in vitro, or even in vivo studies of blunt force trauma to a joint.

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. Dahsin
Liu and Dr. Seungik Baek 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. I would like to thank these students for their hard work and assistance: Marc
Schlaud, Greg Schafer, and Carmen Gacchina. Last but not least, I would like to thank
my fellow graduate students for their help and friendship: Nurit Golenberg, Eugene

Kepich, Eric Meyer, Dan Isaac, Tim Baumer, and Jerrod Braman.

iv

TABLE OF CONTENTS
LIST OF FIGURES ......................................................................................................... VII
LIST OF PUBLICATIONS ............................................................................................ VIII
RESPONSE OF ARTICULAR CARTILAGE TO A BLUNT ACUTE OVERLOAD

CAN BE AFFECTED BY INTERMITTENT CYCLIC PRELOAD AND ALTERATION
OF PROTEOGLYCAN CONTENTS

Introduction ............................................................................................................. 1
References ............................................................................................................... 8
CHAPTER ONE

EFFECT OF INTERMITTENT CYCLIC PRELOADS ON THE RESPONSE OF
ARTICULAR CARTILAGE EXPLANT S UNDER AN EXCESSIVE LEVEL OF

UNCONFINED COMPRESSION
Abstract ................................................................................................................. 12
Introduction ........................................................................................................... 13
Methods ................................................................................................................. 16
Dissection and tissue culture ........................................................................... 16
Indentation testing of explants ........................................................................ 16
Cyclic loading of explants .............................................................................. 18
Unconfined compression tests ........................................................................ l9
Explant surface fissure and cell viability tests ................................................ 20
Determination of proteoglycan and hydroxyproline contents ........................ 21
Statistical analysis ........................................................................................... 21
Results ................................................................................................................... 23
Indentation test ................................................................................................ 23
Unconfined compression tests ........................................................................ 25
Fissure length .................................................................................................. 26
Cell viability .................................................................................................... 28
Proteoglycan and hydroxyproline contents ..................................................... 31
Discussion ............................................................................................................. 34
Acknowledgement ................................................................................................ 39
References ............................................................................................................. 40
CHAPTER TWO

HIGH LEVELS OF GLUCOSAMINE-CHONDROIT IN SULFATE CAN ALTER THE
CYCLIC PRELOAD AND ACUTE OVERLOAD RESPONSES OF CHONDRAL
EXPLANT S

Abstract ................................................................................................................. 43
Introduction ........................................................................................................... 44
Methods ................................................................................................................. 49
Dissection, tissue culture and grouping of explants ........................................ 49
Indentation testing of explants ........................................................................ 51
Cyclic loading of explants .............................................................................. 51

Unconfined compression testing of explants .................................................. 52

Matrix damage and cell viability testing of explants ...................................... 52
Biochemical assays ......................................................................................... 53
Statistical analysis ........................................................................................... 54
Results ................................................................................................................... 55
Indentation testing ........................................................................................... 55
Biochemical assays ......................................................................................... 57
Matrix damage and cell viability .................................................................... 59
Discussion ............................................................................................................. 63
Acknowledgement ................................................................................................ 70
References ............................................................................................................. 71
CHAPTER THREE
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK ..................... 75
Conclusions ........................................................................................................... 75
Recommendations for future work ....................................................................... 78
References ............................................................................................................. 83
APPENDIX A: LOW CONCENTRATION OF SUPPLEMENT .................................... 85
APPENDD( B: PROCEDURE FOR OBTAINING CARTILAGE EXPLANT S FROM
BOVINE METACARPAL JOINTS ................................................................................. 89
APPENDIX C: BOVINE MEDIA STOCK RECIPE ....................................................... 90
APPENDIX D: BOVINE MEDIA HANDLING SOP ..................................................... 91
APPENDD( E: BOVINE EXPLANT INDENTATION SOP .......................................... 92
APPENDIX F: IMPACTING BOVINE EXPLANTS ...................................................... 98
APPENDIX G: LIV E/DEAD VIABILITY STAINING FOR ARTICULAR
CARTILAGE EXPLANTS ............................................................................................ 100
APPENDIX H: PROTEOGLYCAN ASSAY ................................................................ 102
APPENDIX 1: HYDROXYPROLINE DETERMINATION ......................................... 107

vi

LIST OF FIGURES

Images in this thesis are presented in color.

INTRODUCTION
Figure 1 ................................................................................................................... 1
Figure 2 ................................................................................................................... 2
Figure 3 ................................................................................................................... 3
CHAPTER 1
Figure 4 ................................................................................................................. 18
Figure 5 ................................................................................................................. 19
Figure 6 ................................................................................................................. 24
Figure 7 ................................................................................................................ 26
Figure 8 ................................................................................................................. 27
Figure 9 ................................................................................................................. 28
Figure 10 ............................................................................................................... 29
Figure 11 ............................................................................................................... 30
Figure 12 ............................................................................................................... 31
Figure 13 ............................................................................................................... 32
Figure 14 ............................................................................................................... 33
CHAPTER 2
Figure 15 ............................................................................................................... 50
Figure 16 ............................................................................................................... 56
Figure 17 ............................................................................................................... 58
Figure 18 ............................................................................................................... 60
Figure 19 ............................................................................................................... 61
APPENDIX A
Figure 20 ............................................................................................................... 87

vii

LIST OF PUBLICATIONS

Peer Reviewed Manuscripts

Wei F, Golenberg N, Kepich E, Haut R: Effect of intermittent cyclic preloads on the
response of articular cartilage explants to an excessive level of unconfined compression.
J oumal of Orthopaedic Research, In Revision

Wei F, Haut R: High levels of glucosarnine-chondroitin sulfate can alter the cyclic
preload and acute overload responses of chondral explants. Journal of Orthopaedic
Research, In Review

Peer Reviewed Abstracts

Wei F, Golenberg N, Kepich E, Haut R: In vitro exercise affects the response of articular
cartilage to blunt impact loading. Transactions of the 53rd Annual Meeting of the
Orthopaedic Research Society, San Diego, California 32:662, 2007

Wei F, Golenberg N, Kepich E, Haut R: Glucosamine and chondroitin sulfate affect the
response of exercised articular cartilage to blunt impact loading. Transactions of the 31“
Annual Meeting of the American Society of Biomechanics, Stanford, California 31:P4—
21, 2007

Wei F, Golenberg N, Kepich E, Haut R: Alteration of protcoglycan synthesis in a
cartilage explant during cyclic loading can affect its susceptibility to damage in an acute
overload. Transactions of the 54th Annual Meeting of the Orthopaedic Research Society,
San Francisco, California, 2008

viii

INTRODUCTION

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 (PG’s)
(Figure 1). PG’s are protein aggregates having polysaccharide side-chain units known as
glycosaminoglycans (GAGs). As the joint is subjected to load, the cartilage will deform
in order to distribute the load, causing compressive, tensile, and shear stresses throughout
the cartilage (Mow and
Setton, 1998). The function
of the collagen is to provide
the cartilage with tensile

strength, whereas the PG’s

 

are associated more with the
Figure l. The extracellular matrix of articular . _
cartilage is composed mainly of collagen fim stiffness properties of the
proteoglycam, and water. , . .
cartrlage In compressron
(Hehninen et al., 1992). The content and structure of PG’s 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).

Articularsurtace

 

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

Osteoarthritis (0A) is a degenerative joint disease associated with multiple factors,
genetic, metabolic, biochemical and biomechanical, that act to disturb the equilibrium of
anabolic and catabolic events in articular cartilage and adjacent bone (Derfoul et a1.
2007). While the mechanisms responsible for this disease are unknown, the risk of OA is
increased significantly in joints suffering a major injury (Felson 2000 and 2004).
Hereditary defects may predispose to 0A, 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; Helrninen et al., 1992; Gelber et al., 2000; Marsh et al., 2002).
OA 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 OA 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 soft 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.

Mechanically injurious loads can generate articular cartilage extracellular matrix damage
and chondrocyte death, which have been suggested to facilitate development of OA. We
developed a model of post-traumatic OA using the Flemish Giant rabbit (Haut et a1.
1995). In a previous in vivo animal study in our lab, a rigid impact mass was dropped
with 6 J of energy onto the flexed patelIo-femoral rabbit joint, resulting in acute surface
fissures, progressive degradation of retro-patellar surface cartilage, and thickening of
underlying subchondral bone afier 3 years (Ewers et a1. 2002). In another study, this
impact was found to produce patella-femoral contact pressure of approximately 25 MPa
(Newberry et a1. 1998). A more recent study showed that this level of blunt impact load

applied to the patello-femoral joint induces acute chondrocyte necrosis throughout the

retro-patellar cartilage (Rundell et al. 2005). In vitro studies also show that a single blunt
impact load can result in surface fissures and cell death in chondral and osteochondral
explants. Necrotic cell death was also found in situ using the bovine patella subjected to a
single impact of 53 MPa in 250 ms. Cell death occurred largely in the superficial zone
adjacent to fissures, but not in impacted areas away from these cracks (Lewis et al. 2003).
Studies involving explants removed from bone indicated that the extent of matrix damage
would be higher [in high versus low rate of loading experiments, while cell death would
be greater in low versus high rate experiments (Ewers et al. 2001). A critical threshold
stress of 15-20 MPa was found for cell death and collagen matrix damage in unconfined
compression using bovine chondral explants (Torzilli et al. 1999). Significant matrix
fluid pressurization and surface cracking were observed from unconfined compression of
bovine osteochondral explants (Morel and Quinn 2004). Other studies have also
associated articular cartilage physiopathology in 0A with chondrocyte death (Hashimoto

et al. 1998).

The variations in cartilage morphology, mechanical properties (Murray et al. 1998;
Murray et al. 1999), and chondrocyte matrix synthesis (Ackerrnann and Steinmeyer 2005;
Buschmann et a1. 1999) have been reported and may result from the variations in load.
Therefore, exercise would be expected to alter the composition and mechanical behavior
of cartilage. Although mechanical stimulation is necessary for cartilage development and
homeostasis (Buschmann et al. 1995), abnormal loading has also been shown to initiate
cartilage degradation (Farquhar et al. 1996; Jeffrey et al. 1995; Thibault et al. 2002).

Mechanical loading conditions ranging from very high strain rate impact loading

conditions, such as traumatic accidents, to very low strain rate loading, such as obesity or
joint misalignment, may all contribute to cartilage injury and degenerative joint disease
(Morel et a1. 2005). In vitro studies of impact loading (Atkinson et al. 1998; Borrelli et al.
1997; Milentijevic and Torzilli 2005; Milentijevic et al. 2003) and high strain rate
injurious compression (Quinn et al. 1998b; Ewers et al. 2001; Kurz et al. 2001; Quinn et
al. 2001) of cartilage have shown that cell death and matrix damage resulting fi'om fluid
pressurization and collagen network tensile failure (Morel and Quinn 2004) could
represent initiating events for degenerative processes. Cyclic loading can also lead to
chondrocyte death (Levin et al. 2001; Clements et al. 2001; Chen et al. 2003), mechanical
weakening of collagen (Thibault et al. 2002), and decrease of collagen synthesis rate

(Ackerrnann and Steinmeyer 2005) in cartilage.

Since prestrain of cartilage explants prior to injurious loading reduced the occurrence of
superficial cracks and associated cell death (Morel et al. 2005), the short-term loading
history of cartilage can influence its subsequent response to injurious compression due to
modifications in tissue structural organization and mechanical properties. The
extracellular matrix (ECM) consists mainly of PG’s and type H collagen. Based on
experimental evidence, PG’s are primarily responsible for the compressive stiffness of
the cartilage matrix (Mow et al. 1990 and Korhonen et al. 2003). Biosynthesis of PG can
be stimulated by intermittently applied cyclic loading (Sah et al. 1989). Investigators
have provided many fi'arneworks for identifying both the physical and biological
mechanisms by which dynamic compression can modulate chondrocyte biosynthesis. In

one study, cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa

compressions repeated at intervals of 2-60 3 for 1.5 h and simultaneously labeled with
35804. It was found that explants under a 0.5 MPa load showed significantly increased
35804 incorporation by compression repeated at 2- and 4-3 intervals. Therefore, the
stimulation of PG synthesis, as indicated by [3 5 S] sulfate incorporation, is limited to
certain loading frequencies and pressures (Parkkinen et al. 1992). In another study, a
compressive pressure was introduced for 1, 3 or 6 days using a sinusoidal waveform of
0.5 Hz frequency with a peak stress of 0.1, 0.5 or 1.0 MPa. A maximum PG synthesis
was observed at day 3 with 0.5 Hz fi'equency and 0.5 MPa peak stress (Steinmeyer et al.
1999). However, the role of PG synthesized from intermittently loaded cartilage in

response to injurious trauma of articular cartilage has not been fully elucidated.

In Chapter One of the study we hypothesized that physiologically intermittent cyclic
loading would stimulate PG synthesis in cartilage explants, resulting in an increased
matrix stiffness of the tissue, and therefore reduce its susceptibility to matrix damage and
cell death following a single blunt impact loading to approximate 25 MPa. This
hypothesis was tested in the present study by systematically applying different durations

of intermittent loading to cartilage explants before injurious blunt impact loading.

Understanding the changes in collagen and PG content of cartilage due to mechanical
forces is necessary for progress in treating join disorders, such as OA. The use of
potentially chondro-protective agents such as glucosamine (glcN) and chondroitin sulfate
(CS) has been explored to medicate OA. Previous studies by others have shown that

bathing cartilage explants in a supplement of glcN and CS can up-regulate the synthesis

of tissue PG’s, and particularly in stressed tissue (Lippiello 2003). However, the role of
PG’s synthesized from intermittently loaded cartilage in response to an acute
compressive overloading of articular cartilage has not been elucidated. Recent studies
have also shown that this supplement limits tissue degradation in the presence of

inflammatory enzymes in an in vitro setting (Derfoul et al. 2007).

Since previous data showed that PG content directly paralleled with stiffness of cartilage
(Chapter 1), we wanted to experimentally increase tissue PG content to stiffen the
cartilage, making them inhibit the degradation of the tissue after long term of cyclic
compression and reduce the susceptibility of cartilage to a severe level of mechanical
injury, such as an acute overload. The objective of Chapter Two of this thesis was to use
this supplement in an experimental setting to alter PG synthesis during cyclic loading of
chondral explants and document its effect on the response of explants to an acute
overload. The hypothesis was that increased levels of tissue PG will significantly limit

blunt force trauma to the tissue.

