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msszs

Michigan State
University

This is to certify that the

thesis entitled
THE ANALYSIS OF TISSUE RESPONSE FOLLOWING A SINGLE RIGID
BLUNT IMPACT IN AN IN VIVO ANIMAL MODEL

presented by

BRIAN THOMAS WEAVER

has been accepted towards fulfillment
of the requirements for

Master 0: Science degree infinginggr—ing Mechanics

624%“

Major professor

I

Date October 25, 2001

 

0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
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6/01 c2/CIRC/DateDuep65op. 15

 

THE ANALYSIS OF TISSUE RESPONSE FOLLOWING A SINGLE RIGID BLUNT
IMPACT IN AN IN VIVO ANIMAL MODEL

By

Brian Thomas Weaver

A THESIS

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

MASTER OF SCIENCE
Department of Material Science and Mechanics

2001

ABSTRACT

THE ANALYSIS OF TISSUE RESPONSES FOLLOWING A SINGLE RIGID BLUNT
IMPACT IN AN IN VIVO ANIMAL MODEL

By

Brian Thomas Weaver

The Federal mandated requirement for certification of new vehicles limits the
impact forces from the instrument panel to the knee joint in simulated vehicle collisions
to not cause gross visual fracture. Our laboratory has developed a post-traumatic model in
the rabbit to investigate the effects of a single blunt trauma on the patellofemoral joint
without bone fiacture. Each chapter of this manuscript used a similar animal model. In
Chapter 1 the impact energy was increased from our previous 6.0 J to 10.0 J to study the
effects of impact energy on the rate of joint tissue alterations. The results indicated that
the higher intensity blunt impact does result in more significant joint degeneration
furthering our working hypothesis that joint alterations are associated with the severity of
impact energy. In Chapter 2, the long term affects of exercise verses non-exercise on a
stable injured joint were examined. These data suggest that exercise post-trauma may
have a beneficial effect in the long term. In Chapter 3 the possible adverse effects of
blunt trauma on a joint with preexisting surface lesions was examined in the animal
model. This study indicated that mechanical trauma in these subjects did not accelerate
tissue changes. The data presented in this thesis may help in understanding some of the
underlying mechanisms of post-traumatic osteoarthritis and its relationship to impact

energy, exercise and preexisting defects.

DEDICATION

I would like to thank my parents for their support and guidance throughout my life. With
out their support I would never have dreamed this possible, I owe everything to them.
Most importantly I would like to dedicate this work to my Father who has taught me to
strive for excellence and work hard.

iii

ACKNOWLEDGMENTS

I would like to acknowledge my professor, mentor and good fi‘iend Dr. Roger
Haut.

I am extremely grateful to Dr. Hubbard and Dr. Amoczky for serving on my
committee.

I would like to express my gratitude towards Clifford Becket aka. “the
information bank”, without him I would have been lost and confused.

I would also like to thank Jane Walsh for all her hard work and dedication.

Last but not least I would like to acknowledge everyone that works around me for
their help and friendship: Ben Ewers, Vijay Jayaraman, Eric Sevensma, Eric Clack, Jill

Krueger, and Masaya Kitagawa.

iv

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................... vii
LIST OF FIGURES ..................................................................................................... viii
RESEARCH PUBLICATIONS ................................................................................... ix
CHAPTER 1

CHRONIC DEGENERATION OF THE RABBIT RETROPATELLAR CARTILAGE
FOLLOWING AN ULTRA INTENSITY BLUNT INSULT

Abstract .................................................................................................................. 8
Introduction ............................................................................................................ 8
Methods .................................................................................................................. 10
Results .................................................................................................................... 18
Discussion .............................................................................................................. 26
References .............................................................................................................. 31

CHAPTER 2

REGULAR EXERCISE IS BENEFICIAL IN A STABLE JOINT AFTER TRAUMA
Abstract .................................................................................................................. 33
Introduction ............................................................................................................ 33
Methods .................... 35
Results .................................................................................................................... 38
Discussion .............................................................................................................. 45
References .............................................................................................................. 49

CHAPTER 3

THE EFFECTS OF BLUNT TRAUMA IN A JOINT WITH PRE-EXISTING SURFACE

DEFECTS
Abstract .................................................................................................................. 51
Introduction ............................................................................................................ 51
Methods .................................................................................................................. 52
Results .................................................................................................................... 55
Discussion .............................................................................................................. 60
References .............................................................................................................. 64

CHAPTER 4

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK ................... 66
References .............................................................................................................. 69

APPENDIX A

DROP TEST FIXTURE .............................................................................................. 70

APPENDIX B

STANDARD OPERATING PROCEDURES .............................................................. 76

APPENDIX C

HISTOPATHOLOGIC SCORING SYSTEM .............................................................. 88

APPENDIX D

RAW DATA FROM CHAPTER 1 .............................................................................. 98
Transversely isotropic properties ............................................................................. 99
Histopathologic scores ............................................................................................ 102
Subchondral bone thickness .................................................................................... 105

APPENDIX E

RAW DATA FROM CHAPTER 2 .............................................................................. 133
G1: and GR data ....................................................................................................... 134
Histopathologic scores ............................................................................................ 138

APPENDIX F

RAW DATA FROM CHAPTER 3 .............................................................................. 142
Cu and GR data ....................................................................................................... I43
Histopathologic scores ............................................................................................ 145
Subchondral bone thickness .................................................................................... 146

vi

LIST OF TABLES

CHAPTER ONE
Table 1 .................................................................................................................... 17
Table 2 .................................................................................................................... 21
Table 3 .................................................................................................................... 23
Table 4 .................................................................................................................... 25
CHAPTER TWO
Table 1 .................................................................................................................... 42
CHAPTER THREE
Table 1 .................................................................................................................... 57
Table 2 .................................................................................................................... 58
Table 3 .................................................................................................................... 6O

vii

LIST OF FIGURES

INTRODUCTION
Figure l .................................................................................................................. 1
Figure 2 .................................................................................................................. 2
Figure 3 .................................................................................................................. 3
CHAPTER ONE
Figure 1 .................................................................................................................. 10
Figure 2 .................................................................................................................. l 1
Figure 3 .................................................................................................................. 13
Figure 4 .................................................................................................................. 13
Figure 5 .................................................................................................................. 14
Figure 6 .................................................................................................................. 15
Figure 7 .................................................................................................................. 16
Figure 8 .................................................................................................................. 18
Figure 9 .................................................................................................................. 19
Figure 10 ................................................................................................................ 19
Figure 11 ................................................................................................................ 19
Figure 12 ................................................................................................................ 22
Figure 13 ................................................................................................................ 23
Figure 14 ................................................................................................................ 26
CHAPTER TWO
Figure l .................................................................................................................. 38
Figure 2 .................................................................................................................. 39
Figure 3 .................................................................................................................. 43
Figure 4 .................................................................................................................. 43
Figure 5 .................................................................................................................. 44
Figure 6 .................................................................................................................. 44
CHAPTER THREE
Figure 1 .................................................................................................................. 56
Figure 2 .................................................................................................................. 5 6
Figure 3 .................................................................................................................. 5 9
Figure 4 .................................................................................................................. 59

viii

RESEARCH PUBLICATIONS

PEER REVIEWED MANUSCRIPTS

Johnson AL, Probst CW, Decamp CE, Rosenstein DS, Hauptman JG, Weaver BT,

Kern TL: Comparison of trochlear block recession and trochlear wedge recession for
canine patellar luxation using a cadaver model. Journal of Veterinary Surgery 30; 1 40-50,
2001

Ewers BJ, Weaver BT, Newberry WN, Haut RC: Impact shear stresses in an animal joint
predicts chronic sofiening of cartilage but not thickening of underlying bone. Journal of
Biomechanical Engineering, In Review

Ewers BJ, Weaver BT, Sevensma ET, Haut RC: Chronic changes in rabbit retropatellar
cartilage and subchondral bone after blunt impact loading of the patellofemoral joint.
Journal of Orthopaedic Research, In Press

PEER REVIEWED ABSTRACTS

Johnson AL, Probst CW, Decamp CE, Rosenstein DS, Hauptman JG, Weaver BT,
Kern TL: Comparison of trochlear block recession and trochlear wedge recession for
canine patellar luxation using a cadaver model. Proceedings 27'" Conference of the
Veterinary Orthopaedic Society, 2000

Weaver BT, Ewers BJ, Haut RC: Regular exercise is beneficial to a stable joint after
trauma. Proceedings 48“ Annual Orthopaedic Research Society Meeting, In Review.

ix

INTRODUCTION

Lower extremity injuries are a fi‘equent outcome of automobile accidents
comprising nearly 25% of all injures (Luchter et a1. 1995). Knee trauma alone is the most
common lower extremity injury comprising approximately 10% of all injuries recorded
every year (Atkinson et al., 2000). Of all knee injuries, 70% involve a driver impacting
either a right or left knee into the instrument panel (IP) (Atkinson et al., 2000). Currently
the Federal Safety Standard on IP design in new vehicle certification limits femur (thigh
bone) loads to 10 RN (Figure 1). This standard is based on loads required to cause gross
fiacture of the patella (knee cap), femur, or pelvis from hallmark cadaver studies
conducted in the 60’s and 70’s (Melvin et al., 1975; Patrick et al., 1965; Powell et al.,
1975). However, only 50% of severe knee injuries result in patella and femur fractures
(Atkinson et al., 2000). Approximately 75% of knee injuries are of less severity or

“subfracture” injuries such as contusions, abrasions and lacerations.

'{ Load Cell

 

Figure 1: Photograph of a crash test dummy used to determine
the femur loads in a simulated crash.

Epidemiological studies associate major knee injury, such as subfracture injuries,
with secondary osteoarthritis (OA) (States, 1970). 0A is a degenerative disease that
affects both articular cartilage and bone in diarthrodial joints. Articular cartilage is the

white dense material covering the ends of bones to transmit loads and supply a near

frictionless surface for normal joint function (Mow et al., 1997). Cartilage is a biphasic
material with a solid phase (composed of collagen, proteoglycans and other proteins,
glycoproteins and chondrocytes) and a liquid phase (composed of water and electrolytes)
(Mow et al., 1997). The extracellular matrix of cartilage is broken up into three zones:
superficial tangential, middle, and deep (Figure 2). In the early stages of 0A, the cartilage
becomes fissured, sofiened and more permeable, and the underlying subchondral bone
thickens (Figure 3). The end stage of this disease results in full thickness loss of cartilage

with bone on bone contact which causes pain and lameness.

,4 Anterrlar surface

Zones
Superfaoal tangential (10.20%) {’7’
Midcle (40600.10)

-1_- 34'; I : if, j
‘1‘.“ g V I-Ixi 1,, i" ’
Deap<30%j - f: ’ 1’ v

Galahad cartilage ’/

  

Tide mark
Subchondral norm
‘ CanceIIous bone

Figure 2: Sketch of cross section of cartilage collagen nemork illustrating three
distinct regions.

 

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

The investigation of 0A with its relationship to mechanical joint trauma is very
difficult to accomplish in a human model, therefore animal models have been developed.
To investigate the mechanisms of post-traumatic osteoarthritis, both mechanical and
histological studies are needed (Mow et al., 1997; Armstrong et al., 1982). The stiffness
of cartilage is assessed with stress-relaxation indentation testing (Garcia, 1998; Hayes et
al., 1973; Mow et al., 1997). Histology allows the investigation of the cellular response of
the tissues. The pathologic features of osteoarthritis are diagnosed as fissuring of the
superficial layer of cartilage, depletion of matrix proteoglycans, chondrocyte proliferation
(clone formation), endochondral ossification, matrix erosion and thickening subchondral
bone (Colombo et al., 1983; Gallagher, 1996; Pritzker, 1998).

Our laboratory has developed a post-traumatic animal model in the rabbit (Haut et
al., 1995). With the use of a single rigid blunt impact we document acute superficial

lesions on the retropatellar surface immediately following impact. In a recent long term

study with this animal model, we document continual tissue alterations out to 36 months
post-impact. These include an increase in the cartilage permeability, a decrease in the
cartilage thickness and an increase in the subchondral bone thickness with time (Ewers et
al., in review). However, articular cartilage lesions and the material properties of the
cartilage plateau at 12 months and remain constant out to 36 months post-impact. In
contrast, researchers with a similar animal model document full thickness ulceration of
the retropatellar cartilage at 6 months post-impact (Mazieres et al., 1987). The most
significant difference in our models is the impact energy; Mazieres administers blunt
trauma at 10.0 J versus our 6.0 J impact. Chapter 1 of this thesis increased our previously
defined 6.0 J impact to 10.0 J to examine the effects of impact energy on these tissue
alterations.

Along with the underlying mechanisms leading to the development of 0A,
methods of treatment and rehabilitation of this disease are questionable. Exercise as a
treatment has been getting considerable attention (Buckwalter, 1995; Burroughs et al.,
1990; Petrella, 2000; Salter et al., 1980). Clinically, exercise has been documented to
improve osteoarthritic symptoms, joint mobility and the overall health of patients with
full 0A (Patrella, 2000). However, the role of exercise in rehabilitation of joint trauma
patients is not sufficiently documented. Chapter 2 of this thesis examined the long term
effects of exercise and non-exercise on a stable injured joint.

Most of the experimentation conducted in our laboratory is with healthy animals.
This enables studies to focus on the effects of the blunt trauma, while limiting other
factors that could contribute to joint alterations. Currently in litigation the effects of

trauma on joints with some underlying pathology is becoming an important issue (Haut,

personal communication). In the developmental stages of our current animal model, we
document multi-colored Flemish Giant rabbits to have preexisting lesions on the
articulating surface of the retropatellar cartilage versus the fawn-colored rabbits which
have pristine articular cartilage. Chapter 3 of this thesis analyzed the effects of blunt
impact on these multi-colored rabbits and compared the results to the fawn colored

rabbits.

REFERENCES:

1.

10.

11.

12.

13.

Armstrong CG, Mow VC. (1982) Variations in the Intrinsic Mechanical
Properties of Human Articular Cartilage with Age, Degeneration, and Water
Content. J Bone Joint Surg. 64-A:88-94.

Atkinson TS, Atkinson P. (2000) Knee Injuries in Motor Vehicle Collisions: a
Study of the National Accident Sampling System Database for the Years 1979-
1995. Accid Anal Prev. 32:779-786.

Buckwalter JA. (1995) Activity vs. Rest in the Treatment of Bone, Soft Tissue
and Joint Injuries. Iowa Orthopaedic J. 15:29-42.

Burroughs P, Dahners LE. (1990) The Effect of Enforced Exercise on the Healing
of Ligament Injuries. Am J Sports Med. 18:376-378.

. Colombo C, Butler M, O’Byrne, Hickman L, Swartzendruber D, Selwyn M,

Steinetz B. (1983) A new Model of Osteoarthritis in Rabbits. Arth & Rheum.
26:875-886.

Ewers BJ, Weaver BT, Sevensma ET, Haut RC. (In review) Chronic Changes in
Rabbit Retro-Patellar Cartilage and Subchondral Bone After Blunt Impact
Loading of the Patello femoral Joint. J Orthopaedic Res.

Gallagher PJ. (1996) Osteoarticular and Connective Tissues. Underwood JCE
(Eds). General and Sytematic Pathology, 2“d eds. Churchill Livingstone New
York. pp. 783-828.

Garcia JJ, Altiero NJ, Haut RC. (1998) An Approach for the Stress Analysis of
Transversely Isotropic Biphasic Cartilage Under Impact Load. J Biomech Eng
120:608-613.

Haut RC. (Personal Communication) Professor at Michigan State University.

Haut RC, Ide TM, DeCamp CE. (1995) Mechanical Responses of the Rabbit
Patello-Femoral Joint to Blunt Impact. J. Biomech. Eng. 117:402-408.

Hayes WC, Keer IM, Herrmann, Mockros IE. (1972) A Mathematical Analysis
for Indentation Tests of Articular Cartilage. J Biomechanics. 5:541-551.

Luchter S, Walz M. (1995) Long-term Consequences of Head Injury. J
Neurotrauma. 12:517-526.

Mazieres B, Blanckaert A, Thiechart M. (1987) Experimental Post-contusive
Osteoarthritis of the Knee: Quantitative Microscopic Study of the patella and the
Femoral Condyles. J Rheum. 14:1 19-121.

14.

15.

l6.

17.

18.

19.

20.

21.

Melvin J, Stalnaker R, Alem N, Benson J, Mohan D. (1975) Impact Response and
Tolerance of the Lower Extrernities. 19'” annual Stapp Car Crash Conf. 19:543-
559.

Mow VC, Ratcliffe A. (1997) Structure and Function of Articular Cartilage and
Meniscus. Mow VC, Hayes HC (Eds), Basic Orthopaedic Biomechanics, 2'“1 ed.
Raven Press, New York. pp. 113-177.

