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This is to certify that the

thesis entitled

An Analysis of Some of the Effects of
Surgical and Mechanical Trauma on the
Mechanical Stiffness of Rabbit Articular Cartilage

presented by

Dennis Elburn Shelp

has been accepted towards fulfillment
of the requirements for

Masters Mechanics

degree in

 

 

 

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AN ANALYSIS OF SOME OF THE EFFECTS
OF SURGICAL AND MECHANICAL TRAUMA
ON THE MECHANICAL STIFFNESS OF RABBIT ARTICULAR CARTILAGE

By

Dennis Elburn Shelp

A THESIS

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

MASTER OF SCIENCE

Department of Materials Science and Mechanics

1994

ABSTRACT
AN ANALYSIS OF SOME OF THE EFFECTS

OF SURGICAL AND MECHANICAL TRAUMA
ON THE MECHANICAL STIFFNESS OF RABBIT ARTICULAR CARTILAGE

By
Dennis Elburn Shelp

Osteoarthritis is a degenerative joint disease causing painful instability and loss
of motion. Traumatic injury, such as a single blunt impact, has been speculated as a
possible pathogenesis leading to this disease. The results of previous impact studies
performed on a rabbit model, have suggested that traumatic fissures and surgical
synovitis may reduce patellar articular cartilage stiffness possibly leading to degeneration.
In an acute study we found that impact induced fissures caused a reduction in the
instantaneous stiffness of cartilage, while exposure to a surgical synovitis created a
decrease in its equilibrium stiffness. The simultaneous combination of these two factors
not only decreased cartilage stiffness but also its flow viscosity. However, although the
overall load carry capacity of the cartilage was reduced, the existence of an interaction
between the fissures and synovitis could not be proven. A second study was performed
to investigate the possibility of creating a synovitis under closed joint conditions through
blunt impact to the synovial tissue. Six days after synovial trauma, unfissured patellar
cartilage specimens demonstrated an overall increase in stiffness and flow viscosity, while
the fissured specimens demonstrated overall decreases. Again, however, a statistical
interaction between the fissures and synovitis could not be proven. Interestingly,
hyperflexion of the limb during the induction of synovial trauma was found to prevent

changes in cartilage stiffness in both intact and fissured specimens.

ACKNOWLEDGEMENTS

I would like to begin by thanking my advisor, Dr. Roger Haut, for his guidance,
friendship, and occasional words of motivation.

I would also like to thank the following people: Dr. Charles DeCamp for his
surgical expertise, Jean Atkinson for her work on the rabbits, Jane Walsh for her
histological preparations, and Tammy Haut for her help in tabulating data and preparing
figures.

Finally, I would like to thank Brenda Robinson for her long hours of typing and
good sense of humor.

These studies were entirely supported by a grant from the Centers for Disease

Control.

iii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF SYMBOLS

INTRODUCTION
Articular Cartilage
Collagen
Proteoglycans
Water
Osteoarthritis Research
Load Trauma Models
Gross Observable Damage
Arthritis and the Synovium
Synovial Morphology
Synovial Fluid
Mechanisms of Synovitis
Background Studies

THE OPEN JOINT STUDY

iv

xiii

XV

10
13
l7
l8
19
19
22

23

OPEN JOINT MATERIALS AND METHODS

Impacting Device
Pressure Sensitive Film
Preliminary Studies
The In Vivo Study
Surgical Impact Trauma

Test Procedure

OPEN JOINT RESULTS

Blunt Impact
Mechanical Results
Sham Data

Biochemistry

THE CLOSED JOINT STUDY

CLOSED JOINT MATERIALS AND METHODS

The In Vivo (Closed Joint) Study
Blunt Impact

Data Collection

Grip Assembly

Test Procedure

CLOSED JOINT RESULTS

Impact Trauma

23

23

25

26

27

27

28

29

29

30

36

36

37

37

37

38

39

39

41

45

45

Mechanical Testing

Six Days

Three Months

Six Months

One Year

Shams

Comparison of Background Studies

Supplemental Animals

DISCUSSION OF BACKGROUND STUDIES
OBJECTIVES OF THE STUDY
THE ACUTE STUDY
ACUTE STUDY MATERIAL AND METHODS
The Acute In Vivo Study
Surgery/ Blunt Impact
Testing Procedure

(Rigid indenter)

(Porous indenter)
Biomechanical Properties
Elastic/Viscoelastic Analysis
Biphasic Theory
Biphasic Analysis
Statistical Analysis
Histology

ACUTE STUDY RESULTS
Blunt Impact

Gross Observations

vi

45
45
46
47
47
48
48
53
55

59

61
61
62
63
63

65

73
78
82

83

84

Mechanical Test: (Solid Indenter)

Time Zero, High Impact 87
One Day, Shams (No Impact) 87
One Day, High Impact 88
Between Group Comparisons and Interactions 88

Mechanical Test: (Porous Indenter)
Biphasic Results 89
Elastic/Viscoelastic Results 91
Histology 92
SYNOVITIS STUDY 94
The Synovitis In Vivo Study 95
Soft Tissue Trauma/Blunt Impact 96
Testing Procedure 97
Biomechanical Properties 98
SYNOVITIS STUDY RESULTS 99
Blunt Impact 99

PART I: (Synovitis created without the use of the restraining chair) 99

Part 1: Gross Observations

Sham Group 99

Impact Group 100
Part 1: Mechanical Tests (Solid Indenter)

Sham Group 100

Impact Group 101
Part 1: Mechanical Tests (Porous Indenter) 101
Part I: Elastic/Viscoelastic Results 102
Part I: Histology (Articular Cartilage) 103

Part I: Histology (Synovial Lining)
Sham Group 104
Impact Group 105

vii

PART II: (Synovitis created while restrained in the chair)
Part II: Gross Observations
Sham Group
Impact Group
Part 11: Mechanical Tests (Rigid Indenter)
Sham Group
Impact Group
Part II: Mechanical Tests (Porous Indenter)
Part II: Elastic/Viscoelastic Results
Part II: Histology (Articular Cartilage)
Part II: Histology (Synovial Lining)
Sham Group
Impact Group
Chair and Impact Interactions
DISCUSSION
Acute Study
Synovitis Study
SUMMARY
RECOMMENDATIONS
APPENDD(

REFERENCES

viii

105

105
106

106
106
107
108
109
110
111
111
113
114
118
126
129
130

166

Table 1:

Table 2:

Table 3:
Table 4:

Table 5 :

Table 6:

Table 7:

Table 8:

Table 9:

Table 10:

Table 11:

Table 12:

Table 13:

LIST OF TABLES

Impact Intensities

Average Peak Impact Loads and Contact Pressure Ranges for the Open
Joint Study

Six Day Low Impact Force Parameters from the Blunt Impact Procedure
Six Day High Impact Force Parameters from the Blunt Impact Procedure

Three Month Low Impact Force Parameters from the Blunt Impact
Procedure

Three Month High Impact Force Parameters from the Blunt Impact
Procedure

Six Month, Low Impact Closed Joint Force Parameters from the Blunt
Impact Procedure

Six Month, High Impact, Closed Joint, Force Parameters from the Blunt
Impact Procedure

One Year, Low Impact, Closed Joint Force Parameters from the Blunt
Impact Procedure

One Year, High Impact, Closed Joint, Force Parameters from the Blunt
Impact Procedure

Results of Mechanical Tests on the 6 Day, Low Level Impact Closed Joint
Group

Results of the Mechanical Tests on the 6 Day, High Level Impact, Closed
Joint Group

Results of the Mechanical Tests on the 3 Month, Low Level Impact,
Closed Joint Group

ix

Table 14:

Table 15:

Table 16:

Table 17:

Table 18:

Table 19:

Table 20:

Table 21:

Table 22:

Table 23:

Table 24:

Table 25:

Table 26:

Table 27:

Table 28:

Table 29:

Results of the Mechanical Tests on the 3 Month, High Level Impact,
Closed Joint Group

Results of the Mechanical Tests on the 6 Month, Low Level Impact,
Closed Joint Group

Results of the Mechanical Tests on the 6 Month, High Level Impact,
Closed Joint Group

Results of the Mechanical Tests on the 1 Year, Low Level Impact, Closed
Joint Group

Results of the Mechanical Tests on the 1 Year, High Level Impact, Closed
Joint Group

Results of Mechanical Tests on the 6 Day Sham, Closed Joint Group

Three Month, Low Impact, Open Joint, Force Parameters from the Blunt
Impact Procedure

Three Month, High Impact, Open Joint, Force Parameters from the Blunt
Impact Procedure

Results of the Mechanical Tests on the 3 Month, Low Impact,
Supplemental Group

Results of the Mechanical Tests on the 3 Month, High Level Impact,
Supplemental Group

Results of the High Level, Blunt Impacts for the Acute Study Impact

Results of the Solid Indenter Mechanical Tests on the Time Zero, High
Impact, Acute Rabbit Group

Results of the Solid Indenter Mechanical Tests on the One Day, Sham
Operated (No Impact), Acute Rabbit Group

Results of the Solid Indenter Mechanical Tests on the One Day, High
Impact, Acute Rabbit Group

Biphasic Material Properties for the Acute Study Porous Tests, Rate
1.0 sec.

Biphasic Material Properties for the Acute Study Porous Tests, Rate
2.0 sec.

Table 30:

Table 31:

Table 32:

Table 33:

Table 34:

Table 35:

Table 36:

Table 37:

Table 38:

Table 39:

Table 40:

Table 41:

Table 42:

Table 43:

Elastic and Viscoelastic Parameters for the Acute Study, Porous Tests,
Rate = 1.0 sec.

Elastic and Viscoelastic Parameters for the Acute Study, Porous Tests,
Rate = 2.0 sec.

Results of the High Level, Blunt Impacts for the Synovitis Study Impact
Groups

Results of the Solid Indenter Mechanical Tests on the 6 Day Synovitis
Study, Sham Group. (Synovitis created without the use of the restraining
chair).

Results of the Solid Indenter Mechanical Tests on the 6 Day Synovitis,
Impact Group. (Synovitis created without the use of the restraining
chair).

Biphasic Material Properties for the Synovitis Study Porous Tests, Rate
= 1.0 sec. (Synovitis induced without the use of restraining chair).

Biphasic Material Properties for the Synovitis Study Porous Tests, Rate
= 2.0 sec. (Synovitis induced without the use of restraining chair).

Elastic and Viscoelastic Parameters for the Synovitis Study, Porous tests,
Rate = 1.0 sec. (Synovitis created without the use of the restraining
chair).

Elastic and Viscoelastic Parameters for the Synovitis Study, Porous tests,
Rate = 2.0 sec. (Synovitis created without the use of the restraining
chair).

Results of the Solid Indenter Mechanical Tests on the 6 Day Synovitis,
Sham Group. (Synovitis created while restrained in chair).

Results of Solid Indenter Mechanical Tests on the 6 Day Synovitis, Impact
Group. (Synovitis created while restrained in chair).

Biphasic Material Properties for the Synovitis Study Porous Tests, Rate
= 1.0 sec. (Synovitis induced while restrained in chair).

Biphasic Material Properties for the S ynovitis Study Porous Tests, Rate
= 2.0 sec. (Synovitis induced while restrained in chair).

Elastic and Viscoelastic Parameters for the Synovitis Study, Porous tests,
Rate = 1.0 sec. (Synovitis created while restrained in chair).

xi

Table 44: Elastic and Viscoelastic Parameters for the Synovitis Study, Porous tests,
Rate = 2.0 sec. (Synovitis created while restrained in chair).

xii

Figure 1:
Figure 2:
Figure 3:
Figure 4:

Figure 5:

Figure 6:
Figure 7:
Figure 8:

Figure 9:

Figure 10:
Figure 11:
Figure 12:
Figure 13:
Figure 14:
Figure 15:
Figure 16:
Figure 17:

Figure 18:

LIST OF FIGURES

Cartilage layers.

Cartilage under shear loading.

Proteoglycan aggregate.

Micro-damage to collagen increasing cartilage water carrying capacity.

Sagittal (a) and frontal (b) views of the anatomy of the human knee joint
with the synovial lining shown in red.

The gravity impacter.

A typical impact fissure.

Histogram of Gu from the open joint, low level impact groups.
Histogram of Gu from the open joint, high level impact groups.
Histogram of Gr values from the open joint, low level impact groups.
Histogram of Gr value from the open joint, high level impact groups.
Histogram of n values from the open joint, low level impact groups.
Histogram of n values from the open joint, high level impact groups.
Vice-like grip used to hold patellae.

Bath-grip mount assembly.

Patella held in grip.

The six day, closed joint test sites.

The closed joint test sites following six day time point.

xiii

Figure 19:
Figure 20:

Figure 21:
Figure 22:
Figure 23:
Figure 24:
Figure 25:
Figure 26:
Figure 27:
Figure 28:

Figure 29:
Figure 30:

Figure 31:

Figure 32:

Figure 33:
Figure 34:
Figure 35:
Figure 36:
Figure 37:
Figure 38:

Figure 39:
Figure 40:

Open joint study, high vs low level impact, Gu trends.

Closed joint study, high vs low level impact, Gu trends.
Open joint, high vs low level impact, Gr trends.

Closed joint, high vs low level impact, Gr trends.

Open joint, high vs low level impact, 11 trends.

Closed joint, high vs low level impact, 11 trends.

Porous indenter.

Porous indentation test sites.

Indentation test.

A stress-relaxation load-time response curve.

A load-time plot of a thickness test.

A generalized Maxwell model.

The theoretical response of a generalized Maxwell model.

The application of Tobolsky’s analysis (1960) on experimental stress
relaxation data.

The confined compression test.

The unconfined compression test.

Creep relaxation. as a function of tr

Shifting of the theoretical biphasic response curves by S = logw(a2/kHA).

A typical pressure profile from the acute study.

Typical impact fissures created during the acute study. The sites of the
indentation testing are seen as small circular defects to the right (lateral)
of the fissures.

A histological cross section of two impact fissures.

Histological cross section showing the loss of P68 and tidemark observed
in some of the patellae in the acute study groups.

xiv

d

Gr
Gu
h

H».

H(T)

H(T)

I

k

LIST OF SYMBOLS

- radius of the indenter

- fluid flow length

- strain tensor

- exponential integral function

- relaxation time spectrum constant

- Fredholm integral solution

- shear modulus

- relaxed shear modulus

- unrelaxed shear modulus

- thickness of elastic layer (cartilage)
- aggregate modulus

- relaxation time spectrum

- slope of log time relaxation plot

- identity matrix

- permeability

- fluid pressure

- resistive load of elastic layer (cartilage)

- thickness test ramp rate

XV

Ro - rate of compression parameter

s - LaPlace transform variable

S — biphasic curve shift value

t - time

t, - time corresponding to the onset of cartilage loading
t, - time corresponding to contact with subchondral bone
t, - characteristic gel diffusion time

t' - dimensionless time (t/tg)

u - cartilage creep displacement

v' - velocity of solid phase

v‘ - velocity of fluid phase

Vo - indentation rate of loading

w - Simpson’s weights

or - ratio of solid volume to fluid volume

K - finite thickness correction factor

it, - Lame constant of solid matrix

71 - flow viscosity

1). - (1-VJ/(1-SV.)

v - Poisson’s ratio
v, - Poisson’s ratio of the solid matrix
7 - elemental time constant

xvi

rm - minimum relaxation time

1,“, - maximum relaxation time

o’ - stress tensor of fluid phase

0’ - stress tensor of solid matrix
1’ - diffusive force of fluid phase
1r' - diffusive force of solid matrix

- depth of indentation

OS

xvii

INTRODUCTION

Osteoarthritis (CA) is a degenerative disease primarily affecting the
articular cartilage (AC) of load bearing joints (Howell, 1976). OA is the most prevalent
of all joint diseases, responsible for thirty times as many sick leave days as rheumatoid
arthritis (RA) (Kramer, et al., 1983). Diagnosis of this disease is primarily based upon
radiographic assessment and clinical examination of features (Altman, et al., 1986;
Altman, et al., 1987). Although the stages of this disease have been described in detail
(Mankin, 1974), many questions still exist about its pathogenesis and the sequence of
events leading to its formation. Traumatic joint injury has been speculated as a possible
triggering mechanism for the onset of OA (Radin, et al., 1970; States, 1970). It has
been hypothesized that articular cartilage damage sustained during impact may lead to
altered joint loading and progressive degeneration (Repo and Finlay, 1977). It has
further been suggested that the development of CA is not solely due to mechanical
causes, but may involve biochemical interaction with the synovium as well (Shinmei, et

al., 1989; Walker, et al., 1991).

E . l C 'l .
Articular cartilage is a smooth, dense, connective tissue covering the ends of

bones in articulating joints. This tissue provides a low friction, load bearing surface

which is important for the large degree of movement required in diarthral joints

2

(Armstrong and Mow, 1984). Human articular cartilage ranges between 2-4 mm in
thickness (Howell, 1976). This thin layer of tissue must repeatedly withstand loads
between four and five times body weight during normal gait, and loads as high as ten
times body weight during deep knee bends (Solokoff, 1969; Howell et al., 1976). The
resilience of this relatively thin tissue is a product of its composite structure of which

collagen, proteoglycans, and water are the major components.

Collagen
Collagen makes up 40—50% of the dry weight of normal AC (Parson and Black,

1987). Type II collagen is the principal structural component comprising the collagen
framework. Types V, VI, IX and XI are also present, but compared with Type H each
makes up only 3% of the total collagen content (Broom, 1988). The collagen network
is divided into layers described by their depth from the articulating surface and fiber
orientation (Figure l). The collagen rich surface layer is composed of random fibers
which lie tangent to the articulating surface. In the range of physiological loading fluid
flow across this layer is the primary mechanism of viscous deformation (Parsons and
Black, 1987). A healthy surface layer is required to maintain fluid pressure within the
AC during compressive loading (Setton, et al., 1993). Directly below the thin surface
layer lie the middle and deep layers. These layers comprise the majority of the cartilage
thickness. The collagen fibers in these layers are randomly oriented and homogeneously

dispersed (Askew and Mow, 1978; Armstrong and Mow, 1984).

Arficulor Surface

  
        

    

Middle

and

Deep

Zone

. . «Celci fled
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bone

Figure 1: Cartilage layers.

The calcified cartilage zone lies at the cartilage-bone interface. Here the random
collagen fibers come together to form radial bundles which pass through the calcified
cartilage and bind with the underlying subchondral bone (Howell, 1976; Armstrong and
Mow, 1984).

The shear rigidity of AC under compression is produced by tensile forces
generated in these random fibers (Figure 2). The tensile response of the collagen
network is believed to play the major role in controlling the instantaneous deformation
of cartilage (Mizrahi et al., 1986). Enzymes have been used by investigators to disrupt
the collagen network and/or the extrafibrillar matrix in order to isolate their function

within AC. Creep tests performed on cartilage, which has had all or part of its matrix

 

 

 

Figure 2: Cartilage under shear loading.

removed, show no significant change in its instantaneous stiffness (Parsons and Black,
1987; Jurvelin, et al., 1988). After running slow rate tensile tests on control samples of
AC, Schmidt, etal., (1990) enzymatically removed the extrafibrillar matrix and carried
out tensile test on the remaining collagen framework. They found no change in the
stiffness or strength characteristics of the matrix devoid cartilage. Enzyme damaged
collagen was found to have a reduced transient elastic response during damping
coefficient measurements (Bader, et al., 1992). The results of these studies support the
definitive role of collagen as the initial load bearing element within AC. In the
description of the remaining cartilage elements it will become apparent, however, that

the overall response of AC is dependent on interaction between all of its components.

 

mm

Between the collagen fibrils lies a gel-like matrix. The chief components of this
matrix are proteoglycans (PGs). Proteoglycans make up approximately 20~30% of the
dry weight of AC (Mow et al., 1984). Proteoglycan monomers consist of a single
protein core to which 50-100 glycosaminoglycans (GAG) chains are covalently bonded.
These GAG chains are composed of chondroitin and keratin sulfate groups which are
spaced approximately 10-15A apart along the protein core (Buckwalter, et al., 1985;
Bader, 1992; Gu, 1993). In turn, the protein core of these monomers are linked to
hyaluronic acid (HA) to form a proteoglycan aggregate (Figure 3). The sulfate groups
are negatively charged. Due to their ”like" charge, PGs spread out to maximize spacing
and inhibit diffusion through the matrix (Howell, 1976). This large fixed charge density
(FCD) attracts counter ions creating a Donnan osmotic pressure making PGs highly
hydrophilic (Mow, et al., 1984).