REFERENCES

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Atkinson TS, Haut RC, Altiero NJ: Impact-induced fissuring of articular cartilage: an
investigation of failure criteria. Journal of Biomechanical Engineering 120(2):181-
187, 1998

Borrelli J Jr, Torzilli PA, Grigiene R, Helfet DL: Effect of impact load on articular
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Buckwalter J A, Saltzman C, Brown T: The impact of osteoarthritis: implications for
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Buschmann MD, Kim Y], Wong M, Frank E, Hunziker EB, Grodzinsky AJ: Stimulation
of aggrecan synthesis in cartilage explants by cyclic loading is localized to regions of
high interstitial fluid flow. Archives of Biochemistry and Biophysics 366: 1-7, 1999

Chen CT, Bhargava M, Lin PM, Torzilli PA: Time, stress, and location dependent
chondrocyte death and collagen damage in cyclically loaded articular cartilage.
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Clements KM, Bee ZC, Crossingharn GV, Adams MA, Sharif M: How severe must
repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthritis and
Cartilage 9:499-507, 2001

Derfoul A, Miyoshi AD, Freeman DE, Tuan RS: Glucosamine promotes chondrogenic
phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13
expression and matrix degradation. Osteoarthritis and Cartilage 15:646-655, 2007

Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC: The extent of matrix damage and
chondrocyte death in mechanically traumatized articular cartilage explants depends
on rate of loading. Journal of Orthopaedic Research 19:779-784, 2001

Ewers BJ, Weaver BT, Sevensma ET, Haut RC: 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, 2002

Farquhar T, Xia Y, Mann K, Bertram J, Burton-Wurster N, Jelinski L, et al.: Swelling
and fibronectin accumulation in articular cartilage explants after cyclical impact.
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Hashimoto S, Ochs RL, Komiya S, Lotz M: Linkage of chondrocyte apoptosis and
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Haut RC, Ide TM, DeCamp CE: Mechanical responses of the rabbit patella-femoral joint
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Helminen HJ, Kiviranta I, Saamanen AM, Jurvelin JS, Arokoski J, Oettrneier R,
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Jeffrey JE, Gregory DW, Aspden RM: Matrix damage and chondrocyte viability
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16:348-354, 1998

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to sub-impact loading of adult bovine articular cartilage explants: effects of strain rate
and peak stress. Journal of Orthopaedic Research 19:242-249, 2001

10

Quinn TM, Grodzinsky AJ, Hunziker EB, Sandy JD: Effects of injurious compression on
matrix turnover around individual cells in calf articular cartilage explants. Journal of
Orthopaedic Research 16:490-499, 1998

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patellar cartilage after a severe blunt impact to the in vivo rabbit patello-femoral joint.
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response of cartilage explants to dynamic compression. Journal of Orthopaedic
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on proteoglycan metabolism and swelling behaviour of articular cartilage explants.
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Torzilli PA, Grigiene R, Borelli J Jr, Helfet DL: Effect of impact load on articular
cartilage: cell metabolism and viability, and matrix water content. Journal of
Biomechanical Engineering 121 :433-441, 1999

11

CHAPTER ONE

EFFECT OF INTERMITTENT CYCLIC PRELOADS ON THE RESPONSE OF
ARTICULAR CARTILAGE EXPLANTS UNDER AN EXCESSIVE LEVEL OF
UNCONFINED COMPRESSION

ABSTRACT

Mechanical loading of articular cartilage can influence chondrocyte metabolism and lead
to alterations in cartilage matrix composition. Most previous studies have focused on the
effect of cyclic loading on cartilage mechanical properties and proteoglycan synthesis.
However, the role of proteoglycans synthesized from cyclic loaded cartilage in response
to an acute overloading has not been elucidated. We have therefore conducted studies
where low intensity, intermittent cyclic loading was applied to chondral explants prior to
an acute unconfined compression on the tissue. The results of the study showed cyclic
preloading increased the production of proteoglycans and mechanically stiffened the
explants, making them more resistant to matrix damage and cell death under 25 MPa of
acute, unconfined compression up to 14 days. After 21 days of cyclic loading, however,
the explants lost compressive stiffness and suffered more extensive damage in the acute,
unconfined compression test than controls. This study investigated the role of in vitro
cyclic loading on the response of chondral explants to a potentially damaging, acute
overload. In the long term these types of studies may help understand the role of biologic-
based pre-conditioning of articular cartilage for in vitro, or even in vivo studies of blunt

force trauma to a joint.

12

INTRODUCTION

Joints suffering a major acute injury, such as an osteochondral fracture or ligament
rupture, are at risk of developing chronic osteoarthritis (OA) (F elson et al. 2000). In cases
of injury to a knee ligament, early detection of bone bruises is seen in over 80% of cases
(V ellet et al. 1991; Johnson et al. 1998). These bone lesions have also been observed in
clinical cases without acute ligament or osteochondral damage (Snearly et al. 1996).
Associated with these bone lesions recent investigators have documented early
degeneration of overlying articular cartilage and death of chondrocytes (Fang et al. 2001;
Johnson et al. 1998). Damage to articular cartilage overlying MRI detected bone bnrises
may be due to excessive compressive forces generated in the joint during acute injury, i.e.
rupture of the anterior cruciate ligament (ACL) (Fang et al. 2001). These bone lesions
and acute cartilage damage may help explain the basis for a post-traumatic 0A in the
joints of ACL—injured patients, independent of whether they are surgically reconstructed
(Daniel et al. 1994; Myklebust & Bahr 2005). Research on animal models has shown that
acute trauma to articular cartilage causing matrix damage and cell death can lead to
chronic disease in joints (Ewers et al. 2002). A more recent study has also shown that
during these acute trauma experiments contact pressure in the rabbit patella-femoral joint
averages approximately 25 MPa (Rundell et al. 2005), and it induces acute chondrocyte

necrosis throughout the retro-patellar cartilage.

The development of cell death and matrix damage in cartilage is known to vary with the
levels of compressive loading and strains (deformations) applied to the tissue (Torzilli et

al. 1999; Quinn et al. 2001). In particular, direct relationships have been established

13

between the levels of tissue deformation and cellular damage (Torzilli et a1. 2006). In
order to limit or even help mitigate the process that may lead to GA in joints suffering
blunt force trauma, it would seem reasonable to examine factors that might affect the
mechanical properties of cartilage before the acute traumatic event. For example,
increasing the structural stiffness and strength of articular cartilage may alter the long-
terrn consequences of blunt force to the joint. Recently, Quinn et al. have shown that pre-
strain of the tissue by a short-term history of compressive loading helps limit the extent
of damage in chondral explants to an acute blunt force loading (Morel et al. 2005). The
basis for this action may be that these pre-strains cause matrix consolidation and an
increase in structural stiffness of the tissue prior to the blunt impact force. Another
mechanism that could potentially help limit the extent of joint injury during a traumatic
event is to increase the matrix stiffiress of articular cartilage in a material sense prior to
impact. In vitro and in vivo studies have shown that physiologically intermittent cyclic
loading of articular cartilage can up-regulate the synthesis of tissue proteoglycans (PGs)
(Sah et al. 1989; Parkkinen et al. 1992; Saadat et al. 2006). However, the role of PG
synthesized from intermittently loaded cartilage in response to an acute compressive

overloading of articular cartilage has not been elucidated.

The hypothesis of the current study was that low intensity, intermittent cyclic loading of
chondral explants will up-regulate the production of tissue PGs, resulting in an increase
of the explant mechanical stiffness that will help limit the extent of matrix damage and
associated chondrocyte death following a single, severe level of blunt force loading.

These data may have a direct bearing on the issue of pre-impact conditioning of chondral

l4

explants in laboratory experiments dealing with the short-term consequences of impact
trauma. Additionally, these data may also relate to the role of regular exercise on the
long-term consequences of human joints following a traumatic joint injury, such as an

acute ligament rupture.

15

METHODS

DISSECTION AND TISSUE CULTURE

Bovine forelegs from mature animals (18-24 months of age) were obtained fi'om a local
abattoir within two hours of slaughter. The legs were skinned and rinsed with water prior
to exposing the metacarpal joint under a laminar flow hood. A biopsy punch (Miltex
Instrument Company, Bethpage, NY) was used to make twenty-four 6 mm diameter
chondral explants fiom the lower metacarpal surface of the limbs. Each explant was
separated from the underlying bone with a scalpel. All specimens were washed three
times in Dulbecco’s Modified Eagle Media: F12 (DMEM: F12) (Gibco, USA, #12500-
039), and then placed in the media supplemented with 10% fetal bovine serum, additional
amino acids and antibiotics (penicillin 100 U/ml, streptomycin 1 rig/ml, amphotericin B
0.25 rig/ml) in a 24-well plate. The media was replaced every 2 days throughout the study
and harvested and stored fi'ozen at -80°C. The well plate was placed in a mechanical
loading device (the “cartilage exerciser” (described below)) inside of a humidity-
controlled incubator (37°C, 5% C02, 95% humidity). The osmolarity of the media was
300mosM (Osmete 2, Precision Systems), and the pH was 7.4. Physiological and
metabolic stability of this explant system has been demonstrated previously (Phillips and

Haut 2004; Baars et al. 2006; Wei et al. 2006 and 2007).

INDENTATION TESTING OF EXPLANT S
The 24 explants were randomly assigned to three groups, 7, 14 and 21 days cyclic
loading (n=4) versus control (n=4). Prior to and after cyclic loading, each explant was

subjected to mechanical characterization using an indentation stress relaxation test. Afler

l6

allowing a minimum of 30 minutes in media for the explants to equilibrate, mechanical
indentation tests were performed before and after 7, l4 and 21 days of cyclic loading.
Cartilage explant thickness was measured twice at perpendicular orientations across the
center of the explant using a digital vernier caliper (Mitutoyo Corp.: Absolute Digimatic,
Model No. CD-6" CS) with a resolution of 0.01 mm (Steinmeyer et al. 1997 and 1999).
The two thickness values were averaged. 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 4.3
mm diameter hole was placed on top of the explant to secure the edges and help resist
curling of the explants. The explant and fixture were then submerged into a room-
temperature phosphate buffered solution (PBS with pH 7.2) (Figure 1). A 2.39 mm
diameter spherical, non-porous probe was lowered into the cartilage until a preload of
0.03 N was attained and held for 60 s. The indenter was then pressed into the cartilage
25% of the total thickness in 2 s and maintained for 600 5 while resistive loads of
relaxation were measured (Data Instruments, Acton, MA: model JP-25, 25 lb capacity),
amplified and collected at 1,000 Hz for the first second and 20 Hz thereafter. The stress
relaxation curves were obtained and fitted with a fibril-reinforced biphasic FE model
(Soulhat et a1. 1999). Cartilage matrix modulus (Em), fiber modulus (E;) and permeability
(K) were evaluated with a custom-written Gauss-Newton constrained nonlinear least

square minimization procedure.

17

;\

Explant

Magnets

lndenter

\

 

Figure 4. (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.
CYCLIC LOADING OF EXPLANTS
The “cartilage exerciser” consisted of 12 loading chambers simultaneously powered by
air (Figure 2). The control explants rested in the same “cartilage exerciser” as the cyclic
loaded ones, but without cyclic loading on them. Pneumatic cylinders forced the pistons
downward to apply a compressive load to the specimens through 14.6 mm diameter non-

porous Teflon® platens. 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
18

explants would be subjected to an identical culture environment. Intermittently applied,
uniaxial cyclic loading was introduced by using a 0.2 Hz sinusoidal waveform with a
peak stress of 0.5 MPa. The cyclic loads were applied for 10 cycles followed by a load-
free period lasting 3600 5. During the period of unloading the load platen was lifted from

the cartilage surface.

 

Figure 5. The “cartilage exerciser” mechanical loading device witl
12 cylinders to apply compressive loads to the cartilage explants.

UNCONFINED COMPRESSION TESTS

After cyclic loading, all explants (including the control) were taken to 707 N (~25 MPa),
following a 5 N preload, in unconfined compression between two polished stainless steel
plates. A 0.5 Hz (1 s time to peak) haversine loading protocol was programmed for
application onto the explants in a servo-controlled hydraulic testing machine (Instron,

model 1331, retrofitted with 8500 plus electronics, Canton, MA). Peak load, time to peak,

19

and maximum explant compression were documented in each experiment. Immediately
after impact loading, the explants were returned to culture for 24 hours before cell

viability tests.

E)G’LANT SURFACE FISSURE AND CELL VIABILITY TESTS

For matrix damage analysis, the surfaces of all impacted explants were wiped with India
ink and immediately photographed at 25X under a dissection microscope (Wild M5A,
Wild Heerbrugg Ltd, Switzerland) to determine the total length of explant surface
fissures (Baars et al. 2006). The total fissure length was measured with digital imaging
software (Sigma Scan, SPSS Inc., Chicago, IL, USA). One observer (F.W.) digitally

recorded the length of the surface fissures in each photograph.

After fissure length measurement, all explants were washed three times in DMEM:F12
and used to determine cell viability. Each explant was cut through its entire thickness
with a specialized cutting tool with parallel blades spaced 0.5 mm (Phillips and Haut
2004; Ewers et al. 2001). These thin slices were then stained with calcein AM and
ethidium homodimer (EthD-l), according to the manufacturer’s specifications
(Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene, OR, USA). Viable cells were
distinguished by the presence of ubiquitous intracellular esterase activity, determined by
the enzymatic conversion of virtually non-fluorescent cell-permeant calcein AM to
intensely fluorescent calcein. EthD-l enters cells with damaged membranes and
undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby

producing a bright red fluorescence. EthD-l is excluded by the intact plasma membrane

20

of viable cells. Three slices were viewed across approximately 2 mm near the center of
each explant in a fluorescence microscope (Leica DM LB, Leica Mikroskopie und
Systeme Gmgl-I, Wetlzar, Germany) and photographed using a digital camera (Spot
Digital Camera, Diagnostic Instruments Inc.). These images were partitioned into the
superficial zone (top 20%), intermediate zone (middle 50%) and deep zone (bottom 30%)
(Phillips and Haut 2004). An average total cell count was calculated for each zone of the

explants, using image analysis software (Sigma Scan, SPSS Inc., Chicago, IL, USA).

DETERMINATION OF PROTEOGLYCAN AND HYDROXYPROLINE CONTENTS

Cartilage explants were digested overnight at 60°C with papain digestion solution at a pH
6.0. Papain digested cartilage explants and culture media were independently DMB
assayed for sulfated PGs by the reaction with 1,9-dimethylmethylene blue dye solution in
polystyrene 96 well plates and quantitated with spectrophotometry at a wavelength of 530
nm using a Bio Tek rrricroplate reader. Chondroitin sulfate A sodium salt from bovine
trachea (Sigma-ALDRICH GmbH, Steinheim, Germany) was used as the standard
(Steinmeyer et al. 1999). Some explants were used for hydroxyproline (HYP)
measurement. After a guarridine HCl extraction to remove PG, tissue samples were acid-
hydrolyzed in a 70°C water bath for 24 hours and nricroplate assayed to determine the

total HYP content in the tissue (Brown et al. 2001; Bank et al. 1997).

STATISTICAL ANALYSIS
All experiments were repeated three times to obtain consistent results (n=12). The data

obtained from post-cyclic loaded explants were normalized by the pre-cyclic loaded

21

values and subsequently analyzed with two-way repeated measures ANOVA and
Student-Neuman-Keuls post hoc tests to determine differences from control and within
cyclic loading groups. PG and HYP biosynthesis was normalized by tissue wet weight
and reported in pg PG and pg HYP per mg wet weight, respectively. Statistical
significance was indicated for P<0.05. All experimental data were reported as mean :h

standard deviation.

22

RESULTS

INDENTATION TEST

All explants underwent indentation testing for mechanical characterization. Parameters
before and after cyclic loading were compared in terms of “% of before cyclic loading”
as in Figure 3. Fourteen days of cyclic loading significantly decreased the fiber modulus
and permeability, but increased the matrix modulus of the explants. After 21 days,
however, all other parameters reversed, except for the fiber modulus. After 7 and 14 days
of cyclic loading, the matrix modulus increased approximately 22% and 35%,
respectively, while 21 days of cyclic loading decreased the matrix modulus
approximately 12%. After 7 days of cyclic loading, the fiber modulus significantly
decreased nearly 80% and remained at this low level at 14 and 21 days. After 7 and 14
days of cyclic loading, the explant permeability decreased approximately 30% and 45%,
respectively, while 21 days of cyclic loading significantly increased the permeability by

nearly 54%.

23

>

 

 

 

 

 

 

 

 

 

 

 

 

 

 

180 - —D—Matrix Modulus
160 q —<>—Flber Modulus
g, - -A- Penneabilrty *§ IT
'0
.8
.9
'6
8‘
9
.9
8
”5
39 \
23- r r r
O 7 14 21
Duration of cyclic loading (days)
B
180 _ —D—Matrix Modulus
150 - —o—Fiber Modulus
- a- P M“
g 140 _ ermea rrty
3 120 ~
% 100 """ A """" ‘5 """" '5
.. H M
8‘
g 80 ~
E 60 1
'5 _
33 40
20 -
0 . , r
O 7 14 21

Duration of cyclic loading (days)

Figure 6. Up to 14 days cyclic loading significantly decreased the
fiber modulus and permeability, but increased the cartilage matrix
modulus. After 21 days, however, the matrix modulus significantly
decreased and tissue permeability increased (A). In all groups, the
controls (non-cyclic loading) remained constant for all parameters
(B). Statistically significant differences were observed: * fi'om

control, T from day 7, and Y fiom day 14.

