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This is to certify that the
thesis entitled

INVESTIGATIONS ON THE RESPONSE OF KNEE JOINT
CARTILAGE TO BLUNT IMPACT IN A SMALL ANIMAL
MODEL

presented by
DANIEL l. ISAAC

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Engineering Mechanics

ﬂaw z/J%L4

Major Professor’s Signature
3
0 f/gé/o 9 .

Date

MSU is an Afﬁrmative Action/Equal Opportunity Employer

 

 

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5108 K IProi/AccﬁPres/ClRC/Dateoue indd

INVESTIGATIONS ON THE RESPONSE OF KNEE JOINT CARTILAGE
TO BLUNT IMPACT IN A SMALL ANIMAL MODEL

By

Daniel 1. Isaac

A THESIS

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

MASTER OF SCIENCE

Engineering Mechanics

2009

ABSTRACT

INVESTIGATIONS ON THE RESPONSE OF KNEE JOINT CARTILAGE TO BLUNT
IMPACT IN A SMALL ANMIAL MODEL

By

Daniel 1. Isaac

History of joint injury due to participation in SRE, particularly to the knee or hip,
increases the risk of developing a chronic joint disease, osteoarthritis. 0A is one of the
most common and widespread rheumatic diseases responsible for deterioration of
articular cartilage, subchondral bone and synovium, ultimately leading to the failure of
synovial joints. Experimental studies with animal models have sought to understand the
association between acute joint trauma and the development of 0A. The research
presented in the current thesis makes use of an in viva rabbit model to examine the acute
and chronic responses of articular cartilage and subchondral bone to blunt force trauma.
Chapter 2 addressed the issues of acute damage to chondrocytes following a single,
severe insult to the ﬂexed TF joint. Chapter 3 describes chronic studies where a single
impact was again delivered to the TF joint of anesthetized rabbits and the changes in the
mechanical and histological properties of the articular cartilage were evaluated six
months and one year following trauma. Chapter 4 documented the development of an in
vivo model of traumatic ACL rupture. Chapter 5 evaluated the efﬁcacy of a mild non-
ionic surfactant, poloxarner 188 (P188), in ‘repairing’ damaged cells after an in vivo
impact to the rabbit TF joint. Future studies can utilize the data presented to investigate

the progression of chronic joint disease and the efﬁcacy of various intervention methods.

DEDICATION

I would to thank my parents and siblings for their continued guidance and support
throughout my education. Without their constant guidance and support this would have

never been possible.

iii

ACKNOWLEDGMENTS

I would like to acknowledge my professor and mentor Dr. Roger Haut. I am
extremely grateful for his support and guidance throughout my graduate education.
I am grateful to Dr. Mike Shingles and Dr. Seungik Back for serving on my committee. I
would like to express gratitude towards Cliff Beckett for his technical support and
willingness to share his knowledge with me. I would also like to thank Jean Atkinson and
Jane Walsh for their constant support, hard work and dedication. Without their assistance
in these studies none of this would be possible. Finally, I would like to acknowledge my
fellow graduate students for their help and most importantly their friendship: Nurit
Golenberg, Tim Baumer, Eric Meyer, Brian Powell, Feng Wei, Jerrod Bramen, and Mark

Villwock.

iv

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................... vii

LIST OF FIGURES ................................................................................................... x

RESEARCH PUBLICATIONS ................................................................................ xiv

CHAPTER 1

INTRODUCTION ..................................................................................................... 1

CHAPTER 2

CHONDROCYTE DAMAGE AND CHONTACT PRESSURES FOLLOWING

IMPACT ON THE RABBIT TIBIOFEMORAL JOINT .......................................... 14
Abstract .......................................................................................................... 14
Introduction ................................................................................................... 16
Materials and Methods .................................................................................. 17
Results ........................................................................................................... 22
Discussion ...................................................................................................... 25
References ..................................................................................................... 29

CHAPTER 3

CHRONIC CHANGES IN THE MECHANICAL AND HISTOLOGICAL
PROPERTIES OF RABBIT ARTICULAR CARTILAGE FOLLOWING

TIBIOFEMORAL IMPACT ..................................................................................... 31
Abstract .......................................................................................................... 31
Introduction ................................................................................................... 33
Materials and Methods .................................................................................. 35
Results ........................................................................................................... 41
Discussion ...................................................................................................... 45
References ..................................................................................................... 50

CHAPTER 4

A TRAUMATIC ANTERIOR CRUCIATE LIGAMENT RUPTURE MODEL: A

PRELIMINARY STUDY USING THE RABBIT MODEL ..................................... 53
Abstract .......................................................................................................... 53
Introduction ................................................................................................... 55
Materials and Methods .................................................................................. 57
Results ........................................................................................................... 63
Discussion ...................................................................................................... 71
References ..................................................................................................... 75

CHAPTER 5

ACUTE REPAIR OF CHONDROCYTES IN THE RABBIT TIBIOFEMORAL JOINT

FOLLOWING BLUNT IMPACT USING P188 SURFACTANT ........................... 79
Abstract .......................................................................................................... 79

Introduction ................................................................................................... 8 1

Materials and Methods .................................................................................. 84
Results ........................................................................................................... 87
Discussion ...................................................................................................... 93
References ..................................................................................................... 98

CHAPTER 6

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK ............. 102

APPENDICES ........................................................................................................... 106
Appendix A: Raw data ﬁom chapter two ...................................................... 106
Appendix B: Raw data from chapter three .................................................... 111
Appendix C: Raw data from chapter four ..................................................... 128
Appendix D: Raw data from chapter ﬁve ...................................................... 139

vi

LIST OF TABLES

Table 3.1: The mechanical properties (average (1- standard deviation» were extracted

from the relaxation indentation testing across the medial and lateral facet (Site
1 - medial uncovered, Site 2 - medial covered, Site 3 — lateral uncovered and
Site 4 — lateral covered). Statistical differences between the between the 6
month and 1 year group are indicated (b). ............................................... 42

Table 3.2: Histological evaluations of the impacted and control osteochondral sections of

the medial and lateral tibial plateau indicated signiﬁcant increases in surface
ﬁssures, subchondral bone thickness, disruptions (i.e. microcracks) and PG
stain. Statistical differences between the impacted and contralateral, control
limbs are indicated for the 6 month and 1 year group (“) and between the 6
month and 1 year groups (b). ................................................................... 44

Table 4.1: Analysis of pressure sensitive ﬁlm revealed high contact pressures in the

Table A.l:

Table A2:

Table A3:

Table A.4:

Table B.l:

Table B.2:

Table 3.3:

Table B.4:

Table 3.5:

Table 8.6:

Table 8.7:

medial compartment of the TF joint during ACL trauma, and even higher

pressures in the lateral facet ..................................................................... 64
Pressure ﬁlm data for right and left limbs ............................................... 107
Peak pressures on the lateral and medial tibial plateaus .......................... 108
Cell viability data for the impacted (left) and unimpacted (right) limbs. 109
Zonal cell viability data (% dead cells) for the impacted (left) and unimpacted
(right) limbs ............................................................................................. 1 l 10
Histology scores for the impacted (left) limb of the 6 month animals 1 12
Histology scores for the contralateral, control (right) limb of the 6 month
animals ..................................................................................................... 1 13
Histology scores for the non-impacted, control animals of the 6 month grlcﬁlp
................................................................................................................. 1
Histology scores for the non-impacted, control animals of the 6 month glrlosup
Histology scores for the impact limb (left) of the 1 year animals .......... 116

Histology scores for the contralateral, control (right) limbs of the 1 year
animals ..................................................................................................... 1 1 7

Histology scores for the non-impacted, control (left) limbs of the 1 year
animals ..................................................................................................... 118

vii

Table B.8: Histology scores for the non-impacted control (right) limbs of the 1 year
animals ..................................................................................................... 119

Table B.9: Mechanical indentation data for medial uncovered (site 1) in the 6 month

group ........................................................................................................ 120
Table B.10: Mechanical indentation data for medial covered (site 2) in the 6 month group
................................................................................................................. 121
Table 3.1]: Mechanical indentation data for lateral uncovered (site 3) in the 6 month
group ........................................................................................................ 122
Table B.12: Mechanical indentation data for the lateral covered (site 4) in the 6 month
group ........................................................................................................ 123
Table 8.13: Mechanical indentation data for medial uncovered (site 1) in the 1 year group
................................................................................................................. 124
Table B.14: Mechanical indentation data for medial covered (site 2) in the 1 year group
................................................................................................................. 125
Table 8.15: Mechanical indentation data for lateral uncovered (site 3) in the 1 year group
................................................................................................................. 126
Table B.16: Mechanical indentation data for the lateral covered (site 4) in the 1 year
group ........................................................................................................ 127
Table C.1: Cell viability data for the impacted (left) and unimpacted (right) limbs of
animals with acutely torn ACL ................................................................ 129
Table C.2: Pressure ﬁlm data from ACL tear rabbit ................................................. 131

Table C.3: Gross morphological scoring for the traumatic and transected animals. 131

Table C.4: Impact loads and injuries from trial cadaver tests ................................... 132
Table C.5: Isolated joint ACL failure tests performed in the Instron ........................ 134
Table D.l: Cell viability analysis (% dead cells) for the 1-day control animals ....... 140
Table D2: Cell viability analysis (% dead cells) for the 4-day control animals ....... 140
Table D3: Cell viability analysis (% dead cells) for the 4-day P188 animals .......... 141

viii

Table D4: Zonal cell viability analysis (% dead cells) for the 1-day control animals
................................................................................................................. 142

Table D5: Zonal cell viability analysis (% dead cells) for the 4-day control animals
................................................................................................................. 144

Table D6: Zonal cell viability analysis (% dead cells) for the 4-day P188 animals. 146

ix

LIST OF FIGURES

Figure 1.1: Articular cartilage is made up of a solid organic matrix and free intersititial

Figure 1.2:

Figure 1.3:

Figure 2.1:

ﬂuid. The solid matrix consists of Type II collagen and proteoglycans with

chondrocytes irnbedded in the matrix. ................................................... 2
Radiograph of a normal (a) and osteoarthritic (b) knee joint showing

signiﬁcant narrowing of the joint space, a clinical sign of OA ........... 3

Anatomical structures of the knee joint ................................................. 6

The drop tower ﬁxture consisted of a slide track designed to prevent rotation
of the dropped sled during impact. After a single impact the sled was arrested
electronically by an electromagnetic catching device. The impact interface
was a pre-crushed, deformable surface (Hexcel, 3.76 MPa crush strength)
mounted in front of a IOOO-pound load transducer. ............................... 18

Figure 2.2: Impact experiments were performed by dropping a gravity-accelerated mass

Figure 2.3:

Figure 2.4:

Figure 2.5:

Figure 2.6:

onto the ﬂexed tibial-femoral joint with approximately 13 J of potential
energy. The rabbit was oriented such that the deformable interface struck the
distal femur with impact forces oriented axially in the tibia. ................. 19

The posterior half of the subchondral bone was glued to a rectangular
aluminum block which was attached to a rotary microtome. Approximately
7-10 minutes of drying time was allowed, as PBS was continually applied to
the cartilage surface. Approximately 18 slices, each 150 pm thick, was taken
ﬁ'om each facet for analysis. .................................................................. 21

Impact induced contact pressure distributions and contact areas in the tibial
femoral joint were measured by pressure sensitive ﬁlm. Mapping the
pressure distributions onto the tibial plateau showed that the location of
highest contact pressures was largely in the area not covered by the
meniscus. ................................................................................................ 22

The percentage of cells with damaged membranes was manually quantiﬁed
using an image processing and analysis program. Signiﬁcantly more
damaged cells were observed in both the medial and lateral facets of the
impacted samples when compared to the opposite, non-impacted limbs.
Statistical differences in the percentage of cells with damaged membranes are
denoted by an asterisk. Statistical differences were found using a two factors
repeated measures ANOVA with p<0.05 for statistical signiﬁcance. 23

The stained osteochondral explants were imaged and divided into three
zones: superﬁcial, middle, and deep. Cell viability was measured in the thin
sections of cartilage and bone ................................................................ 24

Figure 2.7: Signiﬁcantly more damaged cells were observed in the superﬁcial layer of
the lateral facet when compared to the middle and deep zones. Also,
signiﬁcantly more damaged cells were observed in the superﬁcial zone of the
medial facet when compared to the deep zone; however, no difference was
observed when the superﬁcial zone was compared to the middle zone.
Statistical differences in the percentage of dead cells were denoted by an
asterisk. The statistical analyses were based on two-factor ANOVA’s with
p<0.05 for statistical signiﬁcance .......................................................... 25

Figure 3.1: Impact experiments were performed by dropping a gravity-accelerated mass
onto the ﬂexed tibial-femoral joint with approximately 13 J of potential
energy. The rabbit was oriented such that the deformable interface struck the
distal femur with impact forces oriented axially in the tibia. The tibia was
constrained so as to limit anterior subluxation and prevent ligament damage
................................................................................................................ 36

Figure 3.2: Indentation relaxation tests were performed across the tibial plateau in regions
covered and uncovered by the meniscus. Sites 1 and 3 correspond to regions
uncovered by the meniscus on the medial and lateral facet, respectively. Sites
2 and 4 are located in covered regions on the medial and lateral facet,
respectively ............................................................................................ 37

Figure 3.3: Indentation relaxation testing was performed using a custom built step-motor
device. The tibial plateau was ﬁxed in a specialized camera mounting ﬁxture
and the cartilage was position perpendicular to the spherical indenter..38

Figure 3.4: Histological scoring system used to quantify the characteristics for cartilage
across the tibial plateau .......................................................................... 40

Figure 3.5: Histological analysis showed a signiﬁcant increase in surface lesions for both
the 6 month and 1 year groups (a) and a loss of proteoglycan staining in the 1
year group (b) compared to unimpacted, control limbs. An increase in the
frequency of vertical ((1) and horizontal (c) microcracks at the interface of
articular cartilage and subchondral bone was also documented in both groups
compared to controls .............................................................................. 45

Figure 4.1: Impact experiments were performed by dropping a gravity-accelerated mass
onto the ﬂexed tibial-femoral joint with approximately 13 J of potential
energy. The rabbit was oriented such that the deformable interface struck the
femoral condyle with impact forces oriented axially in the tibia ........... 59

Figure 4.2: 6 mm osteochondral explants were taken in regions uncovered by the
meniscus for cell viability analyses ....................................................... 60

xi

Figure 4.3: Radiograph of the rabbit lower extremity orientation for impacts. The

Figure 4.4:

Figure 4.5:

Figure 4.6:

Figure 4.7:

Figure 4.8:

Figure 4.9:

posterior slope of the tibial plateau creates anterior subluxation of the tibia to
cause ACL rupture ................................................................................. 64

India ink staining revealed acute ﬁssuring and a signiﬁcant amount of
damaged chondrocytes (red) in regions surrounding these surface lesions on
the medial femoral condyle following traumatic ACL rutpure .............. 65

Cell viability analysis showed an increase in the percentage of cells with
damaged plasma membranes in the ACLF joints in all compartments (lateral
femur (LF), medial femur (MF), lateral tibia (LT) and medial tibia (MT))
compared to the contralateral joint tissue .............................................. 66

Severe cartilage erosion was noted on the femoral trochlear ridges in the
traumatic group (a), while only mild erosion was documented in the
transected animals (b). The arrows denote joint osteophytes. The medial
femoral condyles also showed full thickness ulceration of articular cartilage
in the traumatic group (c), but only ﬁbrillation in the transected group ((1).
The medial tibial plateau showed cartilage erosion in the posterior aspect of
the compartment in the traumatic group (C), while the transected rabbits (f)
showed no such erosion in the tibial plateau .......................................... 67

Gross morphological analysis of the medial (M) and lateral (L) (a) femoral
condyle, and (b) tibial plateau after staining with India ink revealed more
cartilage defects in the traumatically injured rabbits. ............................ 68

India ink staining of the lateral menisci highlight meniscal damage as a result
of traumatic ACL injury ......................................................................... 69

Histological sections showed a signiﬁcant increase in the number of vertical
and horizontal microcracks at the articular cartilage/subchondral bone
interface, where (#) denotes statistical signiﬁcance between models. ...70

Figure 4.10: Histological sections of the medial femoral condyles (a & b) and medial

tibial plateau (d) revealed severe surface ﬁbrillation and ﬁssures,
respectively. Proteoglycan loss was noted completely in the femoral sections
(a & b) and at the surface in the medial tibial plateau (c & (1). Horizontal and
vertical micro-cracking was also noted at the ZCC/SB interface as pointed
out ........................................................................................................... 71

Figure 5.1: Cell viability was measured in the thin sections of cartilage and bone. The

stained osteochondral explants were imaged and divided into three zones:
superﬁcial, middle, and deep ................................................................. 86

xii

Figure 5.2: A single, blunt impact to the TF joint produced a signiﬁcant increase in the
percentage of damaged cells in the ‘time zero’ (a) and ‘4 day no P188’ (b)
groups. A ‘*’ indicates a statistically signiﬁcant difference between the
impacted and control limbs .................................................................... 88

Figure 5.3: Administration of P1 88 signiﬁcantly reduced the percentage of damaged cells
in the ‘4 day P188’ group (a) when compared to the ‘4 day no P188’ group

(b) ........................................................................................................... 89

Figure 5.4: P188 reduced the number of damaged cells when compared to the ‘4 Day No
P188’ group (a), while no differences were noted between the ‘4 Day P188’
group and the contralateral, controls (b). A ‘*’ indicates a statistically
signiﬁcant difference between the impacted limbs of the ‘4 day no P188’ and
‘4 day P188’ groups ............................................................................... 90

Figure 5.5: Analysis of zonal data revealed a signiﬁcant increase in the percentage of
damaged cells in the superﬁcial zone of the ‘4 Day No P188’ group
compared to their controls in the (a) LFC, (b) MFC, (c) LTP and (d) MTP.
A’*’ denotes a statistically signiﬁcant difference between the impacted and
contralateral limbs, while ‘+’ denotes a signiﬁcant difference between the
impacted limbs of the ‘4-day no P188’ and the ‘4 day P188’ groups....9l

Figure C. 1: Survival analysis of ACL failure trials indicating probability of ACL failure
at a given load ........................................................................................ 137

Figure C.2: Sample force versus time plot for an ACL failure experiment. The circled
portion indicates ACL failure ................................................................ 138

xiii

RESEARCH PUBLICATIONS

PEER REVIEWED MANUSCRIPTS

Isaac DI, Meyer EG, Haut RC, 2008, Chondrocyte damage and contact pressures
following impact on the rabbit tibiofemoral joint. Journal of Biomechanical Engineering,
130(4): 041018 1-5

Isaac DI, Golenberg N, Haut RC, 2008, Acute repair of chondrocytes using P188
surfactant in the rabbit tibiofemoral joint following blunt impact. In Preparation.

Isaac DI, Meyer EG,'Guillou RP, Dejardin LM, Haut RC, 2008, A traumatic anterior
cruciate ligament rupture model: A preliminary study using the rabbit model. Journal of
Surgical Research, In Review

Killian ML, Isaac DI, Haut RC, Dejardin LM, Leetrm D, Haut Donahue TL, 2008,
Traumatic anterior cruciate ligament tear and its implications on meniscal degradation: A
preliminary novel lapine osteoarthritis model. Journal of Surgical Research, In Press.

PEER REVIEWED ABSTRACTS

Isaac DI, Meyer EG, Kepich E, Haut RC, Acute chondrocyte damage and chronic
changes in the rabbit tibiofemoral joint after impact. 54th Annual Meeting of the
Orthopaedic Research Society, 2008

Killian MK, Lepinski NM, Haut RC, Isaac DI, Haut-Donahue TL, In viva changes in
glycosarninoglycan content in knee meniscal tissue following traumatic injury. 55th
Annual Meeting of the Orthopaedic Research Society, 2009

Lepinski NM, Killian ML, Isaac DI, Haut RC, Haut-Donahue TL, Meniscus tissue
response following tibia-femoral impact. 55th Annual Meeting of the Orthopaedic
Research Society, 2009

Lepinski NM, Killian ML, Isaac DI, Haut RC, Haut-Donahue TL, Characterizing lapine
meniscal tissue: A regional comparison between normal medial and lateral menisci.
ASME Bioengineering Conference, 2009, In Review.

Killian ML, Haut RC, Isaac DI, Dejardin LM, Leetun D, Haut Donahue TL, Traumatic
anterior cruciate ligament tear and its implications on meniscal degradation: A
preliminary novel lapine osteoarthritis model. ASME Bioengineering Conference, 2009,
In Review.

Killian ML, Haut RC, Isaac DI, Lepinski, NM, Regional glcosaminoglycan coverage of
healthy rabbit menisci, ASME Bioengineering Conference, 2009, In Review.

xiv

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Musculoskeletal and joint injuries in the US. have reached epidemic proportions, costing
approximately $300 billion annually. Lower extremity injuries account for approximately
$21.5 billion each year in treatment, rehabilitation and lost work expenses (Miller, 1995).
Furthermore, these injuries are possibly the most predominate cause of disability
resulting from automobile accidents. A recent analysis of the National Accident Sampling
System (NASS) database shows that knee injuries account for nearly 10% of total injuries
resulting from automobile accidents each year (Atkinson, 2000). Injuries can involve
gross fracture of bone (States, 1970; States, 1986; Fife et al., 1984), or so-called
subfracture injuries, such as microtraurna to bone and cartilage (Atkinson & Haut, 2001;
Newberry et al., 1998; Taga et al., 1993; Loomer et al., 1993). As suggested by the
NASS, 75% of knee injuries ﬁ'om automobile accidents are these subﬁacture types,
involving no gross fracture of bone (Atkinson, 2000). These injuries are clinically
relevant to the long-term health of the joint tissues, as both fracture producing injuries as
well as less severe, subfracture types have both been shown to precipitate post-traumatic
joint degeneration (States, 1970; Nagel & States, 1977; Colpin et al., 1990; Upadhyay et
al., 1983).

Osteoarthritis (CA) is one of the most common and widespread rheumatic
diseases responsible for deterioration of articular cartilage, subchondral bone and
synovium, ultimately leading to the failure of synovial joints (Peyron et al., 1984; Badley,
1995; Sangha, 2000). Articular cartilage is a connective tissue that lines the ends of bones

in diarthroidial joints and acts as a “ﬁictionless” surface over which bones can glide.

Articular cartilage consists of a solid organic matrix and free interstitial ﬂuid (primarily
water). The major constituents of the organic matrix of cartilage are collagen (Type II)
and proteoglycans (PGs) (Mow, 1990). Imbedded in the solid matrix are cartilage cells
known as chondrocytes (Figure 1.1). Chondrocytes are responsible for the synthesis and
degradation of the solid matrix constituents. The solid phase (chondrocytes, collagen and
proteoglycans) accounts for 15-30% of the weight of articular cartilage. The remaining
70-85% of the weight is water that maintains the pressurized state of the cartilage.
Collagen provides structural support for the surface tension that is developed by the

pressurized cartilage.

001138511 Proteoglycans
Chondrocyte Articular Cartilage

 

 

Protco glycans

Figure 1.1. Articular cartilage is made up of a solid organic matrix and free intersititial
ﬂuid. The solid matrix consists of Type II collagen and proteoglycans with chondrocytes
imbedded in the matrix.

While the etiology of 0A is currently unknown, the literature suggests that a
breakdown in the homeostasis of the solid matrix and chondrocyte death may contribute
to the progression of this chronic disease. Biomechanically the cartilage material
properties, such as tensile, compressive and shear moduli, change in disease. The
hydraulic permeability of cartilage also changes due to degradation of collagen that
causes an increase in the water content of the tissue and excessive swelling. Clinically,
0A is characterized by joint pain and narrowing of the joint space (Figure 1.2), as
diagnosed by radiological examination (Flores and Hochber, 1998). Pathologically, the
disease exhibits loss of cartilage and sclerosis of underlying subchondral bone. Although
acute injury to cartilage is currently thought to be a factor associated with the
development of 0A, the pathway that leads from a blunt impact load on the joint cartilage

to the development of a chronic disease is yet unknown (Lewis et al., 2003).