The osmotic pressure created by the PCs has a secondary affect on AC
defamation. The collagen network resists the tendency of P63 to swell creating tensile
forces in the collagen fibers and. hydrostatic pressure in the interstitial water (Mizrahi,
et al., 1986; Maroudas, et al., 1979). By enzymatically removing PGs, Bader, et a1.
1992, reduced the swelling pressure of the cartilage and found that larger deformations
were required to achieve the same loads acquired prior to PG removal. Following
confined compression tests on AC samples with various PG contents, Schmidt, et al.,
1989, concluded that the most important function of P63 may be to control the rate at
which the collagen network stretches thereby preventing damage. The Viscoelastic (i.e.

rate dependent) behavior of PGs has also been reported by other investigators. The

 

 

  
   

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Figure 3: Proteoglycan aggregate.

concentration of PGs has been shown to inversely affect the rate of creep and directly
influence the ”flow independent” equilibrium stiffness of AC (Parson and Black, 1987;
Jurvelin, 1988). An inverse relationship between cartilage permeability and PG content
has also been documented (Mow, et al., 1984). The ability of PGs to affect cartilage

deformation stems from the relation between their FCD and the corresponding osmotic

7
pressure it induces (Mizrahi, et al., 1986).

Water

Water makes up 70-80% of the wet weight of normal mammalian AC (Simon and
Wohl, 1982). This water is divided into three types: structural, bound, and free.
Structural water, associated with the crystal, can be described by a specific stoichiometry
much as the hydrates of inorganic salts. The term bound water is frequently used to
describe protein-water interactions. For collagen the amount of bound water is generally
on the order of 0.35 g/g (Nomura, 1977). Torzilli has estimated that approximately 30%
cartilage water is associated with the collagen (Torzilli, 1982). Maroudas has described
interfibrillar water as a function of osmotic pressure difference between extrafibrillar and
interfibrillar space or the equivalent mechanical pressure (Maroudas, et al., 1991).
However, the majority of water inside AC is free to move through the tissue (Mow, et
al., 1984; Maroudas, et al., 1991). As previously noted, fluid flow and the
corresponding mechanical behavior of cartilage are affected by such things as osmotic
pressure and tissue permeability. A proper balance between the interarticular water and
the extra fibrillar matrix is essential for the proper function of AC. Water content has
been shown to be directly related to the permeability and inversely related to the
equilibrium stiffness of AC (Armstrong and Mow, 1982). The affinity of P63 for water
has previously been described. Direct correlations have been made between the presence
of PGs and water in AC (Parsons and Black, 1987; Burton-Wurster and Lust, 1986).
The integrity of the collagen network may also affect the water carrying capabilities of

AC. Donahue, et al., 1983, measured an increase in the water content of canine patellar

 

8

cartilage two weeks after impact. They suggested that micro-damage to the collagen
fibrils may have allowed proteoglycans to spread out increasing their water carrying

capacity, Figure 4.

-4 1‘ .r .
collagen lrbnls 3' D1), ,1“

3"“? ) H .

    
   

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Network
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Figure 4: Micro—damage to collagen increasing cartilage water carrying capacity.

W
The majority of OA research has been carried out with the help of animal models.

Although a gamut of animals have been used, the models themselves typically fall into

9

one of two categories, joint instability or load trauma. Joint instability models usually
involves surgical disruption of the supportive connective tissue in or around the joint.

In 1970, Hulth introduced the first instability model. His intent was to produce a slow
progressive degeneration of the AC. Production of this model involved the removal of
the medial meniscus, and transection of the anterior cruciate, posterior cruciate, and
medial collateral ligaments. Since its conception this model has been used by other OA
investigators (Svalastoga and Reiman, 1985; Lucoschek, et al., 1986). Less disruptive
models such as medial meniscectomy (Hoch, et al., 1983) and anterior cruciate ligament
transection (Altman, et al., 1984; Myers, et al., 1986; Brandt, et al., 1991 (a) and (b))
have also been used. Altman, et al., 1984, measured an increase in the stiffness of
canine femoral cartilage out to 12 weeks post-transection of the ACL. Similarly, Myers,
et al., 1986, also measured an increase in cartilage stiffness out to 12 weeks following
ACL transection in tibial plateau cartilage under the menisci. During these post-
transection test periods neither Altman, et a1. or Myers, et al. reported any signs of
cartilage fibrillation. Interestingly, at 23 weeks Myers, et al., 1986, measured a drop
in cartilage stiffness and noted signs of fibrillation. Shelp, et al., 1993, at 24 weeks
post-transection of the ACL, also reported a significant drop in the stiffness of fibrillated
canine femoral cartilage. The most extensive instability studies were carried out by
Brandt, et al., 1991 (b), where alterations in canine knee cartilage have been tracked out
to 4 1/2 years post-transection of the anterior cruciate ligament. Hypertrophic repair
mechanisms, including increases in cartilage thickness and PG synthesis, were measured
in the unstable joint out to 36 months following transection. These repair trends ceased

by 45 months and a marked loss of cartilage thickness was observed. At 54 months

10

areas of complete cartilage loss and increased subchondral bone thickness, similar to that
observed in human OA, were present. Long term observations such as these seem to
validate the effectiveness of the instability methods as a bonafide model for the study of
OA. The major draw back to instability models are that they require the artificial
alteration of joint loading to create the desired degenerative effects.

Unlike the joint instability models the damaging affects of load trauma models are
aimed directly at the cartilage and bone. These models typically involve either a single
blunt impact or repetitive impulse loading. Due to their direct relevance to the study at

hand, these models and their results are discussed in detail in the following text.

Myriam

Automobile accidents, sports injuries and falls are three common causes of
trauma, with the knee being the joint most often affected (States, 1970). Many times
joint damage does not show up in radiographs immediately following the traumatic event
(Pritsch, 1984). However, it has been hypothesized that a single blunt impact may cause
micro-damage leading to osteoarthritis (Radin, 1970; Insall, 1976).

In 1970, Radin and Paul found that removal of cartilage from bovine stifle joints
did not significantly reduce the force attenuating properties of the joint (Radin and Paul,
1970). Similar observations were made during cyclic loading of human knees (Chu and
Yazdani-Ardakani, 1986). Based on this result it has been hypothesized that bone (in
particular the subchondral bone because of its porosity) is responsible for the majority
of shock absorption across the knee joint. From this conclusion the following chain of

events were proposed as a possible pathogenesis leading to traumatic OA:

 

 

11

Impulse loading
1
Trabecular bone microfracture
I
Bone remodeling
1
Increased stiffness of bone
1
Increase loading on the articular cartilage
l
Cartilage breaks down
1
Joint degeneration
(Radin, 1972). Using a rabbit model and cyclic impulse loading, Radin was able to
measure an increase in the subchondral bone stiffness as well as progressive decreases
in the proteoglycan content of cartilage in the traumatized knee (Radin, et al., 1973;
Radin, et a1. , 1978). In a more recent study, increased bone volume and formation were
reported to have a parallel correlation to the severity of OA degeneration observed in
human AC (Shirnizu, et al., 1993). The results of studies such as these have prompted
many investigators to believe the OA formation is mediated by initial changes in the
subchondral bone. However, in 1977, Repo and Finlay conducted blunt impact studies
on unconfined cartilage-bone plugs. They reported obtaining cartilaginous fissures which

did not involve the underlying bone and determined that an average stress of 25 MPa

12

could result in chondrocyte death. They proposed the following pathogenesis based upon
their findings:
Impulse loading
l
Chondrocyte death
1
Altered cartilage metabolism
1
Structural changes in the cartilage
1
Altered loading
1
Joint degeneration
(Repo and Finlay, 1977). This study suggests that traumatic osteoarthritis may be
initiated without damage to the underlying bone. Currently the definitions of lower
extremity injury are predominantly based upon the occurrence of bone fracture (Nyquist
and King, 1985). However, it has been shown that stresses greater than 25 MPa can be
generated in human patella-femoral joints during impact at force levels below those
required to create bone fracture (Haut, 1986). Results such as these continue to raise
questions about the type and degree of trauma required to produce a degenerative

process.

 

 

13
(33255 ngrvable Damage

The form of joint damage created during load trauma experiment is usually
described by its depth or extent from the articulating surface. Fibrillations are the most
superficial form of structural damage. Their presence is often noted by a dull appearance
of the cartilage or by the increased absorption of india ink (Donohue, et al., 1983).
Fibrillations have been reported in canine and rabbit patellar cartilage subjected to both
blunt trauma and repetitive impulse loading (Radin, et al., 1973; Donahue, 1983; Yang,
et al., 1989). The appearance of these rough surfaces are often associated with
specimens observed days, weeks and years following impact. Ghadially, in 1974, studied
AC surface defects using a scanning electron microscope (SEM). He suggested that
small defects in the articulating surface may lead to increased friction between contacting
joint surfaces. Increased contact loads have been implicated as a cause of cartilage wear
and loss of stiffness (Shelp, et al., 1993).

Acute fissures are the most frequently reported form of impact damage to AC.
These fissures most often begin at the surface and extend downward into the middle/deep
zones of the cartilage (Repo & Finlay, 1977; Broom, 1986; Haut, 1986; Thompson,
1990; Silyn-Roberts and Broom, 1990; Ide, et al., 1991; Thompson, et al., 1991;
Tomatsu, et al., 1992; Thompson, et al., 1993). In some cases, impact fissures are
induced which extend all the way from the surface to the zone of calcified cartilage and
into the underlying subchondral bone (Thompson, 1990; Thompson, et al., 1991;
Tomatsu, et al., 1992; Thompson, et al., 1993). Surface fissures, like the ones
mentioned above, have been observed in cases of human OA (Mankin, 1974). The

average depth of these fissures increase with the advancing state of the disease.

14

The damage induced by impact trauma does not always include the articulating
surface. Step off, or cleft, fractures in the subchondral bone are often seen in
conjunction with fissures that extended up into the cartilage but do not reach the articular
surface (Thompson, et al., 1991; Vener, et al., 1992; Thompson, et al., 1993).
Separation of the cartilage from the underlying bone in the zone of calcified cartilage has
also been reported in response to blunt impact (Armstrong, et al., 1982). These defects
are many times undetectable by surface observation and could possibly be over looked
during a clinical examination leading to future degeneration (Thompson, et al., 1991).

The frequent occurrence of surface fissures has raised questions about the physical
conditions within the joint leading to this form of damage. Staining ruptured cartilage
with india ink has revealed that fissures tend to propagate parallel to the orientation of
the collagen fibrils, i.e. the Hultkrantz lines (Repo and Finlay, 1977; Silyn-Roberts and
Broom, 1990). This corresponds to a path perpendicular to the direction of minimal
strength as determined by tensile tests performed on bovine cartilage (Schmidt, et al.,
1990). Cross sections of impacted AC have also revealed that most fissures begin at a
45 degree orientation to the articulating surface. This observation has lead to the
hypothesis that fissures are the result of shear stress generated during impact (Silyn-
Roberts and Broom, 1990). Elastic math models subjected to impact load profiles have
shown that tensile strains, rather than stresses, may be a more reliable predictor of where
fissures are likely to form (Askew, et al., 1978; Ide, 1992). These models have also
predicted large shear stresses at the cartilage/bone interface which may account for
separations seen experimentally (Askew, et al., 1978; Chin, et al., 1986). Surface

integrity and sufficient stiffness have also been associated with the ability to induce

 

15

impact fissures (Silyn-Roberts and Broom, 1990). The geometry of the traumatized joint
may also control the degree of damage imparted by impact (Vener, et al., 1992). The
fissures reported in uniformly loaded, flat, cartilage/bone plugs are located in the center
of the contact region (Repo and Finlay, 1977; Silyn-Roberts and Broom, 1990). Bimodal
load patterns in the knee joint of rabbits create patellar fissures which occurred at the
edge of contact zones where the pressure gradients are high (Ide, 1992). Due to the
Viscoelastic nature of AC not only the amount of impact energy but the rate at which it
is employed is crucial to the type of cartilage damage elicited (Yang, et al. , 1989). The
condition of matrix itself has been shown to affect the cartilage response to impact.
Human tibial cartilage depleted of P63 by the enzyme papain showed an average
decrease in impact energy absorption of 17.2%. The increase in strain created by such
decreased attenuating properties could result in damage to the collagen network (Finlay,
et al., 1986). It can easily be seen from these various studies that the formation of
traumatic cartilage fissures involves multiple factors. Defining the exact conditions under
which fissures will form continues to challenge researchers using load trauma models.
In recent years, several impact models have been used to study the in vivo
processes leading to the development of OA. These models typically involve a single,
concentrated blow to the test joint in order to inflict trauma. The patello-femoral joint
is frequently used because of its clinical significance and the ability to directly traumatize
the articular surfaces via a transarticular impact. The trauma induced by transarticular
impact can be divided into two categories based on the extent of the damage. These
categories include (1) fracture models which result in subchondral bone fractures (often

including damage to the overlying AC) and (2) sub-fracture models which cause damage

16

to the articular cartilage without disruption of the underlying bone or calcified cartilage.

Bone fracture models have been extensively used at the University of Minnesota
(Thompson, et al., 1991; Thompson, et al., 1993; Pickvance, et al., 1993). In these
studies, non-invasive, transarticular impacts (of approximately 2000 N.) were delivered
to canine patella-femoral joints. These impacts typically result in step-off fractures of
the patellar subchondral bone. The fractures are often accompanied by fissures in the
overlying articular cartilage. In the earliest of these studies osteoarthritic-like changes
were histologically noted out to six months post-impact (Thompson, et a1. , 1991). These
changes included a loss of PG content and thickening of the subchondral bone. In more
recent studies, scanning electron microscopy, magnetic resonance-imaging (Thompson,
et al. , 1993) and biochemical (Pickvance, et a1. , 1993) analysis methods have been added
to the study of this model. Early OA-like changes were again detected, however, by one
year post-impact damaged regions of the patellae showed signs of repair. Bone fractures,
and cartilage fissures which extended into the zone of calcified cartilage, were healed and
the PG content of the cartilage had returned to normal. Only superficial cartilage
fissures remained showing no signs of healing.

OA-like changes have also been reported in sub-fracture models (Donohue, et a1. ,
1983; Ide, 1992). Donahue, et al., 1983, reported increases in cartilage water content
two weeks after transarticular impact. This edema had subsided by 4 weeks post—impact,
but injury responses such as chondrocytes cloning and increased vascularity were still
evident. In 1992, Ide used mechanical methods to study changes in the stiffness of rabbit
patellar cartilage out to one year following transarticular impact. Although it was not

significant, a softening trend was observed in fissured cartilage specimens at one year

l7

post-impact. Except for presence of the fissures, histologically the cartilage appeared
relatively normal. The surface fissures, which did not extend down beyond the middle
zone, showed no signs of healing.

Its unclear from the results of these impact investigations exactly what type of
jury or post-impact duration is required to initiate cartilage degeneration. The longest
in vivo impact studies to date have only been carried out to one year following trauma
induction. The long term fate of these animal models is yet unknown. Of particular
interest are the long term affects of the unhealing surface fissures on the load bearing
capabilities of the cartilage. As previously noted the integrity of the cartilage surface has
been linked to the normal function of cartilage (Setton, et al., 1993). It is possible that
a period of several years, similar to that reported for ACL transection (Brandt, et al.,
1991), may be required for this fissured cartilage to deteriorate and form full thickness

lesions.

Arthritis and the Synovium;

The primary role of the synovial lining (SL) in the development of rheumatoid
arthritis (RA) has long been accepted. RA begins with an acute inflammation of the SL,
i.e. a synovitis. As this synovitis continues a pannus growth develops on the SL and
eventually spreads over the surface of the AC. Concurrently centripetal erosion and
thinning of the cartilage takes place (Beesley, 1992). Unlike RA, OA degeneration is
not proceeded by an acute inflammation of the SL. However, synovial inflammations
of varying degree have been found in human OA joints (Howell, 1976; Peyron, 1981;

Goldberg, 1982). These OA synovitis incorporate all the classic signs of inflammation;

18
hyperplasia, vascular changes and infiltration of leukocytes.

W

The synovial membrane covers the inside of the tissue surrounding the lmee joint
forming a closed sac called the synovial cavity, Figure 5. It is composed of both loose
and fibrous connective tissue. The capsule is thrown into folds or projections which are
composed of connective tissue, adipose tissue, and blood vessels (Goss, 1963). The
inner most layer of this membrane is the synovial lining (SL). In the rabbit the SL is

approximately 1-3 cells thick. This lining consists primarily of two kinds of cells, A and

          
 
 

     
 

      
  
  
 
  

 
   

B.
. - .I J": ‘ \\ ' Tendon of
Af'lCUlOrl.\ :_ ....-‘ \ \\ / _
ganu 1.. \ ~\\, / quadriceps
. x .
.. - — Fol /. ;_ l
V Tibial ”‘7" _ __ __ F'm‘"
Patella collateral
ligament Fibular
collateral
. / ligament
, /
Anterior ‘ ’ Fibrous
cruciate '\ // capsule
ligament mud-A

%*

Tendon of

 

\\ . - , poplileus
/ lnfropatellar ' Alli
.% told \L°'°T°'
‘ \ \ .-,,--. f; “ meniscus
\ “I" r
\ PatFllor ' , ,1 Tibia
\ Mai... ill .
Deep infrapotallar meniscus ‘
bursa / - '7 "Fibula
Cruciote . J
ligament i '

Figure 5: Sagittal (a) and frontal (b) views of the anatomy of the human knee joint
with the synovial lining shown in red.

19

Type A cells are distinguished by their prominent golgi system while B cells are denoted
by their rough endoplasmic reticulum (Ghadially and Ray, 1966). The major role of the

SL is the production and maintenance of the synovial fluid.

Malibu!

The synovial fluid (SF) is a clear, viscous fluid, with a consistency similar to that
of an egg white (Goss, 1963). This fluid provides nutrition for the avascular AC and
lubrication for the articulating surfaces. The main components of this fluid are dialysate
of blood plasma, hyaluronic acid (HA) and protein macromolecule complexes (Hlavacek,
1993). Typically the amount of SF within the joint is just enough to cover the surfaces
of the cavity but human SF levels can range from 0.50 ml to 100 m1 depending on the

state of pathology (Lohmander, 1989).

The pathways to OA inflammation are poorly understood (Peyron, 1981).
However, the role of cytokines and enzymes seems to be promising. Enzymes such as
collagenase, cathepsin (D, G, B and L) and stromelysin all have degrading effects on AC
matrix and have been found at elevated levels in OA joints (Roughley, 1991; Okada,
1992; Pelletier, et al., 1983; Pickvance, et al., 1993; Howell, 1976). In healthy joints
most of these enzymes are kept in check by inhibitors. An imbalance between these
enzymes and their inhibitors may lead to OA (Glynon, 1977). Of the enzymes
mentioned, stromelysin appears to be the most likely candidate because its active at
neutral pH (Okada, et al., 1992). Stromelysin, also known as metalloproteinase (MMP-

3), can be synthesized by both synovial cells and AC chondrocytes. The cytokines IL-lB

 

20

(interlukin) and TNF—a (tumor necrosis factor) are believed to play a key role in the
initiating stromelysin production (Roughley, 1991; Beesley, 1992; Pickvance, et al.,
1993; Ridge, et al., 1980). Like stromelysin, these cytokines can also be produced by
both the synovial cells and the AC chondrocytes. A direct correlation has been measured
between the level of these diffusive cytokines and the degree of synovial inflammation.
However, a similar correlation has not been established between the state of synovitis and
the actual catabolic enzymes. This suggests that cytokines are the predominant mediators
of the synovial response (Pelletier, et al., 1985). Typically proteoglycans inhibit the
diffusion of IL-lB and TNF-a into the AC. However, in fissured areas devoid of P03,
increase permeability to these cytokines may exist (Okada, et al., 1992). Stromelysin,
IL-lB, and TNF-a have all been measured in increased amounts in the middle/deep zones
along the edge of fissures up to two weeks following transarticular impact of canine knee
joints (Pickvance, 1993).