24

UNCONFINED COMPRESSION TESTS

The stress-strain response of all explants had a nonlinear (toe) region (0-10 MPa)
followed by a linear region (IO-25 MPa) during unconfined compression in 1 s (Figure
4A). Explants that were regularly compressed up to 14 days exhibited a stiffened
response, with the longer durations of loading shifted more to the left. However, after 21
days of loading the explants became less stiff than the control. Statistically significant
differences were observed between each test group and the control responses. The slope
of the linear region (modulus) between 10 MPa and 25 MPa was increased approximately
18% of control at day 7 (105.5 :I: 15.3 MPa to 123.0 at 16.2 MPa) and approximately 30%
of control in day 14 (105.5 :I: 15.3 MPa to 133.2 :I: 11.9 MPa). After 21 days of cyclic
loading, however, the modulus of the explant decreased significantly below the 7 and 14

days to 94.2 d: 15.0 MPa (Figure 4B).

25

 

 

 

 

30 >~
I —O—Control
—D-7-Day * * *
25 7i - A- 14—Day l—p—D <>—*
. —<>- 21-Day , / ,/
A 1
N
c.
E
8
9
<73
0 0.1 0.2 0.3 0.4 0.5
Strain (mm/mm)
B
160 —
’«T
c. 150 -
a * *
3 140 -
D
B 130 «
2
‘5 120 -
3
9 110 -
O.
E 100 - I I
O
8 90-
{-5
8 80 —
O
C
D 70-
Control Day 7 Day 14 Day 21

Figure 7. The control shown in this figure is a combination of
controls from all groups. Explants cyclic-loaded up to 14 days
exhibited a stiffer response than controls, resulting in a shift to the
left, with the longer duration of loading shifted more. After 21 days
of cyclic loading, however, the explants became less stiff than the
controls. *: Statistically significant differences from control as
deferrrrined by two-way repeated ANOVA, P<0.05.

FISSURE LENGTH

26

Cyclic loading affected the fissure length of cartilage explants (Figure 5). After 7 days of
cyclic loading and unconfined compression the length of surface fissures decreased 57%
from control (7.9 :t 1.9 cm to 3.4 d: 1.3 cm). After 14 days of cyclic loading and
impaction, the fissure length decreased to 42% of control (3.3 :l: 1.5 cm), not different
flour 7 days but statistically different from control. After 21 days of cyclic loading,
however, there was an increase (approximately 170% of 7 and 14 days) of fissure length
(9.0 d: 2.0 cm). This value was not statistically different fi'om control, but different than

the 7 and 14 day fissure length (Figure 6).

 

Figure 8. a) 7 days; b) 14 days; c) 21 days
cyclic loaded explants alter the
unconfined compression. Explants in
group c) exhibited excessive surface
fissuring across the entire surface.

27

1o- +<>

Average fissure length (cm)
It»
It

 

 

 

   

 

Control Day 7 Day 14 Day 21
Figure 9. Cyclic loading up to 14 days significantly decreased the

explant surface fissure length, while after 21 days of cyclic loading
an increase of fissure length fiom previous groups was observed.

Statistically significant differences were observed: * from control,

7 from day 7, and Y from day 14.
CELL VIABILITY
Cyclic loading prior to the unconfined compression test affected the cell Viability of the
explants (Figure 7). Cyclic loading up to 14 days significantly saved cells in the
superficial zone. After 14 days, non-cyclic loaded (control) cartilage had only 40% live
cells, while cyclic loaded cartilage had 80% (Figure 8). After 21 days of cyclic loading
the average number of live cells decreased significantly in both superficial and deep
zones following impact loading (28% of live cell in superficial zone and 42% of live cell
in deep zone). No significant differences were observed in the middle zone for all groups.
Explant maximum strains from the unconfined compression tests were used to calculate
the percentage of cell death per unit strain. Cell death per unit strain was significantly

decreased versus controls up to 14 days of cyclic loading (70% and 35% of controls at

28

day 7 and day 14, respectively). In contrast, after 21 days of cyclic loading the cell death

per unit strain was not different from control (Figure 9).

a)

b)

(I 1 nm‘

e)

 

7:301 ()1 "WI .73" t I) l r""v

Figure 10. Typical images of cartilage explants stained for cell viability. Live cells
stained green and dead cells stained red. The cracks on the surface were due to the
unconfined compression on the explants. a) 7 day control (left) and cyclic loaded (right);
b) 14 days control (left) and cyclic loaded (right); c) 21 days control (left) and cyclic
loaded (right).

29

Average % live cells

 

Superficial Middle Deep

Figure 11. Cell viability in the superficial, middle and deep zones of
cartilage explants following unconfined compression. Cyclic loading
affected the cell viability mostly in the superficial zone. No
significant differences were observed in the middle zones of all
groups. Cyclic loading, up to 14 days, significantly saved cells in the
superficial zone. After 21 days of cyclic loading, however, the
average number of live cells decreased significantly in both the
superficial and deep zones. Statistically significant differences were

observed: * from control, 7 from day 7, and <> from day 14.

30

*1

% of cell death I unit strain

 

 

 

   

 

 

 

Control Day 7 Day 14 Day 21
Figure 12. Along with cyclic loading period, cell death per unit strain
was significantly decreased for up to 14 days of cyclic loading (70%

and 35% of control at day 7 and day 14, respectively), while after 21
days of cyclic loading there was no difference from control.

Statistically significant differences were observed: * from control,

* from day 7, and <> from day 14.
PROTEOGLYCAN AND HYDROXYPROLINE CONTENTS
Cyclic loading up to 14 days significantly increased PG content of the cartilage explants,
with the largest increase (40%) at 14 days (Figure 10A). After 21 days, however, cyclic
loading resulted in a significant loss in PG’s. After cyclic loading PGs were released
from cartilage explants into the media in all 3 groups (Figure IOB). Cyclic loading also

resulted in a significant decrease in tissue HYP content by approximately 20% in all 3

groups (Figure 11).

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

70 ~-
DControl
60 ~~ ICycllc loading
a
8 5° “ i
3
g 40 -~
3’ 30 l-
: 20 ~—
3.
10 i
0
7 Days 14 Days 21 Days
B
12 -
DContml *+¢
10 ‘ ICyclic loading

 

 

 

ug PG / mg Wet Weight
0)

 

 

 

 

 

4a
2— * *
7Days 14Days 21Days

Figure 13. (A) Total tissue proteoglycan (PG) content per mg
tissue wet weight. Cyclic loading up to 14 days increased PG
content in the tissue sample. After 21 days of cyclic loading,
however, cartilage explants significantly lost PG’s. (B) Total PG
content in media per mg tissue wet weight. After cyclic loading
PG’s were released fi'om tissue samples into media in all 3
groups. After 21 days of cyclic loading PG content in the media
significantly increased 8 times than control. Statistically

significant differences were observed: * from control, Y from
day 7, and <> from day 14.

32

 

El Control
I Cyclic loading

 

 

 

 

*

Total [19 HYP / mg Wet Weight
-L -t N N on 0)
o or c or o 0: c or

 

 

 

 

 

 

 

 

 

7 Days 14 Days 21 Days

Figure 14. Total hydroxyproline (HYP) content per mg tissue wet
weight. Cyclic loading decreased tissue HYP content by
approximately 20% in all 3 groups. Statistically significant

differences were observed: * fi'om control.

33

DISCUSSION

The hypothesis of the current study was that low intensity, intermittent cyclic loading of
chondral explants would up-regulate the production of PGs by matrix chondrocytes to
increase the material properties of the specimens, making them more resistant to blunt
force overloading. The results of the study showed cyclic preloading did mechanically
stiffen the explants, making them more resistant to matrix damage and cell death under
25 MPa of unconfined compression up to 14 days. However, after 21 days of cyclic
loading the explants lost stiffness and suffered more extensive damage in the acute,

unconfined compression test than controls without cyclic preloading.

Numerous aspects of this study parallel the findings of the previous literature. In one
study Sah et al. (1989) reported that multiple 2-hr compressions of calf articular cartilage
explants (at 8-hr intervals) increases the incorporation of ”so. during 1.5-5.5 hr after
release. However, the study was unable to show a significant increase in
glycosaminoglycan (GAG) content of the explants. More recently, Parkkinen et al. (1992)
have shown that a 0.5 MPa load significantly increased 35804 incorporation by
chondrocytes for compressions repeated at 2- and 4-s but not at 20- and 60-3 intervals for
1.5 hr. Steinmeyer et al. (1999) show that peak stresses of 0.1, 0.5 or 1.0 MPa applied for
10 s followed by a load-free period of 10, 100 or 1000 5 increases the incorporation of
35804 after 3 days of cyclic loading, but again no significant change in tissue PG content
was documented in the study. Similarly, Thibault et al. (2002) cyclically loaded
osteochondral explants with 40 ramps of 250 um amplitude (~2-5 MPa) with a 20 5 rest

between each ramp loading without documenting a significant increase in GAG content

34

of the cartilage. In contrast to these previous studies, a significant increase in tissue PG
content was noted at 7 and 14 days following 10 cycles of 0.2 Hz sinusoidal loading at
0.5 MPa followed by 3600 s of unloading in the current study. These data suggest that PG
synthesis in these in vitro experiments is dependent on the intensity, fi'equency and

duration of intermittent cyclic loading.

Fewer studies have investigated alterations in the collagen network following in vitro
cyclic loading of articular cartilage. In Thibault et al. (2002) cyclic loading resulted in a
statistically significant rise in the tissue content of denatured collagen immediately after
the loading period. This collagen was then released to the culture media during a 10 day
post-loading period. In the current study there was a statistically significant decrease in
the tissue content of collagen at 7 days, and that remained at the same level for the 21 day

duration of cyclic loading.

The above changes in the tissue contents of PG and collagen were found to temporally
parallel with the documented alterations in mechanical properties of the explants. More
specifically, the increase in tissue PGs at 7 and 14 days paralleled with an increase in
matrix modulus, Em, and a decrease in tissue permeability K. These results are supported
by a previous study, in another laboratory, that showed enzymatic depletion of PGs in
bovine cartilage results in a parallel decrease in the matrix modulus and an increase in
permeability from a fibril reinforced poroelastic model of cartilage, based on unconfined
relaxation tests of the tissue (Korhonen et al. 2003). Additionally, in the Korhonen et al.

study enzymatic depletion of collagen generated a corresponding decrease in fibril

35

modulus, Ef. A significant decrease in E and increase in tissue permeability was noted
early after cyclic loading in the Thibault et al. study, where they suggested correlation
with an increased content of denatured collagen in the loaded explants. In the current
study there was also a significant decrease in total collagen content at 7 to 21 days that

paralleled with a decrease in tissue Ef.

A dramatic increase in tissue damage to 25 MPa of unconfined compression was noted
after 21 days of cyclic loading in the current study. The effect also involved a dramatic
decrease in the tissue matrix modulus Em, an increase in tissue permeability, and a
decrease in tissue PG content. This may be explained by an increase in the production of
degenerative enzymes, such as matrix metalloproteinase-3 (MMP-3), which is noted to
occur following extended cyclic loading of chondral explants (Lin et al. 2004). This
referenced study documented a significant increase in MMP-3 after 24 hr of cyclic
loading at 1 or 5 MPa. The temporal difference in the previous and current studies could
be due to the frequency and magnitude of the in vitro cyclic loading on the tissue

explants.

In the current study it was also interesting that while the collagen content and fiber
modulus Er of the explants significantly decreased after 7 days, the unconfined
compression modulus of the explants was significantly greater than controls, and it more
directly paralleled with the temporal changes in tissue PG content, the matrix modulus E...
and permeability It. An analysis of the model sensitivity to changes in Er, Em and It

(unpublished) suggested that the stiffness of the explant in the current unconfined

36

compression test should depend on the fiber modulus Er as much as E... A potential
explanation for the lack of Ef response in the current study could be the presence of upper
surface friction in the unconfined test. This could minimize the role of collagen fibers
that are primarily oriented radial in the upper tissue layer that also act to constrain the
explant laterally during the unconfined experiment. And yet, previous unconfined
compression relaxation experiments, reported by Korhonen et a1, displayed sensitivity of
their tests to an enzymatic loss of collagen in the chondral explant. Future studies will be
required to understand more precisely if the lack of E; sensitivity in the current
experiments was due to the test protocol (0.5Hz sinusoidal loading or relaxation testing),
friction or a problem in the validity of the fibril reinforced model for these cartilage

explant tests.

As suggested earlier, the long term health of joint cartilage may relate directly to the
extent of matrix and cell damage that occurs during an acute overloading of a tissue. The
current study indicated that mechanical stiffening of the chondral explant by cyclic
preloads increased the tissue content of PG’s and resulted in less strain during unconfined
compression at 25 MPa. This was correlated with a significant increase in cell survival, as
was hypothesized in the study. This result suggested that cell membrane damage may
directly relate to the amount of matrix deformation, as proposed earlier by others (T orzilli
et al. 2006). Additionally, the percentage of cell damage per unit compressive strain was
also significantly decreased for up to 14 days of cyclic loading. One explanation for this
result may relate to an increase in PG’s surrounding individual cells in the pericellular

matrix as a result of cyclic preloading. This effect has been docmnented previously

37

following low intensity, cyclic loading (Parkkinen et al. 1992; Quinn et al. 1998). Such
deposition of PG’s concentrated in or near the pericellular matrix of chondrocytes may
significantly alter (increase) the local stiffness around cells to help protect them from
damaging distortions during severe levels of unconfined compression, such as that

imposed on the chondral explants in the current study (Guilak et al. 1999).

This study, as understood by the authors, is the first to investigate the role of in vitro
cyclic loading on the response of a chondral explant to a potentially damaging, acute
overload. In the long-term these types of studies may help understand the role of
biologic-based preconditioning of articular cartilage for in vitro, or even in vivo, studies
of blunt force trauma to a joint. The current data would suggest both a positive and a
negative aspect to the idea of cyclic preconditioning or regular exercise in the prevention
of long-term chronic disease in joints suffering an acute overload, such as that proposed
during acute ligament injury. This in vitro study may suggest that the frequency,
magnitude and duration of the preconditioning may play a role in its potential chondral

protective effects. Clearly, additional in vitro and in vivo studies are wanted.

38

ACKNOWLEDGEMENT

This study was supported by a grant from the Centers for Disease Control and
Prevention, the National Center for Injury Prevention and Control (R49/CE000623). Its
contents are solely the responsibility of the authors and do not necessarily represent the
official views of the Centers for Disease Control and Prevention. The authors would like
to thank Mr. Clifford Beckett for technical support of the cartilage exerciser, and Marc

Schlaud for assistance with the biochemical assays.

39

REFERENCES

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poloxamer P188 reduces DNA fragmentation in cells from bovine chondral explants

exposed to injurious unconfined compression. Biomechan Model Mechanobiol 5:133-
] 39.

Bank R, Krikken M, Beekrnan B, et a1. 1997. A simplified measurement of degraded
collagen in tissues: Application in healthy, fibrillated and osteoarthritic cartilage.
Matrix Biol 16:233-243.

Brown S, Worsfold M, Christopher S. 2001. Microplate assay for the measurement of
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Daniel DM, Stone ML, Dobson BE, et a1. 1994. Fate of the ACL injured patient: A
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Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC. 2001. The extent of matrix damage
and chondrocyte death in mechanically traumatized articular cartilage explants
depends on rate of loading. J Orthop Res 19:779-784.

Ewers BJ, WeaVer BT, Sevensma ET, Haut RC. 2002. Chronic changes in rabbit retro-
patellar cartilage and subchondral bone after blunt impact loading of the
patellofemoral joint. J Orthop Res 20:545-550.

Fang C, Johnson D, Leslie MP, et al. 2001. Tissue distribution and measurement of
cartilage oligomeric matrix protein in patients with magnetic resonance imaging-
detected bone bruises after acute anterior cruciate ligament tears. J Orthop Res
19:634-641.

Felson DT, Lawrence RC, Dieppe PA, et al. 2000. Osteoarthritis: new insights. Part I:
The disease and its risk factors. Ann Intern Med 133:637-639.

Guilak F, Jones WR, Ting-Beall HP, Lee GM. 1999. The deformation behavior and
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7:59-70.

Johnson DL, Urban WP, Caborn DNM, et al. 1998. Articular cartilage changes seen with
magnetic resonance imaging-detected bone bruises associated with acute anterior
cruciate ligament rupture. Am J Sports Med 26:409-414.