 

Figure 1.2. Radiograph of a normal (a) and osteoarthritic (b) knee joint showing
signiﬁcant narrowing of the joint space, a clinical sign of OA.

Experimental studies with animal models have sought to understand the potential

association between acute joint injury and the development of 0A. For example, a recent

study shows that a single, 6 J impact can be delivered to the ﬂexed patello-femoral (PF)
joint of the Flemish Giant rabbit leading to progressive degradation of the retro-patellar
surface as well as thickening of the underlying subchondral bone (Ewers et al., 2002).
Radin et al. (1984) has also shown that cyclic loading of the rabbit tibiofemoral (TF) joint
leads to deep ﬁbrillation of articular cartilage along with a stiffening of the underlying
subchondral bone. Furthermore, a study by Rundell et a1. (2005) indicates that a single 6 J
of energy impact to the rabbit PF joint results in lesions on the surface of the retro-
patellar cartilage that is associated with a signiﬁcant number of damaged chondrocytes
surrounding the lesions.

Previous studies have hypothesized that damage to chondrocytes following
tramnatic injury to cartilage may play a key role in the long term development of OA.
Cartilage function is believed to deteriorate as a result of chondrocyte death (Blanco et
al., 1998). Since chondrocytes are required for matrix repair, and chondrocyte death
eventually leads to matrix loss (Simon et al., 1976), chondrocyte death either by
apoptosis or necrosis has become a focus of OA research and more recently, cartilage
trauma research. Necrotic cell death occurs when a cell is severely injured by physical
stress. Damage to the plasma membrane prevents the cell from controlling its ﬂuid and
ion balance. Therefore, a deﬁning feature of necrotic cells is swelling and ultimately,
rupture (Duke et al., 1996). Conversely, apoptosis is programmed cell death in which the
cell undergoes biochemical changes leading to death (Hashimoto et al., 1998). Simon and
Green (1971), studying the short-term effects of chondrocyte death induced by freezing,
noted a signiﬁcant loss of stainable proteoglycans without altered collagen content or

surface ﬁbrillation. After one year the articular cartilage of rabbit knees showed

biochemical and morphological changes typical of degenerative joint disease (Simon et
al., 1976). Normal chondrocytes respond to moderate or low-amplitude dynamic
compression by upregulating biosynthetic activity, a property that may contribute to a
tissue’s ability to withstand compressive loading (Palmoski et al., 1978; Parkkinnen et al.,
1992; Sah et al., 1991). Injured chondrocytes may not respond to dynamic mechanical
stimulation, either because the cells have lost the ability to do so, or because damage to
the extracellular matrix has disrupted the ability for the cells to respond to physical
signals, which stimulate biosynthetic activity (Kurz et al., 2001). These data suggest that
preventing chondrocyte death and/or matrix damage after excessive levels of blunt
loading may help maintain the mechanical integrity of the cartilage; and thereby, helping
to mitigate the onset of post-traumatic CA.

With increasing emphasis on physical ﬁtness and a healthy lifestyle in all age
groups, participation in sports, recreation and exercise (SRE) is increasingly popular and
widespread in American culture. History of joint injury, particularly to the knee or hip
increases the risk of chronic joint disease, particularly OA. Two speciﬁc acute injuries
are strongly associated with development of knee OA; cruciate ligament damage and
meniscal tears (F elson, 2004) (Figure 1.3). The anterior cruciate ligament (ACL) is a
ligament on the interior of the knee joint that restricts anterior motion of the tibia relative

to the femur.

              

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The ACL is the most frequently injured ligament in the knee joint. An estimated
80,000 ACL tears occurred in the year 2000 in the US. alone (Grifﬁn et al., 2000)
costing approximately $2 billion annually (Hewett et al., 2006). Recent studies from our
laboratory on ACL tear mechanisms document that high compressive loading in the
human knee joint, such as that generated during a jump landing, may lead to ACL rupture
with subsequent damage to cartilage and underlying subchondral bone. For example, in a
recent study using isolated human cadaver knees our laboratory has shown that excessive
compression of the TF joint resulted in a signiﬁcant anterior subluxation of the tibia
causing ACL tear (Meyer and Haut, 2005; Meyer et al., 2008). Since the current literature
suggests that the occurrence of post-trauma joint 0A does not seem to depend on whether
the ACL is reconstructed or not (Myklybust and Bahr, 2005), this “micro-trauma” to
subchondral bone and acute cartilage damage may play a large role in the long term
progression of chronic joint disease.

Clinically, ACL ruptures are often associated with subchondral bone lesions
thought to be caused by signiﬁcant axial loads transmitted through the cartilage and
subchondral bone during injury. These bone lesions are thought to be the basis for
geographic bone bruises which are documented in over 80% of patients suffering knee
ligament injury (Johnson et al., 1998). These bone injuries are also associated with visible
damage to chondrocytes in the overlying articular cartilage. Vellet et a1. (1991)
documents an overt loss of cartilage overlying these geographic bone bruises in 48% of
ACL rupture patients within six months of injury. These occult microcracks of
subchondral and/or trabecular bone are thought to be caused by compressive loading

during the acute ligamentous injury. A recent study evaluates the acutely ACL injured

knee with MRI and documents that 57% of all knees suffered from at least one cortical
depression fracture due to large compressive loading in the joint during the acute
ligament injury (Frobell et al., 2008). This study concludes that these cortical depression
fractures, likely to be hallmarks of strong compressive forces, indicate severe injury to
the cartilage and subchondral bone after ACL injury and may represent risk factors for
CA development in the ACL injured knee. Thus, the increased risk of knee OA after
ACL injury might, in part, be dependent on the initial trauma as a contributing cause,
explaining the lack of success in reducing post-trauma CA by surgical interventions
(Frobell et al., 2008).

Currently, the most widely used experimental model of 0A is joint instability via
anterior cruciate ligament transection (ACLT) (Batiste et al., 2004; Yoshioka et al., 1996;
Sah et al., 1997; Vignon et al., 1987; Tiraloche et al., 2005). ACLT has been shown to
lead to progressive changes in the morphology, histopathology, and biochemistry of
articular cartilage and subchondral bone in the rabbit model (Batiste et al., 2004; Vignon
et al., 1987; Tiraloche et al., 2005). Furthermore, MRI evaluation of rabbit knee joints
after ACLT shows mild joint effusion (Batiste et al., 2004). This joint instability model of
CA has proven effective in generating chronic joint changes consistent with post-
traumatic joint disease; however, the model fails to address acute damage to cartilage
cells and subchondral bone as a result of large compressive loads generated in the joint
during the acute injury, as documented in the clinical literature. Acute chondrocyte
damage, subchondral bone lesions and/or instability of the joint generated as a result of

acute ACL rupture are suspected factors in the progression of chronic joint disease.

Understanding the mechanisms that lead from acute cartilage damage and the
chronic progression of 0A are essential in future developments of therapeutic methods to
either prevent or treat joint degeneration. The research presented in this thesis makes use
of an in viva rabbit model to examine the acute and chronic responses of articular
cartilage and subchondral bone to blunt force trauma. Chapter 2 addresses the issue of
acute damage to chondrocytes following a single, severe insult to the ﬂexed TF joint. The
study hypothesized that a single insult delivered to the ﬂexed rabbit TF joint, without
gross fracture to bone or ligament, would result in signiﬁcant damage to chondrocytes.
Chapter 3 describes chronic studies where a single impact was again delivered to the TF
joint of anesthetized rabbits and the changes in the mechanical and histological properties
of the articular cartilage were evaluated six months and one year following trauma.
Chapter 4 documents the development of a “Bona Fide model” for in viva, traumatic
ACL rupture. This “ﬁrst of its kind” model is compared to the widely used ACLT
method. This study hypothesized that compressive loads generated in the joint during
traumatic ACL mpture would result in signiﬁcantly more damage to articular cartilage
and subchondral bone compared to the conventional ACLT model. Chapter 5 evaluates
the efﬁcacy of a mild non-ionic surfactant, poloxamer 188 (P1 88), in ‘repairing’ damaged
cells after an in viva impact to the rabbit TF joint.

The research presented in this thesis provides useful data in regards to the
response of articular cartilage to blunt impact loading in the in viva setting. Furthermore,
a therapeutic treatment has been investigated and found to be effective in preventing
damage to chondrocytes following traumatic injury, which may prevent long term

degradation of articular cartilage. A “Bona Fide” model of in viva, traumatic ACL

rupture has also been developed to address the long term implications of damage to
articular cartilage and subchondral bone during the acute ligamentous injury. Future
studies can utilize the data presented to investigate the progression of chronic joint
disease and the efﬁcacy of various intervention methods to mitigate post-trauma OA

following rupture of the ACL.

10

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Atkinson PJ, Haut RC, 2001 , “Injuries Produced by Blunt Trauma to the Human
Patellofemoral Joint Vary with F lexion Angle of the Knee,” J Orthop Res, 19, 827-833.

Atkinson T, Atkinson P, 2000, “Knee Injuries in Motor Vehicle Collisions: A Study of
the National Accident Sampling System Database for the Years 1979- 1995,” Ace. Anal.
Prev., 32, pp. 779-786.

Bahr R, Myklebust G, 2005, “Return to Play Guidelines after Anterior Cruciate Ligament
Surgery,” Br J Sports Med, 39, pp. 127-131.

Batiste, D., Kirkley, A., Laverty, S., Thain, L., Spouge, A., Holdsworth, D., 2004. Ex
viva characterization of articular cartilage and bone lesions in a rabbit ACL transection
model of osteoarthritis using MRI and micro-CT. 0A Cart. 12, 986-996.

Blanc FJ, Guitian R, Vazquez-Martul E, DeTorro FJ, Galdo F, 1998, “Osteoarthritis
Chondrocytes Die by Apoptosis: A Possible Pathway for Osteoarthritis Pathology,” Arth
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Duke RC, Ojcius DM, Young JD, 1996, “Cell Suicide in Health and Disease,” Scientiﬁc
American, 275, pp. 80-87.

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 Patello-
Femoral Joint,” J Orthop Res, 20, pp.545-550.

Felson D, 2004, “An Update on the Pathogenesis and Epidemiology of Osteoarthritis,”
Radiologic Clinics North Am, 42, pp. 1-9.

Flores R, Hochber M, 1998, “Deﬁnition and Classiﬁcation of Osteoarthritis,” Brandt K,
Doherty M, Lohmander S, editors. Osteoarthritis. Oxford: Oxford University Press, pp. 1-
12.

Frobell RB, Roos HP, Roos EM, Hellio Le Graverand MP, Buck R, Tarnez-Pena J,
Totterrnan S, Boegard T, Lohmander LS, 2008, "The Acutely ACL Injured Knee
Assessed by MRI: Are Large Volume Traumatic Bone Marrow Lesions a Sign of Severe
Compression Injury?,” Osteoarthritis and Cartilage, 16, pp. 829-836.

Grifﬁn L, Agel J, Albohm M, 2000, “Non-contact Anterior Cruciate Ligament Injuries:
Risk Factors and Prevention Strategies,” J Am Acad Orthop Surg, 8, pp. 141-150.

Hashimoto S, Ochs R, Komiya S, Lotz M, 1998, “Linkage of Chondrocyte Apoptosis and
Cartilage Degradation in Human Osteoarthritis,” Arth Rheum, 41, pp. 1632-1638.

Hewett T, Myer G, Ford K, 2006, “Anterior Cruciate Ligament Injuries in Female
Athletes: Part 1, Mechanisms and Risk Factors,” Am J Sports Med, 34, pp. 299-311.

11

Johnson DL, Urban WP, Carbon DNM, Vanarthos WJ, Carlson CS, 1998, “Articular
Cartilage Changes Seen with Magnetic Resonance Irnaging-Detected Bone Bruises
Associated with Acute Anterior Cruciate Ligament Rupture,” Am J Sports Med, 26, pp.
409-414.

Kurz B, Jin M, Patwari P, Cheng DM, Lark MW, Grodzinsky AJ, 2001, “Biosynthetic
Response of Mechanical Properties of Articular Cartilage after Injurious Compression,” J
Orthop Res, 19, pp. 1140-1146.

Lewis J L, Deloria LB, Oyen-Tiesma M, Thompson RC Jr., Ericson M, Oegema TF Jr.,
2003, “Cell Death alter Cartilage Impact Occurs Around Matrix Cracks,” J Orthop Res,
21, 881-887.

Meyer E, Baumer T, Slade J, Smith W, Haut R, 2008, ”Tibiofemoral Contact Pressures
and Osteochondral Microtrauma During ACL Rupture due to Excessive Compressive
Loading and Internal Torque of the Human Knee,” Am. J. Sports Med, 36, pp. 1966-
1977.

Meyer E, Haut R, 2005, “Excessive Compression of the Human Tibio-Femoral Joint
Causes ACL Rupture,” J Biomech, 38, pp. 2311-2316.

Miller TR, Martin PG, Crandell JR, 1995, “Cost of Lower Limb Injuries in Highway
Crashes,” Proc ICPLEI, pp. 47-57.

Mow VC, 1990, “Fundamentals of Articular Cartilage and Meniscus Biomechanics,”
Articular Cartilage and Knee Joint Function: Basic Science and Arthroscopy, J .W.
Ewing, ed., Raven Press, Ltd., New York, pp. 1-18.

Nagel WN, States J, 1977, “Dashboard and Bumper Knee-Will Arthritis Develop?,”
AAAM21, pp. 272-278.

Newberry WN, Garcia JJ, Mackenzie CD, Decamp CE, Haut RC, 1998, “Analysis of
Acute Mechanical Insult in an Animal Model of Post-Traumatic Osteoarthritis,” J
Biomech Eng, 120, pp. 704-9.

Parkkinen JJ, Larnmi MJ, Helminen HJ, Tammi M, 1992, “Local Stimulation of
Proteoglycan Synthesis in Articular Cartilage Explants by Dynamic Compression In
Vitro,” J Orthop Res, 10, pp. 610-620.

Radin EL, Martin RB, Burr DB, Caterson B, Boyd RD, Goodwin C, 1984, “Effects of
Mechanical Loading on the Tissues of the Rabbit Knee,” J Orthop Res, 2, pp. 221-234.
Rundell SA, Baars DC, Phillips DM, Haut, RC., 2005, ”The Limitation of Acute Necrosis
in Retro-Patellar Cartilage after 3 Severe Blunt Impact to the In-Vivo Rabbit Patello-
Femoral Joint,” J Orthop Res, 23, pp.l363-1369. '

12

Sah RL, Yang AS, Chen AC, Hant JJ, Halili RB, Yoshioka M, Amie] D, Coutts RD,
1997, “Physical Properties of Rabbit Articular Cartilage after Transection of the Anterior
Cruciate Ligrnaent,” J Orthop Res, 15, pp. 197-203.

Simon WH, Richardson S, Herman E, Parsons JR, Lane J, 1976, “Long-term Effects of
Chondrocyte Death on Rabbit Articular Cartilage In Vivo,” J Bone Joint Surg Am, 58,
pp. 517-526.

States J, 1986, “Adult Occupant Injuries of the Lower Limb,” Proc Symp Biomech, pp.
97-107.

States JD, 1970, “Traumatic Arthritis: A Medial Dilemma,” Proc 14th Ann Conf Am
Assoc Automotive Med, 14, pp. 21-28.

Tiraloche G, Girard C, Chouinard L, Sampalis J, Moquin L, Ionescu M, Reiner A, Polle
AR, Laverty S, 2005, “Effect of Oral Glucosarnine on Cartilage Degradation in a Rabbit
Model of Osteoarthritis,” Arth Rheum, 54, pp. 1118-1128.

Vellet AD, Marks PH, Fowler PJ, Munro TG, 1991, “Occult Posttraurnatic Osteochondral
Lesions of the Knee: Prevalence, Classiﬁcation, and Short-Tenn Sequelae Evaluated with
MR Imaging,” Radiology, 178, pp. 271-276.

Vignon, E., Bejui, J ., Mathiew, P., Hartrnann, J., Ville, G., Evreux, J ., Descotes, J ., 1987.
Histological cartilage changes in a rabbit model of osteoarthritis. J. Rheum. 14, 104-106

Yoshioka, M., Coutts, R., Anriel, D., 1996. Characterization of a model of osteoarthritis
in the rabbit knee. OA and Cart. 4, 87-98.

13

CHAPTER TWO

CHONDROCYTE DAMAGE AND CONTACT PRESSURES
FOLLOWING IMPACT ON THE RABBIT TIBIOFEMORAL JOINT

ABSTRACT

Epidemiological studies show that tibial plateau ﬁ'actures comprise about 10% of all
below-knee injuries in car crashes. Studies from this laboratory document that impacts to
the tibiofemoral (TF) joint at 50% of the energy producing gross fracture can generate
cartilage damage and micro-cracks at the interface between calciﬁed cartilage and
underlying subchondral bone in the tibial plateau. These injuries are suggestive of the
initiation for a long term chronic disease, such as osteoarthritis. The disease process may
be further encouraged by acute damage to chondrocytes in the cartilage overlying areas
of occult micro-cracking. The hypothesis of the current study was that signiﬁcant
damage to chondrocytes in tibial plateau cartilage could be generated in areas of high
contact pressure by a single impact delivered to the rabbit TF joint, without a gross
fracture of bone. Three rabbits received a single, 13 J of energy blunt insult to the
tibiofemoral joint, while another three animals were used as controls. Cell viability
analyses compared chondrocyte damage in impacted versus control cartilage. Two
additional rabbits were impacted to document contact pressures generated in the
tibiofemoral joint. The study showed high contact pressures in uncovered areas of the
plateau, with a trend for higher pressures in the lateral versus medial facets. A
signiﬁcantly higher percentage of damaged chondrocytes existed in impacted versus the
opposite, non-impacted limbs. Additionally, more chondrocyte damage was documented

in the superﬁcial zone (top 20% of cartilage thickness) of the cartilage compared to

14

middle (middle 50% of thickness) and deep (bottom 30% of thickness) zones. This study
showed that a single blunt insult to the in situ rabbit TF joint, generating large areas of
contact pressure exceeding 20MPa produce signiﬁcant chondrocyte damage in the tibial
articular cartilage, esp. in the superﬁcial zone, without gross fracture of bone. Future
studies will be needed to investigate the long term, chronic outcome of this blunt force

joint trauma.

15

INTRODUCTION

Injuries to the lower extremity are possibly the most predominant cause of
disability resulting from automobile accidents. Analysis of the National Automotive
Sampling System/Crashworthiness Data System (NASS) shows that knee injuries
account for approximately 10% of the total injuries resulting ﬁ'om automobile accidents
each year (Atkinson & Atkinson, 2000). Epidemiological studies show that tibial plateau
ﬁ'actures comprise about 10% of all below-knee injuries in car crashes (Taylor et al.,
1997; Sherwood et al., 1999). These injuries carry a poor prognosis because they disrupt
the articular cartilage in a weight-bearing joint, which can lead to long-term
complications such as malunion and osteoarthritis (Funk et al., 2000). The NASS data
also suggests that 75% of knee injuries result in no gross bone fracture (Atkinson &
Atkinson, 2000). Previous studies on cadaver joints indicate impacts on the tibiofemoral
(TF) joint at 50% of the fracture energy can generate micro-cracks at the cement line
under the tibial plateau (Banglmaier et al., 1999). Interestingly, automobile accident
victims reporting knee pain with no gross bone ﬁacture show bone bruises in
approximately 25% of cases (Atkinson et al., 2008). Bone bruises are also documented in
over 80% of patients suffering knee ligament injury (Johnson et al., 1998). These bone
injuries are also associated with visible damage to chondrocytes in the overlying articular
cartilage. Since the current literature indicates these patients will likely generate a chronic
disease in the injured joint, whether they are reconstructed or not (Bahr & Myklebust,
2005), a working hypothesis of this laboratory is that compressive loads generated in the
knee during ACL rupture may initiate a chronic disease due to acute damage of

chondrocytes in joint cartilage. The objective of the current study was to develop a small

16

animal model for study of the potential for chronic disease following impact loading of
the TF joint, without causing gross bone fracture, that generates signiﬁcant cartilage cell
damage.

Our laboratory has previously developed an in viva impact model using the
patello-fernoral (PF) joints of Flemish Giant rabbits (Ewers et al., 2002). Softening of the
retro-patellar cartilage and thickening of the underlying subchondral bone has been
observed within one year. Signiﬁcant histological changes, such as the loss of
proteoglycan staining, ossiﬁcation and erosion of the retro-patellar cartilage have also
been observed in the impacted limbs within three years. These changes are consistent
with early stages of osteoarthritis (Pritzker, 1998). A recent study by this laboratory has
also indicated that lesions produced on the surface of retro-patellar cartilage are
associated with a signiﬁcant number of damaged chondrocytes (Rundell et al., 2005).
Acute damage to these cells is currently thought to be associated with the long term
development of osteoarthritis (Colwell et al., 2001; Blanoo et al., 1998). The hypothesis
of the current study was that a single, severe level of blunt force delivered to the rabbit
TF joint could produce high contact pressures and a signiﬁcant number of damaged
chondrocytes in the articular cartilage overlying the tibial plateau, without gross fracture
of bone in the joint. The future plan is then to use this model to study long term
consequences of an acute blunt force trauma in a live animal.

MATERIALS AND METHODS

Eight skeletally mature Flemish Giant rabbits (5.7 :t 0.1 kg) were used in the

study. The investigation was approved by the Michigan State University All-University

Committee on Animal Use and Care. All animals were housed in individual cages (152 x

17

152 x 36 cm) prior to the study. Three rabbits received a blunt force insult to the left TF
joint using a previously described drop tower (Ewers et al., 2002; Rundell et al., 2005),
with a newly designed restraint system. All animals were sacriﬁced with 85.9 mg/kg BW
Pentobarbital I.V., prior to impact. Three non-impacted animals served as controls.
Another two animals were used to measure joint contact pressures developed during the
impact.

The drop tower used a sled that was arrested electronically after one impact. A
pre-crushed, deformable impact head (Hexcel, 3.76 MPa crush strength) was used to

ensure uniform loading over the femur (Figure 2.1).

Impact Sled
Load

Transducer Slide Track
Electromagnetic
catching device

Deformable

Interface Animal

restraint ﬁxture

 

Figure 2.1. The drop tower ﬁxture consisted of a slide track designed to prevent rotation
of the dropped sled during impact. After a single impact the sled was arrested
electronically by an electromagnetic catching device. The impact interface was a pre-
crushed, deformable surface (Hexcel, 3.76 MPa crush strength) mounted in front of a
1000-pound load transducer.

The impact interface was mounted in front of a 4.45 kN (1000 lb) load transducer (model
AL311CV, lOOOlb capacity, Sensotec, Columbus, OH). Pilot studies with a 1.33 kg mass
dropped from 0.7 m (9.1 J of potential energy) generated approximately 737 i 68.9 N of

impact force on the joint, but it did not alter the mechanical properties of TF joint

cartilage (Meyer, 2004). In the current study the impact mass was increased to 1.75 kg,
and it was dropped from 0.75 m (~13 J).

The animal was laid supine in the ﬁxture (Figure 2.2). The knee was ﬂexed 90°.
The foot was ﬁxed in a boot with three Velcro straps. Two Velcro straps were crossed
over the femur. The tibia was constrained to limit anterior motion of the tibia during

impact. The leg was positioned so that the dropped mass struck the distal femur and

axially loaded the tibia.
Gravity accelerated
mass Electronically
triggered clap
Load transducer
Deformable interface

 

 

constraint J

Figure 2.2. Impact experiments were performed by dropping a gravity-accelerated mass
onto the ﬂexed tibial-femoral joint with approximately 13 J of potential energy. The
rabbit was oriented such that the deformable interface struck the distal femur with impact
forces oriented axially in the tibia.