The exact trigger mechanism leading to the release of these cytokines and
enzymes is yet unknown. To study the effects of synovitis foreign materials have been
introduced into joints to induce inflammation. Some of the material used include; talcum
powder, teflon, carbon fibers, and even ordinary synovial fluid (Frost and Ghosh, 1984;
Messner, et al., 1993; Gershuni and Kuei, 1984). Natural substances, like blood
(hemarthrosis) can also elicit a synovial response thereby affecting the AC (Ghadially,
1983). It has been suggested that in a natural trauma scenario, substances released by
the AC into the SF may induce a degenerating synovial response (Howell, 1976;
Schumaker, et al., 1981; Pelletier, 1985). As previously mentioned, increased levels of

PG aggregates have been measured in the SF for extended periods following damage to

 

21
AC (Lohmander, et al., 1989). Keratin sulfate, from GAG chains, have also been found

in higher than normal concentrations in patients with OA (Thonar, et al., 1985). It has
been proposed that elevated levels of such substances measured in body fluids (SF, blood
serum and urine) may some day provide a means of detecting cartilage damage following
joint injury (Lohmander, et al., 1992). Interestingly cases of reactive arthritis are
sometimes reported following infections of other parts of the body, although no
microorganisms are detected within the joint (Saxne and Hienegard, 1992).

The exact role of the synovium in the sequence of events leading to a state of
pathology is unknown. It is well documented that direct damage to the synovium during
surgery has a transitory degrading effect on AC (Thompson, 1975; Frost and Ghosh,
1984; Svalastoga and Reiman, 1985; Pelletier, et al., 1985; Walker, 1991; Messner, et
al., 1993). In 1985, Svalastoga and Reiman used the Hulth joint instability model to
study the effects cartilage degeneration with respect of changes in the SL. The Hulth
method was performed on the right leg of each test rabbit. Consecutively, the left leg
underwent arthrotomy and served as a surgical control. In the first two weeks following
surgery the test and control joints both showed signs of cell proliferation in their SL as
well as a surface loss of cartilage PGs. By four weeks post-surgery the control joint had
returned to normal, however, on the test side hypertrophy of the SL continued. Between
4 and 6 weeks the first signs of cartilage fibrillation appeared in the test joint. These
results suggested that the inflammatory response of the SL may have contributed to the
initial breakdown of the AC. In turn, the breakdown of the cartilage may have
stimulated further response by the SL. In 1986, Lucoschek, et al., did a comparison

study between the Hulth instability model and the non-invasive, repeated impulse loading

22
(RIL) model used by Radin (1973). The results of the Hulth model were similar to those

seen by Svalastoga and Reiman (1985). However, in the RIL model early signs of
inflammation did not appear in the SL. Unlike the Hulth model, progressive
inflammatory changes in the SL were not observed until after superficial damage to the
AC appeared. This suggested that damage to the cartilage itself could initiate an
interactive synovial inflammation. The results of these two studies suggest that
interactions between the cartilage and synovium may occur during times of joint injury.
However, both of these studies involved extraneous joint loading scenarios applied over
a period of months. Neither addresses the possibility of an interaction between acute

surface damage to the AC accompanied by a traumatic synovitis.

W

A review of two previous studies will be presented before the current topics of
research are covered. These background studies are key to the development of the
questions which motivated the current investigations. The first study, the open joint
study, will lay out the ground work involved in developing the animal model which was
used. The results of this study will develop important questions about the affects of
mechanical trauma on AC. The second study, the closed joint study, will detail the
continuation of the work begun in the open study, and in addition it will also raise
questions about the effects of arthrotomy on AC. Following this review the reasons for

carrying out the current studies will be clearer.

THE OPEN JOINT STUDY

The purpose of this study was to develop an animal model in order to study the
effects of blunt trauma on AC. The hypothesis being that the altered mechanics of
damaged cartilage would lead to the degenerative stages associated with OA.
Biomechanical tests were used in concurrence with histology and biochemistry in order
to relate changes in the mechanical load response of the cartilage with alterations in its
physical structure. The following sections describe the methods, materials and results
of this study. Only those parts of the study which pertain to the current investigations

are covered. For complete details see reference Ide, 1992.

OPEN JOINT MATERIALS AND METHODS
The rabbit was chosen as the animal model on which to study the effects of blunt
trauma to AC. This impact induced trauma was delivered under the acceleration of
gravity. One patella per rabbit was traumatized. Pressure sensitive film placed within
the patello—femoral space recorded contact pressures. The details of these procedures and

the processes leading up to them will be described in the following text.

Impacfing Devig;

Gravity provided a simple and consistent means of delivering a repeatable impact.

A free falling mass has a constant gravitational acceleration of 9.81 m/s2 (at sea level).

23

24

The energy imparted (E) during impact can easily be calculated by multiplying the mass
of the object (m) by the acceleration of gravity (g) and the relative height from which it
is released above the impact surface (h), i.e. E = mgh.

Due to the surgical nature of the open joint study, the impacter was designed to
operate within a sterile field. The base plate and supportive scaffolding were constructed
of aluminum. The impacting rod was made of steel and was guided by two low friction
bearings. At the lower end of this rod a flat, aluminum impact head (1" in diameter)
was attached by way of a load cell (500 lb. range). At the upper end of the rod a mass
could be added to obtain desired impact energies from any given drop height. Figure 6,
depicts the gravity impacter described.

The load cell output was recorded by an IBM compatible PC. A solenoid held
the impact rod supporting the raised mass. The PC was programmed to disengage the
solenoid following input from the operator. Upon impact the PC began collecting the
load-time data at the rate of 10,000 Hz. When the load readings returned to zero the
solenoid was re-engaged catching the impact mass and preventing it from striking a
second time.

A fixture was needed to properly position the rabbit’s hind limb to be impacted
below the impact mass. It was desired that the patella itself receive the entire force of
the impact. Therefore, a seating structure was constructed out of 3/4" plexiglass. The
animal was placed in the chair supine with the right leg hyperflexed. The right leg
(arbitrarily chosen) was held in place by a spring loaded aluminum bar clamp. A vinyl
strap was used to secure the contralateral limb preventing rotation during impact. This

seat configuration aligned the patella and underlying femur vertically with the impact

25

mass (radiographically confirmed).

MASS —*

1/4' ROD —’

 

l

SOLENOID RELEASE —O

3

 

 

LOAD TRANSDUCER —5 J

 

 

 

 

 

Figure 6: The gravity impacter.

This film consists of a thin sheet of plastic covered with microscopic beads of red
dye. When compressed between two contacting surfaces the beads burst marking the

area of contact. The larger the force over a given area, the greater the number of beads

which break. The intensity of the stain reveals the magnitude of the force. The medium

26

sensitivity, prescale film used (Fuji Co.) provided a linear stain intensity for statically
applied pressures between 10 and 50 MPa. This film, covered by a thin, sterile, plastic
sheath, was placed between the patella and the femur via medial and lateral incision.

The film provided a record of contact pressures over the cartilage surface during impact.
A computer analysis package was used to convert the relative stain intensities to

pressures according to a predetermined calibration curve.

im' i

The rabbit was chosen because of its low cost and ease in handling. Originally
the New Zealand White breed of rabbit was chosen but examination of their patellar
cartilage revealed a commonly occurring, and quite extensive, baseline pathology. The
Dutch Belted species of rabbit also displayed baseline AC pathology. The fawn colored
Flemish Giant breed was found to have the healthiest cartilage. Due to their large size,
these animals had the added advantage of large AC surfaces for testing.

Six Flemish Giants were used to determine the appropriate seat design and impact
energy levels. The levels of impact energy were chosen as follows, the "low" energy
impact level was set based on the largest impact energy that did not result in fissuring
of the AC. The ”high” impact level was set as the smallest impact energy resulting in

AC fissures, but not bone fracture. These levels are shown in Table 1.

T 1 Im a Int n i ies
Impact Intensity Energy (J) Drop Height (m) Mass (kg)
Low 0.90 0.20 0.43

High 6.30 0.46 1.33

27

The medium sensitivity pressure film was found to provide adequate stain intensity at

both levels.

111mm

Once the impacting procedure was established the next step was to devise an in
vivo study. At each impact level, three rabbits were traumatized at each of the following
time points: 1, 3, 6, and 14 days, and 3, 6, and 12 months. When the animals reached
their predetermined test date they were euthanized and their patellas extracted for
biomechanical, biochemical and histologic analysis.

Fifteen animals were also subjected to the same surgery, film insertion and
hyperflexion of the limb, but were not impacted. Three animals received surgery, but
no film or hyperflexion. Another three rabbits received only surgery, but no film or
hyperflexion. These groups served as controls for the procedures itself. All of the

rabbits used were mature (six months or older) and were bought from a single supplier.

W

The rabbits’ right leg was prepared for surgery by a veterinary technician. The
rabbits were anesthetized with ketamine (11 mg/kg) and xyaline (1.1 mg/kg) and
maintained on isoflorine (2-2.5%) per (2L) of oxygen. The rabbit was placed in the
restraining seat with its right leg unflexed. As previously stated bilateral incisions were
made on the medial and lateral sides of the patellar tendon and patella. The pressure
sensitive film was placed in a sterile plastic sleeve and was slid through the incisions
placing it between the patella and femur. The surgical limb was then hyperflexed and

secured by the bar clamp. Impact was then carried out with the gravity impacter

28

previously described. Following surgery the film and sleeve were removed and the knee
was sutured. Resorbable sutures were placed in the bilateral incisions along the tendon
and patella. Non-resorbable sutures were used to close the outer skin incision. All
surgeries were carried out by Charles DeCamp, D.V.M. The animals were returned to

cage activity immediately following surgery.

W

On the day of testing the animals were euthanized and both patellas were removed
for immediate testing. The patellas were potted in an epoxy resin, bone side down,
leaving the untouched cartilage surface exposed. The AC was kept bathed in physiologic
phosphate buffered saline while the epoxy cured and during testing.

The mounted patella was placed directly beneath a plane ended, rigid, cylindrical
indenter, 0.50 mm in radius. This probe was connected to the actuator of a servo-
hydraulic testing machine by way of a load cell (25 lb. range). Actuator displacement
was monitored by an accompanying LVDT. Both load and displacement data were
recorded using a Nicolet storage oscilloscope. The indenter probe was brought into
contact with the surface of the articular cartilage and was preloaded to 0.02 N. The
probe was then indented 0.10 mm into cartilage using a ramp function. The ramp
duration was slightly greater than 50 msec. The indentation was maintained for 100 sec.
while resistive cartilage forces were measured. The hold time of 100 see. was set by the
limited storage capabilities of the Nicolet. Two indentations were preformed per patella;
one at the lateral rim and the other just lateral of the centerline which lies between the

medial and lateral facets.

 

29

Cartilage thickness data was required for the computation of its shear moduli. A
small needle-like probe replaced the larger indenter used during the stress-relaxation test.
The needle probe was brought down into contact with cartilage surface at the same site
as the preceding indentation test. The needle probe was then rammed into the cartilage
at the rate of 1 mm/sec. The nwdle probe was allowed to penetrate the cartilage until
a sharp increase in load signaled that contact with the underlying bone had been made.
The distance the needle probe traveled between the first signs of loading and contact with
the bone (recorded by the LVDT) gave the thickness of the uncalcified cartilage. This
method of thickness measurement, unlike sectioning, left the patella in tack for further
histological and biochemical analysis.

From each stress-relaxation test three mechanical parameters were calculated.
Using the measurements of peak load, equilibrium load and cartilage thickness an
unrelaxed (Gu) and relaxed shear moduli (Gr) were calculated from Hayes solution of
an elastic layer bonded to a rigid half space. From the time dependent relaxation portion
of the cartilage response a flow viscosity (:7) term was calculated based on Tobolsky’s

analysis of a generalized Maxwell material (see Biomechanical Analysis).

OPEN JOINT RESULTS
1312mm
Table 2 shows the maximum loads and contact pressures generated in the patella-
femoral joint during impact. The peak impact loads were determined by the PC data
collection program. The contact pressures were obtained from the Fuji pressure film.

The peak contact pressures on the lateral facet were found to be statistically greater than

30
those on the medial facet by a 8 MPa offset. The average peak pressures generated on

lateral facet during high level impacts were significantly larger than those at low impact

levels.

Table 2. Average Peak Impact Loads and Contact Pressure Ranges for the Open

Joint Study
Impact Impact Contact Pressure Range (MPa)
Lexel Erratum Medial) (Lateral)
Low 200 j; 21 7.5 :1; 4.4 15.6 i 2.9
High 516 :t 73 19.2 3‘; 3.8 26.0 :1; 3.7

Figure 7 shows an impact induced fissure. Typically, these fissures were located at the
proximal end, just lateral of the centerline and ran parallel to the long axis of the patella.
This location corresponded with large shear stress and tensile strains as indicated by the
pressure film. Histological sections revealed that the fissures ran parallel to the major
collagen bundles. Surface fissuring was documented in 6 of 24 low level impacts and

18 of 24 high level impacts.

W115

The unrelaxed shear modulus (Gu) and relaxed shear modulus (Gr) were
computed by Hayes solution (eq. 1), see Biomechanical Analysis. The unrelaxed
modulus was calculated from the peak load and the relaxed modulus was computed after
100 s of stress-relaxation. A two way ANOVA showed no statistical differences between
the test and control moduli (Gu and Gr) with respect to post-impact time. Histograms

of the data did, however, reveal some interesting trends with respect to post-impact time.

31

 

Figure 7: A typical impact fissure.

Figure 8, is a histogram of the post-impact time average response values for Gu,
based on the centerline data from the low level study. A tendency can be seen in Figure
8 for Gu values to decrease below controls after day 1, reaching a minimum at 14 days
and then rising back to control levels by 6 months. The response trend seen in Gu at
high impact levels was different, Figure 9. From 1 to 6 days average test side Gu values

were less than controls. From 14 days to 6 months, Gu values approached control

32

UNRELAXED MODULUS

Open Joint Study

 

 

 

 

 

 

 

 

 

2.57

2 .................................................................
73 ‘ _
E15 .........................................
O
Q T fl
'51 ..................................................... ..
,9

0,5... .................. fl ...... H .............

0

1 3 6 14 90 180 365

Days

Impact Level
Cl Low

Figure 8: Histogram of Gu from the open joint, low level impact groups.

UNRELAXED MODULUS

Open Joint Study

 

 

 

2.5

Test/Control

 

 

 

 

 

14
Days

Impact Level
I High

Figure 9: Histogram of Gu from the open joint, high level impact groups.

33

levels.

Figure 10, shows the time response trends for the relaxed modulus, centerline
values from the low level impact groups. A gradual decrease can be seen in the Gr test

values starting at 1 day, reaching a low at 14 days, and then returning back to control

levels by 1 year.

RELAXED MODULUS

Open Joint Study

 

0|

.......
.........................................................

p Test/Control 7.
J.
J

 

 

 

 

 

 

:—:
:

 

Days

Impact Level

Cl Low

Figure 10: Histogram of Gr values from the open joint, low level impact groups.

At high impact levels the time response of Gr values did not resemble that seen
at low impact, Figure 11. Test Gr values were considerably lower than controls

beginning at 1 day and continuing out to 6 days post-impact. After 6 days they returned

to control levels were they remained.

34

RELAXED MODULUS

Open Joint Study

 

 

.....................................

p Test/Control _.

 

 

  

 

 

 

14
Days

Impact Level
IHigh

Figure 11: Histogram of Gr value from the open joint, high level impact groups.

Flow viscosity (1;) values were calculated from Tobolsky’s analyses of a
generalized Maxwell material, see Biomechanical Analysis. This parameter was
calculated as a means of characterizing the time dependent relaxation portion of the
cartilage response curve. Figure 12 shows the post-impact time viscosity trends for the
low level impact groups. Test side values were basically the same as controls out to 6
days. At 14 days and 3 months the test values dropped below controls and then
demonstrated an increasing trend back toward normal out to 1 year. At high impact

levels an overall decrease was seen in test side viscosity values except at 14 days, Figure

13.

 

35
FLOW VISCOSITY

Open Joint Study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.5
2 ..............................................................
2'9 .............................
g 1.5 ............ , ............ ..
o
3 1 —— ....... 7 ................................
i- a
0.5 . . i ......................... fl ............
0
l 3 6 14 90 180 365
Days
Impact Level
B Low
Figure 12: Histogram of the 11 value from the open joint, low level impact groups.
FLOW VISCOSITY

Open Joint Study

 

Test/Control

 

 

 

 

14 90

Days
Impact Level
I High
Figure 13: Histogram of n values from the open joint, high level impact groups.

36
mm

In the sham group subjected to surgery, film insertion and hyperflexion, decreases
of 25 and 5 percent were seen in test side Gr and Gu values out to 6 days post-impact.
These changes were not significant but they did warrant further investigation. This
prompted the investigation of the other sham groups. The sham group which included
surgery, hyperflexion but no film insertion demonstrated decreases similar to those of the
first sham group. The last sham group which underwent arthrotomy, but was not placed
in the restraining chair with its leg hyperflexed, did not show any signs of change test

versus control.

B l .

Eighteen cases were randomly chosen from the four short term study groups (1,
3, 6, and 14 days). Cartilage samples were cored from the lateral facet of each patellar
pair (test and control). The water content of each sample was determined. The average
water percent of the impacted cartilage was 73.1 :1: 7.9 compared to 72.4 j; 11.0 in
controls. This difference was not statistical. However, in 7 of 10 cases where the test
side water content was greater than controls there were no surface fissures. On the
contrary, of the 8 test specimens which had a water content greater than their respective

controls only 2 of them possessed surface fissures.

THE CLOSED JOINT STUDY

The use of arthrotomy in the first study was necessary for the evaluation of
pressure profiles existing within the joint during impact. With these conditions
thoroughly documented, closed joint trauma was the next step in the development of the
animal model. The closed joint model served a dual purpose. First, the closed joint
scenario more closely represents the joint environment present during an actual in viva
knee injury. Secondly, it prevented the cartilage from being exposed to possible transient
surgical affects which were believed to have altered the mechanical response of the open
joint test specimens. Most of the procedures followed in this study, other than surgery,
were unchanged from those in the open joint study. However, some alterations in the
methods and materials were made to increase the quantity of information available from

each animal.

CLOSED JOINT MATERIALS AND METHODS
W1
In the open joint study the low number of rabbits at each time point (n=6, 3 high
and 3 low) made the statistical differences hard to establish. To increase statistical power
in the closed joint study, additional rabbits were added. Sixteen Flemish Giant rabbits
were placed in each of the following post-impact time periods; six days, three months,

six months, and one year. These time points correspond with postoperative dates used

37

38

in the open joint study. Of the sixteen rabbits at each time point, half were subjected to
high level impacts, the other half low level. These impact energy levels were the same
as those used previously (i.e. low level = 0.9 J, and high level = 6.3 J).

In the interim following impact the animals were individually housed and allowed
cage activity (36" x 24" x 15.6”). On the predetermined test date each rabbit was
anesthetized, using ketamine and xyaline, and then euthanized using Ecklimide (2 ml).
Both the right (test) and left (control) patellas were immediately removed for
biomechanical testing.

Due to the effects of arthrotomy seen in the open joint sham group out to 14 days
post-surgery, a 6 day, closed joint, sham group was added to the study. The six rabbits
in this sham group were placed in the restraining seat with their right leg hyperflexed and
held in place by the bar clamp. The animals remained in the hyperflexed position for
five minutes. They were then returned to their cages. Six days later the rabbits were

euthanized and their patellae tested.

Emma!