Korhonen RK, Laasanen MS, Toyras J, et al. 2003. F ibril reinforced poroelastic model
predicts specifically mechanical behavior of normal, proteoglycan depleted and
collagen degraded articular cartilage. J Biomech 36:1373-1379.

Lin PM, Chen CTC, Torzilli PA. 2004. Increased stromelysin-l (MMP-3), proteoglycan
degradation (3B3- and 7D4) and collagen damage in cyclically load-injured articular
cartilage. Osteoarthritis Cartilage 12:485-496.

40

Morel V, Mercay A, Quinn TM. 2005. Prestrain decreases cartilage susceptibility to
injury by ramp compression in vitro. Osteoarthritis Cartilage 13:964-970.

Myklebust G, Bahr R. 2005. Return to play guidelines after anterior cruciate ligament
surgery. Br J Sports Med 39:127-131.

Parkkinen JJ, Lamrrri MJ, Helminen HJ, Tammi M. 1992. Local stimulation of
proteoglycan synthesis in articular cartilage explants by dynamic compression in
vitro. J Orthop Res 10:610-620.

Phillips DM, Haut RC. 2004. The use of a non-ionic surfactant (P188) to save
chondrocytes fi'om necrosis following impact loading of chondral explants. J Orthop
Res 22:1135-1142.

Quinn TM, Allen RG, Schalet BJ, et al. 2001. Matrix and cell injury due to sub-impact
loading of adult bovine articular cartilage explants: effects of strain rate and peak
stress. J Orthop Res 19:242-249.

Quinn TM, Grodzinsky AJ, Buschmann MD, et al. 1998. Mechanical compression alters
proteoglycan deposition and matrix deformation around individual cells in cartilage
explants. J Cell Sci 111:573-583.

Rundell SA, Baars DC, Phillips DM, Haut RC. 2005. The limitation of acute necrosis in
retro-patellar cartilage after a severe blunt impact to the in vivo rabbit patello-femoral
joint. J Orthop Res 23:1363-1369.

Saadat E, Lan H, Majurndar S, et al. 2006. Long-term cyclical in vivo loading increases
cartilage proteoglycan content in a spatially specific manner: an infrared
nricrospectroscopic imaging and polarized light microscopy study. Arthritis Res Ther
8:R147.

Sah RLY, Kim YJ, Doong JYH, et al. 1989. Biosynthetic response of cartilage explants
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Snearly WN, Kaplan PA, Dussault RG. 1996. Lateral-compartment bone confusions in
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Soulhat J, Bushmann MD, Shirazi-Adl A. 1999. A fibril reinforced biphasic model of
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Steinmeyer J, Ackerrnann B, Raiss RX. 1997. Intermittent cyclic loading of cartilage
explants modulates fibronectin metabolism. Osteoarthritis Cartilage 5:331-341.

Steinmeyer J, Knue S, Raiss RX, Pelzer I. 1999. Effects of intermittently applied cyclic
loading on proteoglycan metabolism and swelling behaviour of articular cartilage
explants. Osteoarthritis Cartilage 7: 155-164.

41

Thibault M, Poole AR, Buschmann MD. 2002. Cyclic compression of cartilage/bone
explants in vitro leads to physical weakening, mechanical breakdown of collagen and
release of matrix fi'agrnents. J Orthop Res 20:1265-1273.

Torzilli PA, Deng XH, Ramcharan M. 2006. Effect of compressive strain on cell viability
in statically loaded articular cartilage. Biomechan Model in Mechanobiol 5: 123-132.

Torzilli PA, Grigiene R, Borelli J Jr, Helfet DL. 1999. Effect of impact load on articular
cartilage: cell metabolism and viability, and matrix water content. J Biomech Eng
121:433-441.

Vellet AD, Marks PH, Fowler PJ, Munro TG. 1991. Occult posttraumatic osteochondral
lesions of the knee: Prevalence, classification, and short-term sequelae evaluated with
MR Imaging. Radiology 178: 271-276.

Wei F, Golenberg N, Kepich E, Haut R. 2007. In vitro exercise affects the response of
articular cartilage to blunt impact loading. Trans Orthop Res Soc 32:0662.

Wei F, Golenberg N, Kepich E, Haut R. 2007. Glucosamine and chondroitin sulfate
affect the response of exercised articular cartilage to blunt impact loading. Trans Am
Soc Biomech 31:P4-21.

42

CHAPTER TWO
HIGH LEVELS OF GLUCOSAMINE—CHONDROITIN SULFATE CAN ALTER THE
CYCLIC PRELOAD AND ACUTE OVERLOAD RESPONSES OF CHONDRAL
EXPLANTS

ABSTRACT

A recent study by our laboratory has shown that 14 days of low intensity, intermittent
cyclic preloading of chondral explants has a positive effect on the tissue by elevating the
concentration of proteoglycans (PGs) to cause a mechanical stiffening of the explants
prior to an acute overload and limit the extent of tissue damage. And yet, longer term
loading to 21 days results in tissue degradation prior to the acute traumatic event and
excessive damage fiom an acute overload. Previous studies, by others, have shown that
bathing chondral explants in a supplement of glucosamine-chondroitin sulfate (glcN-CS)
can up-regulate the synthesis of tissue PGs, particularly in stressed tissue, and the
supplement serves as an anti-inflammatory agent. The hypothesis of the current study
was the supplementation of culture media with a high concentration of glcN-CS would
up-regulate the production of tissue PG and limit or mitigate long term degradation of
chondral explants under cyclic preloading and limit tissue damage in an acute overload.
The current study showed that, in the presence of supplement, cyclic preloading
significantly increased tissue PG content and matrix modulus by approximately 65% and
300%, respectively, at 21 days, resulting in a reduction of matrix damage and cell death
during an acute overload event. These experimental data may help explain the biological
action of high concentration of this culture supplement and its effect on the mechanical

properties of this in vitro tissue model.

43

INTRODUCTION

Traumatic joint injury by blunt mechanical forces has been demonstrated to be a risk
factor for the development of osteoarthritis (OA) (Wilder et al. 2002). In vitro studies of
impact loading (Torzilli et al. 1999; Quinn et al. 2001) of cartilage have shown cell death
and matrix damage. Cyclic loading of articular cartilage has also shown chondrocyte
death (Lin et al. 2004; Sauerland et al. 2003) and mechanical weakening of collagen
(Thibault et al. 2002) which are dependent on the intensity and frequency of loading. In
contrast, other in vitro and in vivo studies have shown positive aspects of low intensity,
intermittent compressive loading of cartilage, such as increased biosynthesis of
proteoglycans (PGs) (Sah et al. 1989; Parkkinen et al. 1992; Saadat et al. 2006). Changes
in the PG and collagen contents of cartilage have been shown to significantly alter the
mechanical properties of this tissue (Korhonen et a1. 2003). Thus, it is reasonable to
assume that cyclic preloading of cartilage can alter the mechanical properties of the tissue
and help determine the extent of matrix damage and cell death that will occur due to the

blunt force overloading of a joint.

Recent studies in our laboratory have shown that low intensity, intermittent cyclic
preloading of chondral explants prior to an acute overload yields an increase in the
synthesis of tissue PGs, resulting in a less severe traumatic insult to an acute overload
event. Interestingly, between 14 and 21 days of cyclic preloading the chondral explants
experience a significant loss of tissue PGs which leads to a dramatic loss in mechanical
properties. An acute overloading of the explanted tissue then yields matrix damage and

cell death significantly greater than non-preloaded controls. The study suggests that low

intensity, intermittent cyclic loading can increase the biosynthesis of tissue PG up to a
point, and thereafter there seems to be a yet unknown biological event that causes tissue
degeneration and compromises the explanted tissue in an acute overload event, such as

that which may occur during an acute knee ligament rupture (Fang et al. 2001).

Previous in vitro studies, by others, have documented fi'equency and intensity dependent
responses suggesting that cyclic compression of articular cartilage modulates the
metabolism and turnover of PGs and collagen in a complex and multifactorial manner.
For example, cyclic intensities of 0.5 MPa in 4 3 intervals for 1.5 hrs stimulates PG
production, while at 1.0 MPa PG production by tissue cells is down-regulated (Parkkinen
et a1. 1992). Another study at 0.5 MPa documents an increase in endogenous PGs at low
and high frequencies of cyclic loading over 6 days, without significant changes in the
total PG content of the tissue (Sauerland et al. 2003). Similarly, an early study at a
frequency of 0.5 Hz and either 0.1, 0.5 or 1 MPa indicates maximum PG synthesis at 3
days and a dramatic release of endogenous PGs and excessive cell death at 6 days
(Steinmeyer et al. 1999). While a more recent study at 0.5 Hz for intensities of l and 5
MPa over 24 hrs shows a significant loss of tissue PG and cell death with production of
the matrix metalloproteinase3 (MMP-3) adjacent to damaged collagen fibers (Lin et al.
2004). Similarly, under 0.5 MPa of 1 Hz cyclic loading significant increases in the
synthesis of MMP-2 and -9 have been indicated after 3 hrs (Blain et al. 2001).
Furthermore, previous in vivo studies have shown that moderate intensities of cyclic
loading can have a positive effect on the mechanical properties of joints by elevating the

concentration of PGs in articular cartilage (Helminen et al. 2000). Saadat et al. (2006)

45

documented that 80 cumulative hours of physiological joint loading led to a 46% increase
of PG synthesis. On the other hand, other studies have shown that a more strenuous
regimen of exercise can also have a deleterious effect on joint cartilage, resulting in a
significant modulus decrease of 13-14% and a reduction of PG content (Helminen et al.
1992 and 2000). Sirrrilar effects have been documented for in vitro experiments using
explanted cartilage wherein low or moderate levels of intermittent cyclic loading has
positive effects on the mechanical properties of explants (Parkkinen et al. 1992, Sah et al.
1989; Steinmeyer et al. 1999), while other studies with more severe levels of cyclic
loading have shown detrimental effects on cartilage explants, with an increase of cell

death and matrix damage (Thibault et al. 2002).

0A is a manifestation of an imbalanced synthesis of articular cartilage matrix and the
associated growth factors. Understanding the changes in collagen and PG content of
cartilage due to mechanical forces is necessary for progress in treating join disorders,
such as OA. The use of potentially chondro-protective agents such as glucosamine (glcN)
and chondroitin sulfate (CS) has been explored to medicate OA. Previous studies by
others have shown that bathing cartilage explants in a supplement of glcN and CS can up-
regulate the synthesis of tissue PGs, and particularly in stressed tissue (Lippiello 2003).
Recent studies have also shown that this supplement limits tissue degradation in the
presence of inflammatory enzymes in an in vitro setting (Derfoul et al. 2007). However,
the role of P63 synthesized fi'om intermittently loaded cartilage in response to an acute
compressive overloading of articular cartilage has not been elucidated. While clinical

trials of oral glcN in OA patients support the contention that it is a disease-modifying

46

agent (Richy et al. 2003), other studies find no clinical effect (Hughes and Carr 2002). In
contrast, more consistent results have been documented in more environmentally
controlled tissue and cell culture experiments. This combination has, indeed, been shown
to firnction as a “biological response modifier” that can boost the natural protective
responses of tissue under adverse environmental conditions, such as mechanical
compression (Lippiello 2003). Using human OA articular cartilage, treatments with glcN
sulfate have shown increased aggrecan mRNA levels, as well as a decrease in the activity
of MMP-3 (Dodge and J irnenez 2003). Other in vitro studies have shown that incubation
of chondral explants in glcN after exposure of the tissue to the proinflammatory cytokine

interleukin-l prevents the increase in PG release and MMP activity (F enton et al. 2000).

Since previously data showed that PG content directly paralleled to stiffness of cartilage,
we wanted to experimentally increase tissue PG content to strengthen the cartilage,
making them inhibit the degradation of the tissue after long term of cyclic compression
and reduce the susceptibility of cartilage to a severe level of mechanical injury, such as
an acute overload. The objective of the current study was to use this supplement in an
experimental setting to alter PG synthesis during cyclic loading of chondral explants and
document its effect on the response of explants to an acute overload. Thus, the
hypotheses of the current study were that, during low intensity, intermittent cyclic
loading, a high concentration of glcN-CS would up-regulate the production of PGs in an
explanted tissue to alter its mechanical properties in relatively short-term experiments. By
increasing the mechanical stiffiress in this fashion, there would be a reduction in the

extent of matrix damage and cell death following an acute overload on the explant.

47

Secondly, because of the documented inhibition effects of glcN on aggrecanase and
MMPs, supplementation of the culture media with this product during cyclic preloading

will limit or even mitigate the degeneration effect noted to occur between 14 and 21 days

in this in vitro model.

48

METHODS

DISSECTION, TISSUE CULTURE AND GROUPING OF EXPLANTS

Bovine forelegs from mature animals (18-24 months of age) were obtained flour a local
abattoir within two hours of slaughter. The legs were skinned and rinsed with water prior
to exposing the metacarpal joint under a laminar flow hood. Biopsy punches were used to
make one hundred and forty-four 6 mm diameter chondral explants from the lower
metacarpal surface of the limbs. Each explant was separated from the underlying bone
with a scalpel. All specimens were washed three times in Dulbecco’s Modified Eagle
Media: F 12 (DMEM: F12) (Gibco, USA), and placed in the media added with 10% fetal
bovine serum, additional amino acids and antibiotics (penicillin 100 U/ml, streptomycin l
rig/ml, amphotericin B 0.25 [lg/ml) in 24-well plates. The explants were randomly
assigned to three groups: control (n=72), cyclic loading (n=36) and cyclic loading with
supplement (n=36). Each group was subjected to 7, 14 and 21 days of testing periods
(Figure l). The concentration of supplement in the media with was 500 [lg/ml glcN
(FCHG49®) and 250 ug/ml CS (TRH122®) to try to maximize the hypothesized effect,
based on previous studies (Rundell 2005). The media was replaced every 2 days
throughout the study and harvested and stored frozen at -80°C. A mechanical loading
device, “cartilage exerciser” (described below), was used to cyclically load the explants
inside of a humidity-controlled incubator (37°C, 5% C02, 95% humidity). The osmolarity
of the media was 300mosM (Osmete 2, Precision Systems), and the pH was 7.4.
Physiological and metabolic stability of this explant system has been demonstrated

previously (Phillips and Haut 2004; Baars et al. 2006; Wei et al. 2007a and 2007b).

49

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50

INDENTATION TESTING OF EXPLANTS

Prior to and after cyclic loading, each explant was subjected to mechanical
characterization using an indentation stress relaxation test (Figure 1), followed by an
equilibration of 30 min in the media. Cartilage explant thickness was measured twice at
perpendicular orientations across the center of the explant using a digital vernier caliper
with a resolution of 0.01 mm (Steinmeyer et al. 1997 and 1999). The two thickness
values were averaged. 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 4.3 mm diameter hole
was placed on top of the explant to secure the edges and help resist curling of the
explants. The explant and fixture were then submerged into a room-temperature
phosphate buffered solution (pH 7.2). A 2.39 mm diameter spherical, non-porous probe
was lowered into the cartilage until a preload of 0.03 N was attained and held for 60 s.
The indenter was then pressed into the cartilage 25% of the total thickness in 2 s and
maintained for 600 s while resistive loads of relaxation were measured, amplified and
collected at 1,000 Hz for the first second and 20 Hz thereafter. The stress relaxation
curves were obtained and fitted with a fibril-reinforced biphasic FE model (Soulhat et a1.
1999) for an assumed Poisson’s ratio of 0.25 and void ratio of 5.67. Cartilage matrix
modulus (Em), fiber modulus (Br) and tissue permeability (K) were evaluated with a
custom-written Gauss-Newton constrained nonlinear least square minimization

procedure.

CYCLIC LOADING OF EXPLANTS

51

The “cartilage exerciser” consisted of 12 loading chambers simultaneously powered by
air. The control explants rested in the same “cartilage exerciser” as the cyclic loaded
ones, but without cyclic loading on them. Pneumatic cylinders forced the pistons
downward to apply a compressive load to the specimens through 14.6 mm diameter non-
porous Teflon® platens. 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. Intermittently applied,
uniaxial cyclic loading was introduced by using a 0.2 Hz sinusoidal waveform with a
peak stress of 0.5 MPa. The cyclic loads were applied for 10 cycles followed by a load-

fiee period lasting 3600 s.