 

 

 

 

 

Hind limbs of another two animals were used to measure TF contact pressures
and contact areas ﬁom the impact. Pressure sensitive ﬁlm packets (Prescale, Fuji Film
Ltd., Tokyo, Japan) were inserted through anterior and posterior joint capsules. After
impact the ﬁlm was removed from the packet and scanned (Scanmaker MRAS-1200E6,
Microtek, Taiwan). The entire area of contact was digitized (Photostyler, version 1.1A,
Aldus Co., Seattle, WA) at 150 dpi in 8-bit gray scale. The gray scale was converted to

pressure (Scion Image 2.0, 2005) and the average pressure, total contact area and total

19

area having pressure over 20 MPa were determined using established protocols (Atkinson
et al., 1998).

For the three impact-control and the three control-control specimens, the knee
joints were cleaned and the articular capsule was opened. The meniscal location on the
tibial plateau was photographed. A diamond saw (Isomet 11-1180 Low Speed Saw,
Buehler, Lake Bluff, IL.) was used to split the tibial plateau into medial and lateral
compartments. The facets were then undercut leaving 1-2 mm of bone and rinsed 3 times
with culture media before being placed in separate wells ﬁlled with ﬁ'esh Dulbecco’s
modiﬁed Eagle’s medium (DMEM): F12 (Gibco, USA #12500-062) supplemented with
10% fetal bovine serum, additional amino acids, and antibiotics (penicillin 100 units/ml,
streptomycin 1 ug/ml, arnphotericin B 0.25 rig/ml), and incubated for 24 hours (3 7°C and
95% humidity) to help maximize the percentage of damaged cells (Ewers et al., 2001;
Phillips et al., 2004).

Following incubation, the specimens were prepared for cell viability analyses.
The posterior half of each facet was ﬁxed to a rectangular aluminum block (Figure 2.3)
attached to a rotary microtome (Model 45; Lipshaw Mfg, Detroit, MI) using glue (Zap-
A-Gap, Pacer Tech., Rancho Cucarnonga, CA). After 7-10 minutes, while the specimen
was sprayed with phosphate buffered saline (PBS), approximately 18 slices, 150 pm thick

were cut and placed in individual wells with fresh media.

20

Blade

 

Figure 2.3. The posterior half of the subchondral bone was glued to a rectangular

aluminum block which was attached to a rotary microtome. Approximately 7-10 minutes

of drying time was allowed, as PBS was continually applied to the cartilage surface.

Approximately 18 slices, each 150 pm thick, was taken from each facet for analysis.
The cell viability analyses followed previous procedures (Rundell et al., 2005). Brieﬂy,
four slices from each facet were rinsed with PBS and stained with calcein AM and
ethidiurn homodirner (EthD-l), according to the manufacturer’s speciﬁcations
(Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene, OR). Prior to imaging, each
sample was rinsed three times with PBS. Viable cells were distinguished by the presence
of ﬂuorescent (green) calcein AM. Damaged cells were distinguished by a bright red
ﬂuorescence. The number of green and red cells was manually counted with an image
analysis program (Image J, National Institutes of Health, 2004).

The number of total cells and the percentages of damaged (red) cells in opposite
limbs were compared. Data from four slices on each facet were averaged, and the data
from each of the three animals was combined. Two factors (limbs, facet) repeated
measures ANOVA (Sigma Stat, SPSS Inc., Chicago, IL) compared the percentage
damaged cells in opposite limbs and as a function of facet. Paired t-tests were used to

evaluate differences in contact pressures and areas between facets. Statistically

signiﬁcant differences were indicated at p<0.05.

21

RESULTS

Examination of joints after impact indicated no gross bone fractures or ligament
damage. The meniscus was always intact. The average peak, inertially-compensated load
and impact duration were 1175 i 29.8 N and 23.0 i 0.2 ms, respectively (n=3).

Post-test mapping of the pressure ﬁlm location onto the plateau indicated high
contact pressures centered largely in the uncovered areas (Figure 2.4). The average
contact pressures on the medial and lateral facets were 19.6 at 1.2 MPa (n=4) and 23.9 i
3.7 MPa (n=4), respectively. While the pressure on the lateral facet was approximately
18% higher than on the medial facet, this difference was not signiﬁcant (p=0.25). The
average maximum peak pressures on the medial and lateral facets were 43.1 :I: 5.8 MPa

and 45.4 :I: 7.2 MPa, respectively.

   

Medial —' Lateral ,

Figure 2.4. Impact induced contact pressure distributions and contact areas in the tibial
femoral joint were measured by pressure sensitive ﬁlm. Mapping the pressure
distributions onto the tibial plateau showed that the location of highest contact pressures
was largely in the area not covered by the meniscus.

Analysis of contact area having pressures between 20 to 49 MPa showed no difference
(p=0.24) between the lateral and medial facets, being approximately 17.1 :t 1.2 mm2 and
13.7 d: 4.4 m2, respectively.

No signiﬁcant differences existed in the percentage of viable cells (n = 3) between

right and left control limbs (p=0.21). No signiﬁcant differences were measured between

right and left limbs in the lateral (p=0.54) or medial (p=0.24) facets. Therefore, these data

22

were combined. The percentages of damaged cells in the lateral and medial facets were
24.9 :I: 12.3% and 22.4 i 9.7%, respectively.

Cell viability analyses in each facet indicated signiﬁcant differences in the
percentages of damaged cells in impacted versus unimpacted limbs for the medial
(p=0.01) and lateral (p<0.001) compartments (Figure 2.5). There was also a trend

(p=.067) for more damaged cells in the lateral than medial facets of impacted limbs.

 

'3 70' * Ilmpacted
3 60. DNon-Im acted
E 3 50. a: p
2 E 4o-
: ‘5’ 30- I

d,
g 2 20-
o 10.
.\°

0. *

 

 

 

 

Lateral Medial

Figure 2.5. The percentage of cells with damaged membranes was manually quantiﬁed
using an image processing and analysis program. Signiﬁcantly more damaged cells were
observed in both the medial and lateral facets of the impacted samples when compared to
the opposite, non-impacted limbs. Statistical differences in the percentage of cells with
damaged membranes are denoted by an asterisk. Statistical differences were found using
a two factors repeated measures ANOVA with p<0.05 for statistical signiﬁcance.

Finally, slices from impacted limbs were photographed and the cartilage layer was
divided into three zones: superﬁcial (top 20%), middle (middle 50%), and deep (bottom

30%) (Figure 2.6).

23

Superﬁcial EIZII'IE = ElIl'i-‘E Elf thicltrlaaa

l'-.-'iil:iI:l|E! ZONE: . 31-“. [If tl'IlIZI-=:ZI'IESS

Deer - Elf tl‘ril::l-:;:r| E :5; :.

 

Figure 2.6. The stained osteochondral explants were imaged and divided into three
zones: superﬁcial, middle, and deep. Cell viability was measured in the thin sections of
cartilage and bone.
Signiﬁcantly more damaged cells existed in the superﬁcial layer of the lateral facet
compared to the middle (p<0.001) and deep (p<0.001) zones (Figure 2.7). In contrast, in

the medial facet the superﬁcial zone had more damaged cells than the deep zone

(p=0.043), but not more than the middle zone (p=0.13).

24

 

80-

 

. Lateral El Medial

jiii

Superﬁcial Middle Deep

 

7o-
60-
50-
4o-
30-
2o-
10-

% Cells with Damaged Membranes

 

 

 

 

0-

Figure 2.7. Signiﬁcantly more damaged cells were observed in the superﬁcial layer of
the lateral facet when compared to the middle and deep zones. Also, signiﬁcantly more
damaged cells were observed in the superﬁcial zone of the medial facet when compared
to the deep zone; however, no difference was observed when the superﬁcial zone was
compared to the middle zone. Statistical differences in the percentage of dead cells were
denoted by an asterisk. The statistical analyses were based on two-factor ANOVA’s with
p<0.05 for statistical signiﬁcance.
DISCUSSION
This study showed that high intensity impacts produced contact pressures on the
medial and lateral tibial facets of approximately 20 MPa and 24 MPa, respectively.
Furthermore, the 13 J of impact potential energy produced signiﬁcant chondrocyte
damage in articular cartilage overlying the tibial plateau. These data were consistent with
Tor'zilli et a1. (1999) using bovine chondral explants where signiﬁcant necrosis was
documented at contact pressures of 15-20 MPa. Repo and Finlay (1977), on the other
hand, show that contact pressures greater than 25 MPa generate signiﬁcant chondrocyte

necrosis using in vitra human osteochondral explants. The current study also showed a

tendency for higher contact pressures and more contact area with pressures Z 20 MPa in

25

the lateral versus medial facet. These data correlated with a statistical trend for a greater
percentage of damaged cells in lateral versus medial facets (Figure 6). Impact loading
also increased the percentage of damaged chondrocytes in lateral and medial facets by
approximately 18% and 14%, respectively. In a similar study using the rabbit PF joint, a
6 J impact, with a rigid interface, resulted in a 15% increase in damaged cells in retro-
patellar cartilage (Rundell et al., 2005). Another study by this laboratory indicates that the
PF contact pressures were ~ 27 MPa for similar impacts (N ewberry et al., 1998).

Our ﬁndings also indicated signiﬁcantly more damaged cells in the superﬁcial
(top 20% of cartilage thickness) than in the middle and deep zones. Other studies
document signiﬁcant damage to chondrocytes in the superﬁcial and middle zones of
bovine chondral explants subjected to low rate (3 5MPa/s) unconﬁned compression at
contact pressures of 15-20 MPa (Torzilli et al., 1999). At pressures greater than or equal
to 20 MPa, the former study also documents cell death throughout the entire thickness of
the explants. Krueger et al. (2003) documents that high rate (~500 MPa/s), unconﬁned
compression experiments to 25 MPa on bovine chondral and osteochondral explants yield
approximately 50% and 30% cell death in the superﬁcial zones, respectively. The former
study also documents cell death throughout the middle zone, but not the deep zone of
either chondral or osteochondral explants. The current study generated contact pressures
of approximately 1000 MPa/s, and it also showed that the deep zone had signiﬁcantly
less cell death than the superﬁcial zone. A recent study, using an open joint with a rigid
impact interface on the rabbit femoral condyle, documents cell death initiating in the
superﬁcial layer of cartilage at ~ 20MPa (for 420MPa/s) near the edges of the impactor

and more unifome across the entire contact area at 25MPa . Thus, the distribution of

26

cell death through the cartilage thickness in the current in situ study generally paralleled
with the ﬁndings of Krueger et al (2003) on osteochondral explants and Milentijevic et a1
(2005) using an open joint, in situ rabbit model. The former study also showed that cell
death increases in depth with increasing contact pressures (2.8 i 2% thickness/MPa),
until full thickness death at contact pressures Z 40 MPa (Milentijevic et al., 2005). Based
on these in situ studies, the investigators conducted in viva experiments at 35MPa (for
420 MPa/s) and document “arthritic” changes in the joint by 3 weeks post injury.

There were a number of limitations in the current study. One limitation was the
potential effect on joint contact mechanics of inserting Fuji Film packets into the rabbit
joint. Theoretical studies have shown that the measured contact pressures may be in error
by 14-28 percent (Wu et al., 1998). But, by using the Fuji Film method, we are able to
compare the current results with previous studies by this laboratory from the in situ rabbit
and the human PF (Atkinson and Haut, 2001) and TF joints (Banglmaier et al., 1999).
Another limitation of the current study was that we did not accurately locate the position
of each slice on the facet with respect to the meniscus. These data would have provided
more information to correlate the exact distribution of damaged cells with respect to the
overlying contact pressure. The sample size of the current study was also small leading to
relatively large standard deviations in chondrocyte viability data. This may have
contributed to a low statistical power between zonal data, for example. While signiﬁcant
cell damage was documented in both impact and non-irnpacted slices, we discounted co-
culturing with bone as the problem since we previously co-cultured osteochondral
explants for a longer period without a problem (Krueger et al., 2003). Rather, while we

attempted to optimize our cutting methods, we still believe the baseline cell damage in all

27

sections was likely due to a cutting artifact in the making of thin slices of cartilage on
bone. But, this existed in all slices and we were still able to measure a statistical effect
due to impact loading. We were ﬁrrther concerned that removal of the thin tibial cartilage
ﬁ'om the underlying bone across a complex contour of the plateau might also cause
signiﬁcant damage to chondrocytes, especially in the deep zone.

While 22-25% cell death in unimpacted, control specimens may seem quite high
it does seem to be in accordance with current literature documenting baseline
chondrocyte death in a variety of animal models. Particularly, Gulata et a1 and Rundell et
al (2005) document approximately 19% dead cells in unloaded rabbit femoral condyle
articular cartilage and 12% cell death in unloaded rabbit patella, respectively. To the
authors knowledge, there currently exists no literature documenting baseline cell death
percentages in the unloaded rabbit tibial plateau. Furthermore, we do not believe there to
be 100% viable cells in unloaded articular cartilage, possibly due to the fact that there is
no blood supply to provide the agents for rapid removal of damaged cells, as in may other
types of tissue (Roach and Clarke, 2000). Most importantly, however, is that this study
does document a statistical increase in the percentage of dead cells in the impacted
articular cartilage when compared to the unloaded controls.

A major outcome of the current study was the establishment of a “closed joint”
model for in viva loading of the TF joint. The model will be utilized in future
investigations to study potential correlations of acute cell damage with the pathogenesis
of a post—traumatic 0A in the joint. Studies can then proceed, for example, to investigate
the long term efﬁcacy of intervention methods to acutely repair damaged cell membranes

in the impacted articular cartilage (Phillips et al., 2004).

28

REFERENCES

Atkinson PJ, Cooper TG, Anseth SM, Walter NE, Kargus R., Haut RC, 2008, “Knee
Bone Bruies Frequencies Vary with Time Post-Injury and Type of Soft Tissue Injury,”
Orthop, 31, pp. 440.

Atkinson PJ, Haut RC, 2001, “Impact responses of the ﬂexed human knee using a
deformable impact interface,” J. Biomech.Eng., 123(3):205-211.

Atkinson PJ, Newberry WN, Atkinson TS, Haut RC, 1998, “A Method to Increase the
Sensitive Range of Pressure Sensitive Film,” J. Biomech, 31, pp. 855-859.

Atkinson T, Atkinson P, 2000, “Knee Injuries in Motor Vehicle Collisions: A Study of
the National Accident Sampling System Database for the Years 1979- 1995,” Ace. Anal.
Prev., 32, pp. 779-786.

Bahr R, Myklebust G, 2005, “Return to Play Guidelines after Anterior Cruciate Ligament
Surgery,” Br. J. Sports Med., 39, pp. 127-131.

Banglmaier RF, Dvoracek-Driksna D, Oniang’o TE, Haut RC, 1999, “Axial Compressive
Load Response of the 90° F lexed Human Tibiofemoral Joint,” 43"d Stapp Car Crash Conf.
Proc., pp. 127-139.

Blanco FJ, Guitian R, Vazquez-Martul E, DeToro FJ, Galdo F, 1998, “Osteoarthritis
chondrocytes die by apoptosis-a possible pathway for CA pathology,” Arth. Rheum,
41 (2), pp. 284-289.

Colwell CW, D’Lima DD, Hoenecke HR, Fronek J, Pulido P, Morris BA, Chung C,
Resnick D, Lotz M, 2001, “In vivo changes after mechanical injury,” Clin. Orthop. Rel.
Res., 3918, pp. $116-$123.

Ewers B, Krueger J, Dvoracek-Dricksna D, Orth M, Haut R, 2001, “Chondrocyte
viability decreases over 24 hours post-impact in a mechanically traumatized cartilage
explant,” 47‘h Mtging Orthop. Res. Society, 2001.

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 Patello-
Femoral Joint,” J. Orthop. Res., 20, pp. 545-550.

Funk JR, Tourret LJ, Crandall JR, 2000, “Experimentally Produced Tibial Plateau
Fractures,” Int. Res. Council Biomech. Injuries, pp. 171-182.

Johnson DL, Urban WP, Caborn DNM, Vanarthos WJ, Carlson CS, 1998, “Articular
Cartilage Changes Seen With Magnetic Resonance Irnaging-Detected Bone Bruises
Associated With Acute Anterior Cruciate Ligament Rupture,” Am. J. Sports Med., 26,
pp. 409-414.

29

Krueger JA, Thisse P, Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC, 2003, “The
Extent and Distribution of Cell Death and Matrix Damage in Impacted Chondral Explants
Varies with the Presence of Underlying Bone,” J. Biomech. Eng, 125, pp. 1-6.

Meyer EG, 2004, “Biomechanical response of tissues to various blunt impact
orientations,” MS. Thesis, Michigan State University, East Lansing, MI.

Milentijevic D, Rubel IF, Liew ASL, Helfet DL, Torzilli PA, 2005, “An in vivo rabbit
model ﬁo cartilage trauma. A preliminary study of the inﬂuence of impact stress
magnitude on chondrocyte death and matrix damage,” J. Orthop. Trauma, 19, pp 466-
473.

Newberry WN, Garcia JJ, Mackenzie CD, Decamp CE, Haut RC, 1998, “Analysis of
Acute Mechanical Insult in an Animal Model of Post-Traumatic Osteoarthrosis,” J.
Biomech. Eng, 120, pp. 704-709.

Phillips DM, Haut RC, 2004, “The Use of a Non-Ionic Surfactant (P188) to Save
Chondrocytes from Necrosis Following Impact Loading of Chondral Explants,” J.
Orthop. Res., 22, pp. 1135-1142.

Pritzker KPH, 1998, “Pathology of osteoarthritis,” In Osteoﬂhritis, Edited by Brandt,
K.D., Doherty, M., Lohmander, L.S., Oxford University Press. Chapter 5, pp. 50-61.

Repo R, Finlay J, 1977, “Survival of Articular Cartilage after Controlled Impact,” J. Bone
Jt. Surg., 59-A (8), pp. 1068-1076.

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, pp. 1363-1369.

Sherwood C, O’Neill B, Hurwitz S, 1999, “Lower extremity Causation in Frontal
Crashes,” Proc. 1999 Int. Conf. Biomech. Impact, Sitges, Spain, pp 513-524.

Taylor A, Morris A, Thomas P, Wallace A, 1997, “Mechanisms of Lower Extremity
Injuries to Front Seat Car Occupants - An in Depth Accident Analysis,” Proc. 1997 Int.
Conf. Biomech. Impact, Hannover, Germany, pp 53-72.

Torzilli PA, Grigiene R, Borrelli J Jr, Helfet DL, 1999, “Effect of Impact Load on
Articular Cartilage: Cell Metabolism and Viability, and Matrix Water Content,” J.
Biomech. Eng, 121, pp. 433-441.

Wu JZ, Herzog W, Epstein M, 1998, “Effects of Inserting Pressensor F ilrn into Articular
Joints on the Actual Contact Mechanics,” J. Biomech. Eng, 120, pp. 655-659.

30

CHAPTER THREE

CHRONIC CHANGES IN THE MECHANICAL AND HISTOLOGICAL
PROPERTIES OF RABBIT ARTICULAR CARTILAGE FOLLOWING
TIBIOFEMORAL IMPACT

ABSTRACT

Characteristic osteochondral lesions have been strongly correlated with ACL trauma, as
well as in the clinical literature of patients with no reported ligamentous injury. These
lesions are associated with occult microcracks of subchondral and/or trabecular bone. Of
concern in today’s clinical literature is that there is evidence of acute injury to articular
cartilage overlying these bone bruises, which may predispose the tissue to degenerative
changes. Previous studies using the rabbit patellofemoral (PF) joint have shown that a
single impact to the ﬂexed rabbit knee results in signiﬁcant damage to articular cartilage
and subchondral bone, reminiscent of early stage OA. Given the frequency of bone
bruising in the tibial plateau and femoral condyles in patients reporting knee injury, the
implications of blunt trauma to articular cartilage in the tibiofemoral (TF) joint has
become an area of interest. The current study investigated the chronic changes in articular
cartilage and underlying bone due to a single, severe impact to the ﬂexed rabbit TF joint.
The hypothesis of this investigation was that a single compressive impact to the rabbit
knee would lead to alterations in the mechanical and histological properties of the
articular cartilage within 1 year. Forty eight (24 test, 24 control) animals received a single
insult to the ﬂexed knee. The animals were sacriﬁced at 6 months (n=24) and 1 year
(n=24) post-trauma, and mechanical and histological properties were documented. The
study showed no sigiiﬁcant difference in the mechanical properties of the articular

cartilage from the impacted versus contralateral, control TF joint at either 6 months or 1

31

year post-trauma. However, a change in the stiffness of cartilage was documented in the
medial uncovered region between 6 months and 1 year. Histological evaluations revealed
signiﬁcant differences in the morphology of subchondral bone between impacted and
contralateral, control joints as well as differences in the proteoglycan content of the
articular cartilage 1 year post-trauma. Future studies should investigate the longer-terrn
implications of these histological effects on the mechanical integity of the articular
cartilage, as well as the long-term implications of cartilage stiffening with time post-

trauma.

32

INTRODUCTION

Long term participation in vigorous physical activities increases the risk of acute and
chronic injuries, such as ligament sprains or osteoarthritis (OA), respectively (Lane,
1996). Axial compressive loading of the knee joint is a key component during a majority
of anterior cruciate ligament (ACL) injuries. Acute injury to the ACL has been shown to
lead to characteristic osteochondral lesions in the postero-lateral aspect of the tibia and/or
antero-lateral aspect of the lateral femoral condyle (Atkinson et al., 2008; Mink &
Deutsch et al., 1989; Speer et al., 1992; Spindler et al., 1993). These lesions are
associated with occult microcracks of subchondral and/or trabecular bone (Speer et al.,
1992; Rangger et al., 1998). Although strongly correlated with ACL trauma, bone bruises
have also been documented in the clinical literature in patients with no ligamentous
damage (Wright et al., 2000; Davies et al., 2004). Since the current literature seems to
suggest that even following ACL reconstruction as many as 50% of patients will develop
radiological degenerative changes within 10-15 years (Myklebust & Bahr, 2007) the
initial injury to articular cartilage and subchondral bone may play a large role in initiating
chronic joint disease.

Of concern in today’s clinical literature is that there is evidence of acute injury to
articular cartilage overlying these bone bruises, which may predispose the knee to
degenerative changes (Mankin, 1982; Thompson et al., 1991; Faber et al., 1999). Recent
studies have focused on the long-term implications of cartilage and subchondral bone
damage generated during the acute joint injury. However, establishing a cause and effect
relationship between acute joint injury and the chronic development of CA has been

difﬁcult. Our laboratory has previously developed a model using the patellofemoral (PF)

33

joint of the Flemish Giant rabbit in which a 6 J energy blunt impact was delivered to the
ﬂexed knee. Acute studies using this model document signiﬁcant damage to
chondrocytes as well as impact induced lesions on the retropatellar surface. Chrornic
studies using this PF rabbit model document signiﬁcant changes in the mechanical and
histological properties of the retropatellar cartilage including a 30% reduction in cartilage
stiffness, a signiﬁcant thinning of the cartilage and an increase in its permeability 36
months post-trauma. Ewers et a1. (2000) also document a signiﬁcant softening of the
retropatellar cartilage within 12 months following blunt trauma to the rabbit PF joint.
However, given the frequency of bone bruising in the tibial plateau and femoral condyles
of patients reporting knee injury, the implications of blunt trauma to articular cartilage in
the tibiofemoral joint (TF) has become an area of active interest.

Vellet et al. (1991) document an overt loss of cartilage overlying geogaphic bone
bruises in 48% of patients within 6 months of the reported injury. In order to investigate
the pathogenesis that leads from the acute bone bruise to this overt cartilage loss animal
models have been developed. Radin et a1. (1984), for example, document severely
ﬁbrillated cartilage, horizontal and vertical microcracks and the interface between
calciﬁed cartilage and bone, and a stiffening of underlying subchondral bone 3 weeks
after impulsive loading to the rabbit TF joint. These authors also conclude that in this
model early bone changes precede changes in articular cartilage. Our laboratory has
recently developed an in viva traumatic injury model involving the rabbit TF joint. We
have shown that a single, severe impact to the ﬂexed rabbit knee results in average
contact pressures of approximately 20 MPa and 24 MPa, with corresponding increases of

22% and 25% in the percentage of cells with damaged plasma membranes in the medial

34

and lateral facets, respectively (Isaac et al., 2008). And, acute trauma to cells has been
thought to be associated with the long-term development of osteoarthritis (Colwell et al.,
2001).