Patrick LM, Koell CK, Mertz HJ Jr. (1965) Forces on the Human Body in
Simulated Crashes. 9" annual Stapp Car Crash Conf. 12:237-259.

Petrella RJ. (2000) Is Exercise Effective Treatment for Osteoarthritis of the Knee.
BrJ Sprots Med. 34:326-331.

Powell WR, Ojala SJ, Advani SH. (1975) Cadaver Femur Responses to
Longitudinal Impacts. 19" annual Stapp Car Crash Conf 19:561-579.

Pritzker KPH. (1998) Pathology of Osteoarthritis. Brandt KD, Doherty M,
Lohmander SL (Eds). Osteoarthritis. Oxford University Press, New York. pp.50-
61.

Salter RB, Simmonds DF, Malcolm BW, Rumble EJ, Macmichael D, Clements
ND. (1980) The Biological Effect of Continuous Passive Motion on the Healing
of Full-Thickness Defects in Articular Cartilage. J Bone Joint Surg. 62: 1232-
1251.

States JD. (1970) Traumatic Arthritis: a Medical Dilemma. In: Proceedings of the
14'” Annual Conference of the American Association for Automotive Medicine.
14:21-28.

Chapter One

CHRONIC DEGENERATION OF THE RABBIT
RETROPATELLAR CARTILAGE FOLLOWING A 10 JOULE
INTENSITY BLUNT IN SULT

ABSTRACT:

Chronic degeneration of articular cartilage and bone in a rabbit has been
hypothesized to occur due-to acute impact overstresses. These stresses are a direct result
of impact energy. In this study, we impacted the rabbit patello femoral joint with an more
severe impact (10.0 I) compared to our previously defined high severity (6.0 J) impact.
The rabbits were sacrificed at 7.5 months and their retropatellar cartilage was
biomechanically and histologically examined. The stiffness of the retropatellar cartilage
was measured by indentation, its pathology was scored histologically, and the thickness
of the subchondral bone was measured. The data indicated that a 10 J impact caused more
significant articular cartilage alterations compared to the high severity impact, furthering
our working hypothesis that chronic tissue alterations are correlated to the severity of
impact energy.

INTRODUCTION:

Our laboratory has previously developed a post-traumatic animal model to study
the effects of subfracture injuries (Haut et al., 1995). A single blunt impact with a rigid
interface to the right patellofemoral joint of Flemish Giant rabbits results in acute
superficial lesions on the lateral facet of the retropatellar cartilage. These acute injuries
are dependent on impact energy (Newberry et al., 1998-A). A rigid high severity (6.0 J)

impact causes a sofiening of the retropatellar cartilage adjacent to superficial lesions and

thickening of underlying subchondral bone at 12 months post-impact. No acute damage,
however, is documented following a rigid low severity (1.0 J) impact. The alterations in
these tissues caused fiom the 6.0 J impact are suggested to be chronic responses to the
impact-induced stresses rather than a direct relationship between the cartilage and
underlying subchondral bone (Newberry et al., 1998-A; Atkinson et al., 1998). Others
suggest that cartilage degeneration is a result of increased stresses caused from the
changes in underlying bone (Radin et al., 1990). In a recent study with our model,
however, the high severity impact with a padded interface generated superficial lesions
and a softening of the retropatellar cartilage, while mitigating the thickening of
subchondral bone (Ewers et al., 2000-B). A long-term study of the rigid, high severity
impact indicates that surface lesions and softening of the retropatellar cartilage plateau at
12 months and remain constant out to 36 months post-impact (Ewers et al., In review).
However, the cartilage permeability increases, the cartilage thickness decreases and the
subchondral bone thickens with time. In a similar blunt impact model, Mazieres et a1.
documents cartilage ulceration with exposure of subchondral bone in White New Zealand
rabbits at 6-months following a 10.0 J immct. We hypothesized that a 10.0 J impact may
accelerate tissue alterations observed in our model compared to the 6.0 J impact.

The current study was conducted to further our working hypothesis that acute
degeneration of the retropatellar cartilage is dependent on the severity of impact stresses
resulting from impact energy. We impacted the right, patellofemoral joint of Flemish
Giant rabbits with a single, rigid impact at 10.0 J. Our hypothesis was that a single 10.0 J
impact would result in more significant tissue alterations than a 6.0 J impact, in our

animal model.

METHODS:

A total of 25, mature rabbits was used in this study. Nine rabbits received a single
10.0 J impact to the right hind patellofemoral joint. Sixteen rabbits were taken from a
previous study; eight were impacted at 6.0 J, while eight others served as controls (Ewers
et al., 2000-A). All animals were sacrificed at 7.5 months post-impact. The All-
University Committee on Animal Use and Care approved this study.

The drop fixture used in all previous studies was designed to drop a mass from a
maximum height of 0.5 m. In this study a new fixture was fabricated to drop a mass from
a height of 1.0 111 (See Appendix A for sketch and design specifications). Blunt impact
was administered to the right
hind patellofemoral joint
(Figure 1) (see Appendix B for
the standard operating
procedure). The animals were

maintained under general

 

 

 

anesthesia using 2% isoflurane

  

and oxygen. Each animal was

 

Figure 1: A) Photograph of the impactset-up. B) Sketch placed in a Specially designed
illustrating the impact load directed onto the patella.

chair that held the right hind

limb rigid while flexed at 120° with the animal supine and the femur aligned vertically. A

strap was placed across the left hind limb, which prevented the pelvis from rotating

during impact. The 10.0 J impact was administered by dropping a 1.0 kg mass from a

height of 1.0 m with the same impact interface. This impact did not result in bone

fracture. The dropped mass was arrested electronically after the fist impact preventing
multiple impactions. A load transducer (model 31/1432: Sensotec, Columbus, OH,
USA) with a 2,224 N capacity was attached behind the impact head to capture the
impact loads. Experimental data were collected at 10 kHz by a personal computer
equipped with an analog-to-digital board. The peak load and time to peak were recorded
from the load versus time curves (Figure 2). The 6.0 J impact was conducted in a
previous study (Ewers et al., 2000-A). This energy is obtained by dropping a rigid 1.33

kg mass from a height of 0.43 m.

 

 

 

  

 

 

 

 

 

 

  

 

 

 

 

 

1200 '.
100° ’ """"""""""""" "up-Peak load
2 800 .
8 600
6 :
u. 400 :
200
0 $ T
0 0.005 0.01
5 Sec
zTimeto peak

Figure 2: Graph of data collected during impact

Pre-impact, animals were exercised 10 min a day, 5 days a week at 0.3 mph on a
treadmill. Following impact, the animals were given two weeks of rest. Regular exercise
was then resumed and continued for the duration of the study. When not exercising, all

animals were housed individually in cages (122 cm x 61 cm x 49 cm).

11

All animals were euthanized with a lethal injection of Pentabarbitol (85.9 g/kg).
Immediately after sacrifice all patellae were excised out, stained with India ink, examined
and photographed. The femurs of the 10.0 J intensity impact group were also removed for
histology and photographed. The patellae were placed in a phosphate buffered solution
(pH 7.2) for mechanical testing (see Appendix B for the standard Operating procedure).
The structural integrity of the cartilage was determined from indentation stress relaxation
tests (Newberry et al., 1997). Briefly, each patella was placed in a clamp attached to a
camera mount (Bogen, Ramsey, NJ) which, in turn, was secured to the base of a fixture
that allowed in plane motion (Figure 3). The camera mount allowed rotation of the patella
while the mounting plate allowed translation. These together insured indentation tests
were performed on a flat location on the patella. The tests were performed with a
computer controlled stepper motor (Physik Instruments, Waldbrom, Germany: model-M-
168.30). A 1mm diameter flat, non-porous probe was displaced 0.1 mm in 30 ms and
maintained for 150 seconds. The resistive loads were measured (Data Instruments, Acton,
MA: model JP-25, 25 lb capacity), amplified, and collected at 1000 Hz for the first
second and 20 Hz for the remainder. The cartilage was then allowed to recover for 5
minutes and the test was repeated with a 1.5 mm diameter flat, non-porous probe. The
thickness of the indentation site was determined by depressing a needle into the cartilage,
again after a five minute period of rest to allow the cartilage to recover. These indentation

tests were repeated at two different sites on the lateral retropatellar facet (Figure 4).

12

 

Mounting
plate

 

Medial Facet

Surface Lesions

  

Figure 4: Photograph of excised patella.
O = indentation sites.

Cartilage has two phases (solid and liquid) with a superficial zone formed by
sheets of tightly woven collagen fibrils (Figure 3 on page 3), which suggests a continuum
model with a Young’s modulus in the plane (En) different than that in the direction
perpendicular to the surface (E33) (Garcia, 1998). Therefore mechanical data was
analyzed using a biphasic (poroelastic) model having a transversely isotropic (TI) solid
structure. The four elastic parameters (EH, E33, G13, and V3,) and two permeability
measures (RI, and kg) were computed using a curve-fitting algorithm (Garcia, 1998)
(Figure 5).

After mechanical testing, each
patella was placed in 10% buffered
formalin for seven days and decalcified in
20% formic acid for another seven days.
Tissue blocks were cut transversely across

each patella in the area of highest

 

“3‘.” 5‘ ”WWW" ofcw’di"f"‘f S-Vs’e’" patellofemoral contact pressure (Haut et al.,
destgnaed to cartilage. Isotropy [S m the plane

[-2.
1995). The blocks were embedded with

paraffin according to an established protocol. Six sections, 8 microns thick, were stained
with Safranin O-Fast Green and examined under light microscopy at 12—400 power. A
histopathologic scoring system was developed based on the literature (Colombo et al.,
1983; Mazieres et al., 1987). This system was utilized to quantify the progression of
degenerative disease in the cartilage. Each aspect was graded from 0 (normal or absent)
to +4 using the guidelines in table 1 (see Appendix B). Two independent blinded readers

(BW & JW) read one representative slide of each patella. Each reader assessed the index

score of each parameter at three locations on the patella: medial, central, and lateral. The
scores of each parameter were then summed across these locations. The mean and range
of each parameter were documented for the impacted and unirnpacted limb of both
impact groups. The thickness of the subchondral bone plate underlying the retropatellar
cartilage was measured at 25X for all 6 histology sections with a calibrated eye-piece at
the center of each facet (medial, central and lateral) by two independent investigators

(BW & JW) using established protocols (Newberry et a1. 1998-B) (Figure 6).

    

_ ,- m. .

Figure 6: Histologic section ofpatella: arrous
indicate the subchondral bone thickness
measurements.

The femurs were histologically processed using previously described protocols. A
pilot study determined the location of the patella on the femur during impaction. This was
conducted by placing a rabbit in the impaction chair and visually locating the
approximate location of the patella on the femur. Two tissue blocks were cut transversely
across the femurs at the location determined from the pilot study for four randomly
chosen (impacted) femurs (Figure 7). On average, thirteen sections were removed from
block A, while an average of 24 sections was removed from block B. The number of
sections removed was determined by the quality of the block. Each section was eight

microns thick and all were stained with Safranin O-Fast Green and examined under light

microscopy at 12-400X. All histologic sections were analyzed to determine the area most
affected from impact. This was determined to be in-between block A and B. Therefore,
only one block was cut for all other femurs at this location. For this, an average of 15
sections, eight microns thick, we stained and examined under light microscopy as

previously described.

Medial
Condyle

 

Figure 7: Photograph illustrating the mo tissue blocks removed for histology.

 

 

 

 

 

 

 

 

 

0 +1 +2 +3 +4
Surface 1ar Slightly Moderately Focally Extensively
Integrity H Leg“ irregular irregular severe severe
Focally
Proteoglycan . Moderate loss sever (loss
staining Normal Slrght loss (to mid zone) beyond mid Total loss
zone)
Moderate,
Chondrocyte . with some Marked loss No recognizable
Organization Normal Notrceable loss of of columns organization
columns
5 or more
1-2 (small) (small), 3 or
Fissures Absent (just under the zlfiflgg l g Eng) more (mid zone)
surface) or 1 (full
thickness)
7 or more
5-6 (small) or
_ 34 - (small).or 5- 7 or more
Clones 1 2 all . :3 $335, or ("mm") 6 (medium) (medium) or 5 or
(sm ) 1_2 (lar e)° or 3-4 more (large)
g (large)
Ossification Absent -------—-- ----------- Present ----------- -
Exposure of
Subchondral Absent ----------- ---------- Present --------------
Bone
Erosion Absent Detectable Moderate Focally Extarsrvely
severe severe

 

a‘Small = 2-4 cells bMedium = 5-8 cells °Large = 9 or more cells

Table 1: Histopathologic scoring system

To establish statistical significance of impact data between 10.0 J and 6.0 J
impacts, an unpaired t-test was used. A two-factor repeated measures analysis of variance
(AN OVA) with Student-Newman Keuls (SNK) post-hoc test was used to evaluate the
differences in transversely isotropic properties and subchondral bone thickness between
limbs within groups and between groups. Limb was the repeated factor, with group being
the independent factor. There is no two or three way test for non-parametric data,
therefore, to establish differences between groups in the histopathologic scores a Kruskal-

Wallis ANOVA on ranks was used. A Wilcoxon-Signed Rank test was also utilized to

document differences between the impacted and unirnpacted limbs within a group.

17

Results:

The peak load and time to peak recorded during impact were significantly
different between groups (Table 2). Examination after impact and ensuing daily
observations by a veterinary technician (JA) indicated no noticeable joint effusions, and
the rabbits did not appear to favor their impacted limbs. Gross examination of patellae
revealed that seven of nine impacted patellae in the ultra intensity group had gross visual
surface lesions in the retropatellar cartilage running proximal to distal (Figure 4) and six
of eight impacted patellae in the high severity group had these lesions as well. Two of
nine impacted patellae from the ultra intensity group had osteophytes proximally on the
medial facet (Figure 8), whereas one impacted and unirnpacted patella (fiom two
different rabbits) had osteophyte formation in the high severity group (Figure 9). Three of
nine impacted femurs had gross visual surface lesions (Figure 10) and one of nine had an

osteophyte proximally on the medial trochlear ridge (Figure 1 1).

   

  

Figure 8: Photographs of two excised patella. Both were impacted at 10. 0 J. Circled
objects are osteophytes.

   

 
  
    

  

Figure 9: Photographs of an (A) impacted patella and (B) unimpacted patella
from the fawn-color group. Circled objects are osteophyte/annations.

 

Surface lesions

Figure 10: Photographs of three impacted femurs illustrating the gross visual surface lesions.

Figure 1]: Illustration of osteophypte
growth on an impacted femur.

The indentation data revealed that the impacted limbs in both impact groups were
softer and more permeable than the contralateral unimpacted limb in the plane of isotrOpy
(the 11 direction) (Table 2). Comparing the impacted limb to the unimpacted limb, E1 1
was reduced by 33% and 35% in the ultra intensity and high severity group, respectively.
In both groups this reduction was statistically significant, however there was no statistical
difference documented between these impact groups. Although no statistical significance
was documented between limbs or groups, E33 was reduced in the impacted limb
compared to the unimpacted limb by 18% and 27% in the ultra intensity and high severity
group, respectively. The in-plane permeability (k1) was also not statistically different
between groups even though the impacted limb in the ultra intensity was 45% more
permeable than the impacted limb in the high severity group. Between the impacted and
unimpacted limb, however, there was a statistical difference in the ultra intensity group.

There were no differences documented between the unimpacted or impacted
limbs of both impact groups to controls. When comparing the left limb of the control
group to the unimpacted limbs of the impact groups there was a slight trend for the
controls to be more permeable in both planes of isotropy than the unimpacted limb in
both impact groups. There was no statistical difference documented, but there was an
average 25% difference between the left control limb and the unimpacted limb of both
impact groups for both permeability values. The impacted limbs of both groups were, on
average, softer and more permeable than the right control limb. All other parameters
(Thickness, G13, and V3!) between controls and the unimpacted and impacted limb of both

groups were similar.

20

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21

There were no differences between readers of histological slides (Friedman
Repeated Measures AN OVA on Ranks). The impacted limb in the ultra intensity group
had significantly higher index scores for surface integrity, fissures, and clones compared
to the contralateral, unimpacted limb (Table 3). The impacted limb in the high severity
group had significantly more fissuring than it’s contralateral unimpacted limb (Figure
12). The high severity group also had more fissuring than controls. Interestingly, the
controls were documented to have less PG staining than the ultra intensity group.
However, the ultra intensity group was different from controls in the surface integrity and
clone formation categories. Although all other index scores were not significantly
different between the ultra intensity, high severity and controls, there was a trend for the
impacted limb in the ultra intensity group to have more extensive disorganized
chondrocytes, ossification (Figure 13A), exposure of subchondral bone (Figure 13D) and
erosion (Figure 13D). In fact no patellae in the high severity or control group were

observed to have these extensive degenerative qualities.