UNCONFINED COMPRESSION TESTING OF EXPLANTS

After cyclic loading, all explants (including the control) were taken to 707 N (~25 MPa),
following a 5 N preload, in unconfined compression between two polished stainless steel
plates. A 0.5 Hz (1 s time to peak) haversine loading protocol was programmed for
application onto the explants in a servo-controlled hydraulic testing machine (Instron,
model 1331, Canton, MA). Immediately after this acute loading, the explants were

returned to culture for 24 hours before cell viability tests were performed.

MATRIX DAMAGE AND CELL VIABILITY TESTING OF EXPLANT S
For matrix damage analysis, the surfaces of all impacted explants were wiped with India
ink and immediately photographed at 25x under a dissection microscope (Wild M5A,

Wild Heerbrugg Ltd, Switzerland) to determine the total length of explant surface

52

fissures (Baars et al. 2006). The total fissure length was measured with digital imaging
software (Sigma Scan, SPSS Inc., Chicago, IL, USA). One observer (F .W.) digitally

recorded the length of the surface fissures in each photograph.

After the measurement of fissure length, all explants were washed three times in
DMEM:F 12 and used to determine cell viability. Each explant was cut through its entire
thickness with a specialized cutting tool with parallel blades spaced 0.5 mm (Philips and
Haut 2004; Ewers et al. 2001). These thin slices were then stained with calcein AM and
ethidium homodimer (EthD-l ), according to the manufacturer’s specifications
(Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene, OR, USA). Three slices were
viewed across approximately 2 mm near the center of each explant in a fluorescence
microscope (Leica DM LB, Leica Mikroskopie und Systeme GmgH, Wetlzar, Germany)
and photographed using a digital camera (Spot Digital Camera, Diagnostic Instruments
Inc.). An average percent live cell count was performed based on the images from each
explant, using image analysis software (Sigma Scan, SPSS Inc., Chicago, IL, USA).
Explant maximum strains from the unconfined compression tests were used to calculate

the percentage of cell death per unit strain.

BIOCHEMICAL ASSAYS

Seventy-two cartilage explants (36 control, 18 cyclic loading and 18 cyclic loading with
supplement) were digested overnight at 60°C with papain digestion solution at a pH of
6.0. Papain digested cartilage explants and culture media were independently DMB
assayed for sulfated PGs by the reaction with 1,9-dimethylmethylene blue dye solution in

polystyrene 96 well plates and quantitated with spectrophotometry at a wavelength of 530

53

nm using a Bio Tek microplate reader. Chondroitin sulfate A sodium salt from bovine
trachea (Sigma-ALDRICH GmbH, Steinheim, Germany) was used as the standard
(Steinmeyer et al. 1999). The other seventy-two explants were used for hydroxyproline
(HYP) measurement. After a guanidine HCl extraction to remove PG, tissue samples
were acid-hydrolyzed in a 70°C water bath for 24 hours and nricroplate assayed to

determine the total HYP content in the tissue (Brown et al. 2001; Bank et al. 1997).

STATISTICAL ANALYSIS

The data obtained from post-cyclic loaded explants were normalized by the pro-cyclic
loaded values and subsequently analyzed with two-way repeated measures ANOVA and
Student-Neuman-Keuls post hoc tests to determine differences between groups. PG and
HYP biosynthesis was normalized by tissue wet weight and reported in pg PG and pg
HYP per mg wet weight, respectively. Statistical significance was indicated for P<0.05.

All experimental data are reported as mean 3: standard deviation.

54

RESULTS

INDENTATION TESTING

All explants underwent indentation testing for mechanical characterization before and
after cyclic loading (Figure 1). Matrix modulus, fiber modulus and permeability of the
explants were compared in terms of “% of before cyclic loading” (Figure 2). Cyclic
loading with the supplement significantly increased the tissue matrix modulus by
approximately 300% up to 21 days (260.2 :I: 47.0%, 300.9 :I: 80.3% and 307.9 :I: 40.6%
higher after 7, 14 and 21 days cyclic loading, respectively, than before). Cyclic loading
without supplement was also different than control, however, the matrix modulus was
decreased by 58.4 :I: 19.4% after 21 days (Figure 2A). Cyclic loading with and without
supplement significantly decreased the tissue fiber modulus by approximately 40% and
60%, respectively, at all time points (Figure ZB). The fiber modulus was significantly
higher with versus without the supplement. Up to 14 days of cyclic loading the
permeability of the explants without supplement significantly decreased (71.1 :L- 8.5% and
31.0 :I: 7.7% of before cyclic loading in 7 and 14 days groups, respectively), and then it
increased dramatically at 21 days (143.0 :I: 9.9% of before cyclic loading). In contrast,
cyclic loading with supplement decreased the tissue permeability significantly at all time
points (46.7 :h 17.4%, 34.9 :I: 16.4% and 35.4 :t 5.2% of before cyclic loading in 7, 14 and
21 days groups, respectively) (Figure 2C). For all three parameters the controls (non-

cyclic loaded) remained at the same level (approximately 100%) for all time points.

55

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approximately 3 times than control (A), and helped inhibit the degradation of the explants
from longer time (21 days) cyclic loading (A and C). Cartilage fiber modulus, however,
decreased approximately 40% and 60% by cyclic loading with and without supplement,
respectively (B). Statistically significant differences were observed fi'om control (1), and
from cyclic loading (§).

56

 

 

 

 

 

 

 

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BIOCHEMICAL ASSAYS

In the presence of supplement, cyclic loading of the explants significantly stimulated
tissue PG synthesis, resulting in an increase of tissue PGs by approximately 65% of
control (fiom approximately 30 pg PG/mg tissue wet weight to approximately 50 pg
PG/mg tissue wet weight). Cyclic loading without supplement also increased PG content
up to the supplemented level by 14 days, however, a significant loss (approximately
30%) in PGs was noted after 21 days (from 34.5 :I: 5.3 to 24.3 :I: 6.0 pg PG/mg tissue wet
weight) (Figure 3A). Cyclic loading without supplement also resulted in an
approximately 20% decrease in tissue HYP content (from 25 pg HYP/mg tissue wet
weight to 20 pg HYP/mg tissue wet weight, approximately). In the presence of

supplement, however, cyclic loading at 14 and 21 days decreased the tissue HYP content

57

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Figure 17. PG synthesis was stimulated by the presence of supplement at all time points,
while without the supplement the PG content decreased at 21 days (A). Tissue HYP
content decreased approximately 10% and 20% by cyclic loading with and without
supplement, respectively (B). Statistically significant differences were observed from
control (1), and from cyclic loading (§).

58

MATRD( DAMAGE AND CELL VIABILITY

Cyclic loading decreased explant surface fissure length induced by the 25 MPa overload
by approximately 37% (from 91.4 :I: 25.7 mm to 56.8 :I: 18.9 m) up to 14 days (Figure
5A). Furthermore, cyclic loading saved nearly 22% more cartilage cells than control up to
14 days (Figure 5B). In contrast, with a longer duration of cyclic loading (21 days)
superficial fissures and associated cell death (Figure 4C-D) increased significantly
compared with controls (fissure length increased by 32% and percentage of live cells
decreased by 15%, approximately) (Figure 5A-B). In the presence of supplement,
however, cyclic loading resulted in a significant decrease in fissure length and more live
cells after overloading than control at all time points (Figure 4E-F and Figure 5A-B). Cell
death per unit strain was significantly decreased versus controls up to 14 days of cyclic
loading (decreasing by approximately 30% and 65% at day 7 and 14, respectively). In
contrast, after 21 days of cyclic loading the cell death per unit strain was not different
from control. In the presence of supplement, however, cyclic loading significantly
decreased the cell death per unit strain at all time points (decreasing by approximately

72%, 68% and 73% for 7, 14 and 21 days of cyclic loading, respectively) (Figure 5C).

59

 

O 1 mm ' O 1 mm

Figure 18. Typical images of cartilage explants stained for cell viability. Live cells
stained green and dead cells stained red. Special filter was used to see only the dead cells
for counting purpose (BDF). The cracks on the surface were due to the 25 MPa
unconfined compression on the explants. All images showed are from explants after 21
days of testing period. AB-control; CD—cyclic loaded; EF-cyclic loaded with supplement.

60

>

 

 

 

 

 

 

 

 

 

 

 

 

 

200 -
-D-Control
A —°-CYC-loadlno t
E - '6- Cyc. loading wl suppl.
E
-1... l
E» /
2 /
2
/
a: \ /
i \xr/
o _ ......................
z 50 T g T
is * rs
r e l r l
O 7 14 21
Duration of cyclic loading (days)
B
120 - -D—Control
—-o—Cye.loadlng
- iA- Cyc. loading w/ suppl.
g 100 ~
8 *5 r
g l ______ #5
g m- _,-—= ----------- I
§ 1?- t \
JL... \
O 60 _. '1' \
2 tr \‘9
\
40 - *I
O 20 - 7 14 21
0 ‘ Duration of cyclic loading (days)

 

Figure 19. Cyclic loading initially decreased explant surface fissure length from the 25
MPa overload and saved approximately 20% more cartilage cells than control up to 14
days, but with a longer duration (21 days) of cyclic loading superficial fissures and
associated cell death increased significantly (AB). In the presence of supplement,
however, cyclic loading inhibited the matrix further damage and cell death at 21 days
(AB), and significantly decreased the cell death per unit strain at all time points (C).
Statistically significant differences were observed from control (I), and from cyclic
loading (§).

61

 

 

 

 

 

 

2 5 _ +CODII'OI
' —0—Cyc. loading
- A- Cyc.loadflgw/suppl.
r:
.- 2 g
g V
t
g /
:5 1.5 — IL /
m /
'8 1; /
= 1 _ \ \ /
s \ \ /
“g \ \ /
0.5 ‘ T ----------- $ ........... e
rs r *5
7 14 21
Duration of cyclic loading (days)

Figure 19. (cont’d)

62

DISCUSSION

The hypotheses of the current study were that a high concentration of glcN-CS added to
culture media during low intensity, intermittent cyclic loading of chondral explants will
increase the production of tissue PGs versus cyclic loading in media alone. With the
increase in tissue PGs the study further hypothesized that there would be a significant
increase in explant stiffiress, resulting in less tissue and cell damage following a single
acute mechanical overload. Additionally, the current investigation hypothesized that this
media supplement would be effective in limiting, or potentially mitigating, a
degeneration in mechanical properties of these explants between 14 and 21 days of cyclic
loading. Each of the above hypotheses was supported by experimental data presented in

the current study.

Mechanical forces in the chondrocyte environment can affect the cellular processes
involved in the biosynthesis of extracellular matrix. In turn, the biomechanical properties
of cartilage depend on its collagen and PG content (Saadat et al. 2006). The results of the
current study showed that supplementation of bovine culture media with glcN-CS did
experimentally stimulate tissue PG synthesis during low intensity, intermittent cyclic
loading of explants and help stiffen the cartilage to inhibit its degradation between 14 and
21 days, making them more resistant to matrix damage and cell death under 25 MPa of
unconfined compression. The study indicated that an alteration (increase) of tissue PG
content significantly limited the extent of blunt overload trauma to cartilage and
dramatically reduced the cartilage susceptibility to damage in a single, severe level of

acute overload.

63

Various aspects of the study parallel findings of other in vitro research investigations on
the biological and mechanical response modifications documented with the
supplementation of culture media with glcN or glcN-CS. Most notably the study
supported one investigation suggesting that these compounds function as biological
response modifiers (BRMs), which can boost responses of cartilage to environmental
stresses, such as mechanical compression (Lippiello et al. 2003). This referenced study
shows a significant increase in tissue glycosaminoglycans (GAGs) with supplemented
(250 pg/ml Cosarrrin® DS) media versus without, following 24 hrs of unconfined
compression at 0.5 MPa using aged bovine cartilage. The notion was presented that
stressed chondrocytes utilize the compounds more efficiently than non-stressed cells.
This effect was supported in the current study which showed a statistically significant
increase in the production of tissue PGs at 7 days in supplemented versus non-
supplemented, cyclically loaded explants (F igure 3A). A possible limitation of the current
study, however, was that these data were not normalized per viable chondrocyte.
However, data from this laboratory (unpublished) indicated that 21 days of cyclic loading
does not statistically alter the percentage of live cells in the explants compared to
unstressed controls. Additionally, cyclic compression did not statistically alter the
specimen thickness, suggesting no change in tissue wet weight over time (Wei et al.
2007a). Interestingly at 14 days the PG content of supplemented versus non-
supplemented, cyclic loaded specimens was not statistically different. This may be due to
some yet unexplained large variance in the non-supplemented, cyclic loading data at this

time point. Furthermore, in the absence of supplement our data indicated that, compared

to the controls, moderate cyclic loading of cartilage explants up to 14 days stimulated PG
synthesis in the tissue, resulting in a significant increase of cartilage matrix stiffness, a
decrease in tissue permeability, and a limitation of superficial cracks and chondrocyte
death resulting from an acute overload. This may be due to less strain (data not shown)
observed under 25 MPa of unconfined compression. Correspondingly, an increase of
compressive strain explained a significant increase in tissue damage from overloading
after 21 days of cyclic loading. In the presence of supplement, however, the high levels of
tissue PG were retained after 21 days of cyclic loading, resulting in maintenance of tissue
stiffness and helped limit matrix damage following traumatic overload. We think this

might be due to the anti-inflammatory properties of the supplement (Derfoul et al. 2007).

Previous studies, by others, have established direct and indirect correlations between the
PG content of cartilage and its matrix modulus and permeability, respectively (Korhonen
et al. 2003). This may help explain the data in the current study which showed a higher
PG content and matrix modulus at 7 and 14 days for supplemented versus non-
supplemented and control specimens. Additionally, the current study indicated a dramatic
drop in fiber modulus versus unstressed controls at 7 days that persisted at 14 and 21
days. Korhonen et al. show that removal of collagen fiom cartilage lowers the fiber
modulus. This effect paralleled with a decreased content of explant collagen versus
unstressed controls at each time point in the current study. Additionally, at 14 and 21
days the collagen content of supplemented explants statistically exceeded that of the non-
supplemented, cyclic loaded group. In the current study, while a statistical reduction in

fiber modulus versus controls was also documented for supplemented explants, the

65

modulus was generally higher than non-supplemented, cyclic loaded explants. The effect,
however small, may be due to an increase in production of collagen by the chondrocytes
in the supplemented, stressed groups (V arghese et al. 2007; Lippiello et al. 2003). More
likely, the higher content of collagen may reflect the fact that the supplemented group
was stiffened because of a rise in PG content and yielded less matrix damage (Thibault et

al. 2002).

While the previous study of Korhonen et a1. established correlations between the contents
of PG and collagen and alterations in matrix modulus and fiber modulus, as well as
permeability, the stiffness of these chondral explants under unconfined compression was
mathematically sensitive to a combination of matrix modulus, fiber modulus and tissue
permeability (unpublished data). At 7 days, the content of tissue PGs for supplemented
explants was greater than that of non-supplemented, cyclic loaded explants, but this lead
to not only an increase in matrix modulus and a decreased tissue permeability, but also an
increase in fiber modulus of the explant. As a result the unconfined compression resulted
in less matrix damage and cell death in supplemented versus non-supplemented explants.
At 14 days, in contrast, more collagen remained in supplemented than non-supplemented
specimens. While the PG content between the 2 groups was not different, the matrix
modulus of supplemented specimens was statistically greater and the fiber modulus was
slightly higher (not statistically) than in the non-supplemented group. The results from
the current study clearly showed that high levels of glcN-CS supplementation between 14
and 21 days of cyclic preload helped inhibit cartilage degradation and mitigate matrix

damage and cell death in an acute overload of the tissue. At 14 days, however, no

66

statistically significant differences were observed between supplemented and non-
supplemented, cyclically loaded explants in terms of fiber modulus, tissue permeability,
PG content, fissure length, percent of live cells and cell death per unit strain (Figure 2-4).
Therefore, we conclude that while the effect of glcN-CS supplementation during cyclic
preload was obvious at 7 days, it is very low at 14 days time point. However, when the
cyclic preload continued long enough to damage the cartilage (somewhere between 14
and 21 days), the supplement, frmctioning as BRM, affected the tissue responses to the
cyclic preload and the later acute overload. In another word, cyclic preloading of
cartilage had positive effect on the tissue up to a certain point, and before that point,
supplementation of glcN-CS would intensify the effect and strengthen the cartilage in
response to a traumatic event. When cyclic preload started to degrade the cartilage,
however, the high levels of supplement would protect the cartilage from being damaged
and alter the response of the tissue to the cyclic preload and the acute overload in terms
of inhibiting the degeneration of cartilage. These data show that a one-to-one correlation
between PG and collagen content of the explant and matrix modulus and fiber modulus,
respectively, may not exactly exist. It would seem reasonable that these tissue

constitution-mechanical property correlations might be more complex.