The objective of the current study was to use this newly developed TF impact
model (Isaac etal., 2008) to investigate chronic changes in articular cartilage and
underlying bone due to a single, severe impact. Based on the results of previous studies
on the PF joint by our laboratory, the hypothesis of the current study was that a single,
severe compressive impact on the ﬂexed rabbit knee would lead to alterations in the
mecharnical and histological properties of the articular cartilage in the TF joint within 1
year. Such a model could then be used to study the efﬁcacy of various methods of
intervention including repair of acutely damaged chondrocytes or addressing trauma to
subchondral and/or trabecular bone.

MATERIALS AND METHODS

Forty eight skeletally mature Flemish Giant rabbits (average mass = 5.5 :I: .08 kg,
9-12 months of age) were used in this study. This investigation was approved by the
Michigan State University All-University Committee on Animal Use and Care. A
licensed veterinary technician (J .A) cared for the arnimals. All animals were housed in
individual cages (60 x 60 x 14in) throughout the duration of the study. Twenty-four
animals were randomly selected for the chronic impact study. These animals received a
single, high-intensity blunt impact to the left TF joint, per the protocol described below.
Twelve animals were selected for either a 6 or 12 month study. Another 24 animals
served as unimpacted, controls for the study and were split into two goups that for either

the 6 or 12 month study.

35

The blunt impact experiments have been described in an earlier study (Isaac et al.,
2008). Brieﬂy, a 1.75 kg mass was dropped from a height of 75 cm (~ 13 J of potential
energy) onto the left TF joint of anesthetized rabbits (2% Isoﬂurane and Oxygen). Each
animal was laid supine in the test ﬁxture, and the knee was ﬂexed 90 degees. Two
Velcro straps were crossed over the femur, and the tibia was constrained in order to limit
anterior motion of the tibia during impact (Figure 3.1). A pre-crushed deformable
interface (Hexcel, 3.76 MPa) was used to ensure uniform loading over the anterior
surface of the femur. The mass was arrested electronically after the ﬁrst impact,
preventing multiple impacts. After trauma the arnimals received one injection of

Buprenorphine (.03 rrnl/kg BW) for early post-irnpact pain.

Gravity accelerated
mass

 

Load transducer

 

Deformable interface

Tibial constraint .._....._.._p I

Figure 3.1. Impact experiments were performed by dropping a gavity-accelerated mass
onto the ﬂexed tibial-femoral joint with approximately 13 J of potential energy. The
rabbit was oriented such that the deformable interface struck the distal femur with impact
forces oriented axially in the tibia. The tibia was constrained so as to limit anterior
subluxation and prevent ligament damage.

 

 

 

36

After either six months (n=24) or 1 year (n=24), in which the animals were
monitored daily for any abnormal movements or behavior, the animals were sacriﬁced
with an overdose of Pentobarbital I.V. (85.9 mg/kg BW). Immediately following sacriﬁce
the hind limbs were opened, removed and examined for abnormalities. The meniscus
was also examined for abnormalities. It was then removed after marking its location on
the surface of the tibial plateau. The material properties of the articular cartilage were
documented at four speciﬁc locations sites the medial and lateral facets using an
indentation relaxation test (Ewers et al., 2002). Sites 1 and 3 on the medial and lateral
facets, respectively, were near the edge of the merniscus in uncovered areas. Sites 2 and 4
were slightly posterior to these sites and located in regions covered by the medial and

lateral meniscus, respectively (Figure 3.2).

Lateral

 

Figure 3.2. Indentation relaxation tests were performed across the tibial plateau in
regions covered and uncovered by the meniscus. Sites 1 and 3 correspond to regions
uncovered by the meniscus on the medial and lateral facet, respectively. Sites 2 and 4 are

' located in covered regions on the medial and lateral facet, respectively.

The tibial plateau was mounted in a specialized clamp attached to a 3-dimensional
camera mounting ﬁxture and bathed in room temperature phosphate buffered saline
(PBS) (pH = 7.2). Prior to indentation testing a needle was slowly penetrated into the

cartilage to measure thickness at two locations around each indentation site. This

measurement was made based on an analysis of the force time plot that showed the

37

needle touching the surface of the cartilage followed by the appearance of a sudden rise
in force as the needle contacted the deep layer of calciﬁed cartilage (Athanasiou et al.,
1991). At each site the surface of the tibial plateau was positioned perpendicular to the

indenter probe (Figure 3.3).

Spherical
indenter

 

Camera mount

3
n

 

Figure 3.3. Indentation relaxation testing was performed using a custom built step-motor
device. The tibial plateau was ﬁxed in a specialized camera mounting ﬁxture and the
cartilage was position perpendicular to the spherical indenter.

At each of the previously marked sites a 1.06 mm diameter, spherical nonporous probe
was pressed into the cartilage to 40% of the cartilage thickness for approximately 30
minutes using a custom built stepper motor driven device (Physic Instruments,
Waldbronn, Germany, Model M-168.3). The resistive loads were measured (Data
Instrrnnents, Acton MA, Model JP-25), ampliﬁed and sampled at 1000 Hz for the ﬁrst

second and at 20 Hz thereafter. Alter indentation, the cartilage was left to rest for 30

38

rrninutes. The indenter probe was then replaced with the needle and thickness
measurements were taken at the indentation site, following the procedure described
above.

The load relaxation curves were ﬁt with a ﬁbril-reinforced, biphasic
computational model (Golenberg et al., 2008) with an assumed Poisson’s ratio of 0.3. In
‘ this model of cartilage tissue attached to bone, a linear variation of voids ratio which was
assumed increased ﬁom 70% ﬂuid at the cartilage-bone interface to 85% in the
superﬁcial zone (Lipshitz et al., 1975). The model allowed for ﬁnite deformations and
was implemented in a commercial ﬁnite element analysis package (ABAQUS v.6.3,
Hibbitt, Karlsson & Sorensen, Inc., Pawtucket, RI, USA). The matrix modulus (Em), ﬁber
modulus (E!) and tissue permeability (k) of cartilage were determined from this model
with a custom-written, Gauss-Newton constrained nonlinear least square mirnirrnization
procedure (Lindstrom and Wedin, 1993).

Following the mechanical tests, the specimens were prepared for histological
evaluations. The plateaus were bathed in 10% buffered formalin for one week and
decalciﬁed in 20% formic acid for an additional week. Comoal tissue blocks were then
cut in the medial to lateral direction across the plateau. The blocks were processed in
parafﬁn and sequential sections, 8 microns thick, were prepared for examination. The
sections were stained with Safranin O-Fast Green and examined under light microscopy.
The thickness of the subchondral plate was determined by averaging across the facets
with a calibrated eyepiece. The overlying articular cartilage was also scored using a
previously established system (Weaver and Haut, 2005; Columbo etal., 1983; Mazieres

et al., 1987) (Figure 3.4).

39

 

 

 

 

 

 

 

 

Normal 0 Present 0
Slightly Irregular 1 Multiple 1
Surface Geometry Moderately Disrupted 2 Tide Mark Focal Loss 2
Focally Disrupted 3 Diffuse Loss 3
Extensively Disrupted 4 Total Loss 4
None 0 Normal 0
1-3 Surface 1 . Slight 1
Articular Cartilage 1-2 Mid-zone 2 $2133 e: Moderate 2
Fissures 3—4 Mid-zone 3 S . ulg Focally Excessive 3
4+ Mid-zone 4 pro ae Excessive 4
1+ Deep zone 4
Normal 0 Normal 0
Slight Loss 1 Calciﬁed Slight 1
Proteoglycan Stain Moderate Loss 2 Cartilage Moderate 2
Focally 3 Stain Dark 3
Total Loss 4
Normal 0 Dense 3
Some Clones 1 Subchon dr a1 Some Small Spaces 1
Articular Cartilage Many Clones 2 B Moderate Spaces 2
one .
Cells Some Clusters 3 Mo holo Some Splits 2
Many Clusters 4 rp gy Numerous Splits 4
Path Cells 4
None 0
Articular Cartilage Compression Ridges 2
Disruptions Horizontal Splits 4
Vertical Splits 4
Articular Cartilage Subchondral
Thickrness Bone
Thickness
Calciﬁed Cartilage
Thickness

 

 

 

 

Figure 3.4. Histological scoring system used to quantify the characteristics for
cartilage across the tibial plateau.

A two-factor ANOVA (limb, goup) with post hoc Student-Newman-Keuls (S-N-

K) tests was used in order to evaluate the differences in mechanical properties between

the impacted and control limbs, as well as for differences between the 6 month and 1 year

data. A one-factor (limb) ANOVA on Ranks was used to test for differences between

40

 

histological parameters in the impacted and control limbs, as well as between the 6 month
and 1 year goups. A statistically signiﬁcant effect was indicated for p<0.05.
RESULTS

The average peak, inertially-compensated impact load and impact duration were
1070 :t 107 N and 23.0 i 0.2 ms in the study. During the post-trauma period no limping
or swollen knee joints were observed in any animal. Some animals did develop a slight
bruise at the impaction site following the insult, but no subsequent consequences were
evident thereafter. Upon necropsy no signiﬁcant joint pathology such as synovitis,
hardening of the joint capsule, cartilage erosion, etc. were noted in any rabbit.
Additionally, no ligament or meniscal damages were documented in any animal during
dissection of the joints.

No signiﬁcant differences were documented in any mechanical parameter
between the riglnt and left limbs of the control animals; therefore, these data were
averaged for this study. Additionally, no signiﬁcant differences in any mecharnical
parameter were documented between the contralateral, control limbs and the combined
control rabbits at any site across the plateau in either goup of animals; therefore, the
current study compared the impacted limbs to the contralateral, control (right).

Analysis of the data from indentation relaxation tests revealed no signiﬁcant
differences in any mechanical parameter between the left and right limbs in the 6 month,
impacted goup. Similarly, the 1 year, impacted goup also showed no signiﬁcant
differences in any mecharnical property between the left and right limbs. Analysis of
indentation data from the impacted limb between 6 months and 1 year, however, showed

a signiﬁcant increase in both the matrix (p=0.009) and ﬁber (p=0.025) moduli at site 1. In

41

addition, a signiﬁcant decrease in tissue permeability was also noted at site 1 in the 1 year
goups compared to the 6 month goup (p=0.002). Interestingly, the contralateral, control
limb also exhibited an increase of approximately 33% and 50% in the matrix and ﬁber
modulus, respectively, at site 1. These differences, however, did not rise to the level of
statistical signiﬁcance. No such differences were noted at site 1, or any other site,
between 6 months and 1 year for the unimpacted, control animals (Figure 3.5).

Table 3.1. The mechanical properties (average (3: standard deviation» were extracted
ﬁom the relaxation indentation testing across the medial and lateral facet (Site 1 — medial

uncovered, Site 2 — medial covered, Site 3 — lateral uncovered and Site 4 — lateral
covered). Statistical differences between the between the 6 month and 1 year goup are

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

indicated (b).
6 Month Group
Site 1 Site 2 Site 3 Site 4
Em 0.65 (1 0.14) b 0.62 (1 0.20) 0.82 (1 0.21) 1.91 (1 1.12)
Impacted Ef 4.4(11.84)" 2.82 (1 1.08) 12.62 (1 3.89) 31.88 (1 16.37)
k 22.7 (1 8.03) b 10.8 (1 4.09) 5.55 (1 3.32) 2.3 (1 1.32)
Em 0.61 (1 0.17) 0.71 (1 0.23) 0.96 (1 0.37) 2.09 (1 1.42)
Control Limb Ef 3.2 (1 1.21) 4.87 (1 3.48) 15.53 (1 4.42) 36.3§ (1 19.51)
k 25.8 (1 8.68) 8.35 (1 3.10) 5.52 (1 1.93) 2.27 (1 1.24)
Em 0.60 (1 0.12) 0.81 (1 0.38) 0.90 (1 0.24) 1.62 (1 0. 75
control Rabbit Ef 4.42 (1 2.09 6.66 (1 4.66) 1_ 4.58 (1 8.03) 31.23 (1 22.09)
k 21.0 (1 6.07) 8.05 (1 2.74) 5.58 (1 2.15) 2.24 (1 0.69)
1 Year Group
Site 1 Site 2 Site 3 Site 4
Em 0.81 (1 0.12) b 0.71 (1 0.13) 0.88 (1 0.09) 1.58 (1 0.45)
Impacted Ef 6.5 (1 22)" 3.98 (1 3.36) 12.7 (1 3.98) 49.0 (1 23.8)
k 12.7 (1 5.18) b 10.0 (1 3.66) 6.26 (1 2.68) 2.12 (1 0.49)
Em 0.81 (1 0.15) 0.76 (1 0.15) 1.10 (1 0.29) 1.50 (1 0.58)
Control Limb Ef 5.12 (1 1.4) 5.89 (1 4.52) 16.1 (1 4.06) 42.7 (1 21.1)
k 14.9 (1 5.05) 7.94 (1 3.08) 4.73 (1 0.99) 2.24 (1 0.79)
Em 0.69 (1 0.15) 0.91 (1 0.25 1.11 (1 0.29) 1.57 (1 0.47)
Control Rabbit Ef 407(1 2.05) 4.97 (1 2.07) 14.6 (1 4.09) 37.0 (1 17.7)
R 19.5 (1 7.18) 8.88 (1 2.42) 5.46 (1 1.02) 2.28 (1 0.66)

 

 

 

 

 

 

 

The analysis of histological sections from the right and left limbs of the

unimpacted, control rabbits showed no signiﬁcant differences in either the medial or

42

 

lateral aspects of the plateau for either the 6 month or 1 year goup. Additionally, no
statistical differences were noted between the contralateral, control limb and the control
animals; therefore, the impacted limbs were compared to the contralateral limbs for this
study.

Histological analysis of sections from the 6 month goup indicated a sigtiﬁcant
increase in the number of surface lesions on the medial aspect of the impacted plateau
(p=0.04) versus the contralateral limb; however, no sigiiﬁcant increase was found in the
lateral aspect. The number of vertical and horizontal micro-cracks at the interface
between articular cartilage and subchondral bone was also documented in the histological
sections. A sigiiﬁcant increase in the frequency of microcracks was evident in the medial
(p=0.003) and lateral (p=0.005) aspects of the impacted limbs, with a slightly higher
ﬁequency of microcracks in the lateral versus medial aspects. A sigiiﬁcant loss of
Safranin-O stain was also evident in the medial (p=0.017) and lateral (p=0.039) aspects
of the impacted plateau. No sigiiﬁcant Change in subchondral bone thickness was
documented for the 6 month goup in either aspect of the plateau (Figure 3.6).

Analysis of histological sections from the 1 year goup also revealed ﬁndings
similar to those of the 6 month goup. A sigliﬁcant increase in the number of surface
ﬁssures was documented in the medal (p=0.025) aspect of the impacted limb compared to
the contralateral. The frequency of vertical and horizontal microcracks was also found to
be sigliﬁcantly geater in the medial (p=0.001) and lateral (p=0.037) aspects of the
impacted plateau versus the contralateral limb (Figure 3.7). In contrast to the 6 month
goup, a sigliﬁcant increase in subchondral bone thickness was documented in the

medial (p=0.029) and lateral (p<0.001) aspects of the plateau for the impacted versus

43

control limbs in the 1 year goup. A statistical decrease in the Safranin-O stain was also
documented in the impacted cartilage at 1 year compared to the control limbs for both the
medial (p<0.001) and lateral (p=0.003) aspects of the tibial plateau (Figure 3.6).

When comparing the data from histological sections of impacted limbs between
the 6 month and 1 year goups there was also a statistical trend for a higher frequency of
microcracks in the medial aspect of the plateau (p=0.092) of impacted limbs. A
sigiiﬁcant increase in subchondral bone thickness was also shown in the medial
(p=0.100) and lateral (p=0.05) aspects of the impacted plateaus. The lateral aspect of the
plateau also showed a sigiiﬁcant decrease in the Safranin-O stain (p=0.017) in the 1 year
compared to the 6 month goups.

Table 3.2. Histological evaluations of the impacted and control osteochondral sections of
the medial and lateral tibial plateau indicated sigliﬁcant increases in surface ﬁssures,

subchondral bone thickness, disruptions (i.e. microcracks) and PG stain. Statistical
differences between the impacted and contralateral, control limbs are indicated for the 6

month and 1 year goup (a) and between the 6 month and 1 year goups (b).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6 Month
SB Thick Fissures Disruptions PG Stain

Average SD Avegge SD Average SD Average SD
Left M 26.58 3.94 2.926‘ 1.68 2.923 2.02 1.00 0.85
L 29.00 5.10 0.58 0.79 3.17 a 2.76 0.33 0.49
Right M 24.42 3.58 1.25 1.06 0.58 0.67 0.09 0.39
L 26.83 5.64 0.67 0.98 0.67 0.98 0.00 0.00
C M 27.23 4.42 1.41 0.78 0.68 0.56 0.36 0.39

ontrol
L 29.00 5.64 0.18 0.25 0.14 0.23 0.14 0.23

1 Year
SB Thick Fissures Disruptions PG Stain

Average SD Averagg SD Average SD Average SD
Left M 28.50 5"" 3.44 3.208 1.99 4.703 2.71 0.90"" 0.32
L 33.55 a’b 5.39 1.27 1.42 2.73"” 2.45 1.36 a 1.12
Right M 25.00 2.24 1.18 1.17 0.36 0.50 0.00 0.00
L 25.18 2.82 0.36 0.67 0.64 1.29 0.00 0.00
M 24.45 1.48 1.20 0.75 0.55 0.55 0.10 0.21

Control
L 24.40 2.28 0.35 0.41 0.25 0.35 0.10 0.21

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.5. Histological analysis showed a sigriﬁcant increase in surface lesions
for both the 6 month and 1 year goups (a) and a loss of proteoglycan staining in the 1
year goup (b) compared to unimpacted, control limbs. An increase in the frequency of
vertical ((1) and horizontal (c) microcracks at the interface of articular cartilage and
subchondral bone was also documented in both goups compared to controls.
DISCUSSION
The current study documented the mechanical and histological properties of articular
cartilage on the rabbit tibial plateau 6 months and 1 year following blunt trauma to the TF
joint. Mechanical tests showed no sigriﬁcant differences in the mechanical properties
between the impacted limb and the contralateral, control limbs at any of the sites on the
medial or lateral tibial plateau. However, the study documented a statistical increase in
both the matrix and ﬁber moduli, as well as a decrease in the permeability, of the articular
cartilage in the medial uncovered region of the impacted limb between the 6 month and 1

year goup. The medial uncovered region of the tibial plateau has been shown to exhibit

statistically more baseline damage than its lateral counterpart (Golenberg et al., 2008).

45

This “baseline” damaged cartilage has also been thought to be similar to a higher
prevalence of clinical 0A in the medial compartment of the knee (Ahlback et al., 1968).
These results are in contrast with those from previous studies by our laboratory on the PF
joint which document a sigriﬁcant softening of the retropatellar cartilage 3 years
following impact (Ewers et al., 2000). Previous investigations on the pathogenesis of OA
have shown deposition of calcium in degenerative cartilage (Radin et al., 1984). Weaver
and Haut (2005) also document histological ossiﬁcation/calciﬁcation in the rabbit PF
joint 2 years after impact. The authors of the latter study conclude that this ossiﬁcation
may lead to a stiffening of the impacted cartilage. It is possible that the articular cartilage
from the 1 year goup may also have exhibited some calciﬁcation which could have lead
to the observed stiffening.

An interesting ﬁnding of the current study was the response of the contralateral,
control limb in the medial uncovered region of the impacted animal goups. Although not
statistically sigriﬁcant, the contralateral limb also showed a slight stiffening of cartilage
between the 6 month and 1 year goups. In contrast, the unimpacted, control rabbits did
not show any Changes in mechanical properties between 6 months and 1 year. There are a
number of possible explanations for stiffening of cartilage in the medial uncovered region
of both the impacted and contralateral limbs in these animals. It is possible that these
rabbits simply had different baseline material properties. However, the rabbits were
randomly assigied to goups, and it seems unlikely that rabbits with different cartilage
properties were all placed in the same goup. It does seem possible, however, that altered
gait as a result of injury could have lead to an increased loading of the contralateral limb.

During normal gait higher loads have been shown to pass through the medial

45

compartment of the rabbit knee joint (Mansour et al., 1998) particularly in the uncovered
regions. This could result in a more advanced disease process in the medial compartment
of these animals. On the other hand, Gaushe et al., (2005) suggests that higher loads pass
through the lateral compartment for the rabbit. This could suggest that medial
compartment 0A in the rabbit is largely due to an unloading effect, which could have
also been additionally provoked by trauma to the opposite, impacted limb. Future studies
will be needed to better understand the loading pathways through the rabbit knee, and
alterations that could be due to trauma in on of the limbs.

The current study also documented sigiiﬁcant histological differences between
the impacted and contralateral limbs in both the 6 month and 1 year goups. Impact
trauma was found to sigriﬁcantly increase the frequency of vertical and horizontal
microcracks at the interface between calciﬁed cartilage and subchondral bone, with a
higher frequency noted in the 1 year goup compared to the 6 month goup. Furthermore,
a trend for higher frequency of these disruptions were noted in the lateral compartment of
the tibial plateau at 6 months compared to the medial compartment. A recent study by
Batiste et a1. (2004) indicates that the bone mineral density (BMD) is sigiiﬁcantly higher
in the medial tibial plateau. This could possibly explain why slightly fewer microcracks
are seen in the medial than lateral compartments 6 months following trauma. A previous
study by Ewers et a1. (2002) documents a sigliﬁcant softening of the retropatellar
cartilage with no subsequent rnicrotrauma at the articular cartilage subchondral bone
interface in the rabbit PF joint following single, severe impact. Initiation of cartilage
damage and progession to end stage CA has been shown to depend on pathophysiology

of cartilage and bone (Burr and Schafﬂer, 1997). Therefore, the occult bone trauma

47

documented in the current TF impact model and not the previous studies using the rabbit
PF joint could have lead to a more accelerated degeneration and therefore, calciﬁcation
and stiffening of the articular cartilage.

There were a number of limitations of the current study. In particular, histological
analysis of osteochondral sections did not allow for evaluation of calcium deposition in
the articular cartilage, which we have implied may possibly have lead to a stiffening of
the articular cartilage at site 1 in the 1 year goup. Additionally, although a statistical
increase in articular cartilage stiffness was documented between 6 months and 1 year in
the medial uncovered regions of the impacted limb it is not yet conclusive as to whether
this increase was due, in part, to an increase in the mechanical parameters of the
contralateral limbs or to impact itself. Although there were no statistical differences
between the contralateral limbs of the two goups there did seem to be a trend for an
increase in both the matrix and ﬁber moduli in the 1 year compared to the 6 month
control limbs. Altered loading mechanics due to injury of the impacted limb could have
affected the contralateral limb. However, post-trauma gait and cage activity was not
quantiﬁed. Future studies should investigate the implications of this change in the
properties of the contralateral limb.

While the current study indicated sigliﬁcant Changes in the mechanical and
histological properties of articular cartilage and subchondral bone in the rabbit TF joint,
the mechanical property Changes that were potentially due to impact trauma seem to be in
contrast to those documented in previous studies on the rabbit PF joint. Future studies
should investigate the mechanism of cartilage stiffening to determine if it may be

associated with a more end stage disease. Longer-term studies should also be performed

48

in order to understand the relationship between articular cartilage degeneration and
subchondral bone remodeling in this model. Investigations can then proceed to study the
efﬁcacy of therapeutic agents aimed at preventing damage to cartilage and subchondral
bone following traumatic loading to the joint.