  

Figure 12: Histologvslide at 7.5 months of
an impacted patella following a 6.0 J impact.

22

     

 

a.
gure 13: Examples of

Histology slides of impacted patellae at 7.5 months flrllowing a

  

 

10.0J impact.
Ultra Intensity I-Iigh Severity

Unim ed Im acted Unim ed Im acted Gum's
Surfaceintegrity 0.8 (0.0-3.0) 2.30.0460)” 0.4 (0.0-1.5) 0.3(o.0—1.0) 0.2 (0.0—1.3)
PG Staining 0.1(0.0-1.0) 1.9 (0.0-5.5) 2.6(1.4-5.0) 2.6 (0.0—5.0) 2.2(l.0-3.8)%
Fissurss 1.2 (0.04.0) 2.9(0.0-6.5)‘ 1.8 (0.0-6.0) 4,200.65)" 1.5 (0.5—4.3)
Chondrocyte
Organization 1.8 (0.0-5.5) 5.3 (0.0—9.5) 2.3 (0.0-7.0) 3.4 (0.5-9.5) 2.7(1.-5.3)
Clones 4.2 (2.0-10.0) 6.6(45-90)“ 5.0 (2.0-8.5) 5.4 (2.5-8.0) 3.6 (2.3-6.3)
Ossification 0.0(0.0-0.0) 0.3 (0.0-3.0) 0.0(0.0—0.0) 0.0(0.0-0.0) 0.0(0.0-0.0)
Exposure of
Subchondral Bone 0.0(0.0-0.0) 0.2 (0-1.5) 0.0(0.0-0.0) 0.0(0.0-0.0) 0.0(0.0—0.0)
Erosion 0.0 (9.0-0.0) 0.8 (0.04.5) 0.0(0000) 0.0(0.0-0.22 0.0 9.0-0.0)

 

.Significantly different from contralateral unimpacted limb by Wilcoxon-Signed Rank Test.
”Significantly different from high seva'ity impacted by Kruskal-Wallis ANOVA on Ranks.
ASignificantly different than connols by Kruskal-Wallis ANOVA on Ranks.
%Significantly different than ultra intarsity impacted limb by Kruskal-Wallis ANOVA on Ranks.

Table 3: Histology scores for the unimpacted and impacted patellae of the ultra intensity and high severity

group and controls (mean (range)).

23

There was no difference between readers of subchondral bone thickness (average
variance between readers values was 1.4%), therefore the measurements were averaged
between readers for both impact groups and controls (Table 4). In the ultra intensity
impact group the impacted limb was on average 23% thicker than the unimpacted limb,
which was a significant thickening response across all three locations on the patella
(medial, central and lateral). For the high severity group, the subchondral bone in the
impacted limb was on average 12% thicker than the unimpacted limb, however there was
not a significant thickening on any location of the patella. The thickness values were
similar comparing the impacted limb of the ultra intensity group to that of the high
severity group. Strangely, the unimpacted limb in the high severity group was thicker
than both the ultra intensity group and controls, with a statistically significant difference
on the medial side. In fact, the subchondral bone across all three locations on the patella
in unimpacted limb of the high severity group was on average 16% thicker than the

unimpacted limb in the ultra intensity group and controls.

24

 

Medial Central Lateral

 

g g Unimpacted‘ 0.60 r 0.14 0.83 i 0.34 0.74 i 0.27
5 E lmpacted‘ 0.77 r 0.13" 1.14 i 0.27’ 0.93 i 0.17"
fig Unimpactedb 0.82 i 0.15 0.98 r 0.37 0.86 i 0.14
E 33 Impactedb 0.90 r 0.23 1.15 r 0.37 0.98 i 0.11
g Leflb 0.64 r 0.11 0.90 :t 0.24 0.77 r 0.15
S Rightb 0.64 i 0.18 0.83 i 0.31 0.80 i 0.20
'N=9
l’N=8

'Significantly different than contralateral unimpacted patella.

Table 4: Subchondral bone thickness of Unimpacted and Impacted patellae
of the ultra intensity impact. the high severity impact and control group (mm (mean i stdev)).
Significance was determined by a two way repeated factor ANOVA with SNKpost—hoc (p < 0.05).

The impact did not appear to affect the femurs in the ultra intensity group. One of
nine femur pairs was not observable due to poor histologic processing (femur in Figure
10C). Three of eight rabbits did not have any surface defects on either the impacted or
unimpacted limb (Figure 14A and B). Interestingly, Figure 14A corresponds to the femur
with the gross visual osteophyte (Figure 11). In another set of three animals, both the
impacted and unimpacted limb had frssures (Figure 14C and D). Two of these correspond
to Figure 9A and B, while the other animal was not observed to have gross visual surface
lesions. The remaining two rabbits were observed to have large chondrocyte clusters in

both the unimpacted and impacted limb (Figure 14E and F).

25

 

     

Hag-.4

Figure 14: Photographs f femur histologic sections. Photos on the left are of
unimpacted femurs with their corresponding impacted limb on the right.

DISCUSSION:

The current study was conducted to further our working hypothesis that joint
degeneration in our animal model is dependent on impact stresses. We proposed that an
ultra intensity 10.0 J impact would result in more cartilage alterations than a high severity
6.0 J impact because the impact induced stresses were assumed to be higher. While the
mechanical data were not statistically different between groups, the impacted limb of the
ultra intensity impact did have more severe histological changes. The impacted limb of

the ultra intensity group had higher histological index scores than the impacted limb of

26

the high severity group including surface integrity, ossification, exposure of subchondral
bone and erosion.

It was hypothesized that there would be significant differences in the material
properties between the ultra intensity and high severity group, however this was not
documented in the current study. It has been suggested that the intrinsic material
properties reflect the compositional and ultrastructural characteristics of the tissue (Mow
et al., 1997). In the transversely isotropic model, E“ is the measure of the instantaneous
response of the cartilage, while E33 is the measure of the relaxed response (Garcia, 1998).
Researchers hypothesize that the instantaneous response is controlled by the quality of
the collagen network (Jurvelin et a1, 1988; Mizrahi et al., 1986) and the relaxed response
is predominately controlled by the quantity of matrix proteoglycans (Armstrong et al.,
1982; Jurvelin et al., 1988; Parsons et al., 1987). From the histological index scores the
high severity group was found to have a slightly higher score for fissures than the ultra
intensity group. It has been suggested that fissures may be reflective of network damage
(Newberry et al., 1998-B). It was also found that the high severity group had a slightly
larger percent reduction in E33 between the unimpacted and impacted limb than the ultra
intensity group, which may be represented by its slightly larger index score for
proteoglycan staining. The reason for these differences is not known, however it may be
possible that the ultra intensity impacted animals were favoring their impacted limb
resulting in increased weight bearing on the unimpacted limb. Increased weight bearing
on normal healthy joints has shown to promote a stiffening effect of the cartilage

(Jurvelin et al., 1986).

27

In solid mechanics, stress concentrations are suggested to concentrate near crack
tips thus causing them to propagate in a predominantly compressive stress field (Altiero,
1974). A similar phenomenon may be present in this study. Blunt impact in this animal
model has been shown to result in surface lesions at time zero post-impact (Haut et al.,
1995). The depth and number of cartilage fissures have been documented to increase with
time out to 12 months post-impact in regularly exercised animals (Ewers et al., in review;
Newberry et al., l998-B). It may be possible that regular exercise results in an increased
compressive stress field in the patellofemoral joint thus propagating the surface lesions
caused from the blunt impact, which may result in more fissuring. If the animals
subjected to a 10.0 J impact were favoring their impacted limb, this would result in a
decrease in the compressive stress field, which might explain why the high severity
impact group had more fissuring. However, one would then expect the unimpacted limb
of the ultra intensity group to differ from the control group.

It has also been shown that increased loading and motion of articular cartilage, up
to a certain level, may increase matrix proteoglycans (Jurvelin et al., 1986; Buckwalter,
1995). The histopathologic scores indicated that the unimpacted limb in the ultra intensity
impact group had significantly more matrix proteoglycans than the unimpacted limb in
the high severity impact and control group. Again, this may be indicative of the ultra
intensity impact animals favoring the impacted limb, however the gait forces of these
animals were not measured, therefore we can not be sure of any favoring of this limb.

Researchers have hypothesized that the remodeling of subchondral bone is in
response to trauma-induced microdamage (Radin et al., 1986). In our animal model we

have never documented microcracks or any other damage that supports this theory. It has

been suggested that the remodeling of subchondral bone in this animal model is caused
from impact induced shear stresses (Atkinson et al., 1998; Newberry et al., 1998-A). In
the current study the subchondral bone significantly thickened in the impacted limb of the
ultra intensity group compared to its contralateral unimpacted limb. In contrast, there was
not a significant thickening response in the high severity group. Frost et a1. (1986)
suggest that the rate of remodeling of subchondral bone is dependant on the size of the
remodeling signal. If shear stress is the remodeling signal in our animal model then these
results may further our hypothesis. However, the subchondral bone in the unimpacted
limb in the high severity group was documented to be thicker than the unimpacted limb
in the ultra intensity group and controls. It was also documented that the thickness values
in the impacted limb of both impact groups were similar. The reason why the unimpacted
limb of the high severity group was thicker than both of the other two groups is unknown,
however it may be possible that the animals in the high severity group came from a
different breeder than those of the control and ultra intensity group, which had thicker
subchondral bone.

The results of this study do difi'er from others. Mazieres et a1. (1987) document
full ulceration and exposure of bone 6-months following a 10.0 J blunt impact to the
patellofemoral joint. They also document ulceration and fibrillation in the femur on the
medial facet of the trochlear ridge. In contrast, the current study did not document full
0A in the patella or femur at 7.5 months post-impact. There were differences in our
models, however. Mazieres used New Zealand White rabbits versus our Flemish Giant
rabbits, therefore there may be a discrepancy due to breed of animal. The New Zealand

rabbits are much smaller than Flerrrish Giant rabbits which may indicate that the impact

29

energy in our model may need to be increased to obtain comparable tissue stresses.
Mazieres also does not utilize a regular exercise regime. The role of exercise in this
animal model has been suggested to increase fissuring and soften cartilage mewberry et
al., 1998-B). However, the role of exercise following joint injury has been a subject of
controversy (Buckwalter, 1995). Recent data may suggest that exercise has a benefit in
the longer term in this blunt impact model (Weaver et al., in review).

In conclusion, this study has shown that a single blunt impact of 10.0 J in
magnitude to the patellofemoral joint does cause more severe histopathologic changes in
the cartilage than those produced from a 6.0 J impact. This study further supports our

hypothesis that joint degeneration is directly correlated to the intensity of impact energy.

30

REFERENCES:

l.

10.

11.

12.

Altiero NJ. (1974) Fracture Initiation and Propagation in Nonuniform
Compressive Stress Fields. University of Michigan, Ph.D. dissertation.

Armstrong CG, Mow VC. (1982) Variations in the Intrinsic Mechanical
Properties of Human Articular Cartilage with Age, Degeneration, and Water
Content. J Bone J Surg. 64-A:88-94

Atkinson TS, Haut RC, Aliero NJ. (1998) Inpact-Induced Fissuring of Articular
Cartilage: An Investigation of Failure Criteria. J Biomech Eng. 120: 18 1-1 87.

Buckwalter JA. (1995) Activity vs. Rest in the Treatment of Bone, Soft Tissue
and Joint Injuries. Iowa Orthopaedic J. 15:29-42.

Colombo C, Butler M, O‘Byrne, Hickman L, Swartzendruber D, Selwyn M ,
Steinetz B. (1983) A New Model of Osteoarthritis in Rabbits. Arth & Rheum.
26:875-886.

Ewers BJ, Haut RC. (2000-A) Polysulphated Glycosaminoglycan Treatments Can
Mitigate Decreases in Stiffness of Articular Cartilage in a Traumatized Animal
Joint. J Orthopaedic Res. 18:756-760.

Ewers BJ, Newberry WN, Haut RC. (2000-B) Chronic Softening of Cartilage
without Thickening of Underlying Bone in a Joint Trauma Model. J Biomech.
33(12):]689-1694.

. Ewers BJ, Weaver BW, Sevensma ET, Haut RC. (In review) Chronic Changes in

Rabbit Retropatellar Cartilage and Subchondral Bone Alter Blunt Impact Loading
of the Patellofemoral Joint. J Orth Res.

Frost HM. (1986) Intermediary Organization of the Skeleton. Vol 1. CRC- Press,
Boca Ration.

Garcia JJ, Altiero NJ, Haut RC. (1998) An Approach for the Stress Analysis of
Transversely Isotropic Biphasic Cartilage Under Impact Load. J Biomech Eng
120:608-613.

Haut RC, Ide TM, DeCamp CE. (1995) Mechanical Responces of the Rabbit
Patello-femoral Joint to Blunt Impact. J Biomech Eng. 117:402-208.

Jurvelin J, Kiviranta I, Tammi M, Helminen HJ. (1986) Effect of Physical

Exercise on Indentation Stiffness of Articular Cartilage in the Canine Knee. Int J
Sports Med. 7:106-110.

31

l3.

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Jurvelin J, Saamanen A-M, Arokoski J, Helminen JH, Kiviranta I, Tammi M.
(1988) Biomechanical Properties of the Canine Knee Articular Cartilage as
Related to Matrix Protoglycans and Collagen. Eng Med 17:157-162.

Mazieres B, Blanckaert A, Thiechart M. (1987) Experimental Post-contusive
Osteoarthritis of the Knee: Quantitative Microscopic Study of the Patella and the
Femoral Condyles. J Rheum. 14:119-121.

Mizrahi J, Maroudas A, Lanir Y, Ziv I, Webber TJ. (1986) the “Instantaneous”
deformation of cartilage: Effects of Collagen Fiber Orientation and Osmotic
Stress. Biorheologv 23:31 1-330.

Mow VC, Ratcliffe A. (1997) Structure and Function of Articular Cartilage and
Meniscus. In: Mow VC, Hayes WC (Eds.). Basic Orthopaedic Biomechanics, 2nd
ed. Lippincott-Raven Publishers, Philadelphia. pp. 113-177.

Newberry WN, Garcia JJ, Mackenzie CD, DeCamp CE, Haut RC. (1998-A)
Analysis of Acute Mechanical Insult in an Animal Model of Post-traumatic
Osteoarthrosis. J Biomech Eng. 120:704-709.

Newberry WN, Mackenzie CD, Haut RC. (1998-B) Blunt Impact Causes Changes
in Bone and Cartilage in a Regularly Exercised Animal Model. J Orth Res.
16:348-354.

Newberry WN, Zukosky DK, Haut RC. (1997) Subfracture Insult to a Knee Joint
Causes Alterations in the Bone and in the Functional Stiffness of Overlying
Cartilage. J Orth Res. 15:450-455.

Parsons JR, Black J. (1987) Mechanical Behavior of Articular Cartilage:
Quantitative Changes with Enzymatic Alteration of the Proteoglycan Fraction.
Bull Hosp Jr Dis Orthop Inst 47:13-30.

Radin EL, Burr DB, Fyhrie D, Brown TD, Boyd RD. (1990) Characteristics of
Joint Loading as it Applies to Osteoarthrosis. In: Mow RC, Ratcliffe A, Woo SL-
Y. (Eds.), Biomechanics of Diarthrodial Joints. Springer-Verlag, New York, pp.
437-451.

Radin EL, Rose RM. (1986) Role of Subchondral bone in the Initiation and
Progression of Cartilage Damage. Clin Orthop Rel Res. 213:34-40.

Weaver BW, Ewers BJ, Haut RC. (In review) Regular Exercise is Beneficial to a
Stable Joint After Trauma. Proceedings 48"I Annual Othopaedic Research Society
Meeting, In Review.

32

Chapter Two

REGULAR EXERCISE IS BENEFICIAL IN A STABLE JOINT
AFTER TRAUMA

ABSTRACT:

Post-traumatic osteoarthritis (0A) is a degenerative joint disease that has been
suspected to initiate with previous joint trauma. The role of exercise as a treatment
following trauma has been a subject of controversy. Previous studies in our laboratory
with an animal model suggest that exercise may accelerate some of the underlying
mechanisms of 0A. However, recent data may indicate that exercise has a benefit in
“normal stable” joints. The objective of this investigation was to document the effects of
exercise and cage activity/non-exercise on a stable, but injured joint out to 24 months
post-trauma. The results indicated that exercise does accelerate fissuring and softening of
the injured cartilage compared to the non-exercise group at 12 months post-impact. These
cartilage alterations in the exercised limbs plateau and do not progress out to 24 months.
On the other hand more, severe degeneration was documented in the non-exercise group
at 24 months. These data indicate that exercise may have a benefit in the longer term and
may play an important role in rehabilitation of this trauma patient.