Patellar impact and data collection were carried out using the same gravity
impacter, load cell, and accompanying computer system previously described (see Open
Joint Materials and Methods). The restraining seat was again used to hold the
hyperflexed right leg, thereby preventing full body movement during impact. Prior to
impact the rabbits were anesthetized with ketamine (15 mg/kg) and xyaline (2 mg/kg) and
were held under during impact with isoflorine (2 %-2.5 %) per 2L of oxygen as previously

described. The right knee was shaved, then swabbed with alcohol and betadine prior to

39

placing it in the restraining seat. Immediately following the impact, the rabbits were
given an injection of butorphenol (0.10 cc) to help reduce pain. The animals were then

returned to their cages.

mm:

As in the previous study, indentation relaxation tests were carried out on the
patellar AC. The indentation procedures were carried out using a servohydraulic testing
machine (MTS models 1330 and 8500). Cartilage response loads were measured by a
load cell (25 lb. range). The load cell outputs were amplified using a Validyne amplifier
prior to data collection. Data collection was carried out with a Commodore Amiga
computer. Data collection software was created for the Amiga (Jim Husch, programmer)
which provided real time display of both the load response and actuator displacement.
Load-time data was collected at a rate of 20 Hz. The raw ascii data was transferred to

a Sun spare station 10 computer for analysis, see Biomechanical Analysis.

mm:

For the closed joint study a new system of holding the patella during mechanical
testing was developed. A single screw, stainless steel, vice-like grip was built. Unlike
the potting method used in the "open joint” study, the grip allowed testing access to both
the medial and lateral facet of the patella. This grip was attached to a 1 1/4 inch,
stainless steel post, 1/2 inch in diameter, with a 1/2 inch threaded section at the opposing
end (Figure 14). A clear acrylic bath with an inner diameter of four inches was designed
to slide up and down on the grip post by way of a hole in the bottom of the bath. A

rubber ’o’ ring lining the hole prevented loss of bath saline. This allowed the bath to be

a .’. . _. ,
. O .. .

.'a ao- : d K

U ~a- - .... v

 

 

r'T'

5"- 4" t ' '4'
‘. uh: ‘. «mun. ‘
a.» u‘ (ft! .‘ a . '4'

~ _ h I n 1

n- 00‘ fl

40

 

Figure 14: Vice-like grip used to hold patellae.

lowered exposing the grip in order to change specimens or align the surface of the
patella. This grip-bath assembly was attached to the head of a camera mount by the
threaded portion at the base of the grip post. The ball and socket joint of the mount head
allowed the bath-grip assembly to be rotated 360° while simultaneously being tilted as
far as 90° from its vertical position. The top of the bath was cut at a 45° angle to allow
a full bath to be tilted without spilling. A trigger release mechanism allowed entire
assembly to be adjusted to the desired position and locked into place. This entire bath-
grip mount assembly allowed the surface of the patellar AC to be visually aligned
perpendicular to the indenter while maintaining the bath environment, Figure 15.

Furthermore, the bath-grip mount was fixed to a double axis slide platform which

 

41

allowed horizontal movement of the entire assembly. This allowed a specific position

on the AC to be directly aligned beneath the fixed position of the indenter.

 

Figure 15: Bath-grip mount assembly.

mm:

The patella was placed in the grip end to end with medial and lateral facets
exposed, Figure 16. The bath, filled with 0.10 M phosphate buffered saline at room
temperature, was then raised immersing the patella. The patella remained immersed
except for brief periods when the indentation site was changed and the articular surface

was aligned.

 

Figure 16: Patella held in grip.

Testing was earried out on the lateral facet. As in the open joint study, stress relaxation
tests were again carried out using a solid, rigid, cylindrical, flat ended indenter 0.50 mm
in radius. Indentations were performed with a ramp rate of 1.20 mm/s to a depth of
0.10 mm where the indenter was held for 150 seconds while resistive cartilage loads
were monitored. The new data collecting system allowed the acquisition time to be
increased from 100 sec in order to assure that the cartilage reached equilibrium. A
nwdle probe was again used to determine the thickness of the cartilage at the site of the
indentation immediately following the stress-relaxation test. At the six day time point,
two stress-relaxation tests were performed in the center of the lateral facet. One on the

proximal half and the other on the distal half, Figure 17. At the later test times, a third

43

o = solid indenter
test site

Lateral ' Medial

Figure 17: The six day, closed joint test sites.

test was added, Figure 18. At six days and three months tests were also performed on
the medial facet of each patella. A visual comparison of the medial test values (Gu, Gr
and 27) revealed spatial differences between the mechanical response of the two facets.
For comparison reasons only the lateral facet results will be presented.

A total of 64 rabbits entered into the closed joint study. Two, one year, low
level, animals died and one, high level, one year, animal was dropped in into the 3
month time period. Two more rabbits, one high level and one law, were removed from
the 3 month time point for an alternate study after it became apparent that their inclusion

in the results would not alter the overall outcome.

44

I
I
I
O
l . .
. I .=solidnidenter
test site
. I
I
l
I
Lateral l Medial

Figure 18: The closed joint test sites following six day time point.

CLOSED JOINT RESULTS

Impactlrauma:

The peak impact loads were determined by the plotting program. The average
impact load for the low and high level impacts group were 158 :I; 20 and 562 i 53,
respectively. At low levels only 2 of the 28 animals impacted were fissured. At high
impact levels 24 out of 30 animals were fissured. These impact results are comparable
to those seen in the open joint study. A complete record of the closed joint impact

parameters are presented in Tables 3 through 10 in the Appendix.

Waxing:

$18.23!:

The following results are based only upon the mechanical tests carried out on the
lateral facet of the patellae. For the low level group the average instantaneous shear
modulus (Gu) was increased 6.3% on the test side compared to the contralateral control
side. The relaxed modulus (Gr) was up 2.5% on the test side and the viscosity (1;) was
15.5% larger on the test side relative to controls. None of these differences, test vs.
control, were found to be significant. The calculated material parameters for the six day,
low level, impact animals are compiled in Table 11, see Appendix.

At high impact levels the average Gu values were increased 17.7% on the test

45

46

side. Both the average test and control values for Gr were 0.200 MPa. The viscosity
values were up slightly an average 14.2% on the test side. Neither of the differences in
Gu and n values were found to be significant. The six day, high impact, results are
tabulated in Table 12 in the Appendix.

Overall, the ANOVA detected no significant difference between the results of the
low and high impact levels for any of the mechanical parameters (Gu, Gr and 1;) within

the test and control groups (e.g. low test Gu vs. high test Gu).

W

Similar to the six day results, little change was found between the test and control
values of the low or high level impact groups. At low levels test values of Gu and Gr
were only slightly higher than those of controls by 4.1% and 3.6%, respectively. Low
impact ’7 values were a mere 1.9% greater on the test side compared with controls.
None of these small differences were found to be significant, see Appendix Table 13.

In contrast to the slight increases in stiffness measured in the low level group, the
high level group showed a slight decrease in test side stiffness. Gu values were down
by an average of 2.4% on the test side and Gr values by 2.6%. However, like the low
level group, a slight increase in ’7 was again seen on the test side, 6.3%. None of these
differences were found to be significant, see Appendix Table 14.

In a comparison between low and high level parameters a significant difference
was detected between the test side values of Gr, and between Gr values on the control
side (P = 0.00 and P = 0.00). No significant difference was detected for either Gu or

n values with respect to impact level.

47

SLXMQMLS

At six months the results continued to show little or no change between test and
control values for either low or high impact levels. The low level average Gu values
showed the largest difference with an increase of 27.3% on the test side. The low level
Gr values were essentially unchanged between test and controls (0.05 %). The n values
seemed to follow the same trend as Gu, increasing 20% on the test side. None of these
differences were found to be significant.

At high impact levels only small variations were measured. Gu values were
slightly down an average of 8.0% on the test side, while Gr were slightly up 4.5%. The
average value for ’7 was down 4.8% on the test side. As has previously been the case,
none of these differences were significant. The mechanical parameters for the high and
low impact groups are presented in Tables 15 and 16 in the Appendix.

The overall ANOVA did reveal a significant difference between high and low
level impacts within parameter Gr at the six month time point. Interestingly, this
difference was seen not only between test side values, but also between control values
P = 0.001 . Multiple researchers were involved in collecting the mechanical test data at
this time point. Small variations in the testing procedure between investigators may

have contributed to some of these differences.

maker
At one year post-impact an overall decrease in test values was measured in the
low level group. The unrelaxed and relaxed shear modulus were down 33% and 14%,

respectively. After removing an extraneous viscosity measurement (rabbit BN3, control,

1‘

48

site 2), the test side was found to be decreased by 19%. None of these decreases were
found to be statistically significant.

At high impact levels, similar decreases were measured in the test side values.
Gu and Gr were down 22% and 12%, respectively. n was reduced by 25%. Both Gu
and 11 were found to be significantly less than control values (P =0.04, P =0.03 ,
respectively). The mechanical results for the low and high impact groups are presented

in Tables 17 and 18, respectively.

Shams
Unlike the open joint study, Gu and Gr test values were virtually unchanged

compared to controls. The test side Gu and Gr values were 5% and 2% higher than
their respective controls. This suggests that early differences in the open joint study

were predominantly an affect of arthrotomy (see Appendix Table 19).

E . IE I l S 1'

Upon completion of the background studies, comparisons were made not only
between the affect of impact level, but also the affects of using either the open or closed
joint model. Histograms of ratios (test/control) were used to visually depict the time
varying trends of the measured mechanical parameters. The ratios in these histograms
were determined from parameter averages and not the average values of the individual
test ratios (i.e. the last 3 columns of Tables 11 through 19, see Appendix). Ratios are
not normally distributed, therefore the mean of these ratios does not accurately represent
the relationship between the average test and control values. Figure 19 represents a

comparison between high and low level impact, Gu ratios of the Open Joint Study.

 

 

 

49

UNRELAXED MODULUS (Gu)

Open Joint Comparison

 

 

 

 

 

 

 

 

 

 

 

 

2.5[
2
73
E 1.5
O
2
‘w‘ 1
1‘2
0.5 -
0 ,
1 3 6 14 180 365
Days
Impact Level
lHigh l:lLow

Figure 19: Open joint, high vs low level impact, Gu trends.

Except for an increase at 6 days, the low level impact groups showed a gradual
decreasing trend after day one which reached a minimum at 14 days. By six months the
test values had returned to control levels where they remained out to one year post-
impact. At high impact levels an immediate drop in stiffness was measured at one day
post-impact. The test values remained at or below control values out to 14 days when
a large increase in the unrelaxed modulus was measured. After 14 days the test values
returned to control levels where they remained. This same comparison was carried out
for the closed joint study, Figure 20. A general decreasing trend was noted in the high
impact groups over the one year test period. In the low impact groups the test values

remained at or above control levels until 1 year post-impact. By 1 year test values at

50

UNRELAXED MODULUS (GU)

Closed Joint Comparison

 

01

......................
....................

p Test/Control _—a

01

......
.................

 

 

 

 

 

 

 

 

6 90 180 ' 365
Days
Impact Level
.High Eltow

Figure 20: Closed joint, high vs low level impact, Gu trends.high impact levels

both impact levels showed a decrease below controls. At this decrease was significant
(P =0.04).

The time trends measured in Gr in the open joint study were similar to those
described for Gu, Figure 21. A gradual decrease in stiffness was noted out to 14 days
post-impact at low impact levels. After 14 days, a gradual increase was noted. By 1
year the modulus values had returned to control levels. On the contrary, at high impact
levels, Gr dropped substantially at 1 day and remained low out to 3 months. At 3

months a sharp increase was noted in the test values. This was followed by a decrease

at 6 and 12 months back towards control levels.

51

RELAXED MODULUS (Gr)

Open Joint Comparison

 

U!

.....................................................................

3 6 i 14 90 180 365

Days

_0 Test/Control :4

O'l

 

 

 

 

 

 

Impact Level
I High EILow

Figure 21: Open joint, high vs low level impact, Gr trends.

In the closed joint model large differences were not noted in Gr between impact
levels, Figure 22. The Gr values were consistently close to control levels until 1 year
post-impact where both low and high impact groups displayed a slight decrease.

Specific time trends were not apparent in flow viscosity in the open joint study,
Figure 23. Except at 1 and 6 days, low level test viscosity values remained below
controls. The high impact test values were also consistently low, except at 14 days
where a large increase in viscosity was measured.

In the closed joint study, the flow viscosity trends resembled those of Gu, Figure
24. At high impact levels an overall decrease in the test values was noted over the one

year test period. At low levels, the average test side viscosities values were at or above

52

RELAXED MODULUS (Gr)

Closed Joint Comparison

 

U!

........................................

.....

_o Test/Control _-i

01

.............................

 

 

 

 

 

 

 

 

1 80 365
Days

Impact Level
I High III Low

Figure 22: Closed joint, high vs low level impact, Gr trends.

FLOW VISCOSITY

Open Joint Comparison

 

Test/Control

 

 

 

 

 

1 3 6 14 i so 180 365
Days

Impact Level
.High ClLow

Figure 23: Open joint, high vs low level impact, 11 trends.

 

53

FLOW VISCOSITY

Closed Joint Comparison

 

 

.......................

p Test/Control .4

 

 

 

 

 

 

 

 

 

180 365
Days

Impact Level
I High [:1 Low

Figure 24: Closed joint, high vs low level impact, 1) trends.

control levels out to 1 year post-impact. At 1 year the test side viscosities at both impact

levels were below their respective controls. At high impact levels this decrease was

significant (P =0.03).

l nim
The results of the open joint study were based upon three observations per group
at each time point. The differences between the closed and open joint results prompted
the addition of further testing. A total of 16 rabbits underwent open joint impact (eight
at each impact level) and were placed into a three month test group. The 3 month test
period was chosen because of large discrepancies between the Gr and 1; results of the

open and closed joint studies at this time point. The mechanical testing procedure carried

54

out on these supplemental groups was the same as that used in the closed joint study.

The results of the supplemental groups closely matched the results of the 3 month
closed joint groups. At both low and high impact levels the test side values were close
to control levels. The large increases measured in Gr and ’7 at high impact levels in the
open joint study were not detected in the supplemental groups (see Appendix Tables 20-
23 for results of the 3 month, supplemental groups).

As a personal point of interest, two rabbits were subjected to high level open joint
impacts which included the insertion of pressure film. These rabbits were euthanized at
six days post-impact and their patellae removed for testing. The unrelaxed and relaxed
moduli were decreased an average of 17.6% and 54.3%, respectively. These results

provided additional confirmation of the 6 day, open joint, high impact results.

DISCUSSION OF BACKGROUND STUDIES

The primary goal of the open joint study was to investigate the degenerative
effects of blunt trauma on AC. An additional goal was to document the load conditions
within the joint during impact. This documentation process required the use of
arthrotomy. In the study that followed, blunt impact was delivered under non-invasive
closed joint conditions. This impact scenario eliminated possible cartilage softening due
to a transient surgical synovitis. In both studies indentation stress-relaxation tests were
performed at predetermined post-impact times. The mechanical parameters, Gu, Gr and
n, were calculated for both studies using identical analysis methods. A comparison of
results revealed differences in the time dependent responses of these mechanical
parameters. These differences were not only found to be a function of impact level, but
also of the model type (i.e. open vs. closed joint).

Within the open joint study, differences were noted in the stiffness results (i.e.
Gu and Gr) between the low and high level impact groups with respect to the post-impact
test time. These differences were most obvious in Gr. At low impact levels, Gr values
gradually decreased out to 14 days post-impact. However, at high impact levels an acute
drop in Gr was noted immediately 1 day after impact and these values remained low out
to 14 days. As previously noted the softening effects of arthrotomy have been

documented «Thompson, 1975; Frost and Ghosh, 1984; Svalastoga and Reiman, 1985;

55

56
Pelleteir, et al., 1985; Walker, 1991; Messner, et al., 1993). This softening may explain

the overall decrease in stiffness measured in both low and high impact levels at early
time points in the open joint study. However, it does not explain the acute decrease
measured at high impact levels. This difference was attributed to the extensive trauma
(i.e. fissures) imparted by the high energy impacts (Ide, 1992). This structural damage
was believed to have compromised the load carrying ability of the cartilage thereby
initiating an irreversible degeneration process. The sharp rise in stiffness measured at
3 months was thought to be the result of cartilage breakdown and compaction. The
gradual decreases measured at 6 months and one year were the result of continued
breakdown.

At the low impact levels, in the open joint study, only 25% of the test side
patellae showed signs of impact fissuring. Therefore, in the majority of the low impact
specimens the structural integrity of the cartilage was unharmed. The gradual softening
observed in the first two weeks following impact was probably solely an effect of
surgical synovitis. Without structural damage this cartilage was able to recover.

Based on the hypothesis that impact fissures have a degenerative effect on
cartilage stiffness, an acute reduction in stiffness was anticipated at high impact levels
in the closed joint study. However, although 75% of the test patellae were fissured, a
marked decrease in stiffness was not measured at the six day time point. In fact, unlike
the open joint study, no large differences were noted between high and low impact levels
in the closed joint study. By one year post-impact all three mechanical parameters at
both impact levels were decreased on the test side. At high impact levels, Gu and ’1 were

significantly decreased. These results suggest a long term degeneration process, like that

57
reported following ACL transection (Brandt, et a1, 1991), may be required for the

development of OA using this closed joint model.

Two possible explanations have been considered to explain differences between
the open and closed joint studies. The first possibility is that the differences between
impact levels observed in the open joint study, may have been caused by animal to
animal variation and the small sample size used at each time point (n=3, per impact
level). The second possibility is that an interaction may have taken place between the
fissured cartilage and damage synovium in the high impact groups. This interaction may
have enhanced the cartilage degeneration resulting in the stiffness differences between the
high and low impact levels of the open joint study. In an attempt to address the small
sample size, the supplemental groups were added. The results of the 3 month, open joint
supplemental tests closely matched the results from 3 month, closed joint study at both
low and high impact levels. Based on these results it would appear that the small sample
sizes was the cause of the differences between impact levels in the open joint study.
However, the results of the two additional high impact, open joint supplemental rabbits
tested at 6 days post-impact overwhelmingly supported the decreases in stiffness
measured at 6 days in the original open joint study. At the conclusion of the
supplemental tests, questions still remained about the differences between the results of
the low and high impact groups of the open joint study, and the differences between the
Open and closed joint studies.

The first question dealt with the effects of impact fissures. It was still unclear
whether the presence of fissuring alone, without a synovial inflammation, could produce

a measurable decrease in cartilage stiffness. Secondly, questions remained as to whether

58

or not an interaction may have occurred between the fissured cartilage and the damaged
synovium which contributed to the large decrease in stiffness measured at high impact
levels in the Open joint study. If such an interaction exists, it would also help explain
the differences between the results of the high impact groups in the open and closed joint
studies. Finally, assuming that transient synovial inflammation can enhance the
degeneration of fissured cartilage, can a synovitis be created by mechanical means under

closed joint conditions.

OBJECTIVES OF THE STUDY

The investigation developed in the following pages will be presented in two
segments. The first segment, the Acute Study, will address questions about the effects
of severe trauma and surgical synovitis. The initial goal of this study will be to define
the effects of severe trauma, in particular surface fissures, on the mechanical response
of AC. After documenting these effects, the study will attempt to measure any enhanced
degradation effects which may occur when fissures are present simultaneously with a
surgical synovitis. The second segment, the Synovitis Study, will investigate the
possibility of inducing a synovial inflammation using non-invasive blunt trauma to the
synovial tissue surrounding the joint. Biomechanical and histological analysis will be
used to determine the effects of this trauma on the AC and synovium. These
investigations will further introduce biphasic theory into the ongoing OA research carried
out in our laboratories. Unlike the elastic and Viscoelastic analysis used in the
background studies, biphasic theory is based upon the microstructure of the cartilage.
It does not incorporate a phenomenologic material model to quantify the mechanical
characteristics of cartilage. By incorporating biphasic analysis in the present studies we
hope to directly correlate changes in the material properties of the cartilage, specifically

permeability, to the presence of trauma (i.e. fissures).