The most dramatic effect of media supplementation in the current study was the reduction
in matrix damage and cell death that occurred between 14 and 21 days with
supplementation. The hypothesis of a previous study (Wei et al. paper in review) was that
the documented degenerative effect in mechanical properties was likely due to the

production of MMPs (possibly MMP-3) as a result of intermittent cyclic compression at

67

0.5 MPa (Lin et al. 2004). The hypothesis of the current study was that media
supplementation would limit or mitigate the effect. The study showed not only a
significant increase in explant PG content with supplementation, but more collagen in the
explants. Again we hypothesize that the increase in tissue collagen likely may be the
result of less damage to this structure during cyclic loading due to a stiffened matrix,
rather that a statistically significant increase in the biosynthesis of new, functional
collagen. However, the mechanism by which supplementation of the culture media with
glcN-CS retained more collagen and PG than cyclic loaded specimens without
supplement is yet unknown and will require further study. And, as suggested in studies by
others that examine the effects of selective and non-selective metalloproteinase inhibitors
on interleukin-l-induced cartilage degradation, the effects of media supplementation on
altering the material properties of cartilage may only be delayed in time (Wilson et al.
2007). Future studies will be needed to determine if the same effects will be seen with
altered frequency, amplitude and with an extended duration of cyclic loading of the

supplemented explants.

The current in vitro study does not directly have utility in answering the question of
whether this supplement will have efficacy in helping to mitigate long term joint disease,
such as post-traumatic OA, resulting from an acute traumatic overload, such as rupture of
a knee ligament (Fang et al. 2001). The major problems with extending these results to a
clinical setting are the short duration of in vitro studies, as well as the elevated
concentration of the supplement. Studies have shown that high doses of glcN-HCI have

detrimental effects on bovine articular cartilage explants cultured in vitro (De Mattei et

68

al. 2002). At concentration of 25 mg/ml glcN a decrease of over 90% in cell viability was
observed in their study. While in our short-term in vitro experiments the dose of
supplement (500 pg/ml glcN and 250 pg/ml CS) was much lower than that documented
above, it is still significantly higher than clinical levels (Persiani et al. 2005). The
concentrations of glcN in serum after oral and intravenous administration range from 1 to
20 pg/ml, while CS concentrations are in the 5-200 pg/ml range (Adebowale et al. 2002;
Du et al. 2004; Volpi 2002). Yet, unpublished studies fi'om this laboratory do show
(Appendix A) some reductions in the cyclic load-induced degradation of a chondral
explant for even a reduced (by 50%) concentration of supplement. Our experimental data
may suggest some long-term utility of the supplement to provide a chondro-protective
effect for joint cartilage suffering an acute overload, such as that which might occur
during knee ligament rupture (Lahm et al. 1998). And yet, it also seems inviting to
propose in vivo studies at clinical concentrations to see if this supplement may have
incremental, positive effects on the already documented effects of physiological levels of
cyclic loading on changes in the mechanical properties of joint cartilage (Saadat et al.
2006). Another potential utility for these types of in vitro studies may be in the area of
tissue-engineered cartilage (Waldrnan et al. 2004; 2006). While recent studies show the
utility of cyclic mechanical loading on engineered cartilage stiffness, supplementation of
the culture media with high concentrations of glcN-CS may show utility in producing
better products (V arghese et al. 2007). As the current study may imply, however, the
intensity, frequency and duration of intermittent cyclic loading may play important roles
in the response of these engineered tissues to media supplementation (Wei et al. paper in

review-b).

69

ACKNOWLEDGEMENT

This study was supported by a grant fiom the Centers for Disease Control and
Prevention, the National Center for Injury Prevention and Control (R49/CE000623). The
nutraceutical was supplied by Nutramax Laboratories, Inc. (Edgewood, MD). The authors
would like to thank Nurit Golenberg for assistance with the mechanical testing, Marc
Schlaud for assistance with the biochemical assays, and Eugene Kepich for building the

computational model.

70

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74

CHAPTER THREE

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK

CONCLUSIONS

In the previous Chapters we have conducted studies where low intensity, intermittent
cyclic preloading was applied to chondral explants prior to an acute unconfined
compression on the tissue. Based on the results of Chapter One, we used a high level of
nutraceutical (glucosamine-chondroitin sulfate, glcN-CS) in an experimental setting to
inhibit the degeneration of cartilage after long duration of cyclic preloading (21 days) and
limit the blunt force trauma to the explanted tissue. The effects of cyclic preload and high

levels of glcN-CS on chondral explants responses to acute overload were documented.

The hypothesis of Chapter One was that low intensity, intermittent cyclic preloading of
chondral explants will up-regulate the production of tissue proteoglycans (PGs), resulting
in an increase of the explant mechanical stiffness that will help limit the extent of matrix
damage and associated chondrocyte death following a single, severe level of blunt force
overloading. The results of Chapter One showed cyclic preloading did mechanically
stiffen the explants, making them more resistant to matrix damage and cell death under
25 MPa of unconfined compression up to 14 days. After 21 days of cyclic preloading,
however, the explants lost stiffness and suffered more extensive damage in the acute,
unconfined compression test than controls without cyclic preloading. These data may
have a direct bearing on the issue of pre-impact conditioning of chondral explants in

laboratory experiments dealing with the short-term consequences of impact trauma (Wei

75

et al. paper in review-a). Additionally, these data may also relate to the role of regular
exercise on the long-term consequences of human joints following a traumatic joint

injury, such as an acute ligament rupture (Lahm et al. 1998).

The objective of Chapter Two was to use a high concentration of glcN-CS (500 pg/ml
glcN and 250 pg/ml CS) in an experimental setting to alter PG synthesis during cyclic
preloading of chondral explants and document its effect on the response of explants to an
acute overload. Thus, the hypotheses of Chapter Two were that a high concentration of
supplement would up-regulate the production of PGs in an explanted tissue to alter its
mechanical properties in relatively short-term experiments. By increasing the mechanical
stiffiress in this fashion, there would be a reduction in the extent of matrix damage and
cell death following an acute overload on the explant. Secondly, because of the
docmnented inhibition effects of glcN on aggrecanase and matrix metalloproteinases
(MMPs), supplementation of the culture media with the product during cyclic preloading
will limit or even mitigate the degeneration effect noted to occur between 14 and 21 days
of the in vitro model in Chapter One. The results of Chapter Two showed that the
supplement of glcN-CS did experimentally stimulate tissue PG synthesis during moderate
cyclic preloading of chondral explants and help stiffen the cartilage to inhibit its
degradation at 21 days, making them more resistant to matrix damage and cell death
under 25 MPa of unconfined compression. The study indicated that an alteration
(increase) of tissue PG content significantly limited the extent of blunt overload trauma to
cartilage and dramatically reduced the cartilage susceptibility to damage in a single,

severe level of acute overload.

76

Since in the short-term in vitro experiments the dose of supplement used in Chapter Two
was much higher than clinical levels (Persiani et al. 2005), more recently, we used a
lower concentration of supplement in the bovine media (250 pg/ml glcN and 125 pg/ml
CS). The studies showed that with long duration of cyclic preloading, a lower
concentration of supplement still inhibited the degeneration of cartilage explants between
14 and 21 days as the high level of supplement did in Chapter Two (Appendix A). These
experimental data, including the data fi'orn Chapter Two, may suggest some long-term
utility of the supplement to provide a chondro-protective effect for athletes suffering an
acute overload, such as that which might occur during knee ligament rupture (F elson et

al. 2000).

77

RECOMMENDATIONS FOR FUTURE WORK

Chapter One was the first study to investigate the role of in vitro cyclic preloading on the
response of a chondral explant to a potentially damaging, acute overload. In the long-
terrn these types of studies may help understand the role of biologic-based pre-
conditioning of articular cartilage for in vitro, or even in vivo, studies of blunt force
trauma to a joint. The current data in Chapter One would suggest both a positive and a
negative aspect to the idea of cyclic preconditioning or regular exercise in the prevention
of long-term chronic disease in joints suffering an acute overload, such as that proposed
during acute ligament injury. This in vitro study may suggest that the frequency,
magnitude and duration of the preconditioning may play a role in its potential chondral

protective effects. Clearly, additional in vitro and in vivo studies are wanted.

In Chapter One a dramatic increase in tissue damage to 25 MPa of unconfined
compression was noted after 21 days of cyclic loading. The effect also involved a
dramatic decrease irf’ the tissue matrix modulus, an increase in tissue permeability, and a
decrease in tissue PG content. This may be explained by an increase in the production of
degenerative enzymes, such as matrix metalloproteinase-3 (MMP-3), which is noted to
occur following extended cyclic loading of chondral explants (Lin et al. 2004). MMP-3 is
a connective tissue matrix-degrading enzyme. It was formerly known as proteoglycanase
and is generally considered to be one of the major proteoglycan degrading enzymes in
cartilage. MMP-3 is implicated in cartilage destruction in osteoarthritis and may also be
involved in tissue remodeling in the physis (Armstrong et al. 2002). Lin et al.

documented a significant increase in MMP-3 after 24 hours of cyclic loading at 1 or 5

78

MPa. Therefore, we hypothesize that the MMP-3 production might dramatically increase
between 14 and 21 days of cyclic loading in current study, and more experiments are

needed.

Chapter One also documented that while the collagen content and fiber modulus of the
explants significantly decreased after 7 days, the unconfined compression modulus of the
explants was significantly greater than controls, and it more directly paralleled with the
temporal changes in tissue PG content, the matrix modulus and permeability. An analysis
of the model sensitivity to changes in tissue matrix modulus, fiber modulus and
permeability suggested that the stiffness of the explant in the current unconfined
compression test should depend on the fiber modulus as much as matrix modulus.
Previous unconfined compression relaxation experiments, reported by Korhonen et a1,
displayed sensitivity of their tests to an enzymatic loss of collagen in the chondral
explant. Future studies will be required to understand more precisely if the lack of fiber
modulus sensitivity in the current experiments was due to the test protocol (0.5 Hz
sinusoidal loading or relaxation testing), fiiction on the explant upper surface, or a

problem in the validity of the fibril reinforced model for these cartilage explant tests.

The long-term health of joint cartilage may relate directly to the extent of matrix and cell
damage that occurs during an acute overloading of a tissue. The studies in Chapter One
indicated that mechanical stiffening of the chondral explant by cyclic preloads increased
the tissue content of PG and resulted in less strain during unconfined compression at 25

MPa. This was correlated with a significant increase in cell survival. The percentage of

79

cell damage per unit compressive strain was significantly decreased for up to 14 days of
cyclic preloading. One explanation for this result may relate to an increase in PGs
surrounding individual cells in the pericellular matrix as a result of cyclic preloading.
This effect has been documented previously following low intensity, cyclic loading
(Parkkinen et al. 1992; Quinn et al. 1998). Such deposition of PGs concentrated in or near
the pericellular matrix of chondrocytes may significantly alter the local stiffness around
cells to help protect them from damaging distortions during severe levels of unconfined
compression (Guilak et al. 1999). Therefore, future studies will be needed to test PG

grain density around individual chondrocytes under the current cyclic loading protocol.

As discussed in Chapter Two, the increase of tissue collagen in supplemented explants
versus non-supplemented, cyclic loaded explants likely may be due to less damage to the
explant structure during cyclic loading fi'om a stiffened matrix, rather than a statistically
significant increase in the biosynthesis of new, functional collagen. However, the
mechanism by which supplementation of the culture media with glcN-CS retained more
collagen and PG than non-supplemented, cyclic loaded specimens is yet unknown and
will require further study. And, as suggested in studies by others that examine the effects
of selective and non-selective metalloproteinase inhibitors on interleukin- 1 -induced
cartilage degradation, the effects of media supplementation on altering the material
properties of cartilage may only be delayed in time (Wilson et al. 2007). Future studies
will be needed to determine if the same effects will be seen with an extended duration

(for example, 28 days) of cyclic explant loading in the presence of supplement.

8O

The current in vitro study does not directly have utility in answering the question of
whether this. supplement will have efficacy in helping to mitigate long term joint disease,
such as post-traumatic OA, resulting from an acute traumatic overload, such as rupture of
a knee ligament (Fang et al. 2001). The major problems with extending these results to a
clinical setting are the short duration of in vitro studies, as well as the elevated
concentration of the supplement. In our short-term in vitro experiments the dose of
supplement (500 pg/ml glcN and 250 pg/ml CS) was higher than clinical levels. The
concentrations of glcN in serum after oral and intravenous administration range from 1 to
20 pg/ml, while CS concentrations are in the 5-200 pg/ml range (Adebowale et al. 2002;
Du et al. 2004; Volpi 2002). Yet, studies from this laboratory do show (Appendix A)
some reductions in the cyclic load-induced degradation of a chondral explant for even a
reduced (by 50%) concentration of supplement. Therefore, more experiments in use of
the current in vitro model are needed in the future to investigate the possible lowest (as
close to the clinical levels as possible) concentrations that will have the same in vitro
effects on chondral explants. And yet, it also seems inviting to propose in vivo studies at
clinical concentrations to see if this supplement may have incremental, positive effects on
the already documented effects of physiological levels of cyclic loading on changes in the
mechanical properties of joint cartilage (Saadat et al. 2006). Another potential utility for
these types of in vitro studies may be in the area of tissue-engineered cartilage (Waldman
et al. 2004; 2006). While recent studies show the utility of cyclic mechanical loading on
engineered cartilage stiffness, supplementation of the culture media with high
concentrations of glcN-CS may show utility in producing better products (V arghese et al.

2007). Finally, as the current study may imply, the intensity, fiequency and duration of

81

intermittent cyclic loading may play important roles in the response of these engineered

tissue to media supplementation (Wei et al. paper in review-b).