INTRODUCTION:

Osteoarthritis is a disease that is classified by the degradation and subsequent loss
of articular cartilage until full bone on bone contact is reached which results in pain and
lameness. Epidemiologic studies have associated the development of this disease with
severe joint trauma (Chapchal, 1978; Rassmussen, 1972; States, 1970). The role of

activity or rest in the treatment of joint injury is a subject of controversy (Buckwalter,

33

1995). Varied tissue effects are documented as a result of exercise following knee joint
trauma. In the anterior cruciate ligament (ACL) transection model, full thickness
ulceration of articular cartilage is documented 54-months post-surgery in canines allowed
ad libitum activity (Brandt et al., 1991). In another “unstable” joint model, surgical
resection of the menisci produces osteoarthritic lesions that are exacerbated by exercise
(Armstrong et al., 1993). Exercise is also found to have an adverse effect on ligament
healing in “unstable” joints, but it has been documented to have beneficial effects in
“stable” joints (Burroughs et al., 1990). In a canine blunt trauma model, signs of early
repair and restoration of some degree of normality are seen in retropatellar cartilage and
underlying subchondral bone after a severe impact to the patellofemoral joint following
one year of regular treadmill exercise (Thompson et al., 1991). This impact resulted in a
fi'acture of the subchondral bone which may have caused hemorrhage and repair by cells
from the bone and the bone blood vessels (Buckwalter, 1995). These studies indicate that
exercise post-trauma may have beneficial effects on “stable” joints, but adverse effects in
“unstable” joints.

Our laboratory has developed a blunt trauma model in the rabbit to study the
underlying mechanisms of post-traumatic OA. Following a severe blunt impact to the
patellofemoral joint we document acute surface lesions on the retropatellar cartilage
without bone fracture (Haut et al., 1995). This acute damage progresses and results in
acute superficial fissuring and softening of the cartilage at 3 months post-impact in a
regularly exercised group (Newberry et al., 1998). These changes are also documented in
a cage-activity, non-exercise group, but not until 12 months post-impact (N ewberry et al.,

1997). Although it is yet unclear whether these tissue changes are indicative of early

34

degeneration or repair of the joint, these short-term studies with our rabbit model might
suggest that post-traumatic exercise accelerates early tissue changes compared to the non-
exercise scenario. Other studies using in vitro cartilage models suggest that cyclic or
intermittent compression of articular cartilage significantly increases biosynthetic activity
(Guilak et al., 1997). This may be reflective of the increased compressive stiffness and
thickening that is apparent following moderate levels of exercise in the “normal healthy”
canine knee joint (Jurvelin et al., 1986). In a recent long term study in our animal model,
the number and severity of cartilage fissures and the softening of the cartilage were
documented to plateau at 12 months post-impact and remain constant out to 36 months in
a regularly exercised rabbit (Ewers et al., in press). This study may indicate that exercise
has a benefit in our post-traumatic animal in the longer term. The objective of the current
study was to follow these chronic cartilage changes in exercise and cage-activity, non-
exercise groups out to 24 months post-impact.

METHODS:

A total of 32 mature Flemish Giant rabbits (6-8 months of age) was used in this
investigation. Seven of the animals were utilized in a 12 month exercise study (N ewberry
et al., 1998), six rabbits were taken from a 12 month non-exercise study (Newberry et al.,
1997), nine additional were documented in a recent 24 month exercise study (Ewers et
al., in press) and the remaining ten are new 24 month non-exercise rabbits. This study
was approved by the Michigan State University all-University Committee on Animal Use
and Care. All 31 animals received a single rigid blunt impact to the right patellofemoral

joint and were housed individually in cages (48” x 24” x 19”).

3S

The impact protocol has been described with detail in Chapter 1. Briefly, the
animals were maintained under general anesthesia (2% isoflurane) with the right hind
limb flexed approximately 1200 with the animal supine. Impact was administered by
dropping a rigid 1.33 kg mass onto the patellofemoral joint from a height of 0.46 m,
resulting in an impact energy of 6.0 J. The impact mass was arrested electronically after
initial impact to prevent multiple impacts.

Two weeks pre-irnpact, all animals were exercised 5 days a week for 10 min. at
0.3 mph on a treadmill. The exercise group resumed the regular exercise protocol after a
2-week rest period post-impact. Immediately after the animals were sacrificed, the
patellae were excised, stained with India ink, examined and photographed for permanent
records. Structural integrity of the retro-patellar cartilage was determined using
indentation stress relaxation tests described with detail in Chapter 1. Briefly, the patellae
were placed in a phosphate buffered solution (pH 7.2) and clamped into a custom-built
test fixture. A 1mm flat non-porous probe was displaced 0.1 mm in 30 ms and maintained
for 150 sec. The thickness of the indentation site was determined by depressing a needle
slowly into the cartilage 5 min after the indentation test. The 1.5mm flat non-porous
probe was not utilized in this study because animals used in previous studies were tested
before the transversely isotropic biphasic analysis was developed. The stiffness of the
retro-patellar cartilage was determined from the results of the indentation and thickness
test by a calculation of the shear modulus from an assumed elastic layer bonded to a rigid
half space (Hayes et al., 1972). The instantaneous shear modulus (GU) and the relaxed
shear modulus (GR) were calculated based on the load at 80 ms and 150 s, respectively,

using equation 1.

36

P(l—v)

G:
4*a*K(%,V)*a)

 

(1)

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

Four pairs of patellae from the lZ-month exercise and non-exercise group were
randomly selected for histological processing. All patellae from the 24-month exercise
and non-exercise group were histologically processed. Patellae were placed in 10%
buffered formalin for seven days and decalcified in 20% formic acid for another seven
days. Tissue blocks were cut transversely across the patella in areas of high contact
pressure (Haut et al., 1995). Six sections, eight microns thick were stained with Safi'anin
O-Fast Green and examined in light microscopy at 12—4OOX.

All patellae were scored based on the histopathologic index scoring system
described in Chapter 1. Two independent, blinded readers read one representative slide of
each patella. The scores were summed over three different locations on the patella:
medial, central, and lateral. The mean and range were documented for all aspects for each
group of animals.

A two way repeated factor analysis of variance (ANOVA) was used to test for
statistical differences between impacted and unimpacted limbs within a group and to test
differences between groups. Limb was the repeated factor and group being independent.
The Friedman repeated measures AN OVA on Ranks was utilized to test differences
between readers of the histological slides. The Wilcoxon-Signed Rank test was used to

establish differences between impacted and unimpacted limbs in all groups for the

37

O

histopathologic index scores, while the Kruskal-Wallis ANOVA was used to check for
differences between groups. Statistical significance was set at p < 0.05.
RESULTS:

From gross examination, three of six impacted patellae in the 12-month non-
exercise group and five of seven impacted patellae in the 12-month exercise group had
surface lesions running proximal to distal on the lateral facet. All of the impacted patellae
in the 24 month groups were observed to have gross visual surface lesions. It was also
observed that five of ten impacted patella in the 24-month non-exercise group had

osteophytes located proximally on the lateral facet (Figure 1), whereas no other group

had osteophytes.

There was no difference in cartilage thickness from
the lZ-month groups to the 24-month groups (Figure 2).
, The impacted limb in the exercise group at 12 and 24
months was 9% and 7% thinner than the unimpacted limb,

respectively though this was not found to be significant.

    

' ' Alternatively, the impacted limb in the non-exercise group
Figure 1: Photograph of a

pafé’l/afromfhe 24-m0mh at 12 and 24 month was 20% and 16% thinner than its
non-exercise group. The

‘ l b' ' . . . . .
we ed 0 lea ‘3 0" as’wphyte contralateral unimpacted limb, respectively and this was a

significant difference.

When similar limbs were compared from 12 to 24 months, there were no
significant differences in Gu or GR between groups. The cartilage was significantly
sofler, however, on the impacted limb versus the unimpacted limb at 12 and 24 months in

the exercise group (Figure 2). These results indicated that GU was reduced 42% from the

38

unimpacted limb to the impacted limb at 12 months and 35% at 24 months. GR was
reduced 22% at 12 months and 24% at 24 months. Interestingly, there were no
differences in GU or GR between the impacted limb and the unimpacted limb at either
time point for the non-exercise group. GU was decreased by approximately 15% in the
impacted limb compared to the unimpacted limb at both 12 and 24 months and GR was

approximately 11% lower at both time points.

 

Cartilage Thickness

 

    

12 month 24 month

 

 

 

 

 

 

 

 

 

 

12 month 24 month 12 month 24 month
7, ,,i . 7 ——~__~_l
I Exercised, I Exercised, I Non-Exercised, Non-Exercised,
Unimpacted Impacted Unimpacted Impacted

+ = Significantly different that contralateral unimpacted limb by two way repeated factor
ANOVA (p < 0.05).
12-month exercise: N=7
12—month non-exercise: N=6
24—month exacise: N=9

24-moth non-exercise: N=9
Figure 2: Histogram of the cartilage thickness and the elastic shear moduli (GU and GR).

39

While the histology data did not change with time in the exercise group, the
surface integrity, PG staining, chondrocyte organization, degree of ossification and
erosion indices significantly increased from 12 to 24 months in the non-exercise group
(Table 1). As expected the lZ-month exercise group had more fissuring than the 12-
month non-exercise group. Figure 3 illustrates typical fissures observed in the impacted
patellae in the 12-month exercise group. Comparing these to Figure 4 demonstrates the
difference in cartilage alterations between these two groups. At 24 months, however, the
cartilage in the non-exercise group had more extensive cartilage alterations compared to
the exercise group. The histological analysis indicated that the 24-month non-exercise
group had significantly larger index scores for surface integrity, ossification and erosion
compared to the 24-month exercise group. Figure 5 is an illustration of the typical
cartilage changes observed in the 24—month exercise group. Figure 6 reveals the more
advanced tissue alterations observed in the 24-month non-exercise group. Figure 6 A and
C are examples of ossification and moderately severe surface integrity. Erosion is evident
in Figure 6 A and B, and examples of large clones with severe protoglycan loss are in
Figure 6 D. These extensive cartilage alterations documented in the impacted limb of the
24-month non-exercise group were also found to be different than its unimpacted limb.
The histological index scores revealed that the impacted limb had significantly larger
scores than the unimpacted limb in all categories except for fissures and exposure of
subchondral bone. The impacted limb in the 24-month exercise group was also found to
have more extensive cartilage alterations than its unimpacted limb, with significantly

large histological index scores for fissures and chondrocyte organization. However, from

40

Figures 5 and 6 it is evident that the cartilage alterations in the 24 month non-exercise

group were more extensive than those in any other group.

41

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42

 

\....r

Figure 3: Examples of histological sections. Each are impacted patellae from the IZ-month
exercise group.

 
     

Figure 4: Examples of histological sections. Each are impacted patellae from the 12-month
non-exercise group.

43

 

Figure 5: Examples of histological sections. Each are impacted patellae from the 24-month
exercise group

 

Figure 6: Examples of histological sections. Each are impacted patellae from the 24—month
non-exercise group.

DISCUSSION:

The objective of the study was to follow the cartilage changes in an exercise and
non-exercise animal out to 24 months following a single rigid impact. We hypothesized
that exercise is beneficial to a “stable” injured joint in the long term. The results indicated
that exercise promotes a softening of the cartilage and increases fissuring compared to the
non-exercise regime at 12 months post-impact. By 24 months the cartilage in the non-
exercise group had more extensive alterations than the exercise group indicating that
exercise was beneficial in the longer term in this stable injured joint.

GU and GR were significantly reduced in the impacted limb compared to the
unimpacted limb at both time points in the exercise group, whereas these parameters were
not different between limbs in the non-exercise group. At 24 months, however, the non-
exercise group had more progressive joint degeneration compared to the exercise group
that showed softened cartilage. It has been suggested that visual or histological
appearance of a cartilage specimen may be a poor indicator of its ability to function as the
bearing material in the intact joint and that direct mechanical testing is a more reliable
indicator of the functional properties (Armstrong et al., 1982). The indentation data in
this study, however, seemed not to give any evidence of the extensive joint alterations
found in the 24-month non-exercise group. The role of cartilage softening in progressive
cartilage disease is yet unclear, but this effect would tend to help distribute the contact
loads over the injured joint. A more distributed stress field may have limited further
cartilage alterations. However, the indentation analysis used in this study is a limitation.
Cartilage is a biphasic anisotropic material (Mow et al., 1997). The superficial zone of

cartilage is composed of tightly woven collagen fibrils oriented in the plane whereas the

45

middle zone is composed of mostly vertical collagen fibrils. A transversely isotropic
biphasic analysis may better predict the intrinsic mechanical properties of this cartilage
(Garcia, 1998). This type of model was not utilized in this study because the 12 month
data came from earlier studies, when elastic analyses were the only method of
determining the mechanical properties. The histological and indentation data may have
better correlated if a more sophisticated biphasic model was used.

The histology data at 12 months compared to previous studies showing that
exercise accelerate early tissue changes Newberry et al., 1997; Newberry et al., 1998). It
was hypothesized that exercise following impact increases the stress around the
subfracture injuries causing them to propagate resulting in softer cartilage and increased
fissuring. This was based on a solid mechanics theory which states that crack propagation
results from increased stress concentrations at the crack tip in a predominantly
compressive stress field (Altiero, 1974). In support of this, moderate exercise exacerbates
the lesions produced in cartilage by meniscectomy more so than an unexercised group
(Armstrong et al., 1993). Interestingly, these changes documented in the exercise group
of the current study did not progress or become more extensive at 24 months, which may
be a sign of repair.

Articular cartilage maintenance and degradation are dependent on the activities of
the chondrocytes (Mow et al., 1997). In studies involving in vitro cartilage, cyclic loading
was found to increase the chondrocyte biosynthetic activity (Guilak et al., 1997). In
support of this, regular moderate exercise in normal healthy joints prevents cartilage
degeneration and maintains normal articular cartilage by promoting chodrocyte

proliferation of matrix proteoglycans (Otterness et al., 1998). In the current study the 24-

46

month exercise group did have a lower histopathological score than the 24-month non-
exercise group in the proteoglycan staining category, which may have indicated a
positive cellular response of the chondrocytes influenced from the exercise. This may
only be observed in stable joints. Unstable joints such as those from disruption of the
ACL or meniscus leads to abnormal articular surface friction and high local surface
stresses (Frankel et al., 1971). Therefore, from the results presented here it might be
possible that cartilage has the ability to maintain itself if normal joint function is not
altered.

These results did differ from others. Mazieres et al. (1987) document full
thickness ulceration of the retropatellar cartilage at 6 months post-trauma following a
single blunt impact. However, Mazieres supplied blunt impact at 10.0 J, whereas in the
current study blunt impact was administered at 6.0 J. In Chapter 1 the impact energy was
increased to 10.0 J and the results did indicate that increased impact energy does causes
more cartilage alterations, however the study in Chapter 1 exercised the animals
following trauma. Combining the 10.0 J impact of Chapter 1 with the non exercise
regime of Chapter 2 may produce similar results as Mazieres. Another difference
between the model of Mazieres et al. and the one presented here is the breed of animal.
Mazieres uses New Zealand white rabbits versus our Flemish Giant rabbit. It may be
possible that their rabbits have an underlying pathology which is accelerated by the
mechanical trauma.

In conclusion, the current study indicated that exercise may have a long term

benefit in a stable joint post-trauma. The 12 month data may also suggest that there exists

47

an optimal time of rest post-impact followed by a regular exercise regime which may

help in a total rehabilitation of this trauma patient.

48

REFERENCES:

l.

Altiero NJ. (1974) Fracture Initiation and Propagation in Nonuniform Compressive
Stress Fields. University of Michigan, Ph.D. dissertation.

Armstrong CG, Mow VC. (1982) Variations in the Intrinsic Mechanical Properties of
Human Articular Cartilage with Age, Degeneration, and Water Content. J Bone Joint
Surg. 64-A288-94.

Armstrong SJ, Read RA, Ghosh P, Wilson DM. (1993) Moderate Exercise
Exacerbates the Osteoarthritic Lesions Produced in Cartilage by Meniscectomy: a
Morphological Study. Osteoarthritis and Cartilage. 1:89-96.

Brandt KD, Stephen ML, Burr D, Albrecht M. (1991) Osteoarthritic Changes in
Canine Articular Cartilage, Subchondral Bone, and Synovium Fifty-Four Months
After Transection of the Anterior Cruciate Ligament. Arthritis & Rheumatism.
34:1560-1570.