59

THE ACUTE STUDY

As previously mentioned this study will address two main objectives. The first
objective will be to measure the affects of impact induced fissures on the mechanical
response of AC. This objective requires the induction of surface fissures which are
unexposed to a possible interaction with the surrounding synovial membrane. The second
goal will be to measure possible changes in the mechanical response of fissured AC
which may occur when exposed to a surgically induced synovitis. In order to detect an
interaction between the fissures and surgical synovitis, the effects of the synovitis on
healthy cartilage will first be determined. Then by combining these two factors (i.e.
fissuring and synovitis) their coupled effect on the mechanical response of AC will be
determined. By defining these effects we hope to discern possible reasons for the short
term response differences noted between the two background studies. For instance,
differences between cartilage which has been subjected only to surgical synovitis, and
that which has been exposed to both synovitis and fissuring, may help explain differences
between the low (unfissured) and high (fissured) level impact groups in the open joint
study. Further comparison between the response of fissured cartilage and fissured
cartilage exposed to a synovitis may provide a possible explanation for differences

between the results of the open and closed joint, high impact groups.

ACUTE STUDY MATERIAL AND METHODS

WW

Three separate treatment groups were used in this study. The first group was the
fissure isolation group. The right patella of this group was impacted to induce cartilage
fissures while the left leg served as a non-surgical control. Both patellae were
immediately removed from the animal following the blunt trauma procedure for
mechanical testing. The immediate removal of the test patella prevented the cartilage
from being exposed to a transient softening affect associated with post-surgical synovitis.
Differences between the test and control responses in this group were directly associated
with the presence of the fissures. This group was referred to as the Time Zero, High
Impact group.

The second group acted as a surgical isolation group. Arthrotomy was performed
on the right leg of these animals. The rabbits were then returned to cage activity for 24
hours. One day after surgery these rabbits were euthanized and their patellae extracted
for testing. Differences between the test and control responses in this group were
attributed to the 24 hour exposure to the surgical disrupted joint. This group was
described as the One Day, Sham group.

The last group combined the affects of impact fissures and surgical synovitis.

Immediately following arthrotomy the test leg was subjected to blunt impact. The rabbits

61

62

were then returned to their cages until the following day when they were euthanized and
their patellae excised for testing. This exposed the mechanically fissured cartilage to a
surgically disrupted joint environment. Differences between the results of this test group
and those of the time zero and sham group would suggest a possible interaction between
the cartilage fissures and surgically damaged joint. This third group was called the One

Day, High Impact group.

SargeniLBliuLLmaast

An open joint surgery was performed on the right hind limb of all 18 rabbits in
the acute study (6 rabbits per group). The left leg served as a non—surgical control. The
time zero, high impact animals were euthanized just prior to surgery. An unsterile
arthrotomy was performed on these animals by the principle investigator. The surgeries
performed on the one day animal groups were done under sterile conditions by Dr.
Charles E. DeCamp. All surgeries included the insertion of pressure sensitive film
between the patella and femur, regardless of the impact status. Both the time zero and
one day, high impact groups received blunt trauma to the right knee during the surgical
procedure. These impacts were carried out using the gravity impacter and data
acquisition system described previously (see Open Joint Materials and Methods). All
impacts were delivered at the high energy level (6.31). As previously stated in the
description in viva study, the patellae from the time zero, high impact rabbits were
immediately removed following impact for mechanical testing. The one day animals

were sutured after surgery and/or impact and returned to cage activity for 24 hours.

63

T 'n Pr r

Three stress-relaxation tests were performed on the test and control patellae of
each rabbit using a rigid, solid, cylindrical indenter 1.0 mm in diameter. All three tests
were aligned proximal to distal along the center of the lateral facet, refer to Figure 17
in the Closed Joint study. A ramp rate of 1.2 mm/s was used to indent the cartilage to
a depth of 0.10 mm. This indentation was maintained for 150 sec. while resistive
cartilage forces were measured. The patella was held in place during testing by a grip
assembly (described in the Closed Joint Materials and Methods). Immediately following
each test a thickness measurement was made at the site of indentation using a needle-like

probe to penetrate through the cartilage to the underlying bone.

(Remindsmen

Two additional stress-relaxation tests were carried out on each patella using a
rigid, porous, cylindrical indenter 1.0 mm diameter. Figure 25, shows a photograph of
this indenter. Both porous indentation tests were indented into the surface of the
cartilage to a depth of 0.05 mm and were held at this depth for 1000 sec. while resistive
forces were recorded. These porous tests were performed at two separate loading rates.
The first was brought to the maximum indentation depth in 1.0 seconds, while the other
was done over a 2.0 second interval. Figure 26, depicts the position of the porous tests
on the surface of the patellar cartilage with respect to the positions of the three rigid
indentation tests. The porous stress-relaxation data from two rabbits in each of the three

test groups was analyzed using biphasic theory.

 

Figure 25: Porous indenter.

o = solid indenter
test site

x = porous indenter
test site

 

Lateral l Medial

Figure 26: Porous indentation test sites.

65

There were two main reasons for the addition of the porous indenter tests. The
first reason was directly related to the boundary conditions required by the biphasic
model for the solution of an indentation stress-relaxation test. In order to achieve a
closed form solution, the articular surface of the cartilage is assumed to be permeable to
water flow during the loading phase (Mak, et al., 1987). The pitted surface of the
porous indenter allows water to escape through the articular surface during the loading
phase. The rigid indenter, does not allow the exudation of water through the surface in
the area of contact between the indenter and cartilage. Without a permeable boundary
condition, a closed form solution does not exist and biphasic analysis requires the use of
a finite element approximation (Spilker, et a1. , 1991). The second reason for the addition
of the porous tests is related to the goals of the acute study. It was anticipated that the
fissures created during impact would cause a decrease in the structural stiffness of the
AC. We further hypothesize that this mechanical disruption of the collagen network
would also result in increased cartilage permeability, specifically at the surface where
fissures are present. As previously noted, the integrity of the cartilage surface layer
plays a key role in restricting fluid exudation during loading (Setton, et al., 1993). The
presence of surface fissures should increase the ability of water to escape through the
cartilage surface. With the permeable boundary conditions incorporated in the porous
indenter, we hope to detect increases in permeability which may have been masked by

the impermeable rigid indenter.

15' I'll? .

The indentation testing method was chosen in the original open joint study because

66

it allows the in situ determination of cartilage material characteristics. The
servohydraulic test machine used for mechanical testing was incapable of applying a
constant load of small enough magnitude to perform a creep test (P < 0.7N, Parsons and
Black, 1977). The testing machine was, however, capable of generating small constant
displacements (<0. 10mm) making it compatible with stress-relaxation testing. Both the
elastic solution of Hayes, et al., 1972, and the Viscoelastic analysis of Tobolsky (1960)
were used to compute the shear moduli (Gu and Gr) and the flow viscosity (1)),
respectively, from the test data. For comparison reasons, these same analysis techniques
were also applied in the current studies. In addition, biphasic analysis was also
incorporated to obtain material properties for the cartilage, the permeability in particular,
based upon its microstructural composition. Due to its relative newness to our OA
investigations, a historic development of the biphasic theory has been included prior to

description of the analysis process.

' Vi l ' i
In 1972, Hayes, et a1. , determined a solution for the indentation of a linear elastic
layer bonded to a rigid half-space. Assuming small strains, the shear modulus (G) for

the case of a rigid, cylindrical, plane-ended indenter is expressed as

= P(1-v)
4amO Ida/hut)

 

(1)
where; (P) is the resistive load of the elastic layer, (a) is the radius of the indenter, (v)

is Poisson’s Ratio, (h) is the layer thickness, and (an) is the indentation depth (Figure

67

27). The variable (K) is a correction factor, which adjusts for the finite thickness of the

elastic layer based on the aspect ratio (a/h) and a predetermined value for v.

P11)

21 +-
CYLINDRICAL —> |
INDENTER

 

 

 

 

 

+ ______ "T- (00

h
CARTILAGE

?\- \- \—\-\-\— \—\—\-\-\R—\—\-\A—\-
SUBCHONDRAL BONE

 

Figure 27 : Indentation test.

Due to the high rate of loading used in our rigid indentation tests, the cartilage
is considered to be instantaneously incompressible (Mak, et al., 1987) and a Poisson’s
ratio of 0.50 is used for the computation of Gu from peak response load. The
computation of the relaxed shear modulus (Gr) was based on the equilibrium load. An
average Poisson’s ratio of 0.4 was used for this calculation. The equilibrium Poisson’s

ratio was based upon prior experimentally determined values for v (Parsons and Black,

68

1977). Figure 28, shows a load-time curve for a typical stress-relaxation indentation test
and depicts the response regions used for the computation of Gu and Gr. The actual

calculation of Gu and Gr was carried out via a computer program.

0.0 H

 

0.3 -<

l

Load (NI

 

 

 

I
60 90 120 ISO
“in. (80°)

0..
w
0

Figure 28: A stress-relaxation load-time response curve.

The thiclmess of the cartilage at the site of the indentation test is required for the
computation of the correction factor (K). This measurement was made using a needle-
like probe described previously. A typical load-time plot from a thickness test is shown

in Figure 29. The thickness of the cartilage (h) is calculated graphically as follows

h=(tf-t-)x(R) (2)

where, t, is the time corresponding to the onset of loading, tf is the time corresponding

69

to the sharp rise in load indicating contact with the subchondral bone, and R is the ramp

rate of the needle-like probe.

 

8 I I I I T T T T _I

 

Force (Newtons)

 

 

 

o tf

 

 

-1 l I 1 l I I I l l I

O 2 4 6 8 10 12 14 16 18 20
Time (seconds)

 

Figure 29: A load-time plot of a thickness test.

The calculation of the flow viscosity is based upon Tobolsky’s analysis of the
rheological behavior of a generalized Maxwell model (GMM), Figure 30. Based on this
analysis the time dependent shear modulus (6(0) of the GM is given in terms of the
time constant (1) as

00) = if; H(T) eXp (-t/r) d (Int) (3)
Where, H(r) is the relaxation time spectrum. In turn the flow viscosity (1;) can be

expressed as

n = I :; Gmdr (4)

7O

 

era an e

p———————-—-—_—————

 

,—————————_———_—_—4
L—————_-————_——~—4

 

 

 

 

<)———-————————-—-—-——~—~t>

Generalized Maxwell Model

Figure 30: A generalized Maxwell model.

By assuming the viscous elements of the GM have relaxation times with a box
distribution, H(r) can be expressed as a constant (13,) within the response region bonded

by rm 5 t s rm. Therefore, the shear modulus is given by

G(t) = E0 [Ext/7....) - Edi/Tm] (5)
where E is the resulting exponential integral function. Outside of this range H(r)=0.
If the values of log(rmh) and log(rm) differ by more than one, which has been shown to
be true for cartilage (Woo, et al., 1980), then the central portion of the G(t) vs. log(t)
plot can be considered linear. The slope of this linear portion (H(r)) is equal to

H(r) = 2.303Eo = 2.303H(r) (6)

71

see Figure 31. By substitution, the flow viscosity (equation 5) can be reduced to

’I = H(T) [Tm-7min] (7)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'2
\
.. F‘s:
g
>" O
i \
g ‘ \
3
i: a
K
E _
0 2 lel l —
l l I
2.303 c, \
o l I l‘\
E

 

 

LOG

Figure 31: The theoretical response of a generalized Maxwell model.

The exact value of rm is not experimentally easy, or practical to determine, considering
cartilage under indentation conditions requires approximately 10,000 seconds to reach
complete relaxation (Mak, et a1. , 1987). However, as a property of the exponential

function rm is related to the intersection time (TB) by the following relation

rm = 1.781(TB) (8)

72

Figure 32, shows the application of Tobolsky’s analysis on an actual stress-relaxation
load-logtime plot. By assuming that the cartilage is loaded instantaneously, and that

relaxation begins immediately following maximum indentation, rm can be assumed to

Cartilage Relaxation Response

 

0.8“

0.6“

Load (N)

0.44

 

 

O i i IlIlIII I I IIIIIII I l IMI III I iUFI-Ti

0.1 1 10 100 1000
Logtime (s)

Figure 32: The application of Tobolsky’s analysis (1960) on experimental stress

relaxation data.
be approximately zero. Thereby, reducing equation (7) to

n = H(1') rm (9)
By fitting a line through the average equilibrium load an intersection time (Tn) was
determined for the experimental data. The calculations of the flow viscosity were carried
out via computer analysis.

The elastic calculations of Gu, Gr, and the Viscoelastic calculation of n, were

carried out for all the rigid indentation tests performed in the acute study. As a point of

73

interest these analysis techniques were also applied to the porous tests of the two rabbits
from each group which were analyzed by the biphasic model. Theoretically, the slower
rates of loading used for the porous tests (1 - 2 seconds) should allow the afflux of fluid
out of the cartilage. This should result in Gu values smaller than those calculated for the
fast, solid indenter tests. Under stress-relaxation indentation conditions, fluid flow
through the cartilage has ceased by equilibrium and the loads are supported by the solid
matrix (Mow and Hayes, 1991). Therefore, the Gr values calculated from the porous
tests should be approximately the same as those calculated for the solid indenter. The
flow viscosity represents the average viscosity of the GM damping elements.
Therefore, increases in 17 represent an increase in the resistance of cartilage to relaxation.
The permeability, measured by the biphasic analysis (see following text), is a measure
of the ease with which fluid moves through the cartilage. Increases in permeability are
directly related to increases in relaxation. Therefore, an inverse relationship between the

flow viscosity and permeability values was expected.

i h i
In 1976, Torzilli and Mow proposed a new model for determining the mechanical
properties of AC. This model describes the response behavior of cartilage in terms of
two phases. The wild phase is "flow independent" and is controlled by the material
properties of the solid matrix components. The fluid phase is controlled by the
movement of fluid through the solid matrix and gives rise to the Viscoelastic properties
of the cartilage. The combination of these two phases describes the biphasic model. The

previous methods described, Hayes, et al., 1972, and Tobolsky (1960), are limited to

74

describing only those portions of the cartilage response which resembled the behavior of
their phenomenological models. Unlike these models, biphasic theory is based upon the
microstructure of the cartilage itself. The material properties determined by this theory
are derived from the overall load response and reflect the physical contributions made
by solid and fluid components.

The solid matrix, which determines the response of the solid phase, consist of
collagen fibrils and proteoglycan aggregates which account for approximately 20% of the
wet weight of human cartilage (Muir, 1979). As an assumption of the biphasic theory
this porous, solid matrix is treated as an isotropic, linear elastic solid. As previously
noted, the fluid phase accounts for the period of cartilage relaxation. At equilibrium the
cartilage resistance force is generated by the closely packed PGs and simple bulk
compression of the solid matrix. The stresses generated in the solid during loading are
described by

a’ = apI + he I -I- 2p,e (10)
where; a‘ is the stress tensor of solid phase, e and e are the strain tensor and dilatation
of the solid matrix, respectively, p is the pressure, a is the ratio of solid volume to fluid
volume, it, and A, are the Lame constants for the solid matrix, and I is the identity
matrix.

The fluid phase is controlled by the Viscoelastic response and is governed by

a‘f = -pl (11)
Under quasi-static conditions the mixture of the two phases is given by
div a"’f + I” = 0 (12)

where 1‘" are the diffusive forces arising from the frictional drag. Under these

75

conditions body forces are given by

1’ = -1r' = K(v‘ - v‘). (13)
where K is the constant drag coefficient and V"f are the velocities of the solid and fluid
phases, respectively. The permeability, k, describes the ease with which the fluid passes
through the solid matrix. The permeability is related to the drag coefficient by

k = 1/(l+ai)2 K (14)
(Lai and Mow, 1980). The continuity equation governing the mixture of the
incompressible solid and liquid is

div vf + a div v' = O. (15)
(Mow, et al., 1980).

Biphasic solutions have been derived for both confined and unconfined
compression-creep tests (Mow, et al., 1980; Armstrong, et al., 1984). Under confined
compression a constant load is applied to a laterally confined cartilage specimen by a
rigid, porous, platen, Figure 33. In an unconfined compression test the cartilage is
compressed between two impermeable platens, Figure 34.

The results of these tests have shown that cartilage creep-relaxation is dependent
on the characteristic gel diffusion time (t), for a biphasic material this diffusion time is
given by

g=rmn 06
where; (d) is the fluid flow length and (HA) is the aggregate modulus of the elastic solid
0., + 2,1,). For confined compression d is the radius of the platen. For unconfined

compression d is equal to the thickness of the cartilage (h).

Exudofion ‘i

Contining

 

Lin-l -l- ‘

\

l lid

76

CONFINED COMPRESSION

 

 

 

 

II
I

 

 

/ Specimen

 

I
I'-

///////////////// /// //// /

 

 

T/7/

77f

///7

COMPRESSION DIRECTION l

FLOW DIRECTION l

Figure 33: The confined compression test.

UNCONFINED COMPRESSION

Rigid

Impervious smooth

Plate

ll

02

GA

 

l/Z/Z/Z

ExudoHon

 

A
V

e—

.- .

 

Specimen/

V.

 

 

.77///.//lfl/

“I.

—-——.

7"

 

:TZ

2221/7/2/77Zl

O'A

COMPRESSION DIRECTION l

FLOW DIRECTION

Figure 34: The unconfined compression test.

e—e

77

When the strain rates created during loading are fast in comparison to t, the
cartilage behaves like a single phase, incompressible, elastic material. At strain rates
very slow in comparison to t: the interstitial fluid pressure does not contribute to the
cartilage response and no relaxation is observed.

In 1987, Mak, et al., published load—deformation solutions for cartilage under
creep and stress-relaxation indentation conditions. These solutions begin with constitutive
equations (10) - ( 15). To account for the in situ geometry of the cartilage under
indentation conditions a cylindrical, porous, rigid indenter was used. The free draining
interface between the cartilage surface and the porous indenter provides an additional
boundary condition required to determine a unique solution. The complex Laplace
transforms required for the solution of these indentation tests are inverted via computer
computation, see Biphasic Analysis.

From the stress-relaxation indentation solution it has be shown that under
conditions of rate controlled loading the relaxation behavior of cartilage is dependent
upon the rate of compression parameter (Ru) where,

R, = H Al</V,,h (17)
and V0 is the rate of loading. Under instantaneous load conditions the biphasic stress-
relaxation solution is identical to that of Hayes, et al., 1972, assuming u = u,.
Agreement between these two solutions occurs again when the cartilage reaches complete
equilibrium (for v = v,). For a porous indentation test complete relaxation requires
approximately 10,000 seconds (Mak, et al., 1987).

Current development is in progress on a triphasic theory (Gu, et al., 1993). The

fixed charge density (FCD) associated with the PCS and the counter ions create a

78

swelling pressure within the cartilage (Lai, et al., 1993). Under a mechano-
electrochemical, triphasic theory the stresses induced by this swelling pressure are

incorporated into the existing biphasic theory.

' h ' An i

To maintain compatibility with our available testing equipment, and retain
the in situ geometry of the patella for histology, biphasic indentations stress-relaxation
tests were employed. Analysis was carried out using the least square, curve fitting
algorithm previously discussed (Mow, etal., 1989). The stress-relaxation response data
was collected in our laboratories using a rigid, porous, cylindrical indenter. Test data
was then transferred via computer to Dr. Wenbo Zhu at the University of Maryland
where the curve fitting analysis was performed. An outline of the curve fitting algorithm
is presented below. Although this outline is based upon the creep solution published in
1989 (Mow, et al.), the methodology used is the same for a stress-relaxation test.

The algorithm begins with the Laplace transform solution for creep displacement
(fi(s)) (Mak, et al., 1987).

f*1s

11(3) = 4832(3‘13-1) [w]7[g*]

 

(18)
where P is the resistive cartilage load; a is the radius of the porous indenter; [w]T are
Simpson’s weights and [g*] is a matrix solution to a Fredholm integml equation. 17, is

defined as

79
n. = (1-VJ/(1-2VJ (19)

where v, is the Poisson’s ratio of the solid matrix.

Due to the extensive computations required for the biphasic solution, standard
curve fitting programs were inefficient. However, all theoretical biphasic solutions are
dependent upon the similarity variable t' = t/t, (i.e. t/(a2/HAk)) and the dimensionless
parameters; indentation load (P/Zu,a’), aspect ratio (a/h) and the Poisson’s ratio of the
solid matrix (v). These variables provided the key to the problem.

