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This is to certify that the

thesis entitled

AN IN VITRO MODEL TO STUDY THE EFFECTS OF
STRESS-DEPRIVATION ON THE MATERIAL PROPERTIES
OF CONNECTIVE TISSUE: A BIOMECHANICAL AND
HISTOLOGICAL ANALYSIS

presented by

Amardeep Kaur Hoonjan

has been accepted towards fulfiilrnent
of the requirements for

Master of Sci. Biomechanics

 

 

degree in

    

Major professor

Date 8‘5; 93

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AN IN VITRo MODEL To STUDY THE EFFECTS OF STRESS-
DEPRIVATION ON THE MATERIAL PROPERTIES OF CONNECTIVE
TISSUE: A BIOMECHANICAL AND HISTOLOGICAL ANALYSIS.
37

Amardeep Kaur Hoonjan

A THESIS

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

MASTER OF SCIENCE

Department of Biomechanics

l 993

ABSTRACT

AN IN VITRO MODEL TO STUDY THE EFFECTS OF STRESS-
DEPRIVATION ON THE MATERIAL PROPERTIES OF CONNECTIVE
TISSUE: A BIOMECHANICAL AND HISTOLOGICAL ANALYSIS

37

Amardeep Kaur Hoonjan

Immobilization is a widely used clinical method of rehabilitation of soft tissue
injury. However, it has many disadvantages including alterations in the material
properties and tissue atrophy. While experimental studies have examined some of the
deleterious effects of immobilization, these in vivo investigations are limited by the
inability of models to be standardized. The goal of this study was to evaluate the effects
of immobilization on the material properties of connective tissue using an in vitro model.
The ability of cyclic loading to reverse the immobilization-induced alterations in

material properties of the tissue was also investigated.

Biomechanical and histological properties degenerated with increasing periods of
immobilization. Cyclic loading of the tissue minimized these changes. Cyclic loading of
previously immobilized tissue was able to arrest the detrimental immobilization-related
alterations. The decrease in material properties seen with immobilization is thought to

be a cell-mediated event, secondary to a lack of mechanical Stimulus.

ACKNOWLEDGMENTS

The author wishes to express sincere appreciation to Dr. Steven Arnoczky, for
his constant encouragement and enthusiasm, as well as his guidance throughout this
research experience.

To jean Kilfoyle, for her wonderful advice, and all of her hours spent on
histology. To Keri Gardner, for her patience in teaching me all about tissue culture.

To my fellow graduate students, Jeff Robbins and Dennis Shelp, for their many
hours spent in getting me started with all of the mechanics involved in this study. To
Clifford Beckett, for his assistance with the computer programming.

To my graduate committee: Dr. Steven Arnoczky, Dr. Roger Haut. and Dr.
Herbert Ross. Thank you for your time spent in reviewing and commenting on my
work.

Most of all, to my wonderful parents jaswant and jasjit, for all of their love and
encouragement. Thank you for instilling the importance of education within me.

This research was supported by the Laboratory for Comparative OrthoPaedic
Research and the Wade 0. Brinker Endowed Chair in the College of Veterinary

Medicine at Michigan State University.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABI FS vi
LIST OF FIGURES vii
CHAPTERS:
I. INTRODUCTION l
Hypotheses (H l' H2, H3) 3
ll. LITERATURE SURVEY S
Stress-Deprivation '5
Mobilization/Remobilization l l
Common Experimental Models
and Procedures I4
"I. MATERIALS & METHODS l9
Tissue Evaluated l9
Tissue Culture Techniques 24
Tissue Preparation 25
Biomechanical Testing
of Tendon: 16
Histology 40
Data Analysis 4|
Statistical Analysis 44
IV. RESULTS 45
H I: Stress-Deprivation Study 45
H2: Mobilization Study 57
H3: Stress-Deprivation!
Remobilization Study 6|

 

 

 

 

V. DISCUSSION 64
Future Research Directions 70
Conclusions _7 I
VI. APPENDIX 72

 

VII. LIST OF REFERENCES

74

 

Table I.

Table 2.

Table 3.