Buckwalter JA. (1995) Activity vs. Rest in the Treatment of Bone, Soft Tissue and
Joint Injuries. Iowa Orthopaedic J. 15:29-42.

Burroughs P, Dahners L. (1990) The Effect of Enforced Exercieson the Healing of
Ligament Injuries. Am J Sports Med. 18:376-378.

Chapchal G. (1978) Posttraumatic Osteoarthritis After Injury of the Knee and Hip
Joint. Reconstr Surg T raumatol. 16:87-94.

. Ewers BJ, Weaver BT, Sevensma ET, Haut RC. (In Press) Chronic Changes in Rabbit

Retro-Patellar Cartilage and Subchondral Bone After Blunt Impact Loading of the
Patello femoral Joint. J Orthopaedic Res.

Frankel VH, Burstein AH, Brooks DB. (1971) Biomechanics of Internal
Derangement of the Knee. Pathomechanics as Determined by Analysis of the Instant
Centers of Motion. J Bone Joint Surg. 53Az945-962.

10. Garcia JJ, Altiero NJ, Haut RC. (1998) An Approach for the Stress Analysis of

Transversely Isotropic Biphasic Cartilage Under Impact Load. J Biomech Eng
120:608-613.

1 1. Guilak F, Sah R, Setton LA. (1997) Physical Regulation of Cartilage Metabolism. In:

Mow VC, Hayes WC (Eds), Basic Orthopaedic Biomechanics, 2"d edition,
Lippincott-Raven, Philadelphia, pp. 179-208.

12. Haut RC, Ide TM, DeCamp CE. (1995) Mechanical Responses of the Rabbit Patello-

Femoral Joint to Blunt Impact. J Biomech Eng. 117:402-408.

49

13.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

Hayes WC, Keer IM, Herrmann, Mockros IE. (1972) A Mathematical Analysis for
Indentation Tests of Articular Cartilage. J Biomechanics. 5:541-551.

Jurvelin J, Kiviranta I, Tammi M, Helminen HJ. (1986) Effect of Physical Exercise
on Indentation Stifness of Articular Cartilage in the Canine Knee. Int. J Sports Med.
7: 106-1 10.

Mazieres B, Blanckaert A, Thiechart M. (1987) Experimental Post-contusive
Osteoarthritis of the Knee: Quantitative Microscopic Study of the Patella and the
Femoral Condyles. J Rheum. 14:119-121.

Mow VC, Ratcliffe A. (1997) Structure and Function of Articular Cartilage and
Meniscus. In: Mow VC, Hayes WC (Eds). Basic Orthopaedic Biomechanics 2nd eds.
Lippincott-Raven Publishers, Philadelphia. pp. 113-17 8.

Newberry WN, Zukosky DK, Haut RC. (1997) Subfracture Insult to a Knee Joint
Causes Alterations in the Bone and in the Functional Stiffness of Overlying Cartilage.
J Orthopaedic Res. 15:450-455.

Newberry WN, Mackenzie CD, Haut RC. (1998) Blunt Impact Causes Changes in
Bone and Cartilage in a Regularly Exercised Animal Model. J Orthopaedic Res.
162348-354.

Otterness IG, Eskra JD, Bliven ML, Shay AK, Pelletier JP, Milici AJ. (1998)
Exercise Protects Against Articular Cartilage Degeneration in the Hamster. Arthritis
& Rheumatism. 41:2068-2076.

Rassmussen PS. (1972) Tibial Condylar Fractures as a Cause of Degenerative
Arthritis. Acta Orthop Scand. 43:566-575.

States JD. (1970) Traumatic Arthritis: a Medical Dilemma. In: Proceedings of the
14" Annual Conference of the American Association for Automotive Medicine. 14:21-
28.

Thompson RC, Oegema TR, Lewis JL, Wallace L. (1991) Oteoarthrotic Changes
After Acute Transarticular Load. J Bone Joint Surg. 73-Az990-1001.

50

Chapter Three

THE EFFECTS OF BLUNT TRAUMA IN A JOINT WITH PRE-
EXISTING SURFACE DEFECTS

ABSTRACT:

The objective of this investigation was to document the effect of joint trauma on a
joint with preexisting surface defects. Multi-color Flemish Giant rabbits have surface
lesions on the retropatellar surface pre-impact. On the other hand, fawn-color Flemish
Giant rabbits do not have these lesions at time zero. In the current study, both of these
animals were traumatized with a single rigid blunt insult to the right patello-femoral joint
to investigate the effects of blunt trauma on a joint with preexisting surface lesions. At
7.5 months post-impact all patellae were biomechanically and histologically evaluated.
The results indicated that the multi-color rabbits do not develop more cartilage alterations
following joint trauma than the fawn-color rabbit.

INTRODUCTION:

Osteoarthritis (0A) is the most common form of arthritis affecting nearly 7
million people in the United States alone (Arthritis National Research Foundation). The
incidence of this disease increases progressively with age (Praemer et al., 1992). Recent
data reveals that this disease can be attributed to genetics (Cicuttini et al., 1996).
Secondary CA, on the other hand, has been associated with environmental risk factors
such as obesity (Felson et al., 1998), mechanical trauma (States, 1970), meniscectomy
(Cooper et al., 1994) and transection of the ACL (Brandt et al., 1991). Currently in
litigation, the possibility that patients can develop OA faster following trauma than

through the normal physiologic pathways is a very important issue (Haut, personal

51

communication). The available evidence indicates that recovery is delayed when a joint
with some pre-existing pathology is injured (Maimaris et al., 1988; Watkinson et al.,
1991). In a clinical study of patients involved in a vehicle collision who had sustained
trauma to the cervical spine without bony injury, 53% had problematic symptoms and
33% had abnormal neurological signs two years after injury when degenerative changes
were diagnosed immediately following trauma (Miles et al., 1988).

The mechanisms of post-traumatic degenerative joint disease have been a subject
of investigation in our laboratory. In the developmental stages of our post-traumatic
animal model, pilot studies were conducted to assess the quality of pre-impacted cartilage
in different breeds of Flemish Giant rabbits. These preliminary data indicate that 26% of
fawn-color rabbits have pre-existing surface lesions on the retropatellar surface, while
65% of multi-color rabbits have these surface defects. The presence of acute surface
fissures may be early indication of a preexisting pathology (Flores et at., 1998).

The fawn-color rabbits have been the subject of our blunt trauma model due to
their statistically “cleaner” cartilage at time of impact. Following blunt trauma we have
documented retropatellar fissuring, soften cartilage, and thickening subchondral bone in
the impacted limb at 12 months post-impact (Newberry et al., 1998). The objective of the
current study was to administer a similar blunt impact to the patella-femoral joint of
multi-color rabbits to investigate its effect on a joint which has pro-existing surface
defects and compare these results to the data of fawn-color impacted rabbits.
METHODS:

A total of twenty mature Flemish Giant rabbits (6-8 months of age) was used in

this investigation. Twelve were of multi-color variety while the remaining 8 were fawn-

52

color from a previous study (Ewers et al., 2000). This study was approved by the
Michigan State University all-University Committee on Animal Use and Care. All
animals received a single rigid blunt impact to the right patella-femoral joint. Two weeks
pre-impact, all animals were exercised 5 days a week for 10 min. at 0.3 mph on a
treadmill. Exercise was resumed after a 2-week period of rest post-impact. When not
exercising all animals were housed individually in cages (48” x 24” x 19”). Animals were
sacrificed at 7.5 months post-impact for biomechanical and histological studies.

The impact protocol has been described with detail in Chapter 1. Briefly, the
animals were maintained under general anesthesia (2% isoflurane) with the right hind
limb flexed approximately 120° with the animal supine. Impact was administered by
dropping a 1.33 kg mass with a rigid aluminum impact interface (2 inches in diameter)
onto the patella-femoral joint from a height of 0.46 m, resulting in an impact energy of
6.0 J. A load transducer (model 31/1432: Sensotec, Columbus, OH, USA) with a 500 lb
capacity was attached behind the impact head to capture the impact loads. The impact
mass was arrested electronically following initial contact to prevent multiple impacts.

Immediately after the animals were sacrificed, the patellae were excised, stained
with India ink, examined and photographed for permanent records. Structural integrity of
the retro-patellar cartilage was determined using indentation stress relaxation tests
described with detail in Chapter 1. Briefly, the patellae were placed in a phosphate
buffered solution (pH 7 .2) and clamped into a custom-built test fixture. A 1mm flat non-
porous probe was displaced 0.1 mm in 30 ms and maintained for 150 sec for all the fawn-
color rabbits and 6 of the multi-color rabbits. The remaining multi-color rabbits were

indented with the same 1mm flat non-porous probe, however due to investigator error

53

(BW) the probe was displaced to a depth of 0.3 mm. The thickness of the indentation
sight was determined by depressing a needle slowly into the cartilage 5 min after the
indentation test. As explained in Chapter 2, the stiffness of the retro-patella cartilage was
determined from the results of the indentation and thickness test by a calculation of the
shear modulus from an assumed elastic layer (Hayes et al., 1972). The transversely
isotropic properties could not be documented here because of the 0.3 mm indentation
depth used for some of the multi-color animals. On the other hand, the equation for
calculating the elastic shear modulus (Equation 1 of Chapter 2) is inversely proportional
to the indentation depth.

All patellae were placed in 10% buffered formalin for seven days and decalcified
in 20% formic acid for another seven days for histological processing. Tissue blocks were
cut transversely across the patella in areas of high contact pressure (Haut et al., 1995).
The blocks were then embedded with paraffm according to an established protocol
(Newberry et al., 1997). Six sections, eight microns thick were stained with Safranin 0-
Fast Green and examined under light microscopy at 12-400X.

All patellae were scored using the same histopathologic index scoring system
described in Chapter 1. Two independent blinded readers read one representative slide of
every patella. The scores were summed over three different locations on the patella:
medial, central, and lateral. The mean and range were documented for all aspects for each
group of animals. The subchondral bone thickness was measured using the same
protocols described in Chapter 1. Two independent, blinded readers measured all six
sections of both the impacted and unimpacted patellae. One measurement was recorded

for each location on the patella (medial, central and lateral).

54

A two way repeated factor ANOVA was utilized to test for statistical differences
in mechanical data between the unimpacted and impacted limbs within groups and for
differences between groups. Limb was the repeated factor, while rabbit breed was the
independent variable. A Friedman repeated measures ANOVA on Ranks was utilized to
test differences between readers of the histological slides. The Wilcoxon Signed Rank
test was used to establish differences between impacted and unimpacted limbs, while the
Kruskal-Wallis ANOVA was used to check for differences between groups. A calculation
of the percent variance was used to measure differences between readers of the
subchondral bone thickness. In all tests, statistical significance was set at p < 0.05 .
RESULTS:

The peak load and time to peak recorded for the multi-color animals were 691 i
119 N and 6.88 :t 1.51 ms, respectively. These impact parameters were documented for
the fawn-color animals to be 624 i 45 N and 4.50 i 0.40 ms, respectively. While the
peak load was not statistically different between groups, the time to peak was larger for
the multi-color group compared to the fawn-color group (t-test p < 0.05).

From gross visual observation, the multi-color group had nine of twelve impacted
patellae and seven of twelve unimpacted patellae with surface lesions (Figure 1).
Typically the surface lesions on the unimpacted patellae would not stain as intense as
those on the impacted patella which indicated that they were not as deep or wide. One
impacted and unimpacted patella from the same animal had an osteophyte proximally on
the lateral facet and another distally on the medial facet (Figure 2A and B). Only one
other impacted patella in the multi-color group had an osteophyte and it was located

distally on the lateral facet (Figure 2C). In the fawn-color group, six of eight impacted

55

patellae and three of eight unimpacted patellae had gross visual surface lesions. There
was also osteophyte formation on one impacted and unimpacted patellae from two

different fawn-color rabbits (Figure 9 in Chapter 1).

   

Surface lesion

figure 1: Tim patella from same rabbit illustrating the ditkrence between surface
lesions on (A) unimpactedpatellae and (B) Impacted patellae. Typically the impacted
patella 's surface lesions stain more intense and appear larger.

 

Figure 2: Photographs of patella from multi-color group with osteophyte formation. (A)
Unimpacted and (B) Impacted patella fi'om the same rabbit. (C) Impacted patella. The
circled objects are osteophyte/emotions.

56

The results of the indentation stress relaxation tests indicated there were no
statistical differences for the instantaneous shear modulus (GU) or for the relaxed shear
modulus (GR) between the impacted and unimpacted limb in the multi-color group (Table
l). GU was 15% stiffer in the impacted limb compared to the contralateral unimpacted
limb in the multi-color group but this was not significant. In the fawn-colored group, the
impacted limb was softer than the unimpacted limb with significant differences in both
GU and GR. G; was 46% and GR was 30% lower in the impacted limb compared to the
contralateral unimpacted limb in the fawn-color group. Between groups there was a 45%
difference in GR recorded between the unimpacted limb in the fawn-color group
compared to the unimpacted limb in the multi-color group, which was statistically
significant (t-test p < 0.05). The GU in the unimpacted limb of the multi-color group was
also approximately 25% lower than the unimpacted limb in the fawn-color group,

however this was not statistically significant. No other parameter was different between

 

 

groups.
, Thickness GU GR
leb
(mm) (MPa) (MPa)
_ a Unimpacted 0.52 :l: 0.12 0.85 i 0.59 0.18 a: 0.07
Multl-color
Impacted 0.53 i 0.12 1.01 i 0.88 0.18 :h 0.09
b Unimpacted 0.56 :1: 0.07 1.14 :t 0.34 0.33 :t 0.13"
F awn-color , ,
Impacted 0.53 d: 0.07 0.61 i 0.16 0.23 :t 0.06
'N = s
t’N=7

+Significantly different from multi—color 1mi1npacted limb (t-test p < 0.05).

.Significantly different from contralateral unimpacted limb (paired t—test p < 0.05).
Table 1: Mechanical indentation data from stress relaxation tests (avg i stdev).

Histopathologic index scores revealed no differences between impacted and

unimpacted patellae in the multi-color group (Table 2). There was, however a statistically

57

larger index score in the fissuring category for the impacted limb compared to the
unimpacted limb in the fawn-color group. The fissure index recorded for the impacted
limb in the multi-color group was slightly larger compared to its contralateral unimpacted
limb, however this was not a statistically significant difference. Interestingly, both the
multi-color and fawn-color rabbits had fissures in the unimpacted limb (Figure 4). No

differences were documented between groups in any category.

 

 

Multi-colora F awn-colorb
Index Category
Unimpacted Impacted Unimpacted Impacted
Surface integrity 0.6 (0.0-2.0) 0.6 (0.0-1.5) 0.4 (0.0-1.5) 0.3 (0.0-1.0)
Proteoglycan
. . g 1.8 (0.0-5.0) 1.9 (0.0-5.5) 2.6 (l.4-5.0) 2.6 (0.0-5.0)

Fissures 1.6 (0.0-7.5) 3.1 (0.0-6.5) 1.8 (0.0-6.0) 4.2 (1.0-6.5).
Chondrocyte
organization 4.0 (1.5 — 11.0) 3.9 (0.5-11.0) 2.3 (0.0-7.0) 3.4 (0.5-9.5)
Clones 4.9 (2.0-8.0) 4.9 (2.0-6.5) 5.0 (2.0-8.5) 5.4 (2.5-8.0)
Ossification 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0)
Exposure of
subchondral bone 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0)
Erosion 0.0 (0.0—0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0)

'N=10

bN=8

*Significantly different than contralateral unimpacted limb by Wilcoxon-Signed Rank

test (p < 0.05).

Table 2: Histopathology index scores for the unimpacted and impacted

limb (mean (range)).

  

  

  

a a: *
Figure 3: Examples of histologic sections of impacted patella in the multi-color
group illustrating the fissuring.

 

 

Figure 4: Examples of histologic sections of unimpacted patellae illustrating the
fissures recorded for these limbs. (A and B are multi-color animals and C and D are

fawn-color animals).

There were no differences documented between readers of the subchondral bone
thickness (average variance of 0.9%). In the multi-color group, the impacted limb was on
average 25% thicker than the unimpacted limb with a significant difference on the medial
and central location on the patella. In the fawn-color group the impacted limb was

approximately 12% thicker than the unimpacted limb. There were no differences

documented between groups, however the unimpacted limb in the fawn-color group was

59

approximately 9% thicker than the unimpacted limb in the multi-color group, while the

 

impacted limbs were similar.
Limb Medial Central Lateral
(mm) (mm) (mm)
, , Unimpacted 0.73 i 0.17 0.85 i 0.34 0.83 i 0.41
Multl-color

Impacted 0.97 i- 0.19‘ 1.22 i- 0.34' 1.01 i 0.32

 

r 1 b Unimpacted 0.82 i 0.15 0.98 i 0.37 0.86 i 0.14
"“0 0' Impacted 0.90 i 0.23 1.15 i 0.37 0.98 i 0.11
‘N=10
bN=8

*Significantly dtflerent that contralateral unimpacted limb by

two way repeated factor ANOVA (p < 0. 05).
Table 3: Subchondral bone thickness (avgztstdev) measured on the unimpacted and
impacted limbs in the center of the medial. central and lateral locations on the
patella of both groups.