ACKNOWLEDGEMENTS

This study was supported by a gant ﬁ'om the Centers for Disease Control and
Prevention, Center for Injury Control & Prevention (CE000623). The contents of this
manuscript are solely the responsibility of the authors and do not necessarily represent
the ofﬁcial views of the Center for Injury Control and Prevention. The authors wish to
acknowledge Ms. Jean Atkinson (JA) for her assistance in animal care, Ms. Jane Walsh
(JW) for her assistance with histological evaluations and Mr. Clifford Beckett for his

technical support.

49

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Ewers BJ, Haut RC, 2000, “Polysulphated Glycosarrninoglycan Treatments can Mitigate
Decreases in Stiffness of Articuar Cartilage in a Traumatized J oint," J Orthop Res, 18,
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Golenberg N, Kepich E, Haut RC, 2008, “Histomorphological and Mechanical Property

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Model,” Int J Exp Comp Biomech, 1(1), pp. 58-

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Gushe DL, Houck J, Lerner AL, 2005, “Rabbit knee joint biomechanics: Motion analysis
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Isaac DI, Meyer EG, Haut RC, 2008, “Chondrocyte Damage and Contact Pressures
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Vellet AD, Marks PH, Fowler PJ, Munro TG, 1991, “Occult Posttraumatic Osteochondral
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on the Response of Articular Cartilage Explants to an Excessive Level of Unconﬁned
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Wright RW, Phaneuf M, Limbird TJ, Spindler KP, 2000, “Clirnical Outcome of Isolated
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52

CHAPTER FOUR

A TRAUMATIC ANTERIOR CRUCIATE LIGAMENT RUPTURB
MODEL: A PRELIMINARY STUDY USING THE RABBIT MODEL

ABSTRACT

Axial compressive loading of the knee is a key component during a majority of
anterior cruciate ligament (ACL) injuries and severe tibiofemoral (TF) contact pressures
have been documented during these events. Clinically, there are characteristic
osteochondral lesions with cellular damage in overlying articular cartilage that occurs in
the tibial plateau and femoral condyles in over 80% of ACL injury cases. A hypothesis of
this study was that compressive loading of the rabbit knee would result in ACL rupture
along with signiﬁcant damage in articular cartilage and underlying subchondral bone.. A
second hypothesis of this study was that this traumatic ACL rupture model (ACLF)
would result in relatively more joint degeneration than a surgical transection model of
ACL injury (ACLT). Six Flemish giant rabbits were anesthetized and received a blunt
force insult to the left TF joint resulting in ACL rupture. One of the animals had pressure
sensitive ﬁlm inserted into the TF joint prior to impact to record the acute magnitude and
contact pressure distribution over the medial and lateral plateaus during rupture of the
ACL. Two other animals were sacriﬁced immediately following impact to document
acute cartilage and chondrocyte damages. An additional three animals underwent
urnilateral surgical transection of the ACL.The ﬁnal three ACLF animals and the three
ACLT animals were sacriﬁced at 12 weeks. The dege of chronic degeneration was
evaluated based on the extent of surface ﬁssureing and morphological changes in
articular cartilage and underlying subchondral bone. Vertical and horizontal microcracks

at the articular cartilage subchondral bone interface were manually quantiﬁed in

53

histological sections. The maximum contact pressures in the tibiofemoral joint were
approximately 50 MP8 on both plateaus. Acute surface ﬁssures on articular cartilage
were also documented, especially in the medial femoral condyle. There was acute
damage to the meniscus in all ACLF animals, primarily in the lateral meniscus. There
were signiﬁcantly more chondrocytes with damaged plasma membranes in the medial
and lateral tibial plateaus and femoral condyles in the impacted than contralateral, control
limbs. At 12 weeks there was full tlnickness erosion of the articular cartilage as well as
severe damage to the menisci in all three of the ACLF animals. Only one of the ACLT
animals had moderate cartilage erosion and meniscal degeneration. Histological sections
revealed sigriﬁcantly more vertical and horizontal micro-cracks at the articular cartilage-
subchondral bone interface in the ACLF goup than the ACLT goup. The ACLT animal
model for post-traumatic osteoarthritis fails to address the acute injuries, such as bone
micro-cracks, that occur in most Clinical cases of ACL rupture and may have important
implications in the long-term development of joint disease. The proposed ACLF model is
more directly relevant to the clinical cases of traumatic ACL rupture by incorporating
acute histological microtrauma at the articular cartilage subchondral bone interface and
meniscal damage that leads to chronic cartilage erosion and joint remodeling after 12

weeks.

54

INTRODUCTION

Participation in sports, recreation and exercise is increasingly popular and
widespread in American culture. Long-term participation in vigorous physical activities
increases the risk of acute and chronic injuries, such as ligament injury or post-traumatic
osteoarthritis (OA), respectively (Lane, 1996). Two speciﬁc types of injuries are strongly
associated with subsequent knee OA: cruciate ligament damage and meniscal tears
(F elson, 2004). In the year 2000 approximately 80,000 anterior cruciate ligament (ACL)
tears occurred in the US. alone (Grifﬁn et al., 2000), with a total cost of nearly $2 billion
(Hewitt et al., 2006).

Many Clinical studies have focused on documenting mechanisms that cause injury
to the ACL. Noncontact ACL injuries occur more ﬁequently than injuries involving
player-to-player contact (Griffen et al., 2000), and these injuries often involve landing
from a jump on one or both legs (Boden et al., 2000). The axial load distribution in
injured legs at the time of injury is estimated to be more than 65%, and in most cases
100% of the total gound reaction force (Olsen, 2004). Axial compressive loading of the
knee during landing from a jump can be approximately six times body weight for males
(Hewett et al., 1996). The tibial plateau has an inherent posterior slope of 10°-15° (Li et
al., 1998) which can produce an anterior shift of the tibia under tibiofemoral (TF)
compressive loading (Torzilli et al., 1994). Since the ACL provides 85% of the retaining
ligamentous force during anterior tibial subluxation (Butler et al., 1989), TF compression
may be an important component in the mechanism of clinical ACL injury. Our laboratory
has conﬁrmed that a pure TF compressive load can generate an isolated ACL injury in

human cadaver knees at knee ﬂexion angles between 30-120° (Meyer et al., 2005; Meyer

55

et al., 2008). Another goup reports similar results using porcine knees at 70° of ﬂexion
(Y eow et al., 2008).

Clinically, in over 80% of ACL injury cases a characteristic osteochondral lesion
occurs in the postero-lateral aspect of the tibia and/or antero-lateral aspect of the lateral
femoral condyle, as these regions are aligned and in contact (Atkinson et al., 2008; Mink
& Deutsch, 1989; Speer et al., 1992; Spindler et al., 1993). A number of Clinical studies
have also described osteochondral lesions existing in the postero-medial tibial plateau
after ACL rupture (Chan et al., 1999; Kaplan et al., 1999). Vellet et al. (1991) provides a
classiﬁcation for these osteochondral lesions using magnetic resonance imaging (MRI),
documenting an overt loss of cartilage overlying geogaphic bone bruises in 48% patients
within 6 months of injury. These lesions are associated with occult micro-cracks near the
interface between calciﬁed cartilage and subchondral bone (Speer et al., 1992; Rangger et
al., 1998). These lesions may play a role in pain after joint trauma (??). Also, of concern
in today’s Clinical literature is that there is evidence of injury to articular cartilage and
chondrocytes overlying these bone bruises, which may predispose the knee to
degenerative changes (Mankin, 1982; Thompson et al., 1991; Faber et a1; 1999; Frobell et
al., 2008). Fang et al. (2001) suggests that damage to articular cartilage overlying MRI
detected bone bruises in patients with ACL rupture may be due to excessive compressive
forces generated in the joint during the acute injury.

Our laboratory has previously shown histological microfractures of subchondral
bone in isolated, ﬂexed human knees under high compressive loads that produce 18 to 21
MPa of contact pressure in the TF joint (Banglmaier et al., 1999; Meyer et al., 2008).

Articular cartilage surface lesions and cell death have also been documented in TF impact

56

studies with the rabbit that generate contact pressures of approximately 20 MPa and 24
MPa on the medial and lateral tibial plateaus, respectively (Isaac et al., 2008). Surgical
transection of the ACL (ACLT) in the rabbit has been used to investigate the
pathogenesis of OA irn the knee joint (Y oshioka et al., 1996; Chang et al., 1997; Batiste et
al., 2004; Vignon et al., 1987). These studies document formation,of osteophytes,
ﬁbrillation of cartilage and synovitis which leads to erosion of articular cartilage in the
joint within 8-12 weeks (Y oshioka et al., 1996; Batiste et al., 2004). These studies,
however, have not documented early damage to cartilage cells or subchondral bone
which might be similar to injuries noted in the Clinical literature.

The objective of the current study was to develop an animal model involving
traumatic ACL failure (ACLF) that includes acute compressive trauma to cartilage and
underlying bone. Since the rabbit knee joint also exhibits a posterior slope of the tibial
plateau which is more pronounced than the human (Crum et al., 2003), the ﬁrst
hypothesis of this study was that compressive loading of the ﬂexed rabbit knee would
result in ACL rupture along with signiﬁcant cartilage and underlying subchondral bone
damage. A second hypothesis of the study was that this traumatic model would result in
relatively more Chronic joint degeneration compared to the ACLT model. The traumatic
model of ACL rupture may have a more direct relevance to the clinical situation than the
ACLT.

MATERIALS AND METHODS

Nine skeletally mature Flemish Giant rabbits (5.5 :h 0.1 kg) were used in the

study. The investigation was approved by the Michigan State University All-University

Committee on Animal Use and Care. The animals were housed in individual cages (60 x

57

60 x 14 in) during the study. Three rabbits received a blunt force insult to the left TF joint
resulting in ACL rupture. Another three rabbits underwent unilateral, surgical transection
of the ACL. Two additional animals were used to study acute cellular trauma in the
cartilage, and one animal was used to document impact induced contact pressures in the
TF joint during rupture of the ACL.

Animals undergoing traumatic ACL rupture (n=3) were place under general
anesthesia (2% Isoﬂurane and oxygen). Following a previously described impact
procedure (Isaac et al., 2008), a 1.75 kg mass was dropped from a height of 75 cm (~ 13 J
of potential energy) striking the femoral condyle on the left leg. The sled was arrested
electronically after one impact. A pre-crushed, deformable impact head (Hexcel, 3.76
MPa crush strength) was used to ensure uniform loading over the condyle. The impact
interface was mounted in front of a 4.45 kN (1000 lb) load transducer (model AL311CV,
1000 lb capacity, Sensotec, Columbus, OH). Prior to impact the left limb was shaved.
With the arnimal lying supine in the ﬁxture, the knee was ﬂexed 90° and the foot was
ﬁxed in a custom designed boot with three Velcro straps (Figure 4.1). An additional
Velcro strap was crossed over the femur. Unlike the former study of Isaac et al. (2008),
the tibia was not constrained so as to allow anterior subluxation of the tibia. In one
arnimal, during setup, a lateral X-ray was performed on the ﬂexed knee in the restraint
ﬁxture. All animals received buprenorphine (0.3 ml/kg BW) every 8 hours for 72 hours

for post-trauma pain. The right limb served as a non-impacted, contralateral control.

58

  
  

Gravity accelerated
mass

Electronically triggered
clamp

Load transducer ' AK
Deformable interface L_.l I

q

 

 

 

 

 

 

Figure 4.1. Impact experiments were performed by dropping a gavity-accelerated mass
onto the ﬂexed tibial-femoral joint with approximately 13 J of potential energy. The
rabbit was oriented such that the deformable interface struck the femoral condyle with
impact forces oriented axially in the tibia.

One animal was euthanized witln 85.9 mg/kg BW Pentobarbital IV immediately
prior to impact in order to document contact pressures in the joint during ACL rupture.
The impact was admirnistered as previously outlined, after pressure sensitive ﬁlm packets
(Prescale, Fuji Film Ltd., Tokyo, Japan) had been inserted through anterior and posterior
joint capsules (Meyer et al., 2008). After impact, the ﬁlm was removed from the packet
and scanned (Scanmaker MRAS-1200E6, Microtek, Taiwan). The entire area of contact
was digitized (Photostyler, version 1.1A, Aldus Co., Seattle, WA) and the average
pressure, total contact area and the area having pressures over 20 MPa were determined
using an established protocol (Atkinson et al., 1998).

Two additional animals were sacriﬁced immediately following ACL ruptnrre in
order to document acute cartilage and chondrocyte damage via a cell viability assay. A 6

mm trephine (TREPH-6, Salvin Dental Specialties, Charlotte, NC) was used to core a

region of the medial and lateral tibial plateau in areas not covered by the meniscus, as

59

these areas were exposed to high contact pressures during impact (Figure 4.2) (Isaac et
al., 2008). A diamond saw (Isomet 11-1180 Low Speed Saw, Buehler, Lake Bluff, IL.)
was used to then undercut the cores leaving approximately 0.5-1 mm of bone below the

articular cartilage.

Lateral

 

Posterior

Figure 4.2. 6 mm osteochondral explants were taken in regions uncovered by the
meniscus for cell viability analyses.

Similarly, the femurs were ﬁxed in the diamond saw and sagittal slices were cut across
the medial and lateral condyles leaving approximately 0.5-1 mm of underlying bone. All
explants were rinsed 3 times with culture media before being placed in separate wells
ﬁlled with fresh Dulbecco’s modiﬁed Eagle’s medium (DMEM): F 12 (Gibco, USA
#12500-062) supplemented with 10% fetal bovine serum, additional amino acids, and
antibiotics (penicillin 100 units/ml, streptomycin lug/ml, amphotericin B 0.25 ug/ml),
and incubated for 24 hours (37°C and 95% humidity). This incubation period has been
found to be required to allow the perﬁrsion of dye in damaged cells post-irnpact (Ewers et
al., 2001; Phillips et al., 2004).

Following incubation, the specimens were prepared for cell viability analyses.
Full depth sections of the femoral and tibial cartilage and subchondral bone were cut

using a specialized cutting device (Ewers et al., 2001). The cell viability analyses

60

followed previous procedures (Rundell et al., 2005). Brieﬂy, slices from medial tibia
(MT), lateral tibia (LT), medial femur (MF) and lateral femur (LF) were rinsed with
phosphate buffered saline (PBS) and stained with calcein AM and etlnidiurn homodirner
(EthD-l), according to the manufacturer’s speciﬁcations (Live/Dead Cytotoxicity Kit,
Molecular Probes, Eugene, OR). Prior to imaging, each sample was rinsed three times
with PBS. Viable cells were distinguished by the presence of ﬂuorescent (geen) calcein
AM. Damaged cells were distinguished by a bright red ﬂuorescence. The number of
geen and red cells was manually counted with an image analysis progarn (Image J,
National Institutes of Health, 2004).

Another three animals underwent unilateral transection of the ACL. Both rear legs
of the animals were shaved from the hock to the hip. The area was prepared using 70%
Betadine scrub and 70% alcohol, alternatively. Once scrubbed, the rabbits were moved to
a sterile surgery suite. The left TF joint was then exposed through a medial parapatellar
incision. Following a medial arthrotomy, the patella was dislocated laterally, exposing the
ACL. With the knee in full ﬂexion the ACL was sharply transected. The joint capsule and
reticulum was sutured immediately after transection using 3/0 PDS. The sub-cuticular
layer and skin was Closed in sequence using 4/0 PDS and staples, respectively. A sham
operation was performed on the right limb. The rabbits were monitored closely by a
licensed veterinary technician (JA) for signs of pain. Post-surgery pain medication
(Buprenorphine 0.3ml/kg BW) was administered every 8 hours for 72 hours following
the procedure. MRI was taken of the ACLT and ACLF animals within 1 week post-injury

in order to verify complete transection or rupture of the ACL.

61

The three ACLF and three ACLT animals were sacriﬁced 12 weeks after injury.
The surfaces of the tibial plateaus and femoral condyles were stained with India ink to
highlight surface ﬁssures, cartilage degeneration, and other irregularities. The surfaces
were digitally photogaphed (Polaroid DMC2, Polaroid Corp., Waltham, MA) under a
dissecting microscope at 12X and 25X (Wild TYP 374590, Heerbrugg, Switzerland).
Gross morphological assessments were made according to the following criteria, after the
application of India ink using the following gading scale (Y oshioka et al., 1996).

Grade 1: Intact cartilage with the surface appearing normal with no ink retention;

Grade 2: Cartilage with few surface lesions that appears normal before staining,

but retains some ink;

Grade 3: Cartilage with moderate ﬁbrillation that retains intense black patches of

ink; and

Grade 4: Cartilage with full thickness erosion exposing underlying bone.
After a morphological assessment, the plateaus and condyles were placed in 10%
buffered formalin for one week and decalciﬁed in 20% formic acid for another week.
Tissue blocks were then cut medial to lateral across both the plateau and femoral
condyles. Tissue blocks were processed in parafﬁn and six sequential sections, 8 rrnicrons
thick, were prepared for analysis. The sections were stained with Safranin O-Fast Green
and examined under light microscopy at 12-40X. The thicknesses of the articular
cartilage, the zone of calciﬁed cartilage and the subchondral plate were determined with a
calibrated eyepiece at 25X by visually averaging across the entire sample by three readers
(JW, DI, EM). These readers also independently scored the cartilage and underlying

subchondral bone using a system documented in the literature and previous studies from

62

our laboratory (Columbo et al., 1983; Weaver et al., 2005). The number of surface
ﬁssures and micro-trauma (vertical and horizontal cracks at the ZCC/ SB interface) were
manually quantiﬁed in one histological section from each animal. Proteoglycan content
was scored by the uptake of Saﬁanin-O stain with normal uptake of stain receiving a 0
and total loss of stain receiving a 4 (Golenberg et al., 2008).

A two-factor, repeated measures AN OVA (limb, plateau) with one-tailed post hoc
Student-Newman-Keuls (S-N-K) tests was used to compare the cell viability data from
the left impacted, limbs with the contralateral, controls. A two-factor ANOVA
(plateau/condyle, ACLF/ACLT) with a post-hoc S-N-K test was used to compare the
frequency of nnicrocracks in the tibial plateaus and femoral condyles of the ACLT and
ACLF goups. Morphological scores from the ACLT and ACLF goups were compared
using a one-factor AN OVA. The contralateral, control limb was analyzed using the same
procedures and statistical analyses as the test limbs. Statistical signiﬁcance was indicated
at p<0.05 in all tests.

RESULTS

At 90° of ﬂexion the rabbit’s tibial plateau displayed a distinct posterior slope on
the order of 20° ﬁom the horizontal (Figure 4.3). The average impact force was 931 :t 27
N. In each case rupture of the ACL and damage to the meniscus were evident eitlner
acutely witlnin one week after trauma in an MRI scan of the joint. The average contact
pressures generated on the medial and lateral plateau were 22.7 MPa and 27.5 MPa,
respectively in the single animal tested (Table 4.1). The peak pressures on the medial. and

lateral plateaus were 50.9 and 48.4 MPa, respectively.

63

Table 4.1. Analysis of pressure sensitive ﬁlm revealed high contact pressures in the
medial compartment of the TF joint during ACL trauma, and even higher pressures in the
lateral facet.

Area over 20 MPa

‘

Foot ﬁxation boot 1

 

Figure 4.3. Radiogaph of the rabbit lower extremity orientation for impacts. The
posterior slope of the tibial plateau creates anterior subluxation of the tibia to cause ACL
rupture.

As a result of traumatic ACL rupture acute surface ﬁssures were noted, especially
on cartilage covering the medial femoral condyle (Figure 4.4). Cell viability analyses of
cartilage/bone slices taken from this region also showed a large percentage of cells with

damaged membranes (stained in red), particularly in regions adjacent to surface ﬁssures

(Figure 4.4).

54

 

Figure 4.4. India ink staining revealed acute ﬁssuring and a signiﬁcant amount of
damaged chondrocytes (red) in regions surrounding these surface lesions on the medial
femoral condyle followirng traumatic ACL rupture.

Statistical analysis of the cell viability data revealed a signiﬁcantly larger percentage of
damaged chondrocytes in the MF compartment of impacted versus control limbs

(p=0.08). While the same trend was noted at other locations, no site had statistically more

damaged cells than the contralateral, control (Figure 4.5).

65

45 _ l ACLF Control

 

 

LF MF LT MT

Figure 4.5. Cell viability analysis showed an increase in the percentage of cells with
damaged plasma membranes in the ACLF joints in all compartments (lateral femur (LF),
medial femur (MF), lateral tibia (LT) and medial tibia (MT)) compared to the
contralateral joint tissue.

The ACLF goup showed severe degenerative Changes that included severely
discolored and viscous synovial ﬂuid, erosion of cartilage on the femoral trochlear ridges,
and the development of periarticular joint osteophytes. Morphological assessment of the
ACLF goup at 12 weeks showed full thickness erosion of articular cartilage on the
medial femoral condyle in all specimens. Cartilage erosion was also noted in the posterior
aspect of the medial tibial plateau in these animals. The animals from the transected
goup also showed signs of synovitis including an increase in synovial ﬂuid viscosity
with discoloration as well as osteophyte formation on the femoral trochlear ridges (Figure

4.6). ACLT animals showed severe ﬁbrillation of cartilage on the medial femoral condyle

of one animal, but no full-thickness erosion of cartilage. Comparative morphological

66

 

Figure 4.6. Severe cartilage erosion was noted on the femoral trochlear ridges in the
traumatic goup (a), while only mild erosion was documented in the transected animals
(b). The arrows denote joint osteophytes. The medial femoral condyles also showed ﬁrll
thickness ulceration of articular cartilage in the traumatic goup (C), but only ﬁbrillation

in the transected goup (d). The medial tibial plateau showed cartilage erosion in the

posterior aspect of the compartment in the traumatic goup (e), while the transected
rabbits (0 showed no such erosion in the tibial plateau.

67

assessments of the two goups indicated statistically signiﬁcant higher gades of

degeneration in the MT, the MF and the LT for the ACLF arnimals compared to the

 

   

    

 

ACLT animals (Figure 4.7).
5 .
IACLF
g 4 ' IACLT
0
an
To 3 '
.2
U?
o
3 2 -
.C
E-
o
E 1 -
0 .
M L M L
ACL Rupture Contralateral Limb
5 I
a, IACLF
3 4 - IACLT
0
tn
To 3 -
.2
8’
B 2 '
.C
E
o .
E 1
0 I
M L M L
ACL Rupture Contralateral Limb

Figure 4.7. Gross morphological analysis of the medial (M) and lateral (L) (a) femoral
condyle, and (b) tibial plateau after staining with India ink revealed more cartilage
defects in the traumatically injured rabbits.

68

Both acute and Chronic goups of animals showed injury to the menisci (Figure
4.8). The lateral meniscus of the acute animals displayed longitudinal tears located in
posterior regions. One acute animal displayed medial meniscal damage. All 12 week
ACLF arnimals showed goss morphological changes to the medial and lateral meniscus
that included ﬁbrillation, degeneration of the central portion of the menisci and erosion of
the cranial and caudal horns. One 12 week ACLT animal displayed a “bucket-handle”
tear of the medial meniscus and degeneration of the lateral meniscus. The other two

ACLT animals lacked signiﬁcant meniscal degeneration.

“Longitudinal” tear

Radial tear

 

Figure 4.8. India ink staining of the lateral mernisci highlight meniscal damage as a result
of traumatic ACL injury.

Signiﬁcantly more occult microcracks appeared in all compartments of the ACLF
limbs compared to the ACLT lirnbs (Figure 4.9). Histological sections of both goups
showed signiﬁcant surface ﬁbrillation in articular cartilage on the medial and lateral

femoral condyles with signiﬁcant losses of proteoglycan stain.