LIST OF TABLES

Groups

 

 

Page
Tendon Summary for To and Stress-Deprived
28
Animal Summary for Loaded (Mobilized &
Remobilized) Tendon Groups 33
Summary of the Elastic Modulus Values for Tendons
in the Stress-Deprivation Study 49

 

vi

Figure I:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

LIST OF FIGURES

 

Okuda and co-workers (58)

 

 

media, prior to incubation

 

in tissue culture

 

 

loading device

 

 

Page

A schematic depiction of the avascular and vascular regions
of the canine flexor digitorum profundus tendon (2|) 20
Anatomical drawing of the ventral (top) and lateral views
(bottom) of the canine flexor digitomm profundus
and the flexor digitomm superficialis tendons, with
reference to the distinct regions as determined by

2|
Schematic depiction of the canine flexor digitorum
profundus tendon, with reference to the Okuda regions 23
Photograph showing the freshly-trimmed canine FDP
tendons in the tissue culture dish with culture

27
A schematic depicting the cyclic loading apparatus used
for mobilization and re-mobilization of the POP tendons

34
Photograph of tendons fastened to the cyclic

35
Schematic depicting of all points at which all measurements
were taken on the FDP tendon prior to mechanical testing.
All measurements were taken with an approximate load
of 0.098 N (l0 grams) on the tendon 38
Photograph of the FDP tendons fixed in grips within
the bath apparatus prior to tensile testing 39
Sample load-deformation curve indicating regions
at which stiffness (K) measurements would be

42

taken for further calculations and analysis

 

vii

Figure IO:

Figure l I:

Figure l2:

Figure l3:

Figure l4:

Figure l5:

Figure l6:

Figure l7:

Figure l8:

Figure l9:

Figure 20:

Load-Deformation curve of the time zero control
canine FDP tendon. Depicts the classic load-
deformation response of the tendon

47

 

Histogram depicting the time-dependent changes
in tensile modulus (E) with increased periods of

 

stress-deprivation

Histological appearance of H&E stained time zero
control canine FDP tendon, as seen with light
microscope (at 40X magnification)

Histological appearance of H&E stained canine
FDP tendon after 2 weeks of stress-deprivation.
Observed with light microscope at 40 X magnification

48

5|

52

 

Histological appearance of H&E stained canine FDP
tendon after 4 weeks of stress-deprivation.
Observed with light microsc0pe at 40X magnification

S3

 

Histological appearance of H&E stained canine FDP
tendon after 6 weeks of stress-deprivation.
Observed with light microsc0pe at 40X magnification

Histological appearance of H845 stained canine FDP
tendon after 8 weeks of stress-deprivation.
Observed with light microsc0pe at 40X magnification

Histogram depicting the effect of freeze-thaw
technique with subsequent stress-deprivation
on the tensile modulus of canine FDP tendon

Histology (H&E stained) of canine FDP tendon
following freeze-thaw technique and 2 weeks stress-
deprivation. Complete absence of cellularity is
seen. (Observed with light microscope at 40 X
magnification

Histogram depicting the variations seen between
tendon groups stress-deprived for 2 weeks and
cyclically loaded for 2 weeks

54

“fl
U1

56

58

 

Histological section of canine FDP tendon cyclically
loaded (with 50 gram weight) for 2 weeks in tissue
culture. Wewed with light microscoPy at 40 X

magnification

 

viii

6O

Figure 2|:

Figure 22:

Figure 23:

Histogram comparing the effects of 2 weeks
cyclic loading. 2 weeks stress-deprivation.

2 weeks stress-deprivatioan weeks cyclic loading.
and 4 weeks stress-deprivation treatments on the

62

 

tensile modulus of the canine FDP tendon

Histological section (H&E staining) of canine FDP
tendon deprived of stress for 2 weeks and then
cyclically loaded (with 50 gram weight) for

2 weeks in tissue culture. Viewed with

light microscope at 40X magnification

63

 

The tensile modulus of the 2 weeks and 4 weeks
stress-deprived controls. Column graph

depicts the effects of absence of cells (freeze/thaw
group) on the tensile modulus of stress-deprived tendon

10‘
U1

I. INTRODUCTION

Immobilization or some form of physical support to minimize load or stress has,
on various connective tissues, been a mainstay of therapy for many musculoskeletal
diseases and injuries. By eliminating stress, the injured tissue is protected from any
disruptive forces, enabling the weakened area to heal, mature and strengthen.
Immobilization of the injury site helps to prevent the dislocation of wound edges.

protects the deveIOping tissues. and prevents disruption of new blood vessels.