DISCUSSION:

The objective of the current study was to document the joint alterations following
a single rigid blunt impact in an animal with (multi-color rabbits) and an animal without
(fawn-color rabbits) the pre-existence of surface defects. The data indicated that
mechanical blunt trauma does not produce more joint alterations in the multi-color rabbits
compared to the fawn-color rabbits. In the multi-color group there was not a sofiening of
the cartilage in the impacted limb compared to the unimpacted limb and there were no
differences in the histological index scores between limbs. It was also documented that
the unimpacted limb of the multi-color group had similar histological index scores as the
unimpacted limb of the fawn-colored group.

The impact data was different between groups. Although the peak load was not
different, the time to peak was statistically different. The reason for this may be due to
the different drop fixtures used to administer impact. The fawn-color rabbits received
blunt impact from a previous test fixture, while the multi-color animals utilized the

fixture explained in Chapter 1. The main difference between these two was the path of

60

the dropped mass. The previous test fixture allowed the impact mass to freefall
independently until impact, whereas the current test fixture used a cart and rail system
(see Appendix A for details). Therefore, it is possible that the interactions of the cart and
rail supplied enough friction to alter the time to peak. Researchers have shown that the
rate of loading on a joint plays an important role in joint degeneration following blunt
insult (Ewers et al., in review). However, in that study they analyzed the difference
between a 5 ms time to peak and a 50 ms time to peak. It is not probable that a difference
of 2 ms documented in the current study would change the impact induced shear stresses
or contact pressures in the patello femoral joint to result in different tissue responses.

The impacted and unimpacted limbs of the multi-color group were not
documented to have statistical differences in the instantaneous (GU) or relaxed (GR) shear
moduli. There was a softening of the impacted limb in the fawn-color group, however in
the multi-color group GU was documented to be slightly larger in the impacted limb
compared to the unimpacted limb. Unfortunately the mechanical data recorded were not
reliable due to the difference in indentation depth of the two groups.

In both groups, the impacted limb had more fissuring than the unimpacted limb.
Although the fissure index score in the multi-color group was not significantly different
between limbs, there was a trend for the impacted limb to have more fissures than the
unimpacted limb. In all the other categories the unimpacted and impacted limbs of both
groups were qualitatively similar. It is therefore possible that the indentation data would
also be similar since it is hypothesized that GU is a measure of the quality of the solid
matrix and GR is a measure of the quantity of the matrix proteoglycans (Jurvelin et al.,

1988; Mizrahi et al., 1986; Armstrong et al., 1982; Parsons et al., 1987). Our hypothesis,

61

however was that the multi-color group would have more joint tissue changes than the
fawn-color group post-impact because they may have a pre-existing pathology.

Our pilot study data did indicate that 65% of multi-color Flemish Giant rabbits
tend to have surface defects pre-impact, however the rabbits of the current study may
have come from a different breeder. These data would be stronger if there was a way to
verify the presence of surface defects pre-impact.

In considering that these rabbits did have acute retropatellar fissures at the time of
impact, it has been suggested that the presence of cartilage damage increases its ability to
withstand crack propagation (Silyn-Roberts et al., 1990). They suggest that once cracks
in the cartilage are present a much higher state of stress can be stored in the cartilage
before further damage is initiated. This may be reflective of the current study if indeed
the multi-color rabbits had these surface defects pre-impact. It has been suggested that the
presence of surface lesions can be attributed to softened cartilage (N ewberry et al., 1998).
Softer cartilage would help to distribute the impact load over the joint possibly limiting
its potential damage. It is possible that if a large impact energy were used similar to
Chapter 1 that these early cartilage lesions would have been increased and more severe.

It is also possible that this study did not allow enough time post-impact for
progressive cartilage alterations to be observed. It has been suggested that less severe
injures take several years post-trauma to develop pathologic symptoms (Wright, 1990). In
Chapter two of this thesis we document that exercise also plays an important role in joint
degeneration in the long term post-trauma. Exercise in this trauma patient may have
helped to facilitate a healing response which limited the development of pathologic

symptoms. Other researches have documented full osteoarthritis in a non exercised

62

animal model at 6 months post-trauma following a 10.0] impact (Mazieres et al., 1987).
If the multi-color rabbits were not exercised it may be possible that a more sever joint
degeneration would have been observed at 7.5 months post-trauma.

There was a limitation to this study, which may have affected some of the
conclusions. If the proper indentation depth of 0.1 mm were used for all of the multi-
colored animals then more evidence may have been presented such as the actual stiffness
of the cartilage. A depth of 0.3 mm is more that 50% of the actual thickness of the
cartilage, therefore it is possible that these indentation tests resulted in matrix damage. To
strengthen this study a new population of multi-color animals would need to be tested. A
control population would also help to understand the total effects of the impact. These
have been tested recently, however were not included due to time constraints of this
thesis.

In conclusion, based on the histology data alone mechanical blunt insult does not
result in more severe joint alterations in the multi-colored animals than the fawn-colored
animals. These data may prove to be very important in litigation for those whom claim
that the existence of an underline pathology is accelerated to a full pathology through

mechanical trauma faster than the normal course of the disease process.

63

REFERENCES:

l.

10.

ll.

12.

Armstrong CG, Mow VC. (1982) Variations in the Intrinsic Mechanical
Properties of Human Articular Cartilage with Age, Degeneration, and Water
Content. J Bone J Surg. 64-Az88-94.

Arthritis National Research Foundation. (2001) www.curearthritisorg.

. Brandt KD, Myers SL, Burr D, Albrecht M. (1991) Osteoarthritic Changes in

Canine Articular Cartilage, Subchondral Bone, and Synovium Fifty-Four Months
After Transection of the Anterior Cruciate Ligament. Arth & Rheum.
34(12):]560—1570.

Cicuttini FM, Spector TD. (1996) Genetics of Osteoarthritis. Ann Rheum Dis.
55(9):666-667.

Cooper C, McAlindon T, Coggon D, Egger P, Dieppe P. (1994) Occupational
Activity and Osteoarthritis of the Knee. Annals of the Rheum Dis. 53:90-93.

Ewers BJ, Jayaraman VM, Banglmaier RF, Haut RC. (In review) Rate of Blunt
Impact Loading Affects Changes in Retropatellar Cartialge and Underlying Bone
in the Rabbit Patella. J Biomechanics.

Ewers BJ, Haut RC. (2000) Polysulphated Glycosaminoglycan Treatments Can
Mitigate Decreases in Stiffness of Articular Cartilage in a Traumatized Animal. J
Orth Res. 182756-761.

Felson DT. (1998) Epidemiology of Osteoarthritis. In: Brandt KD, Doherty M,
Lohmander LS (Eds). Osteoarthritis, Oxford University Press, New York. pp.13-
22.

Flores RH, Hochberg MC. (1998) Definition and Classification of Osteoarthritis.
In: Brandt KD, Doherty M, Lohmander LS (Eds). Osteoarthritis, Oxford
University Press, New York. pp.1-12.

Haut RC. (Personal communication) Professor at Michigan State University.

Haut Rc, Ide TM, DeCamp CE. (1995) Mechanical Responses of the Rabbit
Patello-femoral Joint to Blunt Impact. J Biomech Eng. 117:402-408.

Hayes WC, Keer IM, Gerrmann, Mockros IE. (1972) A Mathematical Analysis
for Indentation Tests of Articular Cartilage. J Biomechanics. 5:541-551.

64

l3.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Jurvelin J, Saamanen A-M, Arokoski J, Helminen JH, Kiviranta I, Tammi M.
(198 8) Biomechanical Properties of the Canine Knee Articular Cartilage as
Related to Matrix Protoglycans and Collagen. Eng Med 17:157-162.

Maimaris C, Barnes MR, Allen M]. (1988) Whiplash Injuries of the Neck: A
Retrospective Study. Injury 19:393-396.

Mazieres B, Blanckaert A, Thiechart M. (1987) Experimental Post-contusive
Osteoarthritis of the Knee: Quantitative Microscopic Study of the Patella and the
Femoral Condyles. J Rheum. 14:119-121.

Miles KA, Maimaris C, Finlay D, Barnes MR. (1988) The Incidence and
Prognostic Significance of Radiological Abnormalities in Soft Tissue Injuries to
the Cervical Spine. Skeletal Radiol 17:493-496.

Mizrahi J, Maroudas A, Lanir Y, Ziv I, Webber TJ. (1986) the “Instantaneous“
deformation of cartilage: Effects of Collagen Fiber Orientation and Osmotic
Stress. Biorheology 23:31 1-330.

Newberry WN, Mackenzie CD, Haut RC. (1998) Blunt Impact Causes Changes in
Bone and Cartilage in a Regularly Exercised Animal Model. J Orth Res. 16:348-
354.

Newberry WN, Zukosky DK, Haut RC. (1997) Subfracture Insult to a Knee Joint
Causes Alterations in the Bone and in the Functional Stiffness of Overlying
Cartilage. J Orth Res. 15:450-455.

Parsons JR, Black J. (1987) Mechanical Behavior of Articular Cartilage:
Quantitative Changes with Enzymatic Alteration of the Proteoglycan Fraction.
Bull Hosp Jr Dis Orthop Inst 47:13-30.

Praemer AP, Fumer S, Rice DP. (1992) Musculoskeletal Conditions in the United
States. In: American Academy of Othopaedic Surgeons. Park Ridge, IL.

Silyn-Roberts H, Broom ND. Fracture Behavior of Cartilage-On-Bone in
Response to Repeated Impact Loading. Conn Tissue Res. 24:143-156.

States JD. (1970) Traumatic Arthritis: A Medical Dilemma. In: Proceedings of
the I4"I Annual Conference of the American Association for Automotive Medicine.
14:21-28.

Watkinson A, Gargan MF, Bannister GC. (1991) Prognostic Factors in Soft
Tissue Injuries of the Cervical Spine. Injury 42307-309.

Wright V. (1990) Post-Traumatic Osteoarthritis — A Medico-Legal Minefield. Br
J Rheum 29:474-478.

65

Chapter Four

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE
WORK

In new vehicle certification the Federal Safety Standard on instrument panel
design limits the impact force in the femur to lOkN in simulated crashes. This standard is
based on the load required to cause gross fi'acture of the patella, femur, or pelvis. The
studies presented in this thesis represent a continuing effort to understand the underlying
mechanisms of post-traumatic joint degeneration without bone fracture. It is still unclear
how or why subfi‘acture injuries progress into a degenerative joint disease, although the
data presented in this thesis may shed some light on the relationship between post-
traumatic joint degeneration with the intensity of impact energy, exercise and pre-existing
surface defects.

In Chapter 1 the effects of impact energy and its relationship to chronic joint
alterations were investigated. Earlier studies have found that joint degeneration in our
model are a direct response to the joint contact stresses produced fiom the induced impact
energy (Atkinson et al., 1998; Newberry et al., 1998). The results of Chapter 1 seem to
further support this hypothesis. There were more cartilage alterations following a 10.0 J
impact at 7 .5 months post-impact compared to a 6.0 J impact. However, the joint contact
stresses were not calculated in this investigation. To accurately document that joint
damage is dependent on impact induced shear stresses finite element modeling should be
performed to investigate the different joint contact stresses produced from the 10.0 J

impacts and the 6.0 J impacts.

66

The role of exercise post-trauma has been presented as a very important issue.
The results of Chapter 1 were in contrast to a previous study which document full
thickness ulceration following a 10.0 J impact to the patellofemoral joint (Mazieres et al.,
1987). One difference between the animal models was the use of exercise post-trauma. In
both Chapter 1 and 2, we hypothesized that exercise promotes the propagation of fissures
similar to a solid mechanics fracture theory (Altiero, 1974). However, in Chapter 2 we
did not document an increase in fissures from 12 to 24 months in the impacted limb of
the exercise group. Other investigators have shown that exercise or cyclic loading of the
cartilage may increase the chondrocyte activity, which to some degree may be a healing
response (Guliak et al., 1997; Otterness et al., 1998). Therefore, it is possible that the
results of Chapter 1 were affected by the exercise, which may have retarded the
progression of full joint degradation following impact. The results of Mazieres and those
of Chapter 1 may better correlate if the animals were not exercised post-impact.

The results of Chapter 1 also revealed that the animals may have been favoring
their impacted limb based on the stiffness of the unimpacted cartilage. Both E .1 and E33
were approximately 13% stiffer in the unimpacted limb of both impact groups compared
to controls. In support of this, researchers have documented that increased use of healthy
joints increases their indentation stiffness (Jurvelin et al., 1986). However the veterinary
technician did not document any joint effusions or favoritism of the impacted limbs. For
this to be documented accurately, gait forces need to be measured during normal exercise
activities.

The results of Chapter 3 may prove to have a large significance in litigation

involving personal injuries in vehicle collisions. A question that is commonly asked is if

67

a person with some baseline pathology may develop complications more rapidly than a
person without any pathology following trauma (Haut, personal communication). The
data in Chapter 3 would suggest not, however there were several limitations of that study.
Without a control population it was hard to investigate the effects of the impact on the
joint tissues. Furthermore there is no method of determining if the multi-colored rabbits
do have pre-existing surface lesions at the time of impact. Our study is only based on the
probability that surface lesions are present.

The goal of most animal studies is to interpolate the findings in the animal to
humans. Although the rabbit model used in these studies may not exactly predict the joint
degeneration associated with the human, these data may provide sufficient information on
some of the underlying mechanisms that occur in the human. The results of Chapter 1
adds another variable to consider when designing safety equipment associated with
protecting joints. Most products such as the instrument panel in vehicles are more
concerned with limiting forces to not cause gross bone fracture. However, we have
shown that there are other force levels beneath fracture loads which may cause long term
pathologic symptoms such as osteoarthritis. However, the data of Chapter 2 may be used
to help rehabilitate these trauma patients. Exercise of stable injured joints may be the
difference between post-traumatic osteoarthritis and healthy cartilage. There are those
with joint pathologies such as osteoarthritis which may cause severe joint pain and
lameness, however the data of Chapter 3 appose those whom claim that their pre-existent
pathology was accelerated by trauma.

Upon completion of this work it is hoped that the results of these studies will

benefit man.

68

REFERENCES:

l.

10.

ll.

12.

Altiero NJ. (1974) Fracture Initiation and Propagation in Nonuniform
Compressive Stress Fields. University of Michigan, Ph.D. dissertation.

Atkinson TS, Haut RC, Aliero NJ. (1998) Inpact-Induced Fissuring of Articular
Cartilage: An Investigation of Failure Criteria. J Biomech Eng. 120: 18 1-187.

Guilak F, Sah R, Setton LA. (1997) Physical Regulation of Cartilage Metabolism.
In: Mow vc, Hayes wc (Eds), Basic Orthopaedic Biomechanics, 2nd edition,
Lippincott-Raven, Philadelphia, pp. 179-208.

Haut RC. (Personal communication) Professor at Michigan State University.

Jurvelin J, Kiviranta I, Tammi M, Helminen HJ. (1986) Effect of Physical
Exercise on Indentation Stifness of Articular Cartilage in the Canine Knee. Int. J
Sports Med. 7:106-110.

Maimaris C, Barnes MR, Allen MJ. (1988) Whiplash Injuries of the Neck: A
Retrospective Study. Injury 19:393-396.

Mazieres B, Blanckaert A, Thiechart M. (1987) Experimental Post-contusive
Osteoarthritis of the Knee: Quantitative Microscopic Study of the Patella and the
Femoral Condyles. J Rheum. 14:119-121.

. Miles KA, Maimaris C, Finlay D, Barnes MR. (1988) The Incidence and

Prognostic Significance of Radiological Abnormalities in Soft Tissue Injuries to
the Cervical Spine. Skeletal Radiol 17:493-496.

Newberry WN, Garcia JJ, Mackenzie CD, DeCamp CE, Haut RC. (1998-A)
Analysis of Acute Mechanical Insult in an Animal Model of Post-traumatic
Osteoarthrosis. J Biomech Eng. 120:704-709.

Norris SH, Watt I. (1983) The Prognosis of Neck Injuries Resulting From Rear-
End Vehicle Collisions. J Bone Joint Surg. 65B:608-6l l.