69

9 . IACLF IACLT
8 - # #

l

Frequenc
N (.0 A OI
l

 

 

 

o .3
l 1

MT LT MF LF
Figure 4.9. Histological sections showed a signiﬁcant increase in the number of vertical
and horizontal microcracks at the articular cartilage/subchondral bone interface, where
(#) denotes statistical signiﬁcance between models.
Severe cartilage ﬁbrillation and erosion were documented in all of the ACLF medial
femoral condyles and in two of the lateral femoral condyles (Figure 4.10). One of the
ACLT animals showed excessive histological ﬁbrillation and cartilage erosion. The

medial tibial plateau displayed more surface ﬁssuring in the ACLF animals compared to

the ACLT arnimals.

70

 

Figure 4.10. Histological sections of the medial femoral condyles (a & b) and medial
tibial plateau ((1) revealed severe surface ﬁbrillation and ﬁssures, respectively.
Proteoglycan loss was noted completely in the femoral sections (a & b) and at the surface
in the medial tibial plateau (c & d). Horizontal and vertical micro-cracking was also
noted at the ZCC/SB interface as pointed out.

DISCUSSION

The current study has outlined data from the development of a small animal
model to study traumatic ACL rupture and the potential for post-traumatic OA. ACLT is
widely used to investigate the patlnogenesis of CA, but the traumatic model involves
more acute injuries that precipitate a more aggessive disease process (Burr and
Schaffler, 1997). Rabbit models involving ACLT have documented localized cartilage
erosion accompanied by bone remodeling and osteophyte formation (Y oshioka et al.,
1996; Chang et al., 1997; Batiste et al., 2004; Vignon et al., 1987). A previous study by
Batiste et al. (2004) documents cartilage ﬁbrillation and full thickness erosion in 22% and
59% of the TF joints 12 weeks post—ACLT, respectively. These studies always document

that the most extensive area of degeneration is the medial femoral condyle (Y oshioka et

al., 1996; Chang et al., 1997; Batiste et al., 2004). In the traumatic ACL rupture model

71

gade 4 disease was documented in all animals in the medial femoral condyle, as well as
the medial tibial plateau. While the lateral compartment did experience cartilage
ﬁbrillation in the traumatic model, no ﬁnll thickness defects were noted. Studies on the
rabbit ACLT model have also documented histological changes that include cartilage
hypertrophy, reductions in cell density and matrix alterations preceding cartilage
ﬁbrillation at 12 weeks (Vignon et al., 1987). In previous studies, our laboratory has
shown that blunt trauma at 6 J of energy to the in viva rabbit patellofemoral (PF) joint
leads to a signiﬁcant increase in the percentage of acutely damaged chondrocytes
(Rundell et al., 2005). Furthermore using the same model, Ewers et al. (2002) documents
surface lesions, progessive degadation of retropatellar cartilage and thickening of the
underlying subchondral bone 3 years post-trauma. The current study documents an
increase in the percentage of cells damaged acutely in tibial plateaus and femoral
condyles following ACLF. These acute injuries may have contributed to the rapid
degeneration of cartilage that has been documented in the current study. Hashimoto et al.
(1998) also suggests that acute injuries to cartilage and cells may play critical role in the
long term progession of Chronic joint degeneration in humans.

A signiﬁcant result from the ACLF model was the histological appearance of
numerous vertical and horizontal microcracks at the interface between articular cartilage
and subchondral bone, without signs of goss fracture in either the tibial plateau or
femoral condyles. The ACLT models have not documented these acute damages to
underlying subchondral bone, but the clinical literature does describe tlnese injuries after
ACL rupture (Frobell et al., 2008) and, while these mechanisms are not well understood,

these osteochondral lesions have been strongly implicated in the development of a post-

72

traumatic OA (Frobell et al., 2008; Fang et al., 2001; Burr and Radin, 2003; Tarnbyah et
al., 2008). The current study also documented a higher frequency of microcracks in the
lateral than medial plateau. This could possibly be due to slightly higher contact pressures
in the lateral facet, or because of differences in the material properties of the two
plateaus. In a previous study by Batiste et al. (2004), the bone mineral density (BMD) of
the rabbit TF joint was found to be signiﬁcantly higher in the medial femoral condyle and
medial tibial plateau than in the lateral compartments. The human literature also
documents the BMD of the tibial plateau for a young, non-osteoarthritic population to be
approximately 15% higher in the medial compartment than in the lateral compartment
(Hurwitz et al., 1998). The lower BMD in the lateral compartment may correspond to an
approximately 50% lower ultimate failure stress for trabecular bone (Goldstein et al.,
1983). This may explain why bone bruises are more commonly documented in the lateral
compartment in clinical studies, as well as in the current ACLF model. Future studies
with this newly developed model may be able to help Clarify the role of underlying bone
trauma on the development of degeneration in overlying articular cartilage.

One limitation of the current study was a small sample size for cell viability
analysis, which may have limited statistical signiﬁcance in the study. However,
histological scoring did show signiﬁcant differences between tlne ACLT and ACLF
goups in the MT, LT, MF and LF, as well as statistical differences in the morphological
scores for the MT, LF and LT. Additionally, the study was conducted for a 12 week
period. A recent study by Papaioannou et al. (2004) suggests 2 phases in the ACLT
model. The early degeneration phase is from 0-8 weeks, followed by a late phase of

regeneration or repair from 8-16 weeks. Batiste et al. (2004) also documents a decrease in

73

the volumetric BMD (vBMD) at 4 and 8 weeks post-ACLT, with a return to control
values at 12 weeks. These studies support the notion of degenerative and regenerative
phases following ACLT. Future work on the ACLF model should be conducted for
various time periods in order to more accurately document the disease process in this new
model.

While previous ACLT models have allowed investigators to study the
pathogenesis of 0A, they have failed to address the acute injuries that occur in a clinical
setting; such as damage to underlying subchondral bone, meniscus and cartilage. These
injuries may have signiﬁcant implications in the long-term development of disease. The
current investigation has outlined a model of traumatic ACL rupture which ultimately
may have direct relevance to the clirnical setting. Future investigations can then focus on
the importance of addressing acute injury to articular cartilage, as well as the efﬁcacy of
various therapeutic agents. The long-term efﬁcacy of intra-articular ACL replacements
should also be investigated in the future with the new model.

ACKNOWLEDGMENTS

This study was supported by a gant from the Centers for Disease Control and
Prevention, Center for Injury Control & Prevention (CE000623). The contents of this
manuscript are solely the responsibility of the authors and do not necessarily represent
the ofﬁcial views of the Center for Injury Control and Prevention. The authors wish to
acknowledge Ms. Jean Atkinson (J .A.) for her assistance in animal care, Ms. Jane Walsh
(J .W.) for her assistance with histological evaluations and Dr. Julien Cabassu for his

assistance in the surgical procedures.

74

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Weaver B, Haut R, 2005, “Enforced exercise after blunt trauma signiﬁcantly affects
biomechanical and histological Changes in rabbit retro-patellar cartilage,” J Biomech, 38,
pp. 1177-1183.

Yeow C, Cheong C, Ng K, Lee P, Goh J, 2008, “Anterior cruciate ligament failure and
cartilage damage during knee joint compression,” Am J Sports Med, 36, pp. 934-942.

Yoshioka M, Coutts R, Anniel D, 1996, “Characterization of a model of osteoarthritis in
the rabbit knee,” OA and Cart, 4, p. 87-98.

78

 

CHAPTER FIVE

ACUTE REPAIR OF CHONDROCYTES IN THE RABBIT
TIBIOFEMORAL JOINT FOLLOWING BLUNT IMPACT USING P188
SURFACTANT

ABSTRACT

Two speciﬁc types of injuries are strongly associated with subsequent knee OA; cruciate
ligament damage and meniscal tears. Damage to Chondrocytes has been documented in
patients suffering ACL tears. Recent studies indicate that there may be a correlation
between acute chondrocyte damage and the chronic progession of OA. P188 surfactant
is able to interact with the bilayer of damaged cell membranes to restore their integity
after injury. The hypothesis of the current study was that a single injection of P188 into
the in viva TF joint following impact would reduce the percentage of damaged
Chondrocytes. A single, 13 J of energy impact was delivered to the left limb of eighteen
rabbits, while the right legs served as contralateral controls. Animals were divided in
three goups of six; ‘time zero’, ‘4 day no P188’ and ‘4 day P188’. The left and right
limbs of the ‘time zero’ and ‘4 day no P188’ animals received an injection of sterile PBS
immediately following trauma. The left limbs of the ‘4 day P188’ rabbits received an
injection of P188, and right limbs received a sham saline injection. Cell viability assays
were performed to quantify the percentage of cells with damaged membranes. A two-way
ANOVA was used to determine statistical differences between goups, and a two-way
repeated measures ANOVA was used to determine differences between limbs. In botln the
‘time zero’ and ‘4 day no P188’ goups, an increase in the number of damaged
chondrocytes was documented in the impacted limb compared to the control. The ‘4 day

P188’ goup showed a signiﬁcant decrease in the percentage of damaged Chondrocytes

79

when compared to the ’4 day no P188’ animals. No signiﬁcant difference was found
between the impacted, P188 limb and the contralateral, control. A single injection of
P188 surfactant into the TF joint immediately following insult resulted in a signiﬁcant
reduction in the percentage of cells with damaged plasma membranes in all
compartments of the TF joint. Future studies should examine the long term consequences
of P1 88 or possibly other interventions to acutely repair cellular membranes following

trauma to the knee.

8O

INTRODUCTION

Participation in sports, recreation and exercise (SRE) is increasingly popular and
widespread in American culture. Furthermore, participation in SRE increases the risk of
musculoskeletal injuries. History of a joint injury, particularly to the knee or hip,
increases the risk of developing chronic joint disease, such as osteoarthritis (0A). 0A
affects over 21 million Americans and is the leading cause of disability in the United
States (US Census Bureau, 2000). Acute knee joint injury has been associated with the
subsequent development of post-traumatic osteoarthritis (Gelber et al., 2000). Although
acute injury to cartilage is currently thought to be a factor associated with the
development of 0A, the pathway that leads ﬁom a blunt impact load on the joint cartilage
to the development of Chronic disease is yet unclear (Lewis et al., 2003). Recent studies
have indicated that there may be a correlation between acute chondrocyte damage and the
chronic pathogenesis of 0A in the joint (Hashimoto et al., 1998; Natoli and Atlnanasiou,
2008). Since chondrocytes are required for matrix repair and Chondrocyte death
eventually leads to matrix loss (Simon et al., 1976), Chondrocyte death by eitlner
apoptosis or necrosis has become a focus of OA research, and more recently cartilage
trauma research.

Two speciﬁc types of injuries are strongly associated with subsequent knee OA;
cruciate ligament damage and meniscal tears (Felson et al., 2004). Clincally, in over 80%
of patients suffering anterior cruciate ligament (ACL) tears, a characteristic
osteochondral lesion occurs in the postero-lateral aspect of the tibia and/or antero-lateral
aspect of the lateral femoral condyle (Atkinson et al., 2008; Mink et al., 1989; Speer et

al., 1992; Spindler et al., 1993). These injuries are also associated with visible damage to

81

the chondrocytes (Johnson et al., 1998) and an overt loss of cartilage within six months
(Vellet et al., 1991) overlying “geogaphic” bone bruises, in particular. These types of
bone lesions have been associated with occult microcracks of subchondral and/or
trabecular bone (Speer et al., 1992; Rangger et al., 1998). There is also evidence that
acute injury to articular cartilage overlying these bone bruises may predispose the knee to
degenerative changes in the joint (Mankin, 1982; Thompson et al., 1991; Faber et al.,
1996). Evidence in the current Clinical literature suggests that these ACL patients will
likely develop Chronic joint disease whether they are reconstructed or not (Bahr et al.,
2005). This may be due to the acute damage of articular cartilage in the joint.

Damage to articular cartilage overlying MRI detected bone bruises in patients
with ACL tears has been suggested to be caused by excessive compressive forces on
cartilage and meniscus generated in the joint during the acute ligamentous injury (Fang et
al., 2001). This acute injury may provide a basis for the initiation of the chronic joint
disease, OA (Fang et al., 2001; Frobell et al., 2008). In a previous study, our laboratory
has shown that a single, 6 J of energy blunt insult to the rabbit PF joint leads to a
signiﬁcant increase in the percentage of acutely damaged chondrocytes (Rundell et al.,
2005), as well as acute surface ﬁssures, progessive degadation of retro-patellar surface
cartilage, and thickening of underlying subchondral bone 3 years post-trauma (Ewers et
al., 2002). The authors of these previous studies hypothesized that the chronic cartilage
degadation may be partly due to acute damage of chondrocytes.

Acute damage to Chondrocytes, necrosis, has been shown to produce degadative
changes chronically in an in viva arnimal model (Simon et al., 1976). A deﬁning feature of

cellular necrosis is swelling of the cell due to a damaged membrane. This damage results

82

 

in the inability of the cell to maintain ionic gadients across its plasma membrane and
ultimately, the necrotic cell ruptures (Duke et al., 1996). Prior to cellular lysis, these cells
may develop an apoptotic pathway or produce degenerative matrix enzymes (Baars et al.,
2006). Due to their arnphiphilic properties, some mild surfactants are able to interact with
the bilayer of cell membranes to restore their integity after injury from physical stress
(Clarke and McNeil, 1992; Papoutsakis, 1991). One such surfactant is poloxamer 188

(PI 88). P188 is an 8400-dalton triblock copolymer containing botln hydrophobic and
hydrophilic regions. Recent studies on brain trauma suggest that P188 can help ‘save’
neurons ﬁom developing early necrotic death following severe mechanical loading
(Barbee et al., 1992; Marks et al., 2001). Furthermore, P188 has been shown to reduce
cell damage after in viva loading of the rabbit PF joint (Rundell et al., 2005), as well as
after in vitra impacts to bovine chondral (Phillips and Haut, 2004) and osteochondral
(Natoli and Athanasiou, 2008) explants.

A previous study, using the rabbit tibiofemoral (TF) joint, documents a signiﬁcant
increase in the percentage of acutely damaged chondrocytes following a single, 13 J of
energy impact on the joint (Isaac et al., 2008). Using this previously developed model,
the hypothesis of the current study was that a single injection of P1 88 surfactant into the
in viva TF joint immediately after impact would signiﬁcantly reduce the percentage of
Chondrocytes in the articular cartilage with acutely damaged plasma membranes.
Ultimately, administration of this therapeutic agent immediately following a suspected

joint injury may aid in mitigating the onset of a Chronic joint disease.

83

MATERIALS AND METHODS

Eighteen skeletally mature, Flemish Giant rabbits (aged 6-12 months) were used in this
study after approval by an All-University Committee on Animal Use and Care. The blunt
impact experiments have been described previously in detail (Isaac et al., 2008). Brieﬂy,
a 1.75 kg mass with a pre-crushed, deformable impact head (Hexcel, 3.76 MPa crush
strength) was dropped onto the left, ﬂexed TF joint of anesthetized animals (2%
isoﬂurane and oxygen). The right limb was not impacted and used as a paired,
unimpacted control. A 4.45 kN (1000 lb) load transducer (Model AL311CV, 10001b
capacity, Sensotec, Columbus, OH) was attached behind the impact interface to record
peak contact load, time to peak, and total contact duration.

Six rabbits were impacted and randonnly selected as “time zero” animals and
received a 1.5 mL sham injection of sterile phosphate buffered saline (PBS) into the joint
capsule of both the right and left limbs. These animals were then sacriﬁced immediately
after impact. The remaining 12 animals were sacriﬁced 4 days post-impact. During these
four days the animals were housed in individual cages (152 x 152 x 36 cm) and permitted
free cage activity. Six of the 4 day old arnimals received a single 1.5 mL injection of an 8
mg/mL concentration of P188 surfactant in sterile phosphate buffered saline (PBS) into
the traumatized TF joint capsule immediately after impact. The concentration level was
established in previous studies by the laboratory (Phillips and Haut, 2004; Rundell et al.,
2005; Baars et al., 2006). The right legs of these arnimals received a 1.5 mL sham
injection of sterile PBS into the joint. The remaining six 4 day animals received sham
injections of 1.5 mL sterile PBS into botln the impacted left limb as well as the

contralateral, right limb. The combination of P1 88 in PBS and PBS sham solutions were

84

ﬁlter sterilized prior to injection using a 0.2 mm vacuum ﬁlter (N algene, Nalge Nunc Int.,
Rochester, NY). After injection, the limb was manually ﬂexed a number of times to
distribute the P188 surfactant and PBS solutions in the joint.

Immediately following sacriﬁce the joint was dissected and examined for
abnormalities. The surfaces of the femur and tibia were wiped with India ink to highlight
surface defects and photogaphed using a digital camera (Polaroid DMCS, Polaroid
Corporation, Waltham, MA) under a dissecting microscope (Wild TYP 374590,
Heerbrugg, Switzerland). The femurs and tibiae were prepared for cell viability analyses.
A 6 mm trephine (#TREPH-6, Salvin Dental Specialties, Charlotte, NC) was used to core
a region of the medial and lateral tibial plateau in areas not covered by the meniscus, as
these were the areas of lnigh contact pressure during impact [29]. A diamond saw (Isomet
11-1180 Low Speed Saw, Buehler, Lake Bluff, IL.) was then used to undercut the cores,
leaving approximately 0.5 mm of bone underlying the articular cartilage. The femurs
were ﬁxed parallel to the diamond saw allowing coronal slices to be taken across the
medial and lateral condyles in a predetermined area of contact, also leaving
approximately 0.5 mm of underlying subchondral bone. Explants were rinsed 3 times
with culture media before being placed in separate wells ﬁlled with ﬁesh Dulbecco’s
modiﬁed Eagle’s medium (DMEM): F12 (Gibco, USA #12500-062) supplemented with
10% fetal bovine serum, additional amino acids, and antibiotics (penicillin 100 units/ml,
streptomycin l ug/ml, amphotericin B 0.25 jig/ml), and incubated for 24 hours (37°C and
95% humidity) using an established protocol (Ewers et al., 2001).

Following incubation, full depth sections of the explants were cut using a

specialized cutting device (Ewers et al., 2001). The cell viability analyses followed

85

previous procedures (Rundell et al., 2005). Brieﬂy, slices from each compartment of the
femur and tibia were rinsed with PBS and stained with calcein AM and etlnidium
homodirner (EtlnD-l), according to the manufacturer’s speciﬁcations (Live/Dead
Cytotoxicity Kit, Molecular Probes, Eugene, OR). Prior to imaging, each sample was
rinsed three times with PBS to remove excess stain. Viable cells were distinguished by
the presence of ﬂuorescent (geen) calcein AM. Damaged cells were distinguished by a
bright red ﬂuorescence due to ethidiurn homodirner passing tlnrough a damaged plasma
membrane. Sections were viewed using a ﬂuorescence microscope (Leitz Dialux 20,
Leitz Mikroskopie und Systeme GmgH, Wetlzar, Germany). Each slice was then
photogaphically divided into three zones: superﬁcial (top 20% of explant thickness),
middle (middle 50%) and deep (bottom 30%) (Phillips and Haut, 2004) (Figure 5.1). Two
blinded observers (MR, BP) manually counted the number of geen and red cells with an
image analysis progam (Image J, National Institutes of Health, 2004). The total
percentage of damaged cells was deterrnirned for each section (for an average of

approximately 4 samples from each compartment).

[Keep zone : :Lil‘itl» 1:1‘ swimmer.

 

Figure 5.1. Cell viability was measured in the thin sections of cartilage and bone. The
stained osteochondral explants were imaged and divided into three zones: superﬁcial,
middle, and deep.

86

Statistical analysis was used to evaluate the percentage of damaged cells in each
compartment and zone. A two-factor, repeated measures ANOVA with a post hoc Tukey
test was used to compare the left impacted limbs with the right controls in each time
goup for botln the total percentage of damaged cells and the zonal data. A two-factor
ANOVA with post hoc Tukey tests was used to compare the total and zonal data for both
the impacted limbs of time zero versus 4 day no P188 goup, and the 4 day no P188
versus 4 day P188 goup. The control limbs of botln 4 day goups were compared using a
t-test. Statistical signiﬁcance was indicated at p<0.05.

RESULTS

Gross inspection of the joints at necropsy showed no signs of joint disease and no
damage to ligaments or menisci. No statistical differences were found in the times to
peak impact load or the magnitudes of the peak load between treatment goups. The
average peak, inertially compensated impact load and impact duration were 1102 1 92 N
and 23.0 :1: 0.2 ms, respectively.

Analysis of damaged cells through the depth of the articular cartilage indicated a
statistically signiﬁcant increase in the percentage of damaged cells in the impacted than
unimpacted joints in the medial compartment of the tibial plateau (MTP) (p=0.003),
lateral tibial plateau (LTP) (p=0.002) and lateral femoral condyle (LFC) (p=0.025), as
well as a statistical trend of a difference in the medial femoral condyle (MF C) (p=0.08)
for the ‘time zero’ goup. A signiﬁcant increase in the total percentage of damaged cells
was observed in the MTP (p=0.003), LTP (p=0.004) and LFC (p<0.001), as well as a
statistical trend in the MFC (p=0.069) between the impacted ‘4 day no P188’ and the

contralateral, controls (Figure 5).

87

60 _ IIMPACT ECONTROL

 

 

 

 

50 -
.9
'3
U
13
0
O)
G
E
N
0
.\°
FL FM TL TM
a
60 - IIMPACT ICONTROL
E 50 - * r
a
O
'0
0
D)
N
E
N
0
.\°
b ,
FL FM TL TM

Figure 5.2. A single, blunt impact to the TF joint produced a signiﬁcant increase in the
percentage of damaged cells in the ‘time zero’ (a) and ‘4 day no P188’ (b) goups. A ‘*’
indicates a statistically signiﬁcant difference between the impacted and control limbs.

A single injection of P1 88 into the TF joint immediately after impact signiﬁcantly
reduced the number of damaged chondrocytes in the MTP (p=0.034), LTP (p<0.001),
MFC (p<0.001) and LFC (p<0.001) in the ‘4 day P188’ goup compared to the ‘4 day no

P188 goup’ (Figure 5.3 & 5.4a).

88

 

Figure 5.3. Administration of P188 signiﬁcantly reduced the percentage of damaged
cells in the ‘4 Day P188’ goup (a) when compared to tire ‘4 Day No P188’ goup (b).

Furthermore, no signiﬁcant differences were noted at any of the four locations between

the ‘4 day P188 goup’ and their contralateral, control limbs (Figure 5.4b).

89

60 — I4-Day Impact IP188

50— * *

ells

0

ed

% Dama

 

 

TL TM

 

60 - IIMPACT ECONTROL

% Damaged Cells

 

 

b FL FM TL TM
Figure 5.4. P188 reduced the number of damaged cells when compared to the ‘4 Day No
P188’ goup (a), while no differences were noted between the ‘4 Day Pl88’ goup and
the contralateral, controls (b). A ‘*’ indicates a statistically signiﬁcant difference between
the impacted limbs of the ‘4 day no P188’ and ‘4 day P188’ goups.
Analysis of the zonal data indicated the most consistent statistical effects of impact and
P188 intervention in the superﬁcial zones. For example, signiﬁcantly higher percentages

of damaged cells were noted in the LFC (p=0.002), LTP (p=0.004), and MTP (p=0.05),

with a statistical trend in the MFC (p=0.1) of the impacted limb when compared to the

90

contralateral, control limb for the ‘4 day no P188’ goup (Figure 5.5). Signiﬁcantly
higher percentages of damaged cells were also noted in the superﬁcial zone of the LTP
(p=0.026), MFC (p=0.016) and LF C (p=0.002), as well as a statistical trend in the MTP
(p=0.077) between the impacted limbs of the ‘4 day no P188’ and the ‘4 day P188’
goups. However, no differences were seen between the impacted and contralateral limbs
in the ‘4 day P188’ goup at any site (LFC p=0.52, MTP p=0.4l, LTP p=0.57, MTP
p=0.91).
Figure 5.5. Analysis of zonal data revealed a signiﬁcant increase in the percentage of
damaged cells in the superﬁcial zone of the ‘4 Day No P188’ goup compared to their
controls in the (a) LFC, (b) MFC, (c) LTP and (d) MTP. A’*’ denotes a statistically
signiﬁcant difference between the impacted and contralateral limbs, while ‘+’ denotes a

signiﬁcant difference between the impacted limbs of the ‘4-day no P188’ and the ‘4 day
P188’ goups.