Despite the need for protection of the injury site, some stress is required to
allow for effective wound maturation and subsequent increase in strength. The
relationship between stress and connective tissue is best described by Wolff‘s law (78)
which essentially states, '...that the form of tissue being given, the tissue elements will
increase or decrease themselves to reflect changes in the direction or magnitude of the
applied stress.” Thus, if a tissue is not subjected to normal mechanical stresses, because
of partial or total immobilization, the strength of the tissue diminisheszov 40. 4" 56. 64
82’ 84. Therefore, a dilemma exists in determining how much stress is required for

maintaining tissue homeostasis, without jeOpardizing wound healing.

The detrimental effects of immobilization (stress-deprivation) on connective
tissue have been documented extensively by many experimental and clinical studiesz' 3'
6. 8, l I,lS, l7, l8, 20, 24. 30. 32, 34~36, 40, 4|, 43, 44, SI. 53-56, 62, 63. 65, 66, 72-76.
80’ 82’ 84. These include atrophy 20’ 24' 40' 4" 56’ 32. 82, change in microstructure 2'
20. 32. 62. 75- 75, decrease in extracellular matrix 20. 34- 75’ 83, loss of water

contentzo. 34. 75. 83. and alterations in the material properties of the tissue 3. 6. l l.
”‘8' 32'35' 44' 5" 55' 63’ 66' 73' 83. The changes in the material prOperties of these

tissues, brought on by stress-deprivation, is manifested by a loss in tensile strengthl 6.
I I, IS, I6, IS, 34. 35, 40. 43. 44, 52. SS. 56, 62. 73-75, 83, 84, tensile stiffness 2, l l, 32.

34. 35, 43, 44, 53, 55. S6, 62. 75. 83, 84, and distensibmt, of the tissue IS, 34, 4o, 44, 56,
73-75. 83. 84.

While stress-deprivation has been shown to be detrimental to connective tissue
the role of exercise in preventing these changes or reversing them is poorly
understood. Some investigators have found exercise to result in the recovery of lost
tissue strength following immobility I I, I5, I6, 23, 34. 35. 40, 4|, 43. 44, SI, 52. 55, 56,
55- 66. 7" 73' 74. 80, 32. 84. Other studies have documented little beneficial effect of
exercise or controlled motion as a rehabilitative method for injured and immobilized
tissue 3’ '6’ 55. 66, 72. 74. Despite this work, the effects of exercise or mobilization
are poorly understood. Following varying periods of immobilization, the exercise
(remobilization) period documented has ranged for 3 weeks to l2 months 4" 43- 44'
5l-56. 80' 82' 84. These studies fail to distinguish how much exercise is beneficial and

to what extent the exercise may become detrimental.

While clinical research has shown that exercise and controlled motion can
reverse some of the detrimental effects associated with immobilization, these studies
do not always have precise scientific control and standardization. Much of the
experimental research regarding the effects of immobilization and mobilization on
connective tissue is done utilizing animal models. Factors such as body weight, caloric
intake, and, to a certain extent. activity of the animal are difficult to standardize, this

may lead to erroneous interpretation of the results of various exercise programs.

Thus, an experimental model which allows for explicit control of as many parameters as

possible is needed.

The primary purpose of this thesis is to examine the effects of stress-
deprivation on the material properties of connective tissue, by biomechanical and
histological evaluation of an in vitro model. In addition, the effects of cyclic loading in
maintaining the normal material properties of tendons as well as arresting or reversing
the effects of previously immobilized tendons will be examined. This will be achieved
using an in vitro tissue culture model32 which allows precise control of the loading

(stress) history.

The main tOpics explored in this thesis can be designated into three hypotheses:

H j: Stress-deprived tendons in vitro will experience a change in
their material properties with time. This alteration will be

reflected by a decrease in the tensile modulus of the tissue.