Otterness IG, Eskra JD, Bliven ML, Shay AK, Pelletier JP, Milici AJ. (1998)
Exercise Protects Against Articular Cartilage Degeneration in the Hamster.
Arthritis & Rheumatism. 41:2068-2076.

Watkinson A, Gargan MF, Bannister GC. (1991) Prognostic Factors in Soft
Tissue Injuries of the Cervical Spine. Injury 4:307-309.

69

APPENDIX A
DROP TEST FIXTURE

70

DROP FIXTURE

All previous impact studies were conducted by dropping the impact mass from a
maximum height of 0.5 m. The new fixture needed to be designed such that the impact
mass could be dropped from a height of 1.0m. To accomplish this, the basic concept of
the previous fixture was used. The old fixture was constructed out of aluminum so it was
light, which made it easier to transfer from one lab to the next. It also had the capabilities
of dropping and arresting the impact mass electronically. All these qualities were utilized
in the new fixture. In the old fixture, the impact mass was fixed to a long rod which was
passed through two pillow block bearings to guide the mass onto the target of interest
(Figure l). The rod was released and arrested by a solenoid controlled break. The rod
could not be used for the 1.0m drop because at least two points of the rod had to be
supported so no out of the plane motion took place. The impact mass needed to be guided
onto the target so that reliable and repeated tests were performed. This would have
required the length of the rod to be so long that it would not fit in any of the testing
laboratories. Therefore, we designed a cart and rail system so that the direction of the
mass could be controlled while eliminating any out of plane motion (Figure 2-4).

The cart was designed so that it weighed exactly 1.0 kg, however additional
weights can be added simply by attaching them at the rear end of the cart. Open pillow
blocks (Thompson, SPB-8-OPN) were attached on the underside of the cart plate for a
near fiictionless interaction between the rail (Thompson, SRA-8-SS) and the cart (Figure
3). The cart is slid over the top of the rail and the edge of the cart plate is held with the

solenoid break (Figure 4). To keep the cart from rotating about the axis along the rail,

71

MSD-filled nylon strips were attached along both sides of the rail (Figure 4). The fixture
was designed such that the cart could be released from any height up to 1.0m. This was

accomplished by allowing the solenoid breaks to be adjustable to any height along the

 

 

 

 

frame.
Front view Side View
1. ‘ '56
4 Rod 9
Solenoid break —> 00
Pillow block bearings

 

 

__ Rigid impact interface N

 

 

 

 

 

 

 

 

 

Figure 1: Rough sketch of previous drop/inure.

72

Front view Side View

Solenoid break

 

Figure 2: Front and side viens of new fixture.

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75

APPENDIX B
STANDARD OPERATING PROCEDURES

76

RABBIT PATELLOFEMORAL IMPACT SOP

Pre-test set up:

1.

Turn on the Validyne strain gauge amplifier and insure that the trigger release switch
is turned off to protect from accidental triggering.
Connect the channel #2 output A of the Validyne strain gauge amplifier, to the A/D
box channel #1 input (Load cell output).
Connect the channel #3 output B of the Validyne strain gauge amplifier, to the A/D
trigger input signal.
Turn on the Computer and make sure the boot disk is in drive A:

Note: the boot disk should contain the Rlinpltbak file
Wait for 15 minutes before calibrating the load cell to allow the electronics to
stabilize.
Spray some LPS greaseless lubrication on a rag and wipe down the rail of the impact
cart.
Clean, with alcohol, the side of the impact cart that is gripped by the brakes.
Zero the load cell using the course or fine adjustment with the small screwdriver.
Calibrate the load cell by depressing the shunt cal on the Validyne strain gauge

amplifier. If needed readjust the set point to —6.78 by using the Gain knob.

 

10. Double check to make sure the load cell is still zeroed, adjust if necessary.

Rabbit preparation:

1.

On the Data Sheet, record the Rabbit name, weight (kg), and sex.

77

8.

Once the rabbit is fully sedated, pull the lefi hind foot through the very bottom hole of
the leather strap of the holding chair and tuck the rest of the strap under the rabbit so
it can be fixated to the underside of the chair.

Place the black strap around the right hind foot and pull tight to insure the foot is fully
constrained. Fix the end of the strap, as well as the leather strap, to the Velcro pad on
the underside of the chair.

Move the clamping bar into position and attach the free end to the distal clamp.
Slowly apply even pressure to both clamps until the clamps lock in place.

Slide the chair into position so the patella is directly under the head of the impacting
cart.

Lower the cart so the head of the impacting cart is just above the patella, checking to
insure the patella is centered under the head.

Raise the cart to the desired height.

Computer set-up:

l.

2.

Start the “CDC RABBIT DROP TEST” program by double clicking on the icon in
the Windows atmosphere.

Answer the A/D set-up questions from the computer

Start channel = 1

End channel = 1

Time as ref. = “Y”
Inverting load cell = “N”

Rotary encoder = “N”

78

3. Change the filename.

4.

8.

9.

Type “F” [enter]
File to change = 1

Filename = rabbit’s name / ID

Check the plot control file data

# of channels to plot = 1
X label = time
Y label = Newtons
Data file name = rabbit’s name being tested
Channel number 0 cal factor = 1
(time calibration must be set to 1)

Channel number 1 cal factor = 222.4 NN

Press E for exit when all values are set correctly
Sample rate 10,000 samples / sec (Hz)

Number “N” = 10,000

Trigger level for data acquisition = 12 (N)

Number of samples to save after the trigger = 400

10. Press space bar

Impacting the rabbit:

79

. Turn on the trigger enable switch on the Validyne strain gauge amplifier.

. Press the red release button on the Validyne strain gauge amplifier to drop the cart.
. Impact cart will drop and the A/D board will trigger and save the data.

. Check status of D.M.A. operation = “‘N‘

. If you select “Y” then it should read:

Op type = 1

Status = 0

Word count = 10000

. Plot using line mode = “L”

. After the data scrolls to screen, it will be plotted. On the data sheet note the peak load
and the time to peak.

. Sketch the graph on the data sheet and write any comments about the test that were

unusual, i.e. if the impact cart hit the rubber stoppers during the test, etc.

. FOLLOWING IMPACTION OF ALL ANIMALS REMOVE DISK FROM DRIVE

 

(ml) AND COPY ALL FILES T 0 THE (g:luserlbimgadz DIRECTORY FOR
PERMANENT DOC UMENT A T I ON I

80

RABBIT PATELLA IN DENTATION SOP

Calibration and Program set up:

1.

2.

Turn on the program selector box

Attach the small hook into the bottom of the load cell

Open the “Rabindent.vi” program located on the desk top of the computer
adjacent to the test fixture.

Using the voltage display in the program, zero the load cell using the small screw
driver. The zeroing controls are located on the Sensotec resistor box located under
the program selector box.

Push in the shunt cal button on the resistor box and verify a voltage reading of

0.6970 1- 0.0005. Adjust the gain accordingly if needed.

Re-zero the load cell and then hang the small 100 gram weight on the hook
attached to the load cell. The voltage display should read -0.4000 1: 0.0005 if the
load cell is calibrated correctly. If not then take off the weight and repeat steps 4 —
6. If the problem persists contact Cliff Becket.

Repeat step 6 using the 200 gram weight, the voltage read out should be -0.8000 :1:

0.0005.

. In the rabindent.vi program, verify that the following settings are accurate:

Block 1: # samples = 1000, rate = 1000
Block 2: # samples = 3000, rate = 20
Block 3: # samples = 0

Channel calibration

81

Units/volt 0 = 1.0000
Units/volt 1 = 1.0000
Units/volt 2 = 1.0000

These settings control the collection of data. Block one will collect data at 1000

samples a second for one second, block two will then collect 3000 samples for an

additional 150 seconds. The data should be in volts because all the material property

programs we run automatically convert volts to Newtons.

9.

10.

11

12.

Minimize this program and open the “rabthick.vi” program, which is also located
on the desktop of the computer.

Verify its setting to be

Block 1: # samples = 3000, rate = 50

Block 2: # samples = 0

Channel calibration

Units/volt 0 = 1.0000

Units/volt l = 1.0000

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

HyperTerminal program. The indenter program is set up to be 10, 1 on the
program selector box, and the thickness program is set up to be 10, 2.

In the miniprogram, the indenter program is # 500. type “q500” then hit the
carriage return 14 times to display the program. It should read the following:
500H 0

5021 5000

82

505V 10000

508K 2

5100 0

514R 400

518W 65535

521W 65535

524W 62140

527R 0

531
Refer to the mini indent manual for a complete description of these commands. If a
line is incorrect, change it by typing “p” then the line number and the appropriate
command letter and number.
13. Check the thickness program. Type “q600” followed by 15 carriage returns. It

should read the following

600H 1

6021 200

605V 200

608K 0

6100 0

614R 4000

618H 1

6201 2000

623V 15000

83

626K 3
628R 0
632
14. Double check these programs by running a simulated test. Place the program
selector switches on 10, 1 for the indent program.
15. Rotate the mini indenter jogging knob, located on the top of the indenter, to 0.
16. Hit the start button on the selector box and make sure the knob rotates from 0 to 2
which translates to 0.1 mm of downward travel. If this is incorrect, recheck the
program in the HyperTerminal and make corrections as necessary.
17. Place the program selector switches on 10, 2 for the thickness program.
18. Hit the start button on the program selector box and make sure the indenter travels
downward at a constant rate. Hit the stop button at the end of the grey cable to
end the test.
Equipment set-up
l. Loosen all the claps on the mounting plate, 6 total. These are the round knobs
located around the edge of the fixture.

2. Attach the small grey base of the camera mount to the center of the horizontal
mounting plate.

3. Attach the camera mount to its base. Note that the small reservoir and clamp
should be attached to the camera mount, if not do so now.

4. Fill the reservoir with PBS, making sure not over fill.

Test procedure:

84

10.

ll.

12.

13.

Remove all soft tissue surrounding the patella with a scalpel, this will ensure a
good hold in the clamp.

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

On the data sheet, record the rabbit name and all other pertinent information.
Sketch all surface lesions and make notes of any other abnormalities such as
osteophytes.

Place patella into the small clamp on the camera mount.

Use a small Allen wrench to tighten the clamp to hold the patella in place. Do
not over tighten but make sure the patella is secured rigidly.

Screw in the flat 1mm indenter into the load cell. Find a flat clean location on
the lateral facet of the patella for testing. Rotate the camera mount to get this
location at horizontal as possible and jog the XY plate so the indenter is
located directly above the testing sight.

Lower the indenter as close as possible with out touching it to ensure the
testing sight is flat.

Adjust the camera mount and plate as necessary.

Once in place, tighten all the clamps.

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

Zero the load cell

Lower the indenter as close as possible.

Verify that the program switches are on 10, 1 and place the mouse pointer on

the start button in the rabindent.vi program.

85

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Lower the indenter very slowly by rotating the knob on top of the motor until
the indenter just touches the cartilage surface. This is determined by watching
the voltage indicator.

Once the indenter touches the surface, back off.

As fast as possible, lower the indenter to a preload of 0.0050, hit the start
button in the rabindent.vi program and hit the start button on the program
selector box. Note that the start button in the rabindent.vi program must be hit
a split second before the start button on the program selector box is hit to
ensure all the data is collected.

Once the test is completed, raise the indenter and replace the 1mm indenter
with the 1.5mm indenter.

Save the data file just collected in its appropriate location

Wait 5 min and then repeat steps 11-18.

Once the indentation tests are completed, measure the thickness of the
indentation sight.

Replace the 1.5mm indenter with the needle.

Put the program switches on 10, 2.

Open the “rabthick.vi” program

Zero the load cell

Lower the load cell until the needle is just touching the surface

Hit the stat button in the rabthick.vi program and the start button on the

program selector box at exactly the same time!

86

27. Stop the test with the shut off switch at the end of the grey cable with the
needle hits the bone. This is determined from the voltage plot in the
rabthick.vi program.

28. Watch the trace of the voltage plot. The voltage will gradually increase as the
needle starts to push on the cartilage, it will then drop suddenly as it breaks
through the surface, and then it will increase dramatically when it hits the
bone. A good point to watch is 5 V, once it reaches this value, stop the test.

29. Repeat for one more sight on the patella making sure to record the two sights

on the data sheet.

87

APPENDIX C
HISTOPATHOLOGIC SCORING SYSTEM

88

      

Figure A.1: Normal Patello
SURFACE INTEGRITY:

What to look for:
The surface integrity is the makeup of the articular surface, the score is based on

the amount of visual disturbance.

 

    

.e

Figure A.3: Focally severe.

     

Figure A.4: Ertenst'velv Severe

The main difference between focally severe and extensively severe is the size of the
disturbance. Figure A.3 shows a very concentrated location of the surface irregularity,
whereas Figure A.4 is an example of a very large disturbance in the surface.
PROTEOGLYCAN STAIN LNG:
What to look for:

Viable proteoglycans stain red. The index score is a description of how much

stain is present. The different scores are a direct correlation with the depth of absent

stain.

 

90

 

CHONDROCYTE ORGANIZATION:

What to look for:
Chondrocytes are the visible clear cells in the cartilage. In normal cartilage they
are aligned in vertical columns. The index score is a quantification of how much these

columns of cells have broken down.

91

 

    

a , .

Figure A.9: N0 recognizable organization

92

FISSURES:
What to look for:
Fissures are defined as vertical “v” shaped clefts in the surface of the cartilage.

The index score is a direct correlation of how many or how deep.

     

Figure A.10: Fissures. from the left: One full thickness followed by
two midzone and one small.
CLONES:
What to look for:

Clones are defined as a proliferation of chondrocytes. These cells multiply when

damaged, and are unable to form in normal columns. The size of the clone is related to

the number of cells.

93

    

 

Figure A.11: The circles objects. from the left. are examples of
large and medium size clones.

Figure A.12: The circles objects are examples of small clones.

94

OSSIFICATION:
What to look for:

Ossification is observed as the presence of bone within the cartilage.

 

     

Figure A.15: Ossification. growth of bone within the cartilage.

95

EXPOSURE OF SUBCHONDRAL BONE:
What to look for:
This index is defined as the total loss of cartilage until the underlying subchondral

bone is present.

    

Figure A.16: Exposure of subchondral bone.

EROSION:
What to look for:
Erosion is defined by the rubbing away of cartilage. The score is a quantification

of how much thinning and scrubbing of the surface took place.

 

96

 

Figure A.18: Extensiwa severe erosion.