Impacted
I Unim pacted

% Damaged Cells

 

 

timed zero 4—day no p-188 4 day p-188

91

Figure 5.5 Continued.

 

 

 

 

Impacted
I Unimpacted
30 .
70 1
2 60 ‘
3 +
0 50 -
8
g) 40 -
g 30 -
o\° 20 .
10 -
0 .
b timed zero 4—day no p-188 4 day p-188
Impacted
I Unimpacted
E
'3
0
'c
e
on
N
E
I!
0
,\°
c timed zero 4-day no p-188 4 day p—188

92

Figure 5.5 Continued.

E11 Impacted

80 _ I Unimpacted

70-

8

50-
40-
30‘

% Damaged Cells

20'
10'
0.

 

 

timed zero 4-day no p-188 4 day p-188

DISCUSSION

The objective of the current study was to determine the effect of P1 88 on cell viability
following a single, traumatic impact to the rabbit TF joint. Impact trauma to the joint
resulted in an increase in the percentage of damaged cells in the articular cartilage for all
compartments. These results compared with a previous study by our laboratory
documenting an increase in damaged cells in the medial (18%) and lateral (14%) tibial
plateaus following a 13 J energy impact (Isaac et al., 2008). Furthermore, the current
study indicated the geatest increase in the percentage of damaged chondrocytes
following impact in the lateral compartments of both the femur and tibia. These results
compared with those from Isaac et al. (2008) documenting slightly higher percentages of
damaged cells laterally, and corresponding to a trend for higher impact induced contact

pressures in the lateral compartment during impact. The results of both studies are

93

supported by the clinical literature in patients suffering anterior cruciate ligament (ACL)
rupture where osteochondral lesions (or bone bruises) and early Changes in the overlying
articular cartilage are typically conﬁned largely to the lateral compartment (Atkinson et
al., 2008; Mink and Deutsch, 1989; Speer et al., 1992; Spindler et al., 1993).
Administration of P188 surfactant immediately after impact reduced the
percentage of damaged cells for all locations in the TF joint. Some lack of statistical
power was noted, however, in the MTP. This could be due to relatively more pre-impact,
baseline damage typically in this compartrrnent of the rabbit stiﬂe joint (Golenberg et al.,

2008). The results of the current study also compared with previous studies by our

 

laboratory that document the ability of P188 to reduce the extent of Chondrocyte damage
following insult to the rabbit PF joint (Rundell et al., 2005), as well in bovine chondral
explants undergoing unconﬁned compression for contact pressures of 25 MPa (Phillips
and Haut, 2004). In a more recent study using bovine osteochondral explants Natoli and
Antlnanasiou (2008) also document that the administration of P1 88 surfactant following a
2.8 J impact reduced the percentage of cell death by nearly 75%.

Cell damage in the current study was measured by membrane disruption,
documented by the ability of etlnidium homodirner to pass through the plasma membrane.
A deﬁning feature of this damage, called necrosis, is cellular swelling due to the injured
cell not being able to maintain ionic gadients across a damaged plasma membrane (Duke
et al., 1996). Previously, Marks et al. (2001) showed that P188 surfactant speciﬁcally
inserts into only the damaged areas of the cell membrane. A limitation of the current
study was that the longer term response of these cells was not monitored. Chondrocyte

death by apoptosis has been shown in human biopsy tissue near sites of chondral fracture

94

(Kim et al., 2002), as well as in carnine cartilage explants following cyclic loads (Chen et
al., 2001). While the mechanism of cell death following traumatic loading of articular
cartilage is largely unknown, Chen et al. (2001) suggests that necrosis is observed 2 hrs
after cessation of loading, whereas apoptosis (TUNEL-positive cells) is not signiﬁcant
until 48 or more hours after loading. These data suggest that mecharnical injury to a joint
may result in both necrotic and apoptotic cell death. Irnportantly, P188 repaired '
chondrocytes may ultimately die or produce excessive amounts of degeneration products
after traumatic injury, via apoptosis. In fact, in a study on human Chondral explants

subjected to 14 MPa of unconﬁned compression D’Lima et a1. (2001) documents 34% of

 

chondrocytes suffered apoptosis in the longer term. The fate of these P188 repaired cells
remains unknown. However, in a previous study performed by this laboratory using
bovine chondral explants subjected to 25 MPa of unconﬁned compression, the
administration of P1 88 surfactant was effective in reducing the percentage of cells with
DNA fragnentation (as measured by TUNEL stain) 7 days following impact.
Interestingly, the percentage of cells “saved” was similar to that “saved” within 1 day in
previous studies using the same model (Phillips and Haut, 2004). The authors proposed
that the acute damage to chondrocytes occurred by necrosis, as suggested by Chen et a1
(2001), and this precipitated a longer term response of the cells where apoptosis develops
with the possible production of various products of matrix degadation (Baars et al.,
2006)

Death of chondrocytes following traumatic injury has been associated with loss of
glyosarninoglycans (GAG) from the tissue and decreased proteoglycan synthesis (Huser

and Davies, 2006; Torzilli et al., 1999; Ewers et al., 2001; Jeffrey et al., 1997). These

95

degenerative Changes have been shown to result in a loss of tissue integity, represented
by a decrease in tissue stiffrness as well as an increase in tissue permeability (Kurz et al.,
2001; Ewers and Haut, 2000; Ewers et al., 2002). Additionally, in a study on porcine
patella Duda et al. (2001) document considerable cellular dysfunction that may act to
promote the subsequent structural tissue damage. This may be particularly important
because the synthesis of cartilage matrix proteins is directly dependant on cell viability
(Duda et al., 2001). Cellular necrosis has been shown to generate early OA-like Changes
in tissue from a Chronic animal model (Simon et al., 1976). With the ability of P188 to
repair damaged cell membranes in the articular cartilage of the rabbit TF joint following
severe blunt loading, the potential use of this surfactant should be explored as an
intervention for the ACL injured patient. If P1 88 is capable of repairing Chondrocytes, the
cells may then function normally in the chronic setting. Surgical reconstruction may then
yield a better long term result for the injured knee. Clinically, P188 has been used
because of its lack of toxicity and has been shown to be ‘squeezed out’ of the cell
membrane after it heals and excreted in the urine of the patient (Schmolka, 1977).

In summary, a 13 J blunt impact to the rabbit TF joint resulted in a signiﬁcant
increase in the percentage of damaged cells in the articular cartilage overlying femoral
condyles and tibial plateaus. A single injection of P1 88 surfactant into the joint
immediately following insult resulted in a signiﬁcant reduction in the percentage of cells
with damaged plasma membranes in all compartments of the joint. The long term
consequences of ‘saving’ these cells from necrotic cell death, in terms of them becoming
apoptotic and producing degadative enzymes, should be the focus of future

investigations. While the exact mechanism leading from acute joint trauma to the Chronic

96

 

progession of long term joint disease is currently unknown, recent evidence has shown
that acute chondrocyte damage may play an important role. Therefore, future studies
should examine the more long term consequences of P1 88, or possibly other
interventions, on joint cartilage following ligamentous and other trauma to the krnee and
other diarthrodial joints of the human body.

ACKNOWLEDGEMENTS

This study was supported by a gand from the Centers for Disease Prevention and
Control, Center for Injury Control & Prevention (CE000623). The contents of this
manuscript are solely the responsibility of the authors and do not necessarily represent
the ofﬁcial views of the Center for Injury Prevention and Control. The authors wish to
acknowledge Ms. Jean Atkinson (J .A.) for her assistance in animal care, Michelle Raetz
(M.R.) and Brian Powell (BP) for their assistance in cell viability analyses, and Eric

Meyer for his assistance in the impact procedures.

97

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Frobell RB, Roos HP, Roos EM, Hellio Le Graverand M-P, Buck R, Tamez-Pena
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Frobell RB, Roos HP, Roos EM, Hellio LeGraverand M—P, Buck R, Tamez-Pena J,
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Golenberg N, Kepich E, Haut RC, 2008, “Histomorphological and mecharnical property
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99

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101

CHAPTER SIX

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK

The previous chapters describe the results of a severe, blunt impact load to the in
viva rabbit tibiofemoral (TF) joint resulting in damage to cartilage and underlying

subchondral bone. A novel, in viva model of traumatic ACL rupture has also been

 

developed and contrasted with conventional ACL transaction models. Additionally, the m.
use of a potential therapeutic agent to restore membrane integity to acutely damage
Chondrocytes was also explored.

In Chapter 2 a single, blunt impact to the rabbit TF joint was found to result in L

high contact pressures located primarily in regions uncovered by the meniscus on the
medial and lateral tibial plateaus. Additionally, higher contact pressures were
documented on the lateral plateau compared to the medial plateau. A single, severe
impact was also found to result in a signiﬁcant increase in the percentage of cells with
damaged plasma membranes in the articular cartilage overlying the medial and lateral
tibial plateaus. A slightly higher percentage of damaged cells was documented in the
lateral plateau corresponding to slightly higher contact pressures. Acute damage to
chondrocytes has been thought to lead to the progession of chronic joint disease. Future
studies should investigate the longer-term implications of chondrocyte death in the
pathogenesis of joint disease in order to establish a cause and effect relationship between
acute trauma to cartilage and chronic joint disease.

Chapter 3 described a study on the rabbit TF joint subjected to a 13 Joule impact.
In this study the Chronic alterations in the mechanical and histological properties of

cartilage and underlying subchondral bone at 6 months and 1 year were investigated. The

102

major ﬁndings of this study included the presence of vertical and horizontal microcracks
at the articular cartilage and subchondral bone interface as well as an increase in the
subchondral bone thickness at 1 year post-trauma. Furthermore, analysis of the
mechanical properties of the cartilage showed no signiﬁcant Changes in any mechanical
parameter at either 6 months or 1 year. However, in comparing the 6 month properties to
the 1 year signiﬁcant stiffening in both the matrix and ﬁber modulus was documented in E
the impacted limbs. Stiffening of the contralateral, control limb was also documented

between the 2 goups; however, increase in these properties were shown in the control

 

animals. Although not quantiﬁed in the current study, this stiffening was attributed to '
calciﬁcation/ossiﬁcation of the articular cartilage. Future investigations should

investigate the calcium content of the cartilage in this model in order to validate the

ossiﬁcation process. In addition, ﬁrture studies should acknowledge the fact that

stiffening was also observed in the contralateral limb and document the implications of

altered gait following krnee joint trauma. Bone trauma documented in the current study

could have also lead to a more advanced progession of the disease, therefore, future

studies should also investigate the implications of acute bone trauma in the Chronic

disease process.

Chapter 4 described the development of a traumatic anterior cruciate ligament
failure model where a single, compressive load was delivered to the TF joint resulting in
ACL rupture. This model was then compared to current conventional OA models via
ACL transaction. Compressive loads generated in the joint during the acute ligamentous
injury were found to lead to signiﬁcant damage to cartilage, including acute surface

lesions and chondrocyte damage, as well microcracking in subchondral bone. Since the

103

Clinical literature documents signiﬁcant damage to cartilage and underlying bone in
patients suffering ACL tears, the current study may provide a more clirnically relevant
model for the investigation of joint trauma. Future investigations should focus on
documenting the biochemical Changes in the joint synovial ﬂuid and cartilage. In
addition, the current literature suggests that reconstruction of the ACL has not proven
effective at mitigating the onset of post-traumatic OA, possibly due to tlne acute damage
to cartilage and subchondral bone. However, future studies should also investigate the
implications of ACL reconstruction coupled with therapeutic treatments aimed at
repairing the acutely injured cartilage and subchondral bone.

Chapter 5 investigated the effects of treating acutely injured cartilage with a non-
ionic surfactant, P188. The major ﬁndings of this study were the presence of acutely
necrotic cells in the articular cartilage of the medial and lateral tibial plateau and a
reduction in the percentage of these damaged cells with the admirnistration of Poloxarrner
188 (P188) directly into the joint immediately following impact. This study did not,
however, assess the long term viability of the ‘saved’ cells. It is possible that these
damaged cells, altlnough repaired within 4 days post-trauma, may still have abnormal
flmctionality and soon die via apoptotic pathways at a later time. In addition, the current
study did not investigate the effects of multiple injections of P1 88 at various time
periods. It is possible that multiple injections may help prevent cells from dying in the
chronic setting. Future studies should investigate the long term viability of these acutely
‘saved’ cells. A different cell viability analysis in order to assess apoptotic cells should
also be included in future investigations. Furthermore, the concentration of P1 88 in this

study was Chosen based on previous work done by our laboratory. Future investigations

104

 

should analyze the effects of various concentration levels and the ability to ‘save’ acutely
injured Chondrocytes. Finally, mechanical response of the cartilage matrix should also be
investigated in order to determine the longer term effects of ‘saving’ acutely damaged

Chondrocytes following traumatic injury.

105

APPENDIX A

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107

Table A2. Peak Pressures on the lateral and medial tibial plateau.

 

 

 

 

 

 

 

Lateral Tibial Plateau Medial Tibial Plateau

Peak Peak
Pressure Pressure

Rabbit (M Pa) Rabbit (MPa)
RSOA04R 44.32 RSOA04R 49.6
RSOA04L 54.88 RSOA04L 36.07
LB737R 44.88 LB737R 45.44
LB737L 37.37 LB737L 41.1 7
Average 45.36 Average 43.07
Standard Dev 7.20 Standard Dev 5.79

 

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119

Table 3.9. Mechanical indentation data for medial uncovered (site 1) 6 month group.

 

 

k0
Rabbit Limb “mm” Em (MPa) E,(M5a) (m‘lmsy
(mm) 10‘")

Tisoc04 Left 09"! 0.65 5.01 20.58
RSOLAB Left 0.90 0.57 2.15 22.83
FM1 Left 0.92 0.84 6.21 12.54
MF15 Left 1.02 0.56 5.15 26.15
M517 Left 0.74 0.75 2.51 21.43
M516 Left 0.87 0.41 3.64 23.17
SM1 Left 1.10 0.66 2.52 42.07
T51 Left 0.93 0.84 7.01 14.88
TF6 Left 0.77 0.47 3.48 23.70
M57 Left 0.98 0.78 4.52 28.39
TF1O Left 0.91 0.59 2.96 24.82
TF11 Left 0.77 1.01 7.67 12.04
RSOCO4 Right 0.86 0.54 3.00 25.19
RSOLAB Right 0.94 0.67 5.11 16.91
5M1 Right 0.88 0.90 4.50 13.72
M515 Right 0.86 0.53 3.37 25.52
M517 Right 0.91 0.58 3.90 24.49
M516 Right 1.05 0.35 3.21 40.50
8M1 Right 0.87 0.51 2.16 34.47
TF1 Right 0.70 0.62 1.63 22.36
T56 Right 0.73 0.46 1.70 32.78
M57 Right 0.93 0.67 3.27 32.88
7510 Right 0.74 0.51 1.78 29.23
T511 Right 0.70 0.94 4.73 12.08
FB4 Left 0.95 0.67 3.72 21.41
M56 Left 0.95 0.51 3.24 34.06
M510 Left 0.93 0.63 4.01 24.27
T518 Left 0.89 0.73 6.95 11.04
T58 Left 0.81 0.51 4.14 23.30
T57 Left 0.93 0.68 5.98 21.68
T514 Left 0.74 0.78 5.23 18.26
T517 Left 0.88 0.54 2.68 23.07
5N3 Left 0.72 0.40 10.62 12.51
ACB71 Left 0.74

T525 Left 0.76 0.54 2.53 19.42
584 Right 1.09 0.56 9.32 19.34
M56 Right 1.02 0.50 4.65 27.43
M510 Right 0.86 0.46 2.86 25.55
TF18 Right 0.88 0.89 7.42 11.86
T58 Right 0.88 0.36 4.18 31.19
T57 Right 0.78 0.51 5.11 16.36
T514 Right 0.82 0.83 3.90 14.22
TF17 Right 0.83 0.68 3.25 27.07
FN3 Right 0.78 0.89 3.50 15.64
ACB71 Right 0.75 0.47 1.05 35.98
TF25 ﬂight 0.69 0.47 1.74 23.09

 

120

 

Table B.10. Mechanical indentation data for medial covered (site 2) in 6 month group.

 

 

*0
Rabbit Limb "km” Em (MPa) E,(M5a) (m‘lmsy
(mm) 104’)

8250004 Left 0.50 0.32 1.93 15.29
RSOLAB Left 0.60 0.73 4.23 8.71
FM1 Left 0.53 0.50 2.08 6.90
MF15 Left 0.56 0.45 1.42 18.13
M517 Left 0.56 1.04 16.98 4.35
MF16 Left 0.61 0.51 4.24 12.96
8M1 Left 0.56 0.82 2.61 8.61
T51 Left 0.54 0.62 1.44 12.62
TF6 Left 0.53 0.54 3.52 11.61
M57 Left 0.73 0.94 3.15 14.74
T510 Left 0.67 1.01 10.27 6.52
T511 Left 0.57 0.80 3.59 9.62
RSOCO4 Right 0.63 0.47 2.97 11.74
RSOLAB Right 0.60 0.68 13.42 5.81
5M1 Right 0.67 0.80 6.86 5.62
M515 Right 0.72 0.46 2.26 12.86
M517 Right 0.67 1.36 51.75 2.97
MF16 Right 0.83 0.71 3.35 10.63
SM1 Right 0.56 0.60 0.98 14.31
T51 Right 0.59 0.66 5.11 6.56
T56 Right 0.46 0.56 4.59 7.69
M57 Right 0.59 0.71 2.29 11.78
T510 Right 0.57 0.85 7.78 7.50
T511 Right 0.55 0.63 3.96 8.66
584 Left 0.56 0.72 10.26 7.26
M56 Left 0.57 0.92 14.80 6.44
M510 Left 0.60 0.62 9.58 8.20
TF18 Left 0.61 1.02 10.87 4.55
TF8 Left 0.63 0.67 4.41 11.60
TF7 Left 0.48 0.49 5.91 7.57
TF14 Left 0.53 0.41 3.22 15.52
T517 Left 0.59 0.74 3.51 10.61
FN3 Left 0.54 1.00 7.24 6.98
A0871 Left 0.67 0.65 2.29 12.21
T525 Left 0.57 0.77 11.96 6.90
FB4 Right 0.56 0.54 2.10 10.82
M56 Right 0.56 2.64 29.23 1.47
M510 Right 0.47 0.49 4.72 9.11
TF18 Right 0.52 0.98 2.97 7.07
TF8 Right 0.61 0.36 4.83 12.14
T57 Right 0.53 0.41 5.66 8.96
TF14 Right 0.55 0.83 1.95 12.01
T517 Right 0.66 0.88 5.33 9.60
5N3 Right 0.52 1.13 8.29 4.78
A0871 Right 0.69 0.59 2.30 11.18
T525 Right 0.48 0.95 25.72 3.56

 

121

Table B.11. Mechanical indentation data for lateral uncovered (site 3) 6 month group.

 

 

*0
Rabbit Limb man’s" Em (MPa) E,(M5a) (m‘uusy
(mm) 10'")

7280004 Left 0.72 0.45 9.26 8.18
RSOLAB Left 0.66 0.68 13.46 4.76
FM1 Left 0.63 0.93 7.99 3.98
M515 Left 0.60 0.73 11.14 5.23
MF17 Left 0.49 0.99 16.57 2.67
M516 Left 0.68 0.53 5.22 15.11
8M1 Left 0.93 1.33 15.63 5.88
T51 Left 0.79 1.08 17.46 4.72
T56 Left 0.76 0.87 14.28 6.31
M57 Left 0.84 0.97 15.76 8.56
T510 Left 0.76 0.96 12.07 7.53
TF11 Left 0.75 1.54 23.82 3.30
RSOCO4 Right 0.81 0.44 11.89 10.59
RSOLAB Right 0.72 0.87 18.11 4.70
5M1 Right 0.64 1.04 11.91 4.85
MF15 Right 0.44 0.46 2.30 10.77
M517 Right 0.49 1.25 24.15 2.24
MF16 Right 0.86 0.70 14.64 7.54
8M1 Right 0.76 0.83 7.82 7.59
T51 Right 0.85 0.89 12.55 7.37
T56 Right 0.74 1.02 16.68 5.24
M57 Right 0.78 0.91 16.20 7.71
TF1O Right 0.76 1.41 18.50 4.44
T511 Right 0.69 1.73 18.41 3.55
584 Left 0.67 0.90 14.92 3.62
MFG Left 0.71 0.68 7.73 9.55
M510 Left 0.72 0.80 16.98 4.41
T518 Left 0.69 1.03 11.20 5.12
TF8 Left 0.57 0.79 13.39 6.22
T57 Left 0.55 0.69 7.08 4.56
T514 Left 0.62 1.32 16.69 4.08
TF17 Left 0.70 0.71 9.35 9.08
FN3 Left 0.78 1.12 18.81 4.61
ACB71 Left 0.78 0.56 1.55 19.03
T525 Left 0.56 1.26 29.76 2.40
FB4 Right 0.58 0.86 10.77 4.28
MF6 Right 0.79 0.70 8.09 10.67
MF1O Right 0.69 0.82 15.09 5.99
TF18 Right 0.63 0.79 9.98 5.05
T58 Right 0.78 0.72 23.16 6.60
TF7 Right 0.79 0.75 10.34 7.49
T514 Right 0.62 1.00 11.83 4.01
TF17 Right 0.75 1.05 13.99 5.74
FN3 Right 0.70 0.97 14.22 6.18
A0871 Right 0.80 0.65 13.28 7.63
T525 Right 0.51 1.66 42.67 1.98

 

122

VIII-“"5““?

.ll

 

Table B.12. Mechanical indentation data for lateral covered (site 4) in 6 month group.