This will be tested by examining the tensile modulus of stress-deprived tendons in vitro
over weekly intervals spanning a total of 8 weeks. The second part of the thesis is two-
fold. It involves the evaluation of mechanical stimuli via cyclic loading on maintaining the

normal material properties of the tendon. The respective hypotheses are as follows:

H2: Cyclic loading (mobilization) of tendons in vitro will

reduce loss of material properties (tensile modulus).

H3: The harmful effects ( i.e. the decrease in material properties) of
stress-deprivation in tendons can be reversed to a certain extent by a

specified cyclic loading regime.

The second hypothesis will be tested by examination of the tensile modulus of
the tendons involved in a mobilization procedure in vitro. These values will be
compared with controls and stress-deprived tendons to observe any significant trends.
The final hypothesis will be examined by determining the tensile modulus of tendons
that have been involved in a cyclic loading procedure following stress-deprivation for an

identical period of time.

II. LITERATURE SURVEY

This section is presented in a manner so as to provide sufficient background and
rationale to support the goal behind the topic of this thesis. It will discuss the effects of
stress-deprivation, the effects of mechanical stimuli (controlled motion and/or exercise),

and various experimental models used to date.

Of all the tissues in the body. the connective tissues are thought to have the
greatest mechanical strength. It is known that connective tissue requires a certain
degree of mechanical stress to remain healthy and retain this strength, however the
precise amount of stress required to maintain homeostasis is not known. An overview
of some of the studies that have been done over the past 20 - 30 years reveals an

extensive search for the exact mechanism by which connective tissue maintains itself.

Stress-Deprivation (Immobilization)

Immobilization of connective tissues (bones, ligaments, tendons) following injury
has been a mainstay of therapy for many musculoskeletal diseases. Immobilization
protects the injured tissue from disruptive forces, especially during the early period of
the healing response, in order to ensure effective healing. Immobilization allows for
stabilization of wound edges, which in turn permits the in-growth of new blood vessels

and the normal progression of wound repair (I7, l8).

Despite the benefits of immobilization in the initial healing process of injured
connective tissue. it is well documented that immobilization results in degradative
changes within the tissuezv 3. 6, 8, I I,I5, l7, I8, 20. 24, 30, 32, 34-36, 40, 4I, 43, 44, SI.
53’55' 62’ 53' 65. 66’ 72-76. 80- 82' 84. These changes are attributed to the fact that
some form of mechanical stimuli is necessary for the normal maintenance of healthy
tissue (78). Immobilization has been seen to have detrimental effects on various aspects

of tissue morphology, biochemistry, and biomechanical properties 3' 5» l I. 20: 30. 34.
35. 44. 56. 62, 66. 72. 76. 82, 84.

The effects of immobilization and stress-deprivation on the mechanical integrity
and tensile properties of soft connective tissue have been documented by many groups
over the past two decades. A majority of the investigators have reported decreased
mechanical prOperties with increased periods of immobilization. in both ligament and

tendon.

Noyes (56) examined the effects of immobilization on the mechanical and
histological properties of knee ligaments. The immobilized group showed a 3| %
decrease in linear slope, and a 40 % decrease in maximum load to failure with tensile
loading to failure, when compared to the controls. The energy stored to failure was
significantly less than control. and the elongation to failure increased with the
immobilized groups. Amiel (6) and associates showed similar decreases in stiffness and

strength of the lateral collateral ligaments in rabbits immobilized for 9 weeks.

Larsen and co-workers (4|) studied the effects of 4 weeks of immobilization on
the tensile stiffness of the anterior and posterior cruciate ligaments in rats. They saw a

25 % and 38 96 decrease in stiffness. respectively. when compared to controls.

Loitz and coworkers (44) observed the effects of short-term immobilization on
the biochemical and biomechanical properties of rabbit Achilles tendon. While a
significant decrease in the linear tensile stress was seen, the tensile stiffness did not
differ notably for the 3 week immobilization period. However. overall they found the

short immobilization period (3 weeks) to result in a mechanically inferior tendon.