82

REFERENCES

Adebowale A, Du J, Liang Z, Leslie J L, Eddington ND: The bioavailability and
pharmacokinetics of glucosamine hydrochloride and low molecular weight
chondroitin sulfate after single and multiple doses to beagle dogs. Biopharmaceutics
& Drug Disposition 23:217-225, 2002

Armstrong AL, Barrach HJ, Ehrlich MG: Identification of the metalloproteinase
stromelysin in the physis. Journal of Orthopaedic Research 20:289-294, 2002

Du J, White N, Eddington ND: The bioavailability and pharmacokinetics of glucosamine
hydrochloride and chondroitin sulfate after oral and intravenous single dose
administration in the horse. Biopharmaceutics & Drug Disposition 25:109-116, 2004

Fang C, Johnson D, Leslie MP, Carlson CS, Robbins M, Cesare PE: Tissue distribution
and measurement of cartilage oligomeric matrix protein in patients with magnetic
resonance imaging-detected bone bruises after acute anterior cruciate ligament tears.
Journal of Orthopaedic Research 19:634-641, 2001

Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helrnick CG, Jordan JM, et al.:
Osteoarthritis: new insights. Part I: The disease and its risk factors. Annals of Internal
Medicine 133:637-639, 2000

Guilak F, Jones WR, Ting-Beall I-IP, Lee GM: The deformation behavior and mechanical
properties of chondrocytes in articular cartilage. Osteoarthritis and Cartilage 7:59-70,
1999

Korhonen RK, Laasanen MS, Toyras J, Lappalainen R, Helminen HJ, Jurvelin JS: Fibril
reinforced poroelastic model predicts specifically mechanical behavior of normal,
proteoglycan depleted and collagen degraded articular cartilage. Journal of
Biomechanics 36: 1373-1379, 2003

Lahm A, Erggelet C, Steinwachs M, Reichelt A: Articular and osseous lesions in recent
ligament tears: arthroscopic changes compared with magnetic resonance imaging
findings. Arthroscopy: The Journal of Arthroscopic & Related Surgery 14:597-604,
1998

Lin PM, Chen CTC, Torzilli PA: Increased stromelysin-1 (MMP-3), proteoglycan
degradation (3B3- and 7D4) and collagen damage in cyclically load-injured articular
cartilage. Osteoarthritis and Cartilage 12:485-496, 2004

Parkkinen JJ, Larnmi MJ, Helminen HJ, Tammi M: Local stimulation of proteoglycan
synthesis in articular cartilage explants by dynamic compression in vitro. Journal of
Orthopaedic Research 10:610-620, 1992

Persiani S, Roda E, Rovati LC, Locatelli M, Giacovelli G, Roda A: Glucosamine oral
bioavailability and plasma pharmacokinetics after increasing doses of crystalline
glucosamine sulfate in man. Osteoarthritis and Cartilage 13:1041-1049, 2005

83

Quinn TM, Grodzinsky AJ, Buschmann MD, Kim YJ, Hunziker EB: Mechanical
compression alters proteoglycan deposition and matrix deformation around individual
cells in cartilage explants. Journal of Cell Science 111:573-583, 1998

Saadat E, Lan H, Majumdar S, Rempel DM, King KB. Long-term cyclical in vivo
loading increases cartilage proteoglycan content in a spatially specific manner: an
infiared nricrospectroscopic imaging and polarized light microscopy study. Arthritis
Research & Therapy 8. R147, 2006

Varghese S, Theprungsirikul P, Sahani S, Hwang N, Yarema KJ, Elisseeff JH:
Glucosamine modulate chondrocyte proliferation, matrix synthesis, and gene
expression. Osteoarthritis and Cartilage 15:59-68, 2007

Volpi N: Oral bioavailability of chondroitin sulfate (Condrosulf®) and its constituents in
healthy male volunteers. Osteoarthritis and Cartilage 10:768-777, 2002

Waldman SD, Couto DC, Grynpas MD, Pilliar RM, Kandel RA: A single application of
cyclic loading can accelerate matrix deposition and enhance the properties of tissue
engineered cartilage. Osteoarthritis and Cartilage 14:323-330, 2006

Waldman SD, Spiteri CG, Grynpas MD, Pilliar RM, Kandel RA: Long-term intermittent
compression stimulation improves the composition and mechanical properties of
tissue-engineered cartilage. Tissue Engineering 10:1323-1331, 2004

Wei F, Golenberg N, Kepich E, Haut R: Effect of intermittent cyclic preloads on the
response of articular cartilage explants to an excessive level of unconfined
compression. Journal of Orthopaedic Research (submitted in review-a)

Wei F, Golenberg N, Kepich E, Haut R: High levels of glucosamine-chondroitin sulfate
can alter the cyclic preload and acute overload responses of chondral explants.
Journal of Orthopaedic Research (submitted in review-b)

Wilson CG, Palmer AW, Zuo F, Eugui E, Wilson S, Mackenzie R, Sandy JD, Levenston
ME: Selective and non-selective metalloproteinase inhibitors reduce IL-l-induced
cartilage degradation and loss of mechanical properties. Matrix Biology 26:259-268,
2007

84

Appendix A: Low Concentration of Supplement

Since in the short-term in vitro experiments the dose of supplement used in Chapter Two
was much higher than clinical levels, we recently used a lower concentration of
supplement in the bovine media (250 pg/ml glcN and 125 pg/ml CS). Twenty-four
explants were used in the test, with 12 controls and 12 cyclic loading with low
concentration of supplement (n=4 in each of 7, 14 and 21 days groups). Data of cyclic
loaded explants without supplement were combined fiom Chapter Two. The

experimental design and procedure were the same as described in Chapter Two.

Matrix modulus, fiber modulus and permeability of the explants were compared in terms
of “% of before cyclic loading”. Cyclic loading significantly increased the tissue matrix
modulus by approximately 145% and 180% at 7 and 14 days, respectively. No
differences were observed between cyclic loading with and without low concentration of
supplement. With longer duration of cyclic loading at 21 days, however, cyclic loading
without supplement decreased the matrix modulus by 58.4 :I: 19.4%, while cyclic loading
with low concentration of supplement significantly increased the matrix modulus by
184.5 :I: 64.9% (Figure 1A). Cyclic loading significantly decreased the tissue fiber
modulus by approximately 65% at all time points (32.6 :I: 17.8%, 39.8 i 27.4% and 36.8
a; 14.3% of before cyclic loading at 7, 14 and 21 days, respectively). No significant
differences were observed between cyclic loading with and without low concentration of
supplement (Figure 1B). Up to 14 days of cyclic loading the permeability of the explants
without supplement significantly decreased (71.1 d: 8.5% and 31.0 :L- 7.7% of before

cyclic loading in 7 and 14 days groups, respectivelY). and then it increased dramatically

85

at 21 days (143.0 t 9.9% of before cyclic loading). In contrast, cyclic loading with low
concentration of supplement decreased the tissue permeability significantly at all time
points (61.4 :I: 19.3%, 35.4 :I: 28.3% and 26.4 :I: 16.0% of before cyclic loading in 7, 14
and 21 days groups, respectively). No significant differences were observed between
cyclic loading with and without low concentration of supplement up to 14 days (Figure
1C). For all the three parameters the control (non-cych loaded) remained at the same

level (approximately 100%) for all time points.

To conclude, up to 14 days of cyclic loading with low concentration of supplement, tissue
matrix modulus significantly increased, while both tissue fiber modulus and permeability
significantly decreased, but no significant differences were observed between cyclic
loading with and without low concentration of supplement. With longer duration of
cyclic loading, however, low concentration of supplement still inhibited the degeneration
of cartilage explants between 14 and 21 days as high level of supplement did in Chapter

Two.

86

 

250 _ -D— CODUO'
—<>—Cyc. loading :I:

 

 

 

 

 

 

 

 

% - A- Cyc. Loading w/ suppl.(L)
5‘
2
.2
8
“5
ii
(I)
2
:r
8
2
.x
1%
2
0 I ' '
0 7 14 21

Duration of cyclic loading (days)

 

 

 

 

 

 

 

 

 

A —D—Control

g 120 1 —0—Cyc. loading

'0 - A- Cyc. Loading w/ suppl. (L)

r: \ T

5‘ so - \ ==

2 \

~§ \

”5 60 a \ 1; :I:

a2 \ I

v \ ' a ‘ ‘A ........

3 40 “ ‘ __, g:— -— — — — _

§

2 20 A 1:- t

3

u— 0 r r r
0 7 14 21

Duration of cyclic loading (days)

Figure 20. Cyclic loading with low concentration of supplement strengthened the
cartilage matrix modulus by approximately 200% of the control (A), and helped inhibit
the degradation of the explants from longer time (21 days) cyclic loading (A and C).
Cartilage fiber modulus, however, decreased approximately 60% by cyclic loading,
independent of supplement (B). No significant differences were observed between cyclic
loading with and without low concentration of supplement up to 14 days in all the three
parameters. Statistically significant differences were observed from control (I), and from
cyclic loading (§).

87

Permeability (% of before cyclic loading)

180 .

 

 

 

 

 

 

 

 

-D-Control
160 - —0—Cyc. loading 1
140 _ - d- Cyc. Loading w/suppl. (L) I
120 -
100 I
so i: Put? \ r /
eo— \Iéé‘ //
40 ~ 1: . \x- 1 ,l. - _ -
2o — 1T ' ' ’ ' ifs
0 l i r
0 7 14 21
Duration of cyclic loading (days)

Figure 20. (cont’d)

88

 

Appendix B: PROCEDURE FOR OBTAINING CARTILAGE EXPLANTS FROM
BOVINE METACARPAL JOINTS

1. Call Bellinger’s meat packaging/slaughter house Monday’s and ask for 2 pairs of
Bovine Knees (1-800-778-4577). Pick up the knees on Tuesday’s.

2. Prepare all media and confirm availability of supplies.

3. After obtaining the knees, wash the outside of the leg in a sink to remove mud. Use a
scalpel to remove skin from leg down to hoof. Remove hoof. Place waste in
biohazard bag. Wash to remove any contaminants.

4. Set up the Laminar flow hood. Lay down laboratory paper in hood, place scalpel,
biopsy punches, tweezers, and a petri dish with media (no F BS) in hood.

5. Take leg into hood. Dissect open the metacarpal joint (lower of two joints at top of
removed leg) using a scalpel.

6. Using a biopsy punch, punch several explants in the lower (articular) surface of
cartilage of the joint. Use the scalpel to remove the explants by slowly cutting under
the punched area along the bone surface. Use the tweezers to place the explant in the
petri dish of media. Repeat until you have more than enough explants.

7. In hood, perform three washes by using tweezers to place the explants in new petri
dishes with media. Swirl around in each dish. Place an explant into each well of a 24
well plate with 1 ml of media (with 10% PBS) in each well. If explants are cultured

for more than 2 days, change media every two days.

89

Appendix C: BOVINE MEDIA STOCK RECIPE

1.

2.

8.

9.

Measure 1L of ddH20. Pour 600mL of ddH20 into 2L Erlenmeyer flask with stir bar.
Add 1 package of powdered media DMEM:F12 (Gibco #12500—062). 1 package

makes 1L of media.

. Add 20mL AA solution (Gibco #11130-051).

Add 3.89g Sodium Bicarbonate (NaHC03) (for final concentration of 44mM).

. Add 2mg lactalburnin hydrolysate (for final concentration of 2 pg/mL).

Add 1 pL of diluted sodium selenite stock solution to the media (for final
concentration of lpg/mL).

Add 10pL of ascorbic acid stock solution.

Add 10pL dexamethasone stock solution (for final concentration of 100pg/mL).

Add 10pL of manganese sulfate stock solution to the media.

10. Bring media up to 900mL with ddH20 and pH to between 7.3 and 7.4, then bring

volume to 1L and sterile filter.

11. Before using bovine media for cell culture, add 10mL antibiotics (Biochem stores

#15240-062) to the 250mL sterile filtered jar.

Stock Solution Concentrations

Sodium selenite (lmglmL) - dilute the stock solution 1/1000 for 1 pg/mL.

Ascorbic acid (5mg/mL)

Dexamethasone (lOmg/mL)

Manganese sulfate (16.9mg/mL)

9O

1.

APPENDIX D: BOVINE MEDIA HANDLING SOP

Remove FBS and antibiotics (if needed) from fieezer and thaw by placing tube in
warm water.

Set up laminar flow hood by turning on the fan and lights. Sterilize by wiping down
the inside surfaces with 70% ethanol. Sterilize 101-1000pL pipetter, marker, 24-well
plate, and anything else placed in hood with 70% ethanol.

Remove two 250mL jars of bovine media from refrigerator and place in hood with
thawed FBS and antibiotics.

If needed, pour 10mL antibiotic into 250mL of bovine media. Gently mix by swirling.
This should protect media from contamination for two weeks.

For a standard 24-well plate, 1mL of culture media is needed per well. Pipette 2.5mL
FBS into a conical tube. Bring to 25mL by adding antibiotic-treated media (final
concentration of 10% F BS by volume media). Vortex to mix.

Pipette 1mL of 10% FBS antibiotic-treated bovine media into each well of the 24-
well plate. Cover.

Set up three petri dishes with bovine media from the untreated jar. Rinse explants in
each dish before placing in the flesh 24-well plate.

Put remaining FBS in fi'eezer. Sterilize hood again before shutting off.

Change media in well plate every 2 days throughout an experiment.

91

APPENDIX E: BOVINE EXPLANT INDENTATION SOP

Calibration and Program set up:

1.

2.

Turn on both the program selector box, and the Validyne strain gage amplifier.

Attach the small book into the bottom of the load cell.

Open the “J &Jpreload3.vi” program by double clicking on its shortcut, labeled as
“J &Jpreload.vi” on the desktop.

Using the “Preview” setting on the LabVIEW screen, which displays the current force
in Newtons, zero the load cell to 0 :I: 0.001 using the small screwdriver. The zeroing
control is located in the center of the DANA box.

Hang the small 100 gram weight on the hook attached to the load cell. This should
read -0.9807 :1: 0.002 on the Preview display if the load cell is calibrated correctly.
This value corresponds to -0.4000 on the voltmeter. If the value is off more than :I:
0.002 then use the gain control (just below the zero control) to adjust the value. Then
take off the weight and repeat steps 4 and 5. If the problem persists contact Cliff
Becket.

Repeat step 5 using the 200 gram weight, the “Preview” read out should be -_1.fil_:l:_
L99; N, which corresponds to a value of -0.8000 on the voltmeter.

In the J &Jpreload3.vi program, verify that the following settings are accurate:

Block 1: # samples = 3000, rate = 10

Block 2: # samples = 600, rate = 100

Block 3: # samples = 9600, rate = 10

Preload N = 0.02

92

Channel calibration

Units/volt O = 1.0000

Units/volt l = 2.4517

Units/volt 2 = 1.0000

Analog Trigger = Disabled

PreloadHold Time in Seconds = 300

These settings control the collection of data. The data will be displayed in Newtons.

Run the “Reset MinIndenter” program, located on the desktop, by double clicking.

(This must be done any time the program selector box is turned on, before you can

reprogram the Mini Indenter).

Open up the “Miniprograrn” on the desktop of the computer. This is a HyperTerminal

program. The indenter program is set up to be 30, 1 on the program selector box.

10. In the Mirriprograrn, the indenter program is # 1300. Type “q1300” then hit the

carriage return 8 times to display the program. It should read the following:

1300 H

13021

1305 V

1308 K

13100

l3l4R

1318

1

ID

IQ-

Where g = thickness of specimen x 100

b=2xg

93

So for example, if the specimen being tested is 0.43 rmn thick, program 1300 will read as the

following:

1300 H 1
13021 43
1305 V 43
1308 K 0
1310 O 0
1314 R 86
1318

Refer to the mini indent manual for a complete description of these commands. If a
line is incorrect, change it by typing “p” then the line number and the appropriate
command letter and number. Press Esc to exit edit mode, and type p on the last blank
line to specify the end the program.

11. Double check the program by running a simulated test. Place the program selector
switches on 30, 1 for the indent program.

12. Rotate the mini indenter jogging knob, located on the top of the indenter, to O.

13. Push the red start button on the program selector box. Make sure the knob rotates
item 0 to 2, which translates to 0.1 mm of downward travel. If this is incorrect,
recheck the program in the HyperTerminal and make corrections as necessary.

Equipment set-up

1. Check to make sure that the black cable labeled “Mini Indenter Trigger Out” (with
the BNC connecter) is 1191 connected to the A2D interface box. The program will

not run correctly unless this cable is unattached.

94

l‘

*However, the trigger signal is still functional and could be used on another A2D
interface box hooked up to another computer if so desired.

Loosen all the claps on the mounting plate, 6 total. These are the round knobs located
around the edge of the fixture.

Attach the steel rod with a small washer glued on top to the center of the horizontal
mounting plate. Note that the small reservoir and clamp should be attached to the rod,
if not do so now.

F ill the reservoir with PBS, making sure not over fill. Be very careful not to splash
any PBS up onto the load cell, or to spill any PBS near the circuit card located outside

of the program selector box.

Test procedure:

1.

Explant desired number of samples fi'om bovine joint. Record weights of each off the
john.

Dab the cartilage surface with India ink to highlight any surface fissures, wipe off
excess, and photograph for records.

Place samples in numbered well plate filled with bovine media solution. Let explants
swell for a minimum of 15 minutes prior to indentation testing.

Measure thicknesses of each specimen using a micrometer, and record. These values
will be used in the Mirriprograrn, q 1300, as explained in program setup # 10.