Figure 35, demonstrates how changes in tI effect the creep solution. For any
given set of dimensionless parameters there exists a theoretical displacement curve u(t')
which will provide the solution for experimental displacement curve u(t) when t' = t (i.e.
a2/HAk = 1). Therefore, master curves can be generated and shifted to other t, values

along the logarithmic time axis. The amount of shift (S) is given by

10810 (t) ' 10810 (t’) = S (20)

By the definition of t’ this can be expressed as,

S = 10810 (az/kHA) (21)

This shift is demonstrated graphically by Figure 36.

 

Non-Dimensional Displacement

 

8O

 

 

 

 

ux'h
II
015 P'- I I l
U(oo)/h--Equrlibrium Value
U(:n)/h
0I2 —
009 ~
I;
0 I I IO 100 lOOO I0000
Time (s)

Figure 35: Creep relaxation as a function of t,.

Non-Dimensional Displacement

u/h
I

015—

U(.x)/h - - Equilibrium Value

 

U(oo)/h

0.12 l-

  

1's

 

 

 

increasing
Experimental Data
009 L Master Solutions and CurvelIt
I, 1 _ L . l 1 4——-> i
0 I 1 I0 100 t000 10000
Time (s)

Figure 36: Shifting of theoretical biphasic response curves by S = log,o(a’/kHA).

81

From equation (20) it can be seen that t’ can be written as a function of t and S.
Therefore, for a given set of dimensionless parameters, u(t') is a function of log(t) and
S.

The last constraint of the curve fit requires that at t' = co the curve must fit the

dimensionless equilibrium equation,

]

 

 

u(oo) _ 3 [ Po (21.-Vs)
h h 25.2“, 2K(a/h,v,)

(22)

From equation (22) it can be seen that for a given set of experimental values for
P, h, and a the displacement is only dependent on the relationship between pt, and 9,.
Therefore, if a value for u, could be determined, then the theoretical curve fit would
depend on v, and the amount of shift, S. By forcing S = 0 this dependence can be
reduced to only v,.

Master solutions are first begun by determining values for u. from equation (22)
based on ten discrete values for u, (from 00.499) and experimental values for P, u(oo)
and a/h. Each of the paired values of u, and v, (n=10) creates a unique dimensionless
variable group (P/2p,a’, a/h, y). In turn, each of these dimensionless groups are placed
back into the displacement solution, u(log t, 3). Master solutions are then calculated at
15 values of log(t) for each dimensionless group. This procedure provides master
solutions defined with respect to v, and t, for a given aspect ratio (a/h), leaving (P/2p,a2)
as the parameter.

Due to the large number of repetitive calculations required, a bicubic spline was

chosen for curve fitting because of its simplicity. The curve fitting process is achieved

82

by minimizing the error between the actual experimental curve and the spline of the
creep solution. From Figures 35 and 36 it can be seen that the shape of the master
curves are dependent on v,, and the position of these curves on S. Fitting the
experimental data is achieved by minimizing the least square sum of the differences with
respect to the v, and S values.

Once v,, S and u, have been determined the in situ aggregate modulus (HA) and
the permeability (k) can be calculated from

HA = 2,1, (1-v,)/(1-2v,) (23)

and

k = (aleA) 10s (24)

A large amount of computer computation time was required to perform the curve fitting
routine. For practical purposes the experimental curve data was condensed. Forty
representative load-time points were selected via computer from each raw data file.
Among these points were the peak resistive load and the final equilibrium load.
Experimental values for the cartilage thickness (h) and loading rate were also required

by the algorithm and were also included in the information sent to Dr. Zhu.

Stag’stjfi Analysis

Within group comparisons (test vs. control) were carried out on the three
parameters (Cu, Cr and n) for both the acute and synovitis studies using a Student’s ’t’
test for paired samples. Based upon the results of the background studies, and an
extensive literature review, the effects of fissuring and synovitis were assumed (a priori)

to have a degenerative affect on both the stiffness and flow viscosity of AC. Therefore,

83

a one tailed hypothesis (test < control) was used for these statistical comparisons.
Differences were considered to be statistical when a one tailed P value less than 0.05 was
calculated.

Between group comparisons were carried out on results of the acute study groups
(e.g. time zero, high impact, test side Gu vs. one day, high impact, test side On). These
comparisons were performed using the Student’s ’t’ test for random variables, with a
Bonferroni’s adjustment for multiple comparisons. The null hypothesis assumed equal
means between the compared groups. These between group comparisons were used to
detect the possibility of an interaction between the cartilage fissures and surgical
synovitis. All of the student’s ’t’ tests were carried out on a personal computer using a
spreadsheet program (Excel).

Statistical analysis was not performed on the biphasic results of the porous test
beeause of the small number of observations (n=2) from each test group. However
visual assessments and comparisons were made with results from previous biphasic

studies.

1211512122!

Upon the completion of mechanical testing, the patellae were placed in 10%
buffered formalin for histology. After examining the results of the rigid indenter tests,
patellar pairs which represented the mean results of each group were chosen for
histologic examination. Using routine methods, cross sections were cut from these

patellae and the cartilage was stained with safranin-O to reveal PG content.

ACUTE STUDY RESULTS

Bluntlmnae‘t

As previously noted all impacts were high level. The average peak load for
both the time zero and one day, high impact groups was 6311102 N. This is
approximately 12 percent higher than the average peak loads recorded in the previous
closed joint study and approximately 22 percent greater than those recorded in the open
joint study. The average impact parameters and fissure status are provided for the time
zero and one day impact groups in Table 24 in the Appendix.

Impact pressure profiles were obtained from the pressure sensitive Fuji film.
Figure 37 shows a typical pressure profile from the acute study. Bimodal loading
patterns were indicated by two distinct areas of staining. A visual comparison of loading
patterns was made between the pressure film results of the acute and open joint studies.
This comparison revealed no apparent differences in impact loading between the two

studies.

r rv ' us
The time zero joints inspected during removal of the patellae appeared normal.
No visible differences were observed in the synovial lining, synovial fluid or AC between
the test and control sides. Staining the AC surface with india ink revealed impact

fissures in four of the six test patellae. These fissures typically ran proximal to distal

84

I'IO

Syr
flui

TEI'I

85

 

Figure 37: A typical pressure profile from the acute study.

along the lateral side of the patella centerline. This fissure formation is consistent with
those reported in the background studies. The occurrence of these fissures are
documented in Table 24 in the Appendix.

The tissue surrounding the test joints of the one day, high impact group was
noticeably damaged. The tissues adjacent to the sutures were visibly filled with blood.
Upon opening the joint capsule a similar appearance could be seen from the inside. The
synovial fluid appeared orange in color and traces of blood were observed. The synovial
fluid within the control joints appeared normal. An initial comparison of the cartilage

revealed no visible differences between the test and control patellae. After staining the

86

AC with india ink, five out of six of the impacted patellae were found to have fissures,
see Table 24 in the Appendix. These fissures were similar in appearance to those in the
time zero impact group. Figure 38 shows a typical impact induced fissure from the acute
study, the indentation sites are visible as small circles to the right (lateral) of the fissure

which were left by the thickness needle probe.

 

Figure 38: Typical impact fissures created during the acute study. The sites of the
indentation tests are visible as small circular defects to the right (lateral)
of the fissures.

The solid indenter tests, those farthest to the right in Figure 38, form a vertical line

down the lateral facet of the patella. By visual inspection it was determined that the

average distance between the site of indentation and the impact fissures was between 2

and 3 indenter radii (i.e. 1-1.5 mm.). (See Appendix Table 24).

87

The joint tissue and synovial fluid of the one day, sham group had the same
appearance as those described in the one day, high impact group. Upon inspection of
the AC no differences were detected between the test and control patellae. Staining with
india ink failed to reveal any differences in the surface integrity between the test and

control cartilage.

W (Solid Indenter)

Ti e Zero, fligh Impact

A comparison of instantaneous stiffness (Gu) values showed that the test side
eartilage was on average 11.5% softer than control. The relaxed stiffness (Gr) was an
average 6.8% less on the test side, while the flow viscosity (:1) was increased by an
average of 54.6%. Within group comparisons revealed that the difference between Gu
values (test vs. control) was significant (P = 0.04). The differences in parameters Gr
and n , however, were not statistically significant. A complete list of the solid indenter

test results for the time zero, high impact group are presented in Table 25 in the

Appendix.

h Im
Unlike the time zero, high impact group, the average Gu value for the one day,
sham group was basically unchanged on the test side (1.5% decrease). Test side Gr
values, on the contrary, were decreased by an average of 13.2%. The n values were also
decrease by 14.7% on the average. A within groups comparison revealed that Gr values
were significantly lower on the test side, P = 0.024. The differences in Cu and n were

not found to be significant. A complete list of results for the one day, sham group is

88
provided in Table 26 in the Appendix.

Que Day, High Impact

All three mechanical parameters were reduced on the test side. On was decreased
an average of 21.9%, Gr by 14.3%, and n by 23.4%. These decreasing trends are
similar to those observed in the open joint study at high impact levels one day post-
surgery. Within group comparisons showed that the average test side Gr and 71 were
significantly lower than their respective control values, P = 0.011 and P = 0.042. Gu
was borderline with a P value of 0.050. A complete list of results for the one day, high

impact group are compiled in Table 27 in the Appendix.

n r m ' n Inte ion

Multiple comparisons revealed no significant differences between the time zero,
high impact parameters and those in the one day, high impact group. This result
statistically suggests that the 24 hour exposure of the fissured cartilage to the surgically
disrupted joint environment had no additive degenerative affect on its mechanical
response. Similar comparisons were made between the one day, sham group and one
day, high impact group. These comparisons also revealed no significant differences.
Statistically this suggests that the fissures and surgical synovitis did not interact to
enhance the degeneration of the cartilage beyond that which would have been created by
each of them working simultaneously alone.

Further comparisons were also carried out between the time zero, high impact
group and the one day, sham group. These comparisons revealed the test side Gr values

to be significantly lower in the sham group with a P = 0.008. This statistical

89

significance suggests that the individual softening effects of fissures and surgical synovitis

affect the mechanical stiffness differently, specifically the equilibrium stiffness.

West; (Porous indenter)

mm

The biphasic analysis provided three material properties; shear modulus of the
solid matrix (p), Poisson’s ratio (v,), and the permeability of the cartilage (k). For
direct comparison with previous biphasic studies, the aggregate modulus was also
calculated (11,) using equation (22), see Biomechanical Properties. The biphasic results
of the porous indentation tests are compiled in Tables 28 and 29 in the Appendix. In
several cases the biphasic algorithm was unable to simultaneously fit the large peak load
and fast rate of relaxation of the stress-relaxation data. For these cases the analysis was
aborted. For these aborted cases the biphasic material properties are not available, and
are represented by dashed lines in Tables 28 and 29. The material properties calculated
for both the 1 and 2 second porous tests were combined for presentation in this results
section.

The aggregate modulus values for the control patellae of all three acute study
groups ranged from 0.400 to 0.968 MPa. This range of values is comparable to
average values for bovine femoral condyle cartilage (0.47-0.90 MPa) (Mow, et al. ,
1989). The Poisson’s ratio (v) for the control patellae were essential 0.00 ( except for
rabbit 9326 which was 0.058). This value is 100% smaller than the range of 0.25—0.40
measured for bovine cartilage by Mow, et al., 1989. This value is, however, close to

the average Poisson’s ratio of 0.08 reported for normal human cartilage (Whipple, et al. ,

90
1985). The permeability values (k) for the control patellae ranged from 0.205 to 1.040

m‘le(x10“). These control permeability values are 1.4 - 7.2 times larger than the
range of average values measured for bovine cartilage (Mow, et al., 1989). However,
they do fall within the 0.05-1.95 range measured for human cartilage by Armstrong and
Mow (1987).

The test side HA values for the time zero group ranged from 0.3320864 MPa.
The test side ranges for the one day, sham and impact groups were 0.400-0.540 and
0.842—0.968 MPa, respectively. These ranges are all comparable to average values
measured for bovine cartilage. Like controls, the test side values for Poisson’s ratio
were essentially 0.0 (except rabbit 9326 which was 0.019). The test side permeability
values for the time zero group ranged from 0.836 to 2.370 m‘le(x10“). The test side
ranges for the one day, sham and impact groups were 0.2030870 and 0.896-3.620
m‘le(x10“), respectively. These values are 1.4 to 25.5 times greater than the largest
average bovine permeability measured by Mow, et al., in 1989. While the permeability
values for the one day, sham group do fall within the range for human AC permeability
(0.05-1.95, Armstrong and Mow, 1987), the permeability values for the time zero and
one day impact groups are both relatively high in comparison.

Overall, the control and test side values for the aggregate modulus were
comparable to previously determined bovine values. For both impact groups (time zero
and one day) the test side modulus values were lower than their controls. For the one
day, impact group the test side moduli were consistently 50-60% lower than their
controls. Under biphasic theory the Poisson’s ratio reflects the ability of fluid to move

through the cartilage (Mow and Hayes, 1991). A biphasic Poisson’s ratio of 0.50 would

91

suggest no fluid flow through the cartilage and no relaxation. Therefore, p, values near,
or at 0.0, indicate a large afflux of fluid through the cartilage. These low p, values
reflect the algorithms attempt to match the fast rate of relaxation in the indentation tests.
This rapid relaxation is also evident in the large permeability values. The test side
permeability values for all of the acute study groups demonstrated a general increase on
the test side. In the time zero group this increase suggests that the presence of impact
fissures alone can increase cartilage permeability as hypothesized. Interestingly, 3 out
of 4 of the test side permeability values from the sham group were also greater than their
controls. This suggests that exposing cartilage to a surgically damaged joint environment
may also increase permeability. The combined effects of surgery and fissures, may have

both played a role in the increased permeability measured in the one day, impact group.

El '3? l'Rl

The elastic (Gu and Gr) and Viscoelastic (1;) results for the porous indentation tests
are compiled in Tables 30 and 31 in the Appendix. The material parameters calculated
for both the 1 and 2 second porous tests are combined for presentation in this results
section.

In general, the test and control On values for all three of the acute study groups
were consistently 50—60% lower for the porous indenter tests than those calculated for
the solid indenter. This decrease is consistent with the predicted response. The test and
control values for Gr were also visibly decreased by approximately 50% in comparison
to those from the solid indenter. According to the biphasic model, if the cartilage were

truly at equilibrium no difference in the experimental Gr values would occur between the

92

solid and porous indentation tests (for v=u,=0.40). These differences may be due to the
closer proximity of the porous tests to the site of fissuring and/or their relative relaxation
periods (i.e. 1000 sec. for the porous tests vs. 150 sec. for the solid). The large
variations between individual flow viscosity values made comparisons between the porous
and solid indenter results difficult. No apparent increasing or decreasing trend could be

discerned for either the test or control side values.

1111101ch

The surface integrity of all 6 control patellae in the acute study appeared
histologically smooth and healthy. In both the time zero and one day, impact groups
impact fissures were observed. These fissures were oriented at 45 degrees to the
articulating surface and extended down into the middle zone of the cartilage (Figure 39).
No distinct loss of PGs could be accessed in any of the test side patellae. It had been
anticipated that exposure to a surgically damaged synovial environment would result in
a loss of P63. Failure to observe such a loss may be due to the small number of paired
observations in each group (n = 2) and/or the short period of exposure (24 hours).
Some pre—existing pathologies were noted in patellae from each acute group. These
pathologies included a loss of PG staining in the deep zone and a loss of tidemark (i.e.
the boundary line where the cartilage and subchondral bone meet), see Figure 40. These
pathologies were typically bilateral, appearing on both the test and control sides in equal
intensity, and therefore are not believed to have played a part in the mechanical

differences measured between the test and control sides.

93

 

Figure 40: Histological cross section showing the loss of PCS and tidemark observed
in some of the patellae in the acute study groups.

SYNOVITIS STUDY

Recent research involving cytokines and catabolic enzymes has raised questions
about the possible role of the synovium in OA pathogenesis. Under natural impact
conditions, damage to the peripheral joint tissues, such as the synovial lining, may
provoke the release of such substances. Such a transient injury response may result in
a softening affect similar to that created by surgical arthrotomy. In the open joint study
the release of cytokines and enzymes by the surgically damaged synovial lining is
believed to have contributed to the decreased stiffness measurements at early post-surgery
time points. Under the experimentally controlled conditions of the closed joint study, the
test patellae alone suffered trauma. The surrounding soft tissues were unharmed.
Without damage to the synovial lining an acute biochemical response may not have been
induced. This might explain the differences in stiffness responses between the open and
closed joint studies, particularly at early time points (e. g. 6 days post-impact). It is
hypothesized that loading the cartilage during this transient softened period may increase
the rate of cartilage degeneration.

The purpose of the synovitis study was to address the possibility of inducing an
inflammatory synovial response in the rabbit model without the artificial use of
arthrotomy. In this study, blunt trauma to the synovial tissue was investigated as a

means of producing synovitis by mechanical means. This will help us assess whether or

94

95

not a synovitis can be produced during a natural impact scenario which in turn softens
the cartilage.

Further investigations were also carried out to determine whether an interaction
occurs between the impact fissures and traumatized synovium enhancing the degeneration
of AC. In the acute study, such an interaction could not be statistically proven.
However, due to the overall decreases measured in the one day impact group of the acute

study, we felt that further investigation of a possible interaction was warranted.

vi ' I V‘v

The original synovitis study (Part I) included two treatment groups. The first
group, the sham group, received blunt trauma to the soft tissue surrounding the right
patella. Following the induction of this trauma these rabbits were then returned to their
cages. This group was used to determine whether or not trauma to the synovial tissue
alone could create a secondary effect on the mechanical response of AC. In the second
group, after traumatizing the synovial tissue, the rabbits were placed in the restraining
chair and a high level impact was delivered to the patella of the traumatized joint. This
group was used to determine whether cartilage fissures interact with the traumatized
synovium to produce an additional degradation in the mechanical response of AC. This
may help answer the question of whether or not synovitis played a role in the difference
between the low and high impact groups of the open joint study.

Both groups, sham and impact, were exercised daily following the induction of
synovial trauma and/or impact, until being euthanized for testing. By exercising the

animals, we hoped to further stimulate and maintain a synovial response. Six rabbits

96

were used in each of these two treatment groups.

The open joint study sham results suggested that use of the restraining chair
during surgery may have had degenerative affects on the test side cartilage (Ide, 1992).
To address this, two additional groups were added to the synovitis study (i.e. Part II).
Both groups received blunt trauma to the synovial tissue surrounding the test joint while
restrained in the chair. The rabbits in the first group (Shams) were returned to their
cages following the synovial trauma. The second group received a high level impact to
the test patella following the synovial trauma. Like the two groups in Part 1, these test
groups also received daily exercise following the induction of trauma to stimulate and
maintain the synovial response.

All of the rabbits in the synovitis study were housed individually and allowed cage
activity. The rabbits were exercised for 15 minutes daily on an enclosed treadmill
beginning the day after trauma induction. Six days after trauma induction the rabbits
were euthanized and their patellae removed for mechanical testing. At this time tissue
samples were removed from the synovial tissue medial and lateral to the patellae. This

tissue was placed in 10% buffered formalin and prepared for histological examination.

Spit Tissue TgapmaZBlpnt Impapt

The rabbits were placed under anesthesia using the ketamine, xyaline and were
maintained by isoflorine during trauma induction. In the original study, Part I, the
rabbits received synovial trauma without the use of the restraining chair. The right hind
limb of these rabbits was held in a flexed position (approximately 45° of flexion) by an

assisting veterinary technician. Blunt trauma was then induced in the synovial tissue

97

adjacent to the patella by repeated tapping with a common household hammer. The area
targeted for damage was the synovial lining directly lateral and medial to the patella. In
these areas the synovium is unprotected by superior ligament and muscle cover. The
trauma was continued until cutaneous hemorrhaging was observed in the tissue around
the patella (approximately 50 taps). Care was taken to not strike the patella during this
procedure. Five minutes after the induction of synovial trauma the sham rabbits were
returned to their cages. For the impact group, five minutes after the induction of
synovial trauma the rabbits were placed in the restraining chair where they received a
high level patellar impact.