Many studies have correlated the biochemical changes occurring with the
changes in tensile properties secondary to immobilization 2» 6. I l. '5» 34. 43' 44. 75. 84.
The significant changes have been associated with alterations in collagen content.
synthesis. and cross-linking 6» 34. 35’ 43. 44' 75- 34. Collagen turnover serves as an
important indicator of fibroblastic activity in terms of collagen dynamics. Increases in
soluble collagen cross-links is often seen in immobilized connective tissue 5» 34- 35’ 75'
34. An increase in cross-links is usually associated with decreased strength of the tissue
(75); the increase in cross-links are thought to cause interference of movement in and
between the collagen molecules by inhibiting the free gliding of the collagen fibers (84).
However, contrary to this some studies have reported a decrease in collagen cross-

Iinking with long term immobilization (6).

A significant increase in collagen synthesis and degradation with immobilization
was reported by Woo et al. (82) in a study which examined the effect of immobilization
on Medial Collateral Ligament (MCL)-bone complexes. The groups that were
immobilized for 9 weeks and I2 weeks time showed significant decreases in the
stiffness. and ultimate load values. In another related study, Woo et al. (83) observed
the biomechanical changes in rabbit MCL-bone complexes following 9 weeks stress-
deprivation. Stress-deprivation was seen to result in linear slope, ultimate load, and
energy absorption capacity values approximately one third that of the contralateral

controls.

In an immobilization study done to observe biomechanical and ultrastructural
properties of the rat medial collateral ligament. Binkley and Peat (I I) saw a 56.7%
decrease in the tensile stiffness and a 60 % decrease in linear stress after 5 weeks. A
decrease in collagen synthesis, as indicated by a decrease in small diameter collagen

fibers in the immobilized tissue was also observed.

Harwood and Amiel (35) examined the different metabolic responses for the
periarticular tissue in rabbit knees, with 9 and I2 weeks of immobilization. They
demonstrated that stress-deprivation secondary to internal immobilization had
profound affects on collagen metabolism of the patellar tendon, medial collateral
ligament, and anterior cruciate ligament (ACL). They found that the ACL was the most
susceptible to immobilization-related changes. These results are similar to those

reported by Larsen et al. (4|).

Woo et al. (84) examined the biomechanical and biochemical effects of
immobilization in order to gain insight on why joint stiffness occurs with immobilization.
They found significant decreases in water and glycosaminoglycan (GAG) content after 9
weeks of immobilization, with no changes in total collagen. GAG content is examined
as an indicator as to what is occurring in the ground substance, or extra-cellular matrix.
of the tissue. A correlation was made between hexosamine content (GAG indicator)
and the torque required to move the joint. It was found that with the loss of

hexosamine, the torque required to move the joint increased.

Tencer and associates (72) studied the effect of immobilizing rabbit knees in full

extension versus traditional flexed position. The knees immobilized in full extension

showed degeneration of the articulating surfaces; those immobilized in flexion showed
no deterioration. However there was no difference seen in joint stiffness between the

two groups.

In some cases, usually after tendon surgery in which suturing has been done,
immobilization or partial immobilization as a rehabilitative measure has been seen to
result in better healing than its counterpart of controlled passive motion '4’ 37. Similar
results have been seen with the healing capacity of articular cartilage 8, '7' '8' 54. Cast
immobilization was examined as a rehabilitative measure for the healing of articular
cartilage by Athanasiou and associates (8). They reported cast immobilization to result
in stiffer cartilage and better healing of articular cartilage than what was seen with a

controlled passive motion regimen.

Namba et al. (52) evaluated the effect of immobilization on joint stiffness. After
having induced intra-articular defects in the talar joints of rabbits, one joint was
immobilized while the other was treated with continuous passive motion (CPM). The
immobilization resulted in significantly higher joint stiffness than pre-injury, whereas
with CPM the joint stiffness was maintained at levels similar to pre-injury levels. These
results only reinforced those seen by Salter and associates (65) in which CPM was seen

to prevent joint stiffness.

Pyne and associates (62) found that immobilization of ligament under tension
helped in preserving the strength and stiffness of the tissue. Medial collateral ligaments
of rabbits were immobilized with a valgus tension, and compared to immobilization of
other groups maintained in positions of flexion and extension, for an 8 week period.
The valgus tension resulted in a ligament which has higher stiffness, cross-sectional area,
and breaking strength than the groups immobilized in flexion or extension.