97

APPENDIX D
RAW DATA FROM CHAPTER 1

98

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MM
CONTROL TEST

Animal M C L L C M
8842 0.5166 1.196107 0.940267 1.03648 1.158933 0.8528
A35 0.451 0.4592 0.366267 1.0988 1.128867 0.7298
ZVC3 0.580013 0.70192 0.6396 0.894893 1.368307 0.821093
8841 0.707933 1.038667 0.656 1.104267 1.333867 0.9184
V1 0.486533 0.5576 0.481067 0.677867 0.912933 0.560333
8F315 0.833667 1.49896 1.249133 0.9512 1.0414 0.738547
M86 0.5412 0.625933 0.683333 0.8856 1.137067 0.970333
A36 0.456467 0.661467 0.940267 0.653267 0.636867 0.653267
8F483 0.784467 0.765333 0.6888 1.087867 1.558 0.6724
AVE 0.59532 0.83391 0.738304 0.932249 1.141804 0.768553
SD 0.144296 0.340454 0.266715 0.172801 0.2691 0.13291

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JANE 9.100962
MAX
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MM
CONTROL TEST

Animal M C L L C M
A4 0.9348 1.503333 0.923867 0.945733 1.292867 0.825467
A2 0.798133 1.011333 1.068733 1.107 1.394 1.0086
BF206 0.64616 0.747567 0.809613 1.048507 1.158933 1.112467
A5 0.7626 1.01188 0.861 0.923867 0.8856 0.691533
BF173 0.740733 0.6888 0.710667 0.973067 0.604067 0.642333
BF236 0.722147 0.599147 0.740733 0.76752 0.746747 0.745653
297 0.822733 0.7462 0.759867 0.973067 1.415867 1.317467
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SD 0.147249 0.368696 0.14301 0.106897 0.374341 0.230126

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JANE
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Animal M C L L C M
286 0.615 0.71996 0.608987 0.5986 0.505667 0.5166
SAID 0.574 1.3038 0.839133 0.6273 0.5904 0.492
BF238 0.825467 0.932067 0.863733 1.076933 0.978533 0.899267
287 0.451 0.628667 0.697 0.7544 0.707933 0.464667
Z85 0.650533 0.7134 0.574 0.63304 1.0865 0.61336
BF319 0.740733 1.1726 1.0414 1.0988 1.3858 0.8692
SA12 0.612267 0.761507 0.75112 0.724333 0.5084 0.497467
BF239 0.675133 0.994933 0.81836 0.8692 0.858267 0.768067
AVE 0.643017 0.903367 0.774217 0.797826 0.827688 0.640078
SD 0.111668 0.241232 0.150924 0.198851 0.311322 0.179305

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APPENDIX E
RAW DATA FROM CHAPTER2

133

 

 

 

 

 

 

134

12mo. HIGH EX RAW DATA
SITE 1
Animal 60 test Gr test T test 60 control Gr control T control
rb16 0.516 0.100 0.33 1 .533 0.194 0.350
rb1 1 0.790 0.310 0.43 1 .824 0.375 0.330
ckcool 1 .541 0.315 0.35 1 .980 0.294 0.350
rb21 1 .156 0.315 0.38 1 .865 0.292 0.430
r020 0.916 0.262 0.4 1 .718 0.325 0.390
ksb83 1.060 0.336 0.6 1 .805 0.384 0.760
30317 1 .125 0.329 0.55 1 .471 0.383 0.720
mean 1 .015 0.281 0.434 1 .742 0.321 0.476
stdev 0.322 0.083 0.102 0.183 0.069 0.184
SITE 2
Animal Gu test Gr test T test Gu control Gr control T control
rb16 0.261 0.143 0.38 0.475 ' 0.175 0.43
rb1 1 0.761 0.339 0.45 1 .013 0.341 0.38
ckcool 0.735 0.314 0.4 1 .431 0.407 0.5
r021 0.814 0.304 0.4 1 .1 30 0.493 0.5
rb20 0.668 0.286 0.56 1 .304 0.468 0.5
ksbB3 0.780 0.399 0.58 1 .580 0.529 0.7
sc317 0.617 0.229 0.71 1 .193 0.362 0.57
mean 0.662 0.288 0.497 1 .161 0.396 0.51 1
stdev 0.189 0.082 0.123 0.357 0.1 19 0.103
averaged
Animal (in test Gr test T test 60 control Gr control T control
rb16 0.389 0.121 0.355 1 .004 0.184 0.390
rb1 1 0.775 0.324 0.440 1 .418 0.358 0.355
ckcool 1 .138 0.315 0.375 1 .705 0.350 0.500
rb21 0.985 0.309 0.390 1 .498 0.392 0.465
rb20 0.792 0.274 0.400 1 .51 1 0.397 0.445
ksbBS 0.920 0.367 0.590 1 .693 0.456 0.700
sc317 0.871 0.279 0.550 1 .332 0.373 0.570
mean 0.839 0.284 0.443 1 .452 0.359 0.489
stdev 0.234 0.078 0.091 0.239 0.084 0.1 17

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12 month N0 EX SITE 1 RAW DATA
Animal Gu test Gr test T test Gu control Gr control T control
i1 9 0.414 0.196 5 0.860 0.890 0.161 ' 0.390
gag 0.409 0.177 0.510 0.667 0.177 0.460
i8 0.619 0.212 0.630 1.311 0.127 0.670
i12 0.649 0.188 0.400 0.918 0.231 0.560
i2 0.787 0.229 0.330 0.726 0.205 0.350
005 1.531 0.144 ' 0.510 1.578 0.255 0.550
mean 0.735 0.191 0.540 1.015 0.193 0.497
stdev 0.416 0.029 0.188 0.356 0.047 0.119
SITE 2
Animal 60 test Gr test T test 60 control Gr control T control
i1g 0.457 0.205 0.610 0.478 0.174 7 0.350
gag 0.631 0.289 0.450 0.905 0.270 0.420
i8 0.847 0.252 0.610 0.883 0.218 0.570
H2 0.470 0.191 0.330 1 .268 0.362 0.320
i2 0.336 0.237 0.460 0.494 0.172 0.370
bc5 1 .300 0.284 0.440 1 .636 0.351 . 0.390
mean 0.674 0.243 0.483 0.944 0.258 0.403
stdev 0.354 0.040 0.109 0.449 0.084 0.088
averaged
Animal 60 test Gr test T test Gu control Gr control T control
i1 9 0.684 0.1675 0.37 0.4355 0.2005 0.735
gag 0.52 0.233 0.48 0.786 0.2235 0.44
i8 0.733 0.232 0.62 1 .097 0.1725 0.62
i12 0.5595 0.1895 0.365 1 .093 0.2965 0.56
i2 0.5615 0.233 0.33 0.61 0.1885 0.36
bc5 1.4155 0.214 0.44 1.607 0.303 0.55 .
mean 0.745583 0.2115 0.434167 0.9380833 0.23075 0.544166667
stdev 0.338272 0.027463 0.106227 0.4195381 0.056015846 0.132076367

135

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24mo. EX RAW DATA
SITE 1
Animal 60 test Gr test T test 60 control Gr control T control
221 1.083 0.309 0.455 1.308 0.369 0.495
224 Corrupt 1 .285 0.209 0.49
BC 0.544 0.080 0.63 1.384 027 0.67
R28 0.697 0.216 0.545 0.729 0.28 0.635
BI 1.131 0.416 0.52 2.256 0.679 0.565
BF 1 15 0.6523 0.2289 0.46 0.8348 0.3223 0.54
BF73 0.5433 0.2391 0.56 1.0836 0.4203 0.645
RABBIT 1.2478 0.3722 0.42 1.1987 0.5625 0.5
R48 0.9675 0.6396 0.72 1.3465 0.5913 0.72
mean 0.861 0.313 0.539 1.270 0.41 1 0.584
stdev 0.283 0.168 0.100 0.436 0.164 0.065
SITE 2
Animal 60 test Gr test T test Gu control Gr control T control
221 0.699 0.278 0.56 1 .421 0.451 0.625
224 0.629 0.267 0.59 0.967 0.306 0.63
BC 0.445 0.153 0.61 1.13 0.432 0.535
R28 0.962 0.319 0.48 0.932 0.269 0.635
BI 1.883 0.593 0.69 Corrupt
BF1 15 0.7126 0.2888 0.45 0.7408 0.2768 0.545
BF73 0.3682 0.174 0.68 1 .1687 0.389 0.68
RABBIT 1.1808 0.4529 0.53 1.3908 0.564 0.59
R4B 1.0836 0.603 0.65 1.2715 0.5255 0.64
mean 0.685 0.348 0.582 1.128 0.402 0.610
stdev 0.464 0.166 0.085 0.236 0.1 12 0.050
averaged
Animal 60 test Gr test T test 611 control Gr control T control
221 0.891 0.294 0.508 1.365 0.410 0.560
224 0.629 0.267 0.590 1.126 0.258 0.560
BC 0.495 0.1 17 0.620 1 .257 0.351 0.603
R28 0.830 0.268 0.513 0.831 0.275 0.635
BI 1.131 0.416 0.520 2.258 0.679 0.565
BF 1 15 0.682 0.259 0.455 0.788 0.300 0.543
BF73 0.456 0.207 0.620 1.126 0.405 0.663
RABBIT 1.214 0.413 0.475 1.295 0.563 0.545
R48 1.036 0.621 0.685 1.309 0.558 0.680
mean 0.818 0.318 0.554 1.262 0.422 0.595
stdev 0.273 0.147 0.078 0.426 0.147 0.052

136

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137

24mo. NO EX RAW DATA
SITE 1
Animal 60 test Gr test T test Gu control Gr control T control
A7 1.57383 0.29131 9 0.765 1 .88067 0.489704 0.74
A8 0.575667 0.14006 0.36 1 .06025 0.232048 0.41
A9 0.786855 0.1 15149 0.385 0.849099 0.3417202 0.61
A1 0 1 .01404 0.259315 0.475 0.740476 0.138798 0.57
A12 1 .84658 0.207939 0.435 1 .26036 0.195884 0.55
A15 1.24723 0.08628 0.65 0.880823 ' 0.0971685 0.565
RB1 2.41593 0.361612 0.485 0.525653 0.151786 0.38
R32 1 .4945 0.3407 0.485 1 .2912 0.309738 0.495
R33 1.37416 0.366963 0.585 ‘ . 0.866694 . 0.279777 . 0.425
R34 1 .89234 0.293019 0.54 1 .44436 0.164288 0.48
mean 1 .422 0.246 0.517 1 .080 0.240 0.523
stdev 0.551 0.104 0.123 0.396 0.118 0.108
SITE 2
Animal Gu test Gr test T test Gu control Gr control T control
A7 0.814448 0.28704 0.46 1 .34588 0.400761 0.735
A8 Corrupt file 1 .69772 0.3573131 0.59
A9 0.78334 0.156999 0.515 0.821093 0.332926 0.59
A10 0.366893 0.154835 0.56 1 .031 1 0.250289 0.71
A12 1 .55129 0.246537 0.425 1 .38091 0.330227 0.615
A15 0.723708 0.133611 0.745 Corrupt file
R31 1 .38333 0.385559 0.615 0.982746 0.202209 0.475
R32 1 .24704 0.313266 0.48 Corrupt file
R33 1.18586 0.405068 0.615 1.73224 0.461003 0.67
R34 1 .92502 0.305998 0.595 1 .66283 0.321223 0.52
mean 1.109 0.265 0.557 1.332 0.332 0.613
stdev 0.481 0.100 0.099 0.354 0.081 0.090
averaged
Animal 60 test Gr test T test 60 control Gr control T control
A7 0.814 0.287 0.460 1.613 0.445 0.738
A8 0.576 0.140 0.360 1 .379 0.295 0.500
A9 0.785 0.136 0.450 0.835 0.337 0.600
A1 0 0.690 0.207 0.51 8 0.886 0.195 0.640
A12 1 .699 0.227 0.430 1 .321 0.263 0.583
R31 1 .383 0.386 0.485 0.983 0.202 0.475
R32 1 .371 0.327 0.483 1 .291 0.310 0.495
R33 1 .280 0.386 0.600 1 .732 0.461 0.670
R34 1 .909 0.300 0.568 1 .554 0.243 0.500
mean 1 .167 0.266 0.484 1 .288 0.306 0.578
stdev 0.472 0.095 0.072 0.325 0.096 0.092

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APPENDIX F
RAW DATA FROM CHAPTER3

142

6.0 J Impacts (Fawn-color rabbits)

 

 

 

 

 

 

 

raw data
Animal Site Thickness Gu Gr Site Thickness Gu Gr

296 C1 0.47 0.9 0.2178 T1 0.53 1.242 0.357
CZ 0.54 0.888 0.274 T2 0.68 0.608 0.245

A4 C1 0.49 1.42 0.461 T1 0.53 0.442 0.241
C2 0.55 1.665 0.587 T2 0.68 0.465 0.25
A5 C1 0.45 1 .216 0.333 T1 0.505 1.288 0.356
C2 0.58 1.084 0.347 T2 0.6 0.81 0.333

BF 1 73 01 0.42 1.721 0.363 T1 0.4 0.818 0.221
CZ 0.65 1 .65 0.55 T2 0.4 0.665 0.255

297 C1 0.64 0.778 0.284 T1 0.42 0.612 0.17
C2 0.69 0.881 0.305 T2 0.55 0.4 0.141
BF236 C1 0.52 0.888 0.121 T1 0.49 0.555 0.244
C2 0.7 ' 0.461 0.098 T2 0.6 0.247 0.121
BF206 C1 0.5 1.066 0.314 T1 0.4 0.856 0.198
C2 0.59 0.856 0.298 T2 0.545 0.643 0.235
A2 C1 0.4 1 .71 0.62 T1 0.42 1 .525 0.458
C2 0.43 2 0.467 . T2 0.4 1.11 1 , 0.391

 

These values are considered-outliersl‘ " ‘

Averages of Raw Data

 

Control Test
Animal Gu Gr Gu Gr
296 0.894 0.2459 0.608 0.245
A4 1 .5425 0.524 0.4535 0.2455
A5 1 .15 0.34 0.81 0.333
BF173 1 .6855 0.4565 0.741 5 0.238
Z97 0.8295 0.2945 0.506 0.1555
BF236 0.888 0.121 0.401 0.1825
BF206 0.961 0.306 0.7495 0.2165
Average 1.135786 0.326843 0.609929 0.230857
Stdev 0.344486 0.133078 0.161114 0.056453

F-..— ""1““! I

6.0 J Impact (multi-color rabbits)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

raw data
Control Test
Name site thickness Gu Gr site thickness Gu Gr
809 c1 0.419 .4888. 0.12571 11 0.419 0.495624 0.1666
c2 0.394 0.49037 0.199611 12 0.515 0.46716 0.19712
deo c1 0.405 0.58516 0.109808 11 0.585 0.436684 0.07494
c2 0.52 0.385509 0.080388 12 0.635 0.314131 0.069408
Grey c1 0.379 0.4845 0.10405 11 0.359 0.578879 0.131147
c2 0.394 0.321686 0.12945 12 0.389 0.470176 0.14415
Ks369 c1 0.379 0.508285 0.150475 11 0.359 0.368377 0.089571
c2 0.384 0.244847 0.10453 12 0.384 0.184391 0.087039
K5399 c1 0.399 0.447413 0.21287
c2 0.394 0.577786 0.249819
R5104 01 0.4 0.658907 0.127429 11 0.635 0.921095 0.163746
c2 0.67 0.778916 0.264432 12 0.785 0.535198 0.153825
m1 c1 0.486 3.00237 0.335464 11 0.444 1.28058 0.201015
c2 0.742 2.9648 0.347832 12 0.592 3.15295 0.336315
m2 c1 0.618 2.74513 0.333093 11 0.574 1.97781 0.26398
c2 0.702 2.84024 0.352635 12 0.62 2.10973 0.307207
m3 c1 0.316 2.40695 0.275561 11 0.31 3.09486 0.205095
c2 0.396 2.84266 0.23631 12 0.626 1.01404 0.108112
13 0.514 2.43775 0.179546
m4 c1 0.638 2.4868 0.317387 11 0.518 1.96034 0.176503
c2 0.574 1.46668 0.195434 12 0.576 1.67167 0.281619
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m6 c1 0.678 1.73723 0.285875 11 0.522 2.91827 0.380983
c2 0.654 1.32294 0.284634 12 0.578 2.67038 0.352639
ml c1 0.674 0.734501 0.159174 11 0.746 1.05068 0.231048
c2 0.5 0.758679 0.183154 12 0.488 2.25229 0.136588
blacked out cells represent data that does not exist
Highlighed data are considered outliers
Averages of raw data
Name Thickness Gu Gr thickness Gu Gr
bb9 0.4065 0.49037 0.162661 0.467 0.481392 0.18186
ckdo 0.4625 0.485335 0.095098 0.61 0.375408 0.072174
grey 0.3865 0.403093 0.11675 0.374 0.524528 0.137649
k5369 0.3815 0.376566 0.127503 0.3715 0.276384 0.088305
r5104 0.67 0.778916 0.264432 0.71 0.728147 0.158786
m6 0.666 1.530085 0.285255 0.55 2.794325 0.366811
ml 0.587 0.74659 0.171164 0.617 1.05068 0.183818
m4 0.606 1.97674 0.256411 0.547 1.816005 0.229061
Average 0.52075 0.848462 0.184909 0.530813 1.005858 0.177308
Stdev 0.124722 0.589669 0.073869 0.119809 0.875902 0.092229

144

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5

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0

Multicolor rmbrts
Hlsto scores
art-1
PG suing
Fesures
Disorp at Chondrocytes
Clones
Ossiticabm
Exposure oi Bone
Erosion

Section Surtaoe

GREY K8039

CKDO
Unim
0

889
Unsm inn
0 1 0

ML

Un'nv irm
4

luv
0

Unrw
0

lm
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Unmp
0 1

Un'rnp irm
1

Unlro
0

Jane

Section Surtaoe

PG Sunny

F csures

Disorg oi Chondrocytes

12

1O

3

12

1O

Clones

Ossifrcatron

Exposure oi Bone

Eroston

R8104
Imp
1s

Untrp
0

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imp
1 5

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0

KSO399
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05

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1

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Unrm lrrp Unim lrm
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3.5

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2

15

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Section Sudsos

55
4.5
65

25

05

3.5

1 5
05
3.5
35

05

PG 810an

145

75

11

1 5 0.5

35

45
05

05

Fbsures
Dream 01 Chondrocytes

25
35

15

35

35
35

5.5

2
55

55

35

6.5

4.5

Clones

Ossrttcabon

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6.0 J Impacts (Multi-color)
MM

CONTROL TEST
Animal M C L L C M

. 1
865556 1 11 1.064178
0.

 

STDEV 0.165885 0.345479 0.407779 0.32146 0.337571 0.193336

within variance between variance
Brian 19.62296 5.492962
JANE 47.43126
MAX 47.43126
AVE 4.844297 0.898336

 

 

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