In Part II, induction of the synovial trauma was carried out while the rabbits’ hind
limb was held hyperflexed in the restraining chair. The sham rabbits remained in the
restraining chair for five minutes following the induction of the synovial trauma, and
were then returned to their cages. The impact rabbits remained in the restraining chair
for five minutes following the synovial trauma, and then they received a high level
patellar impact.

All of the patellar impacts in the synovitis study were carried out using the gravity
impacter and data collection system previously described (see Open Joint Study Materials

and Methods).

W
Indentation tests were carried out using both solid and porous indenters. The test

sites and procedures were identical to those used in the Acute Study. After being

mechanically tested, the patellae were prepared for routine histology.

98
Wham

The biomechanical analysis methods used were the same as those described in the
Acute Study, Materials and Methods. A 2x2 ANOVA was performed on the paired
results of the solid indenter tests of all four synovitis study groups. This ANOVA was
performed to detect the effects of impact fissures, and/or the use of the restraining chair
on the mechanical response of the AC.

Within group comparisons were performed on the results of the solid indenter
tests to detect differences between the mechanical response characteristics of the test and
control side cartilage. These within group comparisons were performed using a Student’s
’t’ test for paired samples. It was anticipated, a priori, that both synovitis and impact
fissures would cause a decrease in the shear moduli and flow viscosity of the cartilage.
Based on this assumption, a one tailed hypothesis (test < control) was used for the
within group comparisons.

Due to the small number of rabbits analyzed by the biphasic theory, n=2 per
study group, statistical analysis was not performed on the results of the porous tests.
However, comparisons were drawn between the results of these tests and the results of

previous biphasic studies.

SYNOVITIS STUDY RESULTS

We

As previously noted, all patellar impacts were of high level. The average peak
impact load for the synovitis impact groups was 549 j; 124. This is approximately equal
to the average high level impact value reported for the closed joint study and is
approximately 12% higher than that reported in the open joint study. Impact fissures
were observed in 6 of the 9 rabbits impacted. The impact parameters and fissure status
of these rabbits are tabulated in Table 32 in the Appendix.

The following results are presented in two parts. In part one, the results of the
original two synovitis groups (Part I) will be presented. In these groups the synovial
trauma was induced without the use of the restraining chair. In Part II, the results of the
second study groups will be presented. In these two groups the synovial trauma was

induced while the rabbits were restrained in the chair.

PART I: (Synovitis created without the use of the restraining chair)

 

P'r rv'n
mm

On the day of testing, six days after traumatizing the synovial tissues, the test

joints appeared normal when compared with their control. Upon opening the joints, the

99

100

synovial tissue and fluid of the test and control legs appeared normal. No abnormalities
were noted in the test or control patellar cartilage prior to staining their articular surfaces
with india ink. Upon staining the cartilage baseline pathology was detected in the AC
of five sham rabbits. This pathology included surface roughness and some fissuring.
Typically the patterns of pathology were bilateral, that is to say that they were present

on both the test and control patellae in equal intensity.

1mm

Six days after trauma, the external joint tissues appeared normal on the test side
when compared to controls. Upon opening the joints no abnormalities were nOted in the
synovial lining or fluid of either the test or control joints. An initial examination of the
cartilage revealed no signs of fissuring on the test side. After staining with india ink,
fissures were detected in five of the six test patellae, Table 32 in the Appendix. These
fissures were present on the lateral facet running proximal to distal along the centerline

of the patella.

W (Solid indenter)
Mama

The results of the solid indenter test are presented in Table 33 in the Appendix.
A visual comparison of the average values revealed that in gt cases the test side
parameters were larger than their controls. Gu test values were an average of 18.3 %
greater, Gr values 10.9%, and n was up by 20.3%. Based on the a priori one tailed
hypothesis (test < control) it was obvious that none of the test values were statistically

different than their respective controls.

101
mm
All test side mechanical parameters were visibly decreased in comparison to their
controls, Table 34 in the Appendix. Test side On values were down by 29.1%. Or was
decreased an average of 17.0% and '0 an average of 25.3%. All three test side

parameters; Gu, Gr, and n, were found to be significantly less than their controls with

P values of 0.027, 0.013, and 0.011, respectively.

W (porous indenter)

The biphasic material properties for the sham and impact groups are presented in
Tables 35 and 36 in the Appendix. The material properties for the porous tests
performed at the 1 and 2 second loading rates have been combined in this results section.

The control side, aggregate modulus (11,) values for both the sham and impact
groups ranged from 0.57 1-1.243 and 0549-0792 MPa, respectively. These values are
comparable to the range of average bovine modulus values, 0.47-0.90 MPa, measured
by Mow, et a1. , 1989. The Poisson’s ratios for the sham and impact groups ranged from
0.0-0.288 and 0.0—0.298, respectively. These ranges are low compared to the average
range of 0.25-0.40 measured for bovine cartilage (Mow, et al., 1989), but are
comparable to the average values 0.08 and 0.14 determine for normal human and porcine
cartilage (Spilker, et al., 1992; Whipple, et al., 1989). The control side permeability
values for the sham and impact groups ranged from 0138-352 and 0.300—1.818
m‘le(x10“). These ranges are 3 to 25 times larger than the average range 0043-0. 142
measured for bovine cartilage (Mow, et al., 1989). Although these permeability values

are high, they do fall within the 0.05-1.95 range measured for human AC by Armstrong

 

102
and Mow (1987).

The test side aggregate modulus values ranged from 0521-0828 MPa for the
sham group, and from 0348-0728 MPa for the impact group. These ranges, like their
respective controls, are comparable to the range of average bovine values (Mow, et al.,
1989). The test side Poisson’s ratios ranged from 0.0-0.288 for the sham group. In the
impact group all Poisson’s ratios were 0.00. Like controls, these test side values are low
compared to the range of bovine values, and are closer in comparison to those values
calculated for human and porcine cartilage. The test side permeability values for the
sham and impact groups ranged from 0.103-1.101 and 0.2790792 m‘le(x10"‘),
respectively. These ranges, like controls, are higher than the average range of bovine
values but fall within the range of values measured for human cartilage.

Overall, aggregate modulus values were comparable to previously determined
bovine values. Control and test side Poisson’s ratios, in general, were low in
comparison to previously determined values. In fact in ten of the sixteen porous tests
a Poisson’s ratio of 0.00 was measured. This reflects the algorithms attempt to fit the
fast rate of relaxation occurring during the indentation tests. This high rate of relaxation
was also reflected in the large permeability values measured in both the test and control
patellae. No definitive increase in permeability was noted in the test side cartilage of the
impact group as anticipated. This lack of change in the surface permeability of the test

side cartilage may be due to the small number of observations (n = 2).

PE] 'Vi l

The mechanical parameters (Gu, Or and 1;) calculated for the porous indenter test

 

103

are presented in Table 37 and 38 in the Appendix. For discussion purposes the results
of the l and 2 second porous tests were combined.

Overall, the test and control values of Cu for both the sham and impact groups
were decreased by 50% or more compared with the average solid indenter values. This
is consistent with a priori predictions. Test and control values for Gr in general were
also below the average values of the solid indenter results. This decrease is contrary to
the equivalence between the porous and solid indented values which had been predicted.
As noted in the acute study, this may be due to the closer proximity of the porous test
sites to the area of fissuring, and/or the longer relaxation period available in the porous
tests. The small number of observations and large variations in flow viscosity values
made comparisons between the porous and solid indenter results difficult. No discrete

increasing or decreasing trends were noted.

W (Articular cartilage)

Two animals were selected from each synovitis study group for histological
analysis. These subjects were Chosen based on the results of their solid indenter tests.
The rabbits with results which most closely represented the overall mean values of the
group were chosen for analysis.

One of the four control patellae (n = 2 each from the sham and impact groups)
contained preexisting surface fissures. In the remaining three, the surface integrity was
smooth and healthy. The sham test patella, which corresponded to the fissured control
patella, also possessed preexisting pathological fissures. No impact fissures were visible

in either of the two test patellae from the impact groups. A discemable loss of PCs

104

could not be determined in any of the test side patellae as an effect of their treatment.
A preexisting loss of PGs was, however, observed in 3 of the 4 sham patellae. These
patellae also showed a loss of tidemark definition. Interestingly, in these 3 patellae the
pathology appeared to be limited to the medial facet. These pathologies were similar in

appearance to those shown in Figure 40.

Males! (Synovial lining)

Isolating synovial samples for examination proved not to be a simple task. The
tissues collected in the first 2/3 of the specimens contained little or no trace of synovial
lining. These specimens were taken from the tissue attached to the femoral head after
removal of the patella. The specimens collected in the last third of the experiments were
removed from tissue attached to the lateral and medial edges of the recovered patella.
This method seemed to work better, but some specimens still contained no synovial

lining. The findings from these histologic sections are presented below.

Mam

Synovial samples were examined from two sham rabbits. Only small sections of
synovial lining were visible in the first rabbit examined. The medial and lateral control
joint specimens were similar in appearance. The synovial lining on both sides consisted
of a single layer of cells. No signs of inflammation such as hypertrophy or lymphocyte
invasion were noted. The lateral and medial test samples were identical to those of their
contralateral control.

The control sections from the second animal contained no synovial lining.

However, large areas of synovial lining were visible on the lateral and medial specimens

105

of the test joint. In comparison to the control sections from the first animal, segments
of the lining appeared to be thickened. These hypertrophic sections lay between stretches
of normal appearing synovial lining. These thickened areas were at times three or more

cells deep. No lymphocytes were noted in these hypertrophic areas.

W

Again two animals were histologically analyzed. The first rabbit contained no
synovial membrane in the medial control specimen. The small section of synovial lining
which was present on the lateral side appeared to be normal.

Both lateral and medial test specimens from the first rabbit contained synovial
membrane. One area on the medial side appeared to be thickened. On the lateral side
two villi which appeared to be detached from the main body of tissue appeared
hypertrophic. No leukocytes were observed.

The control specimens from the second animal contained no synovial membrane.
In the corresponding test specimens, only the medial side contained a small section of

synovium. This small section appeared normal.

PART II: (Synovitis created while restrained in the chair)

 

Six days after trauma induction the test limbs appeared normal. Upon opening

the test joints the synovial lining and fluid appeared normal compared with controls.

106

Prior to staining the cartilage with india ink, both test and control patellae appeared
healthy. Staining revealed surface anomalies in only two of the six animals. Again, the
pathology was typically present in both paired patellae.

11:12am

Like shams, no external differences were detected in the test side limb when
compared with their controls. Upon opening the joints the synovial lining and fluid
appeared normal in all the control and test legs. No observable fissures were initially
detected in the test side cartilage. Upon staining with india ink, fissures were detected

in two of the three test patellae. These fissures were similar in appearance to those

described in the Part I impact group.

W (Rigid indenter)

Sham grpup

From Table 39, it can be easily seen that only small differences exist between test
and control parameters. The Cu and Gr values were a mere 5.1% and 1.2% lower than
their controls, and n is essentially unchanged with an increase of only 0.6%. None of

these small differences were found to be significant.

1mm
Similar to the shams, the impact group also showed little change in the test side
parameters, Table 40. All three were only slightly decreased on the test side. Gu was

down 1.6%, Gr 3.0%, and ’1 12.3%. None of these differences were found to be

statistically significant.

107

MW (Porous indenter)

The biphasic material results for the sham and impact groups are presented in
Tables 41 and 42. As in Part I, the results of the 1 and 2 second porous tests are
combined for discussion in this results section.

The control side aggregate modulus (HA) values for the sham and impact groups
ranged from 0.3940672 and 0.5340669 MPa, respectively. These values are
comparable to the range of average bovine values, 0.47-0.90 MPa, measured by Mow,
et al., 1989. The Poisson’s ratios for the sham and impact groups ranged from 0.0-
0.015 and 0.0-0.116, respectively. These ranges are low compared to the average range
of 0.25—0.40 measured for bovine cartilage (Mow, et al., 1989), but are comparable to
the average values 0.08 and 0.14 determine for normal human and porcine cartilage
(Spilker, et al., 1992; Whipple, et al., 1989). The control side permeability values for
the sham and impact groups ranged from 0150-1003 and 0.223-l.410 m‘le(x10“).
These ranges are 1.1 to 10 times larger than the average range of 0.0430142 found for
bovine cartilage by Mow, et al., 1989. Although, these permeability values are high,
they do fall within the 0.05-1.95 range measured by for human AC by Armstrong and
Mow (1987).

The test side aggregate modulus values ranged from 0626-0917 MPa for the
sham group, and 0494-1. 171 MPa for the impact group. These test side ranges, like
their respective controls, are comparable to the range of average bovine values (Mow,
et al., 1989). The test side Poisson’s ratios range from 0.0-0.101 for the sham group

and 0.0-0.166 for the impact group. Like the control side values, these test side ratios

108

are low compared to the range of bovine values, but are comparable to the values
calculated for human and porcine cartilage. The test side permeability values for the
sham and impact values ranged from 0262-0688 and 0.603-0.1.05 m‘le(x10“),
respectively. These tests ranges, like controls, are higher than the average range of
bovine values but fall within the range of values measured for human cartilage.
Overall, aggregate modulus values were comparable to previously determined
bovine values. Control and test side Poisson’s ratios, in general, were low in
comparison to previously determined values. In fact in 8 of the 14 porous tests (which
had values) a Poisson’s ratio of 0.00 was measured. This reflects the algorithms attempt
to fit the fast rate of relaxation occurring during the indentation tests. This high rate of
relaxation is also reflected in the large values of permeability measured on both the test
and control sides. No definitive increase in permeability was noted on the test side of
the impact group. This lack of change in permeability on the test side may be due to the

small number of observations.

1 . . l . R
The mechanical parameters (Gu, Or and 1)) calculated for the porous indenter test
are presented in Table 43 and 44 in the Appendix. For discussion purposes the results
of the 1 and 2 second porous tests are combined.
Overall, the test and control Gu values for both the sham and impact groups were
decreased by 50% or more compared to the average solid indenter values. This is
consistent with the a priori prediction. Test and control values for Or, in general, were

also- below the average values reported for the solid indenter. This decrease is contrary

109

to the equivalence between the porous and solid indenter results which had been
predicted. As in Part I, this decrease may be due to the closer proximity of the porous
test sites to the area of fissuring, and/or the longer relaxation period available to the
porous tests. The small number of observations and large variations made comparisons
between the porous and solid indenters flow viscosity values difficult. No discrete

increasing or decreasing trend were noted.

W (Articular cartilage)

Two animals were selected from each synovitis study group for histological
analysis. These subjects were chosen based on the results of their solid indenter tests.
The rabbits with results which most closely represented the overall mean values of the
group were chosen for analysis.

The surface integrity of 3 out of 4 of the control patellae (n=2 each from the
sham and impact groups) were smooth and healthy. The fourth control patella contained
3 pathological fissures. The surfaces of the sham group test patellae were all smooth
and healthy. Impact fissures were, however, noted in one of the two test side patellae
of the impact group. These fissures began at a 45° angle to the articular surface and
extended down into the middle zone of the cartilage. No discemable loss of PG content
was detected in the test side cartilage as an effect of treatment in any of the sham or
impact patellae. However, a pathological loss of P65 was noted in three of the four
sham patellae, as well as a loss of tidemark definition. These preexisting pathologies

appeared to be limited to the medial facet of these three patellae.

 

 

110
MEI—98! (Synovial lining)

As in Part I, the specimens collected in the first 2/3 of the synovitis study
contained little or no trace of synovial lining. These specimens were taken from the
tissue attached to the femoral head after removal of the patella. The specimens collected
in the last third of the experiments were removed from tissue attached to the lateral and
medial edges of the recovered patella. This method seemed to work better, but some
specimens still contained no synovial lining. The findings from these histologic sections

are presented below.

EMBLEM

The synovial lining of two rabbits were examined. The medial and lateral
specimens from the control joint of the first rabbit appeared normal. The synovial lining
consisted of a single layer of cells. No signs of inflammation (hypertrophy or
leukocytes) were noted.

Of the specimens taken from the test joint of the first rabbit, only the medial side
contained synovial lining. Several large villi were observed which contained
hypertrophic areas three or more cells thick. These villi were not observed in the medial
specimen of the control joint. No leukocytes were visible in these thickened synovial
villi.

The lateral and medial specimens from the control joint of the second rabbit
contained only small sections of synovial membrane. In these sections the lining
appeared to be normal. On the test side both the lateral and medial specimens showed

thickened areas. No signs of leukocyte invasion were noted.

111

Im ro

Of the three animals in this group two were histologically examined. The control
specimens from the first rabbit contained small sections of synovium on both the lateral
and medial sides which appeared normal. The corresponding test specimens, however,
did contain areas on both the lateral and medial side which appeared to be thickened.
No leukocytes were observed. The control specimens from the second rabbit examined
contained synovial lining only in the medial specimen. This small section of synovial
lining appeared normal. The corresponding test specimen from this rabbit also contained
synovial membrane only in the medial specimen. This section did display a thickened

cellular layer. No leukocytes were noted in the vicinity of this hypertrophy.

h 'r Im In ' n

The ANOVA detected an effect of placing the rabbits in the restraining chair
while inducing synovial trauma. This difference was detected in both the test and control
Gr values, P = 0.006 and P = 0.018 respectively. That is to say that a significant
difference was detected between the combined Gr values from the Part I sham and impact
groups and those of the combined sham and impact groups from Part 11. Overall the
combined Gr values from Part II were on average 17.4% greater than those from Part
I. The contrast tests performed following the AN OVA revealed that the effect of the
restraining chair was significant only between the Part I and II sham groups control
values (P = 0.041) and the impact groups test values (P = 0.007).

The ANOVA also detected an effect of impact trauma on Or values. Contrast

tests performed following the ANOVA revealed that this difference was statistical only

112

between the sham and impact groups in Part I (P=0.024). The impact Gr values were
lower by 26.9%. This difference is similar to that seen between the low and high level

impact groups in the open joint study.

DISCUSSION

Both the acute and synovitis studies arose out of questions raised by the results
of the open and closed joint studies. These questions were based on differences in the
mechanical response of cartilage as of function of both impact trauma (fissured vs.
unfissured) and model type (open vs. closed joint). Differences in mechanical response
were measured between the low and high level impact groups at early time points in the
open joint study. At low impact levels a gradual decrease in stiffness was observed over
a period of two weeks, this was followed by a gradual recovery back to control levels
by one year. At high impact levels an acute drop in stiffness was noted one day
following induction trauma. By one year, although stiffness values had returned to
normal, a gradual decreasing trend was observed that suggested a path of long term
degeneration. The differences in stiffness response between the low and high level
groups were attributed to impact fissures present in the cartilage of the high level groups
(Ide, 1992).

Upon completion of the open joint study, a second year long impact study was
carried out. During this study patellar trauma was performed under closed joint
conditions. Results similar to those observed in the open joint study were anticipated.
However, although cartilage fissures were again present in the high impact specimens,
differences in stiffness responses between the two impact levels were not observed. In

fact, prior to one year post-impact none of the low or high level impact groups in the

113

114

closed joint study showed any substantial decrease in cartilage stiffness. It was
hypothesized that differences between the open and closed joint results were either caused
by the small sample size of the open joint study, or by a surgical synovitis induced by
arthrotomy. While the effects of surgical synovitis are believed to account for some of
the differences between the open and closed joint results, it does not explain differences
in stiffness response between the low and high level impact groups within the open joint
study. Upon completion of the open and closed joint studies the following questions
remained: (1) can impact fissures alter the mechanical response of cartilage, (2) are the
degenerative effects of fissures further enhanced in the presence of a surgical synovitis,
and (3) if synovitis does enhance the degeneration of cartilage, can it be created under
closed joint impact conditions without the use of invasive arthrotomy? To answer these

question the acute and synovitis studies were developed.

mm

The acute study had two main objectives. The first was to measure the isolated
effects of fissuring on the mechanical response of the cartilage. It was hypothesized that
disruption of the collagen network created by impact fissures would result in a decrease
in cartilage stiffness. It has been previously reported that the integrity of the cartilage
surface layer plays a key role in creating resistive forces during the loading phase of
indentation by maintaining fluid pressure in the tissue (Setton, et al., 1993). It was
hypothesized that damage to the surface layer, created by the presence of impact fissures,
would result in an increase in permeability. To quantify the effects of fissuring on

cartilage permeability, biphasic theory was added to the analysis procedure.