9

In a similar type of study, Schaberg et al. (66) compared the effects of non-stressed
immobilization, stressed immobilization and mobilization on the alignment of collagen
fibers in healing ligament. Stressed immobilized ligaments demonstrated better collagen

fiber alignment that both mobilized and non-stressed immobilization groups

Walsh an associates (76) evaluated the effects of immobilization on cell function
of ligament in growing rabbits. They demonstrated a 25% decrease in cell number in all
immobilized medial collateral ligaments, with a 50-75% decrease in collagen synthesis
following 2 weeks of immobilization. Plasminogen activator, which can potentially
degrade extracellular matrix components was seen to increase with 4 weeks

immobilization.

Gamble, Edwards and Max (20) evaluated the cellular events responsible for
ligament atrophy following immobilization by investigating enzymatic activity during this
time. The enzymes evaluated were located in the cytOpIasmic, mitochondrial, and
lysosomal compartments of the cell. They saw enzymatic changes due to
immobilization which caused cellular metabolism to switch from an anabolic, synthetic

state to a catabolic, degradative state.

Hannifin and Amoczky (32) observed stress-deprivation to result in a
progressive decrease in the mechanical prOperties of tendon. An alteration from the
normal spindle-shaped fibrocyte was observed upon stress-deprivation. This change in
cell shape, number, and tissue morphology was theorized to be a result of the stress-

deprivation.

Thus, it has been established that stress-deprivation is accompanied by
detrimental changes in tissue morphology. Because it is well known that connective

l0

tissues require some form of mechanical stress to maintain tissue homeostasis. the use
of various exercise regimes to attenuate the effects of immobilization has been
increasing. The effects of mobilization and exercise on tissue health are discussed

below.

Mobilization (Exercise) I Remobilization

The phenomenon of increased stresses resulting in increased collagen synthesis
in connective tissue has been shown in many studies (66, 7S). Woo (84) provided a
general relationship between the level of stress and the maintenance of tissue
homeostasis. He documented that decreased stresses result in tissue atrophy and
inferior mechanical strength: while increased stresses result in hypertrophy of tissue and

mechanical properties somewhat superior to normal controls.

Exercise is accepted as beneficial in the prevention of muscle atrophy,
maintenance of joint motion, and promotion of circulation '89 27. Numerous studies
have evaluated the effects of exercise on connective tissue '5 . 4" 73' 74. 84. The
biological notion of continuous passive motion (CPM) was developed as an alternative
rehabilitative method by Salter (65) in the late l970's. This type of therapy, which
involves passive application of motion to the afflicted area, has been proven to stimulate
healing and regeneration of articular tissues. In a series of basic research studies
conducted by Salter and co-workers. it was determined that CPM is a well-tolerated,
seemingly painless method which stimulates healing and regeneration of articular
cartilage, prevents joint stiffness. and permits normal healing of arthrotomy wounds.
CPM has been seen to decrease and in some cases completely avoid the deleterious
effects of immobilization 3» 23' 32. 42. 43' 5" 55» 58’ 7" 80. It becomes obvious why
the use of CPM has so rapidly increased and become almost standard procedure in

certain rehabilitation programs in the clinical community. Both CPM and other forms

of exercise will be discussed in this section.

It has been suggested that mobilization for connective tissue structures be
initiated in the very early stages of the repair phase of healing (I8). Tipton and his
associates (40, 73, 73) were among the first to document the importance of mechanical
stimuli in the maintenance of the mechanical integrity of ligaments. They saw an
increase in tissue strength with increased training, while immobilized ligament showed
significantly lower strengths. They were able to see differences between different

periods of exercise; more exercise correlating with greater ligament strength.

When Tipton et al. (73) induced injury and subsequent repair to the medial
collateral ligaments of dogs, those which has undergone the regimen of 6 weeks of
immobilization followed by 6 weeks of exercise showed strength values similar to the

control, non-injured ligaments.

Woo. Gomez, Woo, and Akeson (84) examined the effects of 3 months of
moderate exercise versus l2 months of the same with swine extensor tendons. Short
term exercise showed minimal effects on mechanical and structural properties of the
tendon. However l2 months of exercise increased both the strength and stiffness of
the tendons. Dahners and Burroughs (l6) found an exhaustive exercise regime