 

 

 

*0
Rabbit Limb Th‘°""°ss 5,, (MPa) E,(M5a) (m‘/(Ns)*
(mm) 10'“)

=550004 Left 0.40 12.60 135.94 0.30
RSOLAB Left 0.41 0.95 34.45 2.72
5M1 Left 0.38 1.04 21.65 2.27
M515 Left 0.37 3.03 38.13 1.07
M517 Left 0.35 3.74 33.71 1.34
MF16 Left 0.44 3.93 65.22 1.34
SM1 Left 0.57 1.51 15.65 2.53
T51 Left 0.45 0.92 4.64 5.54
TF6 Left 0.52 1.78 48.77 2.11
M57 Left 0.43 1.34 36.18 3.01
TF10 Left 0.46 1.18 21.58 3.09
TF11 Left 0.48 1.55 30.68 2.33
RSOCO4 Right 0.32 14.84 28.70 0.69
RSOLAB Right 0.33 1.74 32.06 1.73
5M1 Right 0.40 1.34 35.98 2.32
M515 Right 0.53 5.65 128.79 0.94
M517 Right 0.39 3.89 78.62 0.99
MF16 Right 0.46 2.13 52.60 1.56
8M1 Right 0.49 1.36 43.97 1.93
T51 Right 0.50 1.10 5.43 5.01
T56 Right 0.50 1.10 31.91 2.68
M57 Right 0.50 1.38 47.94 3.15
TF10 Right 0.44 1.27 26.60 3.30
TF11 Right 0.45 2.04 16.02 2.95
FB4 Left 0.36 1.56 21.09 1.74
M56 Left 0.43 2.71 59.45 1.14
M510 Left 0.34 1.35 37.41 2.26
TF18 Left 0.39 1.25 24.93 1.95
T58 Left 0.34 0.94 37.22 2.26
T57 Left 0.31 3.55 18.92 1.50
T514 Left 0.47 1.28 40.80 2.22
TF17 Left 0.51 1.19 10.49 3.72
FN3 Left 0.42 1.19 14.28 2.13
A0871 Left 0.47 0.93 14.83 2.18
TF25 Left 0.37 3.81 96.45 1.15
584 Right 0.28 1.81 21.92 2.18
M56 Right 0.36 0.94 8.99 3.23
M510 Right 0.33 1.31 23.53 2.29
T518 Right 0.39 1.40 16.64 1.84
TF8 Right 0.33 1.61 31.74 1.48
T57 Right 0.32 1.37 16.98 2.37
TF14 Right 0.49 1.45 49.48 2.25
TF17 Right 0.47 1.06 9.91 4.40
FN3 Right 0.41 1.10 20.99 2.36
A0871 Right 0.47 0.73 25.94 3.14
TF25 Right 0.37 3.12 85.04 1.44

 

123

ii

 

Table B.13. Mechanical indentation data for medial uncovered (site 1) in the 1 year

 

 

 

group.
*0
Rabbit Limb “MM” 5... (MPa) E,(MPa) (m‘I(Ns)*1o'
(mm) 12
___.l.__
A3 Left 0.92 0.70 4.67 ' ' 19.78
M512 Left 0.81 0.75 4.77 19.40
5N1 Left 0.82 0.69 2.74 22.07
583 Left 0.86 0.99 7.26 10.17
CKDOC Left 0.84 0.97 11.54 6.85
A2 Left 0.95 0.91 10.59 8.66
82 Left 0.76 0.69 7.66 11.19
T521 Left 0.96 0.71 8.28 11.93
M59 Left 0.81 0.75 5.53 11.08
CKSAM Left 0.77 0.96 6.45 8.98
T524 Left 0.80 0.80 7.05 9.71
A3 Right 0.98 0.77 6.88 19.72
M512 Right 0.78 0.99 6.62 10.07
FN1 Right 0.72 0.55 1.97 23.31
583 Right 0.84 0.95 6.03 12.66
CKDOC Right 0.68 0.84 4.32 10.53
A2 Right 0.88 0.83 6.38 11.24
82 Right 0.78 0.67 5.59 15.98
TF21 Right 0.83 0.67 3.74 23.04
M59 Right 0.71 0.72 3.49 14.85
CKSAM, Right 0.70 1.05 3.13 12.93
TF24 Right 0.74 0.85 4.98 9.51
BCW2 Left 0.90 0.57 . 5.58 16.30
GM1 Left 0.64 0.73 2.58 17.35
A81 Left 0.62 0.44 0.73 34.32
5N2 Left 0.75 1.03 3.93 13.30
T55 Left 0.77 0.73 2.45 20.25
T523 Left 0.86 0.71 3.36 31.63
T527 Left 0.86 0.62 6.95 18.97
543RL Left 0.89 1.38 14.63 6.67
551R5 Left 0.89 0.72 4.87 19.03
T535 Left 1.00 0.59 6.03 20.18
BCW2 Right 0.89 0.67 7.61 11.34
GM1 Right 0.70 0.67 2.12 20.13
AB1 Right 0.62 0.42 1.00 26.79
FN2 Right 0.72 0.94 2.39 20.21
T55 Right 0.91 0.86 4.04 17.89
TF23 Right 0.83 0.55 2.82 31.70
T527 Right 0.87 0.79 7.16 15.03
543RL Right 0.97 1.45 12.19 8.80
551R5 Right 0.86 0.70 4.63 16.80
T535 Right 0.99 0.58 4.99 23.64
124

 

Table 3.14. Mechanical indentation data for media] covered (site 2) in the 1 year

 

 

group.

*0
Rabbit Limb "km” Em (MPa) E,(M5a) (mﬁmsy

(mm) 42

10 i
A3 Left 0.60 0.77 3.26 10.72
M512 Left 0.64 0.87 7.84 8.04
FN1 Left 0.50 0.75 2.64 7.83
583 Left 0.55 0.88 4.35 8.95
01000 Left 0.47 0.60 1.00 14.73
A2 Left 0.62 0.82 1.78 12.44
82 Left 0.50 0.48 1.94 12.68
T521 Left 0.64 0.71 1.75 16.20
M59 Left 0.52 1.19 15.48 4.45
CKSAM Left 0.51 0.56 3.43 8.38
TF24 Left 0.57 0.69 11.79 5.92
A3 Right 0.63 0.73 4.06 10.81
MF12 Right 0.61 0.87 6.42 9.09
PM Right 0.53 0.60 1.16 13.67
583 Right 0.66 1.03 5.45 11.26
CKDOC Right 0.47 0.87 13.76 4.05
A2 Right 0.62 0.81 5.24 7.35
82 Right 0.55 0.58 3.13 10.25
T521 Right 0.62 0.57 2.75 11.54
M59 Right 0.50 1.31 19.42 3.77
CKSAM Right 0.54 0.75 2.78 6.86
TF24 Right 0.53 0.79 14.13 4.43
80W2 Left 0.55 0.63 3.10 10.85
GM1 Left 0.55 1.22 7.22 4.74
A31 Left 0.51 0.46 0.50 28.29
5N2 Left 0.52 0.80 1.93 12.92
T55 Left 0.56 0.67 2.54 9.26
T523 Left 0.67 1.00 10.09 6.42
TF27 Left 0 0.54 0.66 9.00 6.54
543RL Left 0.73 1.28 5.81 9.55
551RF Left 0.59 0.91 7.86 5.29
T535 Left 0.74 0.69 2.96 16.89
BCW2 Right 0.55 0.65 2.60 10.70
GM1 Right 0.55 1.02 3.70 8.81
A81 Right 0.48 0.51 0.89 16.67
5N2 Right 0.60 1.16 2.83 12.97
TF5 Right 0.58 0.80 2.10 9.38
TF23 Right 0.69 0.72 6.26 9.79
T527 Right 0.60 0.75 4.69 7.51
543RL Right 0.61 1.60 6.79 6.29
551R5 Right 0.61 0.99 9.03 5.92
T535 Right 0.68 0.90 9.41 5.95

 

125

Table 3.15. Mechanical indentation data for lateral uncovered (site 3) in the 1 year

 

 

group.
. *0
Rabbit Limb "mm” 5,, (MPa) E,(M5a) (m‘I(Ns)*10'
(mm) 12)

A3 Left 0.82 0.99 17.88 4.97
MF12 Left 0.57 0.87 7.84 8.04
FN1 Left 0.61 0.92 7.88 7.88
583 Left 0.66 1.58 19.07 2.68
0000 Left 0.60 0.94 13.72 3.33
A2 Left 0.73 0.85 13.11 4.93
82 Left 0.80 0.81 12.10 8.46
T521 Left 0.81 0.72 13.84 7.21
M59 Left 0.70 1.44 13.84 4.45
CKSAM Left 0.75 0.93 6.59 11.82
T524 Left 0.81 1.26 14.28 5.12
A3 Right 0.82 1.13 20.24 4.52
MF12 Right 0.68 1.35 18.45 3.87
PM w Right 0.68 0.84 14.25 5.52
FB3 Right 0.62 1.61 23.02 3.19
CKDOC Right 0.63 0.88 13.67 4.57
A2 Right 0.70 1.04 15.82 4.54
82 Right 0.84 0.92 11.71 8.01
T521 Right 0.83 0.75 19.72 5.65
M59 Right 0.72 1.43 16.43 4.21
CKSAM Right 0.63 0.81 15.15 4.58
TF24 Right 0.79 1.32 8.89 6.68
BCW2 Left 0.78 0.58 14.73 6.28
0M1 Left 0.65 1.24 5.39 6.27
A81 Left 0.65 0.99 8.19 9.15
5N2 Left 0.67 1.56 11.01 4.00
TF5 Left 0.69 1.41 22.67 2.81
T523 Left 0.74 1.03 16.03 8.51
TF27 Left 0.70 0.99 13.36 5.29
543RL Left 0.82 1.47 16.82 5.79
551R5 Left 0.84 1.02 15.02 6.63
T535 Left 0.71 1.00 13.14 6.11
80W2 Right 0.65 0.55 21.62 4.58
GM1 Right 0.64 1.43 11.86 4.37
A81 Right 0.63 0.78 6.83 12.66
5N2 Right 0.63 1.52 15.99 3.30
T55 Right 0.77 1.34 10.46 5.84
TF23 Right 0.68 1.00 25.51 3.96
T527 Right 0.75 1.03 16.31 5.14
543RL Right 0.80 1.37 18.13 5.44
551R5 Right 0.81 0.85 14.56 6.70
TF35 Right 0.85 1.04 14.70 7.31

 

126

 

Table B.16. Mechanical indentation data for lateral covered (site 4) in the 1 year

 

 

group.

*0
Rabbit Limb “km” 8,, (MPa) E,(M5a) (m‘I(Ns)*

(mm) 42

10 i
A3 Left 0.40 1.86 49.88 1.96
M512 Left 0.35 1.59 59.31 2.02
5N1 Left 0.48 1.02 11.99 3.09
FB3 Left 0.49 1.85 30.84 1.61
CKDOC Left 0.38 2.22 75.14 1.92
A2 Left 0.33 4.91 70.30 1.44
82 Left 0.45 2.19 85.39 1.75
TF21 Left 0.54 1.03 24.78 2.81
M59 Left 0.44 1.54 33.02 2.04
CKSAM Left 0.42 1.18 65.57 2.19
TF24 Left 0.56 1.32 32.28 2.46
A3 Right 0.53 1.62 48.28 1.78
MF12 Right 0.42 1.25 30.32 4.17
5N1 Right 0.45 0.80 17.17 3.57
583 Right 0.38 1.94 39.44 1.99
CKDOC Right 0.39 1.79 69.32 1.94
A2 Right 0.38 2.63 57.64 1.25
82 Right 0.46 0.88 18.78 3.53
T521 Right 0.46 0.98 38.96 2.55
M59 Right 0.43 1.89 37.54 1.47
CKSAM Right 0.39 5.20 85.41 1.81
TF24 Right 0.50 1.22 26.79 2.49
BCW2 Left 0.41 1.52 61.12 1.66
GM1 Left 0.48 1.61 51.19 1.50
A81 Left 0.49 0.88 15.64 3.81
FN2 Left 0.45 1.37 17.61 2.96
T55 Left 0.41 1.76 58.75 1.73
TF23 Left 0.45 1.97 34.65 2.37
TF27 Left 0.53 1.19 27.90 2.50
543RL Left 0.52 1.59 20.25 2.62
551RF Left 0.54 1.39 25.24 2.50
T535 Left 0.40 2.66 34.29 1.32
BCW2 Right 0.41 0.81 67.18 1.96
GM1 Right 0.48 1.82 30.10 1.64
A81 Right 0.45 1.30 41.76 1.94
FN2 Right 0.46 1.13 6.98 5.18
T55 Right 0.46 1.55 57.36 1.84
T523 Right 0.43 2.08 72.25 2.04
T527 Right 0.46 0.94 22.79 3.39
543RL Right 0.50 2.12 14.59 3.98
551R5 Right 0.55 1.33 26.96 2.57
T535 Right 0.50 2.34 53.31 1.69

 

127

.3

APPENDIX C

RAW DATA FROM CHAPTER FOUR

128

 

 

 

 

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130

Table C.2. Pressure ﬁlm data from ACL tear rabbit

 

Sﬂimen

[ateral Plateau
Test

# Pixels =

Area =

Ave Pressure

Ave Force =

Max. Pressure

Min. Pressure

Percent of total
Medial Plateau

# Pixels =

Area =

Ave Pressure =

Ave Force =

Max. Pressure

Min. Pressure

Percent of total

Medium Total Force

NSUQ3R

1 (1059N)

1 127
32.266
27.5058
887.5
50.87
10.1
46.3%

1 580
45.2354
22.725
1027.98
48.37
10.1
53.7%

1915.48

Table C.3. Gross morphological scoring for the traumatic and transected animals

 

 

 

 

 

Fansected Tramatic
TF40 TF42 TF4? TF16 TF19 TF34
Femur Tibia Femur Tibia Femur Tibia Femur Tibia Femur Tibia Femur Tibia
LM 4 2 2 3 2 2 4 4 4 4 4
RM 2 2 1 2 1 2 1 2 1 2 2 2
LL 2 1 2 1 2 3 3 3 3 4 3 2
RL 2 2 1 2 1 i 1 1 2 1 2 1 1

 

 

131

M

 

Table C.4. Impact loads and injuries from trial cadaver tests.

 

 

 

Cadaver Drop Test
Rabbit Hei ht (cm) Load Injury

LB737 60 1008 None
RSOA04 L 60 1332 None
RSOA04 R 60 1264 None
RSOTO6 60 920 Partial ACL & Posterior L meniscus
AF 1 70 1 130 None
B0268 1215 None
FB2 874 Tibia Fracture at boot
GM3 883 None
WE 80 858 None
RSOOQ4 90 979 Tibia Fracture at boot
BC108 80 844 L meniscal tear
AC5 90 656 None - Lower weight
BB1 L 80 650 Fractured Tibia
BB1 R 75 645 Fractured Tibia
MG1 L 120 1227.8 Fractured Tibial Plateau - ACL Avulsion
MG1 R 110 715 Tibia Fracture at boot
MF2 L 110 1442 Fractured Tibial Plateau - ACL Avulsion
MF2 R 100 1062 None
RSOA17 La 110 670 None
RSOA17 Lb 130 907 None
RSOA17 Lc 130 945 None
RSOA17 Ra 110 1086 None
RSOA17 Rb 110 1022 None
BU63 L 110 850 Tom Acl - 1.33 kg mass used
GN1 70 960 Tom ACL and L meniscus
MF5 L 70 1264 Fractured Tibia at boot
MF5 R 65 1134 None
RSO|6 L 70 1108 Fractured tibial plateau
RSO|6 R 65 1213 None
TF9 L 70 935 Fractued Tibial Plateau, partial ACL tear,

torn L meniscus
TF9 Ra 60 845 None
TF9 Rb 70 920 None

Tibial plateau fracture, torn M meniscus,
TF9 Rc 75 900 ACL tear, Fibula fracture
FF1 L 70 894 PCL tear torn M&L meniscus
FF1 Ra 75 879 None
FF1 Rb 75 856 None
FF1 Re 75 1038 None
FF1 Rd 75 911 None
FF1 Re 75 985 Tom ACL, torn M meniscus
W 70 1223 Tom ACL

132

Table C.4 Continued

 

 

 

Cadaver Drop Test
Rabbit Hei ht (cm) Load Injury

NFUQB La 120 984 None
NFUQS Lb 130 1073 Fractured Tibial Plateau - ACL Avulsion
NFUQ3 R 130 1059 Tom ACL - Tom L meniscus
RSOF2 100 1081 Torn ACL - Torn L&M mensicus
AC 100 1229 Tom ACL - Torn L&M mensicus
TF3 L 80 976 Tom ACL - Torn L&M mensicus
TF3 R 80 784 Tom ACL - Torn L&M mensicus
SF2 La 70 888 None
SF2 Lb 70 992
SF2 Lo 70 936
SF2 Ld 65 1041
SF2 Le 70 1010 Tom ACL - Tom L meniscus
SF2 R 70 759 Tom ACL - Tom L meniscus
TF2 L 70 1084 Tom ACL - Torn M meniscus
TF2 R 70 964 None
TF32 70 1119 Tom ACL
TF41 70 1010 Tom ACL - Torn L&M mensicus
544 70 1076 Tom ACL
BF738 70 857 Tom ACL
TF19 950
TF16 912
TF34 928

 

133

 

 

Table C.5. Isolated joint ACL failure tests performed in the Instron.

 

 

 

Isolated Joints
Rabbit lngut Load 5N! Actual Load {Ni Injury
K211 2000 630.54 Femur Fracture
MF1 2000 1 150 None
F E1 1500 1235 None
BU64 1000 883
BU64 1200 1071
BU64 1400 1 181
BU64 1600 1275
BU64 1800 1514 Tibial plateau fracture
M63 1200 934
M63 1500 1 180 None
MG3B 1500 1164 None
TP 1500 1375 None
426 L 500 463
426 L 700 635
426 L 900 792
426 L 1100 979
426 L 1300 1 154
426 L 1500 1312
Tom ACL, M 8. L meniscal tears
426 L 1700 1453 posterior
426 R 500 487
426 R 700 677
426 R 900 822
426 R 1100 997
426 R 1300 1 181
426 R 1500 1363 Femur Fracture
T24B L 500 250
T24B L 700 380
T24B L 900 550
T24B L 1100 720
T24B L 1300 850
T24B L 1500 1050
T24B L 1700 1230
T2 48 L 1900 1290 ACL Avulsion & Tibial plateau
racture
T24B R 500 230
T24B R 700 340
T24B R 900 450
T24B R 1100 620
T24B R 1300 780
T2 4B R 1500 810 Tarn ACL - small fracture at posterior
A1 L 500 265
A1 L 700 420
A1 L 900 570
A1 L 1100 720
A1 L 1300 900

134

Table C.5 Continued

 

 

 

Isolated Joints
Rabbit Input Load (N) Actual Load (N) Injury
A1 L 1500 1050
A1 L 1700 1200
A1 L 1900 1350 ACL Tear and TP fracture
A1 R 500 215
A1 R 700 320
A1 R 900 455
A1 R 1 100 600
A1 R 1300 750
A1 R 1500 900
A1 R 1700 1050
A1 R 1900 1175 TP fracture
M57 R 500 280
M57 R 700 410
M57 R 900 540
M57 R 1100 700
M57 R 1300 650 ACL Tear
M57 L 500 280
M57 L 700 410
M57 L 900 560
M57 L 1100 700
M57 L 1300 850
M57 L 1500 950 ACL Tear - Midsubstance
M51 L 300 176
M51 L 500 317
M51 L 700 682
M51 L 900 912
M51 L 1100 1 100
M51 L 1300 1300

ACL Tear - buckethandle tears of

M51 L 1500 1492 M &L meniscus
M51 R 300 170
M51 R 500 320
M51 R 700 481
M51 R 900 634
M51 R 1 100 827
M51 R 1300 1038
M51 R 1500 1262
M51 R 1700 1423
M51 R 1900 1568 ACL Tear

135

 

Table C.5 Continued

 

 

 

Isolated Joints
Rabbit Ingut Load 1N! Actual Load gNj Injury
M49 R 300 210
M49 R 500 350
M49 R 700 510
M49 R 900 700
M49 R 1 100 920
M49 R 1300 1 1 10
M49 R 1500 1 300
M49 R 1700 1 500
M49 R 1900 1448 ACL Tear
M49 L 500 350
M49 L 700 500
M49 L 900 690
M49 L 1 100 850
M49 L 1300 1090
M49 L 1500 1250
M49 L 1700 1448 ACL Tear - Partial
M62 2000 1031 Partial ACL Tear

 

136

 

 

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138

APPENDIX D

RAW DATA FROM CHAPTER FIVE

139

Table D.1. Cell viability analysis of l-day controls (% dead cells).

 

1 -DAY CONTROL

 

 

 

 

LFL RF L LFM RFM LTL RTL LTM RTM

BF733 32.85 13.51 13.66 23.41 26.48 34.99 32.02
BF733 21.61 19.87 35.46 18.46 28.96 22.97 50.86
BF733 25.93 27.14 17.41 20.82 27.98 27.34

DB1 18.70 13.46 8.75 17.86 32.20 7.03 24.02 14.08
DB1 9.13 11.30 9.77 9.00 16.87 9.39 23.06 28.47
DB1 11.96 13.81 7.92 6.95 11.27 11.35 36.67 23.60
TF31 23.14 17.75 16.64 17.99 7.50 9.56 27.76 29.15
TF31 34.49 20.83 19.50 24.10 17.75 13.84 28.25 21.24
TF31 24.13 10.23 31.02 11 .99 24.24 8.52 32.94 21.82
TF31 37.37 22.58 18.88 11.20 20.84 13.72 28.92 15.07
TF30 30.62 27.14 24.91 34.57 38.61 37.31 39.65 17.20
TF30 35.43 29.39 29.09 41.41 43.92 21.73 48.58 19.39
TF30 27.19 27.96 13.64 54.26 25.42 29.61

TF28 24.40 9.72 11 .02 11.17 23.28 11.68 21.27 16.84
TF28 16.31 10.14 24.95 5.20 15.32 10.04 43.46 14.50
TF28 18.48 10.42 6.04 8.91 30.09 6.25 17.38 12.16
Average 24.48 17.83 18.33 17.29 26.22 16.95 30.89 22.60
St. Dev 8.34 7.21 9.11 9.96 12.13 10.01 9.09 10.17

 

 

Table D.2. Cell viability analysis of 4-day controls (% dead cells).

 

 

 

 

 

4-DAY CONTROL

LFL RF L LFM RFM LTL RTL LTM RTM
532RF 24.60 4.01 27.30 5.79 21.32 6.27 16.54 13.43
532RF 21.60 6.26 24.57 3.32 4.88 10.74 9.63 7.45
532RF 17.73 7.36 13.84 13.26 12.29 8.09 17.10 3.99
BF731 31.59 26.54 39.97 17.01 46.97 25.76 52.15 26.76
BF731 44.51 21.91 47.52 30.83 45.15 37.33 41.91
BF731 37.74 29.06 30.53 25.47 29.88 36.40
62881 17.83 9.34 16.97 14.48 15.35 25.04 28.07 24.26
62881 18.45 12.33 13.68 10.82 19.49 19.18 35.08 15.71
62881 16.43 12.21 8.18 8.27 25.69 19.73 31.44 10.67
TF45 21.22 16.36 29.23 26.00 32.22 13.13 16.11 8.97
TF45 20.33 22.12 25.88 17.84 29.15 11.83 23.12 11.73
TF45 19.13 14.71 32.02 19.99 37.41 7.96 33.09 14.46
TF39 47.78 31.85 22.44 30.60 29.86 17.12 44.29 27.19
‘TF39 45.75 33.42 22.77 31.36 36.70 17.36 35.60 22.88
TF39 39.17 23.85 18.36 45.41 27.89 42.54 17.52
ZIBS 57.02 21 .42 28.75 33.84 54.39 38.68 38.61 37.25
ZIBS ‘ 54.54 25.89 49.96 26.27 45.39 37.65 41.98 45.51
ZIBS 55.97 36.68 30.29 23.09 51.79 37.23 51.19 29.75
Average 32.85 19.74 26.79 19.90 32.56 20.23 32.43 21.99
St. Dev 14.92 9.88 11.11 9.54 14.62 10.86 12.23 12.45

 

140

 

Table D.3. Cell viability analysis of 4-Day P188 (% dead cells).

 

 

 

 

 

4-Day P188
LFL RF L LF M RFM LTL RTL LTM RTM

STID 9.58 12.96 13.34 21.81 22.60 24.11 15.15 28.57

STID 20.81 13.10 16.82 11.83 23.13 36.32 27.16 29.13

STID 14.92 16.91 15.82 16.41 25.09 27.23

TF36 14.42 12.56 10.45 11.02 16.80 16.55 31.15 17.46

TF36 20.26 13.58 12.01 14.62 15.27 21.47 43.23 21.15

TF36 15.03 10.70 8.64 21 .41 20.48 12.41

DB4 14.76 14.14 6.57 5.21 16.38

DB4 10.37 17.24 5.17 11.94 12.57

DB4 9.69 13.34 11.81 17.65 13.26

TF37 14.13 11.14 7.48 4.48 23.37 12.22 28.13 16.24

TF37 16.45 9.95 17.58 8.81 17.47 8.61 24.20 14.51

TF37 12.56 13.73 12.51 4.68 20.74 12.01 24.45 18.66

BF739 21.49 20.93 27.95 19.52 27.92

BF739 20.89 11.43 21.63 23.19 18.51

BF739 21.58 16.31 23.33 29.28 25.04

TF46 18.64 18.95 13.72 1 1.70 16.59 17.49 13.07 27.80

TF46 22.18 16.10 12.50 16.72 17.08 19.13 15.96 11.14

TF46 19.63 22.17 12.08 14.39 19.75 26.83 12.44 18.89
' Average 16.62 14.63 14.23 13.96 20.14 18.84 23.83 19.63

St. Dev 4.41 3.43 5.99 6.72 3.71 7.19 9.24 6.38

 

141

 

 

 

 

 

 

 

 

 

 

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