Place explant on top of the rod, in the center of the washer.

Verify that the load cell is attached in the correct orientation. The end where the gray

cable is attached must be located at the top half of the load cell.

95

7.

9.

10.

ll.

12.

13.

14.

Screw in the flat 1mm indenter into the load cell. Jog the XY plate so the indenter is
located directly above the center of the explant. This is the desired testing site. Once
the location is roughly where desired, there is a round knob available in order to
manually adjust the plate in both the X and Y directions in a much smaller, more
accurate amount.

Lower the indenter as close as possible with out touching the explant. Once in place,
tighten all the clamps.

Raise the reservoir containing the PBS so the explant is submerged.

Zero the load cell with the indenter tip in the PBS.

*Note: Make sure the entire indenter tip is not, or will not be submerged. Lowering
the top, larger portion of the indenter into the PBS will cause the zero to be off.
Lower the indenter head so that it is very close, but not touching the surface of the
explant, (0.5-0.1 m away).

‘Note: Use the Fast and Slow actuator jog buttons (located on the selector box) to
move the indenter up and down while the Z-axis clamps are tightened down.

Verify that the program switches are on 30, l and that the “J&Jpreload3.vi” program
is in the run mode, (the run arrow in the top left is black). Then click the green start
button on the LabVIEW screen and let it run.

I""‘(As soon as the green START button is triggered the indenter will
IMMEDIATELY begin running).

Start a stopwatch once the preload is reached.

Be sure not to move or bump either the Mini Indenter or the desk it sits on in order to

avoid unnecessary jolts or noise in the graph.

96

15. The program will start acquiring data once the preload of 0.02 N is reached. 5 min
after that, it will automatically indent 10% of the thickness. 10 min after the indent,
press the red stop button on the “J &Jpreload3.vi” screen. Use the fast actuator jog up
to get the indenter off of the explant. Loosen the Z-clamps, and raise the indenter with
the Z up button if more room is desired.

16. Save the data file just collected in its appropriate location, and repeat steps for next

sample explant.

97

APPENDIX F: IMPACTING BOVINE EXPLANT S (INSTRON INSTRUCTIONS)

1) Turn on main power (red switch).

2)

3)

4)

5)

6)

7)

8)

9)

Check load cell (20001bs).

Warm up hydraulic system:

Move cylinder up so it has room to drop (actuator jog up).

Position -) Waveform: le 25mm sine wave

Position '9 Waveform: Start (bottom left button). Run ~ 200 cycles. 3

Press finish. (Not stop!)

Assemble Instron setup for bovine explants.

Plug in load cell. Calibrate: Set up-) Cal. -)Auto-)Go.

Green light will flash faster, then stops when done.

Lower cylinder ~ 1/2 in fiom sample platform. Loosen clamp-) lower-) clamp.
Check PIDL values:

Position-98etup9loop 13 l 0 1

Load-9 10 l 2 1

Place sample on platform. Lower cylinder. (Actuator on low or high.) Touch cylinder
to sample w/ cut applying force.

Create folder- My Docs- Flaps 5.

10) Open Flaps 5 Program- Follow program instructions to Run. Press Remote. Yes, use

control limits.

11) After all samples have been impacted:

> Unclamp-) raise cylinder-)clamp.

> Turn hydraulics off.

98

> Main switch off.
> Dis-assemble completely.

> Shut down computer.

> Fill out log book.

99

l

APPENDIX G: LIVE/DEAD VIABILIT Y STAINING FOR ARTICULAR

CARTILAGE EXPLANTS (Manufacturer and Part #: Molecular Probes L-3224)

*Allow kit reagents to warm to room temperature before use*

1.

Remove media from explants and save it for analysis if desired.

*This does not have to be done in a sterile environment.

Replace media with DPBS (PBS made with distilled water) — wash tissue 3 times
with 1mL DPBS. This removes dilute serum esterase activity which may increase
fluorescence by hydrolyzing Calcein AM. After third wash leave ~1mL PBS (or
enough to cover tissue) in each well to keep tissue moist.

Cut cartilage explants into thin strips using the mechanical slicer (which will cut 4
sections, each 0.5mm thick). Place tissue back in appropriate well.

Turn out the lights — work with as little light as possible fi'om now on; stain loses
fluorescence if exposed to light.

Add enough stain to cover all tissue (usually ~0.5mL per well for a 24 well plate).
Prepare stain daily — Keep out of direct light.

Add 10pL of supplied 2mM EthD-l to 5mL DPBS. Vortex to mix. Combine 2.5 pL of
supplied 4mM Calcein AM with the 5mL of EthD-l solution. Vortex to ensure
mixing. Simply use multiples of these numbers if more stain is needed. Refer to Excel
doc viability stain amounts.

Incubate with stain ~15min at room temperature. Keep covered — light decreases
fluorescence intensity.

Remove stain and wash 3 times with PBS, after third wash leave ~1mL (or enough to

cover tissue) in each well to keep tissue moist.

100

8. Place on a glass slide. View under fluorescence microscope immediately. If you are
viewing the cross-section, you should see many “columns” of cells with some
scattered. If you are accidentally viewing the top or bottom, they will appear totally

scattered. Keep covered — light decreases fluorescence intensity.

Polyanionic calcein produces a green fluorescence in live cells and EthD-l produces a

red fluorescence in dead cells.

DPBS: KCl (200rng/L). KHzPO4 (200mg/L), NaCl (8000mg/L), NazHPO4 (1150 mg/L)

101

 

APPENDIX H: PROTEOGLYCAN ASSAY

Papain Tissue Digest Protocol (for 24 samples)

1) Take papain from fridge and heat to 37C.

2) Heat water bath to 60C.

3) Prepare papain solution just before use.
0 14 ml PBS
0 26 mg EDTA
0 24.5 mg cysteine
o 39.5 pl papain

4) Add 500p] to each sample.

5) Add 500pl to ~45 mg Chondroitin Sulfate A to create standard solution (record exact
value of Chondroitin used)

6) Incubate at 60C (water bath) overnight (until tissue is gone).

7) Boil samples (in four 50 ml beakers with ~20 ml water) on hot plate for 5 mins to
inactivate the papain.

8) Place vials in freezer until ready to complete assay.

9) Vortex samples for a few seconds.

DMB Tissue Sample Assay

Chondroitin Dilution: standards

1'” Vortex between each step!!!

1) Mix 21.11 of the chondroitin standard solution + 398pl of dilution buffer in vial l.

2) Add 200 pl dilution buffer to vials 2-8.

102

3) Take 200111 from 1“t vial and add to 2““.

4)

5)

Take 200pl from 2“d vial and add to 3'“. Continue for all remaining vials.

Toss 1"t dilution.

Dilute samples

1'“ Vortex between each step!!!

1)

2)
3)
4)

5)

Mix 4pl of sample + 396p] of dilution buffer in vial 1. (use 6p] + 394p] if only part of
sample was digested for PG assays).

Add 200 pl dilution buffer to vials 2-5.

Take 200111 from 1" vial and add to 2'”.

Take 200p] from 2“d vial and add to 3'“. Continue for all remaining vials.

For each sample, repeat step 1—4.

Prgpgg Microplate 8; Run Elgte Reader

1)

2)

3)

4)

5)

6)

Add 50p] of standards to wells All-G11 & A12-G12 (columns 11 & 12 are
duplicates).

Each sample will occupy a single row. Add 50pl of each concentration of diluted
samples to two wells (duplicates). Ex: add the 1‘” dilution to Al & A2, the 2"‘1 dilution
toA3&A4,andsoon.

Turn on plate reader (switch on back).

Add 200pl DMB dye to every well.

Place microplate in reader (line up A1). The assay is time-sensitive so achieve a
reading immediately following the addition of the DMB.

Plot known standard concentration against standard absorption and fit with a linear

regression. Want R42 for control > .98 (996-999 obtainable).

103

7) Use the regression equation to solve for the unknown sample concentrations (x) by

plugging in their absorptions (y).

DMB Media Sample Assay

Base Standard Media Crea_tign

1) Measure out ~5mg Chondroitin Sulfate A.

2) Dilute with lml Bovine Media to every 5mg chondroitin.

3) Add 44p] of the resulting solution to 1156p] Bovine Media to create chondroitin
standard. Toss 1"t solution.

Chondroitin Media Standard Dilution

*** Vortex between each step!!!

1) Mix 100p] of the chondroitin media standard solution with 300p] of dilution buffer in
vial 1.

2) Add 200p] dilution buffer to vials 2-8.

3) Take 200,11 flour 1” vial and add to 2"“.

4) Take 200p] from 2"d vial and add to 3'”. Continue for all remaining vials.

Dilute samples

“* Vortex between each step!!!

1) Mix 100pl of sample + 300p] of dilution buffer in vial 1.

2) Add 200 pl dilution buffer to vials 2-5.

3) Take 200,11 from 1st vial and add to 2““.

4) Take 200,11 from 2“d vial and add to 3'“. Continue for all remaining vials.

5) For each sample, repeat step 1—4.

104

l'.

6) From here, follow the above procedure under “Prepare Microplate & Run Plate
Reader”.

Stock Solutions:

Dilution buffer (500ml): Mix 2.05g sodium acetate + 250 pl Tween 20 + water. pH 5.3.

DMB Reagent (0.5 L):
1) Weigh 0.008g 1,9-dimethylene blue. Using a 50ml tube, dissolve dye in 2.5ml of
100% ethanol.
2) In a separate tube, mix 1 ml formic acid and 1 g sodium forrnate in 20 ml water.
(Work with formic acid in hood!).
3) Add mixture to 50ml tube of ethanol-DMB mixture.
4) Pour DMB mixture into a 1L graduated cylinder (rinse 50ml tube 3 X to transfer all
chemicals) and add water to 500ml.
5) Transfer to a 0.5L bottle and label with date.

”*Shelf life 1 month in the dark.

Sample Calculations:

From the spreadsheet in OBL’s z-drive we can get x ug/ml PG (unadjusted) by solving
the straight line equation, this sample calculation shows how to get the PG content (pg
PG / mg Wet Weight) from x. For convenience, here we use “a” to represent the “x” in

the spreadsheet.

a gglml PG

105

l. 0.25 x a = PG amount (pg) in well, where 0.25 (ml) comes from 50 pl diluted sample

 

 

 

plus 200 pl DMB.
0.25 x a . .
0 05 = concentration (pg/ml) of dlluted sample, where 0.05 comes from 50 p1
diluted sample.
025xa . .
3. x 0.4 = PG amount (pg) 1n dlluted sample, where 0.4 (ml) comes from 4p] of
sample plus 396pl of dilution buffer.
0.25xax0.4
4. 0-05 0 004 = concentration (pg/ml) of sample, where 0.004 comes from 4p1

of sample.

0.25xax04
5. 0-05 0 004 x 0.5 = PG amount (b pg) in sample, where 0.5 comes from 500

 

pl papain solution and “b” represents the final PG amount in sample.

6. b / mg Wet Weight = PG content (pg PG / mg Wet Weight).

106

APPENDIX 1: HYDROXYPROLINE DETERMINATION

Proteoglycan extraction:

1) gay; Record wet weight of samples.

2) Add 2m] 4M Guanidine HCl and 2mL incubation buffer to each sample well (12 well
plate).

3) Place the plate on a roller bank for 24hrs at 4°C.

4)D 2 Wash the explants with lml incubation buffer two times (on roller bank for
3hrs at 4°C).

Digestion/hydrolyzation of collagen:

5) Add each sample to a centrifuge vial containing 500pL of incubation buffer with
lmg/mL oCT. Allow the denatured collagen to digest overnight in a 37°C water bath.

6)D 3 Pipette 500pL of the supernatant (liquid) from each vial into new vials and
dilute 1:1 with 12M HCl.

7) Place the remaining tissue samples into new vials and immerse in 1mL of 6M HCl.

8) Allow both the tissue and the supernatant to hydrolyze in a 70°C water bath for 24hrs.

9) 9314 Dry the tissue and supernatant hydrolyzates overnight (possibly two nights) in
an oven.

10)D_a\L_§ Add 300pL of dH20 to the samples, vortex, and dry overnight again to ensure
the removal of any residual HCl.

11) m Add 300pL of dH20 to the samples again, vortex.

Pr_eparation of microplate wells:

"* Vortex between each step!!!

107

1) Mix 4pL of the 1mg/ml hydroxyproline standard solution + 286pL of 10mM HCl in
vial l.

2) Add 150pL 10mM HCl to vials 2-8.

3) Take 150pL from lSt vial and add to 2“.

4) Take 150pL from 2"d vial and add to 3”. Continue for all remaining vials.

5) Mix 5pL of sample + 495 pL of 10mM HCl in vial 1.

6) Add 250pL 10mM HCl to vials 2-5.

 

7) Take 250pL from 1“ vial and add to 2"“.

8) Take 250pL from 2"d vial and add to 3”. Continue for all remaining vials.

9) For each sample, repeat step 5—8.

10) Add 50p] of standards to wells All-H11 & A12-H12 (columns 11 & 12 are
duplicates).

11)Each sample will occupy a single row. Add 50pl of each concentration of diluted
samples to two wells (duplicates). Ex: add the 18‘ dilution to A1 & A2, the 2'1d dilution
toA3&A4,andsoon.

12) Add lOOpL of oxidizing solution to each of the sample wells.

13) Place plate on shaker or roller bank for 5 minutes.

14) Add lOOpL of Erlich’s Reagent to each well. Place an adhesive seal on the plate and
incubate in a water bath at 60°C for 45 minutes.

15) Place microplate in reader and run test. Absorbance readings are taken at a
wavelength of 570 nm.

Stock Solutions:

a 4M Guanidine HCl:

108

Mix 250mL dH20 with 95.53g Guanidine HCl.

0 Incubation buffer:
In 800mL dHZO, dissolve 15.76g Tris HCl, 184.96mg iodoacetamide, 372.24mg
EDTA, and 10mg pepstatin A. add dI-I20 to 1L.

0 Citrate/acetate buffer:
Mix 6.8g sodium hydroxide, 6.8g citric acid monohydrate, and 24g sodium acetate
trihydrate in 180mL deO. Adjust to pH 6.0 with glacial acetic acid (<5mL), make up
to final volume of 200mL.

- Oxidizing solution:
Mix 22.727mL isopropyl alcohol, 12.5mL deO, and 14.773mL citrate/acetate
buffer. Dissolve 300mg chloramine T in solution. Store in dark.

0 Ehrlich’s reagent:
Mix 52mL isopropyl alcohol with 5.7mL 70% perchloric acid and 10.3mL dH20.
Dissolve 6g p-DAB into the mixture.

0 10 mM HCl:
Mix 50mL dH20 with 40.5pL Hydrocloric acid (37.9% HCl)

0 1mg/ml hydroxyproline standard:

Mix 1mg hydroxyproline in 1mL 10mM HCl in a centrifuge tube. Store fi'ozen.

Sample Calculations:
From the spreadsheet in OBL’s z-drive we can get x pg/ml HYP (unadjusted) by solving

the straight line equation, this sample calculation shows how to get the HYP content (pg

109

f.-

HYP / mg Wet Weight) fiom x. For convenience, here we use “a” to represent the “x” in

the spreadsheet.

a

1.

ml HYP
0.25xa = HYP amount (pg) in well, where 0.25 (ml) comes from 50p1 diluted

sample plus 100p! of oxidizing solution and lOOpL of Erlich’s Reagent.

 

0'32?“ = concentration (pg/ml) of diluted sample, where 0.05 comes fiom 50 pl
diluted sample.
025xa

 

0 05 X 0-5 = HYP amount (us) in diluted sample, where 0.5 (ml) comes from 5p]

of sample plus 495p] of 10mM HCl.

 

 

0.25 x a x 0 5
0°05 0 005 = concentration (pg/ml) of sample, where 0.005 comes from Spl
of sample.
0.25 x a x 0.5
0-05 0 005 x0.3 = HYP amount (b pg) in sample, where 0.3 comes from
300pL of dH20 and “b” represents the final HYP amount in sample.
b / mg Wet Weight = HYP content (pg HYP / mg Wet Weight).

 

 

   

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