115

To isolate the effects of fissuring on the mechanical response of cartilage a time
zero, high impact group was used. The patellae were impacted following arthrotomy and
insertion of pressure film to simulate the open joint study. By removing the patellae
immediately following the impact procedure transient post-surgical softening was
avoided. Although the average peak impact loads for the acute study were 12 and 22%
higher than those reported in the background studies, the impact pressure profiles and
fissure sites suggest that the impact conditions within the joints were similar to those seen
in the background studies. Histologic analysis of the time zero group detected no
discemable loss of PGs in the test side cartilage as an effect of the impact procedure.
However, the mechanical tests performed on the time zero, impact group did detect a
significant decrease (11.5%) in the unrelaxed shear modulus (i.e. instantaneous stiffness)
of the test side cartilage. The instantaneous stiffness of cartilage has been shown to
depend on the integrity of the collagen network (Mizrahi, et al., 1986; Parsons and
Black, 1987; Jurvelin, et al., 1988). This suggests that a disruption in the collagen
network, created by the impact fissures, caused the measured decrease in instantaneous
stiffness.

The exact affect of fissuring on the permeability of the cartilage was not
completely obvious. The small number of biphasic observations in each acute group
(n=4) were further reduced in the time zero group by the inability of the biphasic
algorithm to fit the experimental data. This made it impossible to draw a decisive
conclusion about the effects of fissuring on the permeability of cartilage. In the two (test
vs. control) comparisons that were available, however, the test side permeability values

were higher. This suggests that impact fissures may increase the permeability of

116
cartilage.

The second goal of the acute study was to determine whether an interaction exist
between the cartilage fissures and surgically damaged synovium which might enhance
their individual degradation affects. It has been suggested that this type of interaction
may occur (Walker, et al., 1991). Of particularly interest were the effects of such an
interaction one day post-surgery, since large differences in stiffness were noted at this
time point between the high and low impact groups of the open joint study. To
determine the interactive effects of fissuring and surgical synovitis it was necessary to
first define their individual effects. The effects of fissuring were determined in the time
zero impact group. However, the effects of surgical synovitis alone on the mechanical
response of cartilage were yet unknown. To determine these effects the one day, sham
group was devised. This group exposed the test cartilage to a surgieally damaged
synovium for 24 hours before mechanical tests were performed. This group was
analogous to the 1 day, low impact group of the open joint study. That is to say that
both these groups exposed unfissured patellar cartilage to surgical synovitis for 24 hours
before testing. The results of the mechanical tests performed on the sham specimens
revealed that the test side relaxed modulus (Gr) had been significantly reduced by
(13.2%) due to the 24 hour exposure to the damage joint environment. This reduction
was shown to be significantly larger than the decrease in Gr values measured in the time
zero group. As previously mentioned the equilibrium stiffness of cartilage has been
shown to be directly related to the PG content of cartilage (Parsons and Black, 1987;
Jurvelin, et al., 1988). The histological analysis of the one day, sham group was unable

to detect any loss of PGs as an effect of the surgical exposure. This may have been due

117

to the small number of histologic comparisons from each of the acute study groups
(n=2). Another possibility may be that surface PGs were not removed but were altered
by exposure to blood or enzymes from the damaged synovium (Ghadailly, et al., 1983;
Walker, et al., 1991). It has been suggested that not only the quantity, but also the
quality, of P63 can affect the load carrying capabilities of cartilage (Jurvelin, et al.,
1988). Changes in the functional structure of PCs caused by surgical damage may
account for the drop in Or values measured in the one day, sham group. Interestingly
a decrease in Or was not observed in the one day, low impact group of the open joint
study. This may have been due to the small number of observations made at this time
point (n=3).

The biphasic results for the one day, sham group, although based on a small
number of observations, showed that in 3 out of 4 cases the test side permeability was
increased. It has been suggested that a partial cleavage of PGs may decrease the
viscosity of the cartilage matrix allowing it to flow under stress (Parsons and Black,
1987). This may explain the increased permeability trend, as well as the decreased flow
viscosity values measured on the test side.

With the individual effects of fissuring and surgical synovitis determined these
two factors were next combined for analysis in the one day, high impact group. This
group was identical in procedure to the 1 day, high impact group of the open joint study.
The mechanical test results of this group showed a significant decrease in the modulus
values (On by 21.9% and Gr by 14.3%) as well as the flow viscosity (23.4%). Although
the decrease in Or was not as substantial as those measured at one day in the open joint

study, the overall decreasing trends were similar. A visual examination of the results

118

from all three acute study groups suggests that the mechanical response of the one day,
high impact group was a combination of the individual effects of fissuring and synovitis.
However, the reductions in Gu and 17 were larger than those measured in the time zero,
impact and one day, sham groups. This suggests that an interaction between the fissures
and damaged synovium may have taken place. It has been suggested that fissured
cartilage may be more permeable to cytokines and enzymes present in synovial fluid
(Walker, et al., 1991). Although, histology was unable to detect a loss of PGs on the
test side (even adjacent to fissured areas), it is possible that an alteration in PG structure,
as mentioned previously, may have occurred. The number of P63 altered by cytokine
and enzyme exposure may have been increased by an increase in permeability created by
the presence of impact fissures. Although this interaction could not be statistically
proven, such a phenomena may have contributed to the larger decreases in Gu and I)
measured in the one day, impact group.

The biphasic results from the one day, impact group demonstrated an increase in
test side permeability in all 3 available comparisons. These increases in permeability
also seem to support the decrease measured in flow viscosity. However, due to the small
number of observations, it was not apparent whether these increases in permeability were

greater than those observed in the time zero impact and one day sham groups.

n vi '
The primary objective of the synovitis study was to determine whether a synovial
inflammation, similar to that produced by arthrotomy, could be created by mechanical

trauma to the synovial tissue lining the joint. The results of the acute study suggest that

119

the presence of synovitis can have a degrading affect on the mechanical response of AC.
The acute study also suggests, although not statistically, that if cartilage fissures are
present simultaneously with a synovitis that their degenerative affects may be enhanced.
If a synovitis can be induced experimentally through blunt impact, then its possible that
such inflammation may also occur following a natural impact situation. Loading of the
cartilage during this softened state may act to further degrade mechanical properties and
accelerate the degenerative processes.

The original synovitis sham group was developed to investigate the possibility of
inducing a synovitis through the use of blunt trauma. This trauma was carried out on the
synovial tissue directly medial and lateral to the patella. This synovial trauma was
performed without the use of the restraining chair (Part I). For six days following
synovial trauma the rabbits were exercised to aggravate the injured tissue in order to
elicit and/or maintain a synovial response. Histologic analysis of the cartilage revealed
no change in the PG content of the test side patellae. However, examination of the
synovial lining revealed areas that appeared to be thickened (i.e. hypercellular) in the test
side joints. This suggests that a traumatic synovitis was produced as a result of blunt
impact. The results of the mechanical test showed that none of the test side parameters
(Gu, Gr and II) had been decreased as anticipated. In fact they had all increased
(Gu=18.3%, Gr=10.9% and n=25.3%). These results are similar to those reported
by Altman, et al., 1984, and Myers, et al., 1986, in canine cartilage following ACL
transection prior to any signs of surface fissuring. Altman, et al., measured increases
in the shear moduli (Gu, Gr) and retardation spectrums (creep equivalent of H(-r)) in the

cartilage during this time period. These increases in stiffness were measured

 

120

concurrently with a decrease in the depth of creep indentation and an increase in cartilage
swelling. Altman, eta1., attributed these increases to tissue edema. A similar swelling
may have occurred in the synovitis sham group. This edema may have resulted from
enzymatic exposure. Collagenolytic activity in cartilage has been shown to be directly
related to the degree of synovial inflammation (Pelletier, et al. , 1985). It is possible that
in response to the blunt trauma cytokines and/or enzymes may have been released from
the damaged synovium. Typically the large negative charge associated with the P63
prevents the penetration of these degradive substances into the cartilage (Okada, et al.,
1992). However, these substances may be able to penetrate the surface layer of the
cartilage, where collagen content is high, but PG content is low. This may have allowed
enzymatic weakening of the superficial layer without compromising the overall tensile
strength of the collagen network. Under the strain of daily exercise this weakened
superficial layer may have been loosened. This disruption of the surface collagen may
have allowed underlying PGs to imbibe more water (Donahue, et al., 1983). This
swelling in turn would create increased tension in the collagen network and hydrostatic
pressure in the interstitial water (Maroudas, et al., 1979; Mizrahi, et al., 1986). These
increases in collagen tension and fluid pressure would account for the increases measured
in stiffness and flow viscosity measured in the sham group.

No definitive increase or decrease in test side permeability was apparent from the
biphasic results for the sham group. These biphasic results are similar to findings by
Myers, et al., 1986. As previously mentioned, like Altman, et al., 1984; Myers, et al.,
also measured an increase in canine cartilage stiffness following ACL transection prior

to the development of surface disruptions. However, the results of their confined creep

121

tests were unable to detect any changes in permeability of the intact cartilage.

In the second group from the original synovitis study (Part I) synovial trauma was
again induced without the use of the restraining chair. Five minutes after synovial
trauma, however, the rabbits underwent a high level patellar impact. This group was
used to determine whether or not the presents of fissures would altered the mechanical
responses seen in the sham group. A histological examination of the test side synovial
lining revealed thickened areas (hyperplasia) in one of the two animals analyzed.
Histological examination of the AC, however, did not reveal a loss of PGs as a result of
the synovial trauma and/or patellar impact. The solid indenter test results revealed that
all three mechanical parameters had been significantly reduced (Gu=29.1%, Gr=17.0%
and n=25.3%). These decreasing trends were similar to those reported in the one day
impact group of the acute study. These decreases, although less in intensity, were also
similar to those seen in the six day, high impact group of the open joint study. The
similarity between these groups suggests that the affects of synovial trauma on the AC
were similar to, although less severe than, those created by arthrotomy. The differences
between the impact and sham groups in Part 1 suggests that synovial trauma may have
an increased degenerative effect on cartilage when fissures are present. Upon
comparison with the results of the time zero, impact group from the acute study, it would
appear that the decrease in instantaneous stiffness measured in the synovitis impact group
is probably an effect of the cartilage fissures. These fissures may have increased the
permeability of the cartilage allowing synovial enzymes and cytokines to penetrate deeper
into matrix of the cartilage. This increased penetration by enzymes and cytokines may

have allowed further degradation of the cartilage matrix, especially in the fissured regions

122

(Walker, et al., 1991). A scenario such as this may account for the secondary decreases
measured in Gr and n.

The biphasic results of the synovitis impact group (Part 1) revealed no changes
in the permeability of the tests cartilage. The reasons for this lack of change are not
quite clear. These results appear to be contradictory to the increased cartilage
permeability measured in the fissured specimens of the acute study. The inability to
measure a decrease may be due to the small number of observations (n=2 rabbits).
Interestingly, however, Myers, et al., 1986, was also unable to detect any changes in the
permeability of canine cartilage following ACL transection at later time points when
cartilage fibrillation was noted in conjunction with decreases in stiffness. Myers, et al. ,
provided no explanation for these results.

The same two groups, sham and impact, were used in Part II of the synovitis
study. Unlike Part I, however, synovial trauma was induced while the rabbits test leg
was held in hyperflexion by the restraining chair. The solid indenter results of both the
sham and impact groups in Part 11 demonstrated no significant decrease in Gu, Gr or 17
in the test cartilage. The increases which had been measured in the sham group during
Part I were also not detected in Part II. From these results it would appear that the
synovial response was somehow altered by placing the rabbit in the restraining chair
during trauma induction. The exact cause of this altered response is not clear. Although
some areas of hyperplasia were noted in the synovial lining of the test joints, the extent
of synovial damage is unknown. Spacial constraints imposed by the sides of the
restraining chair made the delivery of the synovial trauma more difficult. The inability

to make a normal swing with the hammer may have reduced the amount of damage

123
imparted by each tap. Another possibility is that a damage shielding effect may be

created by hyperflexion of the knee. In Part I the synovial trauma was carried out while
the leg was manually held at 45 degrees of flexion. During impact, the synovial tissue
was relatively free to deform. It is likely that under these conditions the synovial lining
was not only damaged by compressive forces, but may have suffered damage due to
stretch deformation. When hyperflexed, as in Part H, the synovial tissues surrounding
the knee are stretched tightly over the underlying bone. Striking the synovial tissue
under this scenario may have allowed the impact force to be more directly absorbed by
the underlying bone, decreasing synovial damage due to excessive stretch. This may
have resulted in a decreased level of synovial trauma which was not strong enough to
alter the mechanical properties of the cartilage. This reduction in synovial damage may
explain the lack of change measured in the sham group, however, it does not explain the
lack of stiffness change in the impact group. This is probably due to the small number
of observations in this group (n= 3 rabbits) and the fact that only one of the three test
patellae was fissured.

As in Part I, the biphasic results of Part II also did not demonstrate any definitive
increase or decrease in the test side permeability in either the sham or impact group.
This maybe due to the small number of observations in each group. Especime the
impact group where the small sample size was further reduced by the inability of the

algorithm to fit the experimental data from two of the porous tests.

The elastic and Viscoelastic results of the porous tests were purposely excluded

from the discussion of the acute and synovitis study due to their lack of additive

124

information. Overall porous Gu values were reduced approximately 50% compared to
the average values calculated for the solid indenter tests. This reduction was anticipated.
The relatively slow indentation rates (1 and 2 seconds) used for the porous tests allowed
cartilage relaxation during loading resulting in low peak forces (P). From equation (1)
it is apparent that a decrease in peak load would directly results in a decrease in Gu.
The porous Gr values were also decreased below solid indenter values. This change was
not initially expected. It was anticipated that at equilibrium the solid and porous results
would agree, just as the elastic solution of Hayes, et al., 1972, and the biphasic stress-
relaxation solution of Mak, et al. , 1987, agree at equilibrium for v=v,. Two possible
reasons have been proposed to explain these differences. The first being that both the
solid and porous indenter Gr values were determined at different relaxation times, 150
and 1000 seconds, respectively. According to Mow, et al. , 1989, both of these times are
less than the average time required for complete cartilage relaxation. Therefore, the
longer relaxation time associated with the porous test should allow a larger amount of
relaxation resulting in smaller relaxation loads. Based on previous comparison tests
carried out in our lab, this may account for approximately 10% of the decrease in the
porous Gr values. However, the porous Gr values were typically more than 10% lower
than the solid indenter values. A second contributing factor may be due to the permeable
boundary conditions created by using the porous indenter. Spilker, et al., 1992,
performed a finite element comparison between the stress-relaxation behavior of cartilage
indented by both a porous and solid indenter. Under similar loading conditions the
reaction forces generated in cartilage by the solid indenter were greater than those of the

porous indenter at all time points along the response curves, except equilibrium. This

125

difference was due to larger fluid pressure within the cartilage created by the
impermeable surface condition of the solid indenter. Therefore, at time points short of
true equilibrium the calculation of Gr for the solid indenter would be greater than that
for the porous indenter due to larger resistive loads. The small number of observations
and variations in the flow viscosity values made comparisons with the permeability values
difficult. It could not be determined whether or not increases in permeability

corresponded with decreases in flow viscosity as hypothesized.

SUIVIMARY

The current investigations rose out of questions generated by the differing results
of the previous open and closed joint studies. The acute study was devised to address
two main objectives. The first objective was to define the effects of impact induced
fissures on the mechanical behavior of AC. The second goal was to determine whether
the degenerative affects of these fissure were enhanced by the presence of a surgical
synovitis. Otherwise stated, to determine whether or not an interaction exist between the
fissures and synovitis. The results of the acute study revealed that the presence of impact
fissures can significantly reduced the unrelaxed shear modulus of AC. This reduction
in stiffness is probably due to a disruption of the collagen network created by the
fissures. The results also revealed that by exposing the cartilage to a surgical damaged
joint environment for 24 hours a significant reduction in the relaxed shear modulus can
be created. Although histological analysis revealed no loss of PGs, this decrease in
equilibrium stiffness may be the result of a partial breakdown of the cartilage matrix
invoked by exposure to enzymes and/or blood from the surgically damaged synovium.
Statistically an interaction between the two factors, fissures and surgical synovitis, could
not be proven. However, exposure of fissured cartilage to a surgical synovitis did result
in an overall significant decreases in shear moduli and flow viscosity. The presence of

fissures may have enhanced the affects of the cytokines and enzymes by allowing deeper

126

127

penetration into the cartilage.

It was hypothesized that by loading the cartilage (e. g. exercise) during the
softened state created by synovitis, the rate of cartilage degeneration could be
accelerated. The synovitis study was developed to investigate the possibility of inducing
a synovitis by mechanical means under closed joint conditions. The synovitis was
induced by blunt trauma to the synovial tissue surrounding the patella. In Part I,
synovial trauma was induced while the rabbits hind limb was hand held at 45 degrees of
flexion. Following synovial trauma, half of these rabbits received a high level patellar
impact to induce cartilage fissures. The rabbits were exercised to maintain and provoke
a synovial response. Histological examination of the traumatized synovial lining revealed
areas of hyperplasia. Although histological analysis was unable to detect any loss of
cartilage proteoglycans in the traumatized joints, the results of the indentation tests did
reveal mechanical changes in the test cartilage. In the sham group, which received only
synovial trauma, the intact test cartilage showed an increase in shear moduli (Gu and Gr)
and flow viscosity (1;). These increases were believed to be an effect of edema created
by the exposure of the intact cartilage to a trauma induced synovitis. Five of the six
rabbits which received patellar impact in addition to synovial trauma had observable
cartilage fissures. The indentation results of this group revealed significant decreases in
all three mechanical parameters (Gu, Gr and fl)- These reductions in stiffness and
viscosity were attributed to both the disruption of the collagen network created by the
fissures, and exposure of the cartilage to the damage synovial environment. These
fissures are believed to have increased the permeability of the cartilage surface layer.

This increased permeability may have allowed deeper penetration by enzymes and

128

cytokines released from the damage synovium, thereby enhancing their degradive affects
on the cartilage.

Two study groups were also used in Part II of the synovitis study. In both groups
the rabbits received synovial trauma while their hind limb was held in hyperflexion by
the surgical restraining chair. In addition to synovial trauma, one of the two groups also
received patellar impact to induce cartilage fissures. Histological analysis revealed areas
of hyperplasia in the traumatized synovial linings. The mechanical results of both the
impact and sham groups revealed no significant changes in the stiffness or viscosity of
the test cartilage. This lack of change may be the result of a reduction in the extent of
synovial trauma created by hyperflexion of the test limb. Hyperflexion stretches the
synovial membrane tightly over the underlying bone. In this stretched state the brunt of
impacts may have been directly transmitted to the bone diminishing the extent of synovial
damage. The spacial constraints created by the use of the restraining chair prevented full
length hammer strokes further reducing the ability to induce synovial trauma. In the
impact group a lack of cartilage fissuring (1 out of 3) was also believed to be responsible

for failure to measure a reduction in the unrelaxed modulus.

RECOMMENDATIONS

The results of both the acute and synovitis study suggests that the load carrying
capabilities of impact fissured cartilage can be significantly reduced by exposure to a
synovitis. The synovitis study further suggested that a traumatic synovitis can be created
by mechanical means under closed joint conditions. Extraneous loading of the cartilage
in this softened condition may enhance its degeneration. Future long term, closed joint
studies should include further investigation into the use of synovitis as a means of
accelerating cartilage degeneration. To more closely simulate a natural impact scenario
a trauma method should be developed to induce both cartilage fissuring and synovial
damage simultaneously. This may require redesign of the restraining chair in order to
prevent possible damage shielding effects created by hyperflexion of the test limb. In
addition to the histological analysis used in the synovitis study, future studies should also
include biochemical determination of synovial enzyme and cytokine levels, and cartilage
water content. This will provide a means of verifying the existence, degree and effects

of synovial inflammation.

129

APPENDIX

 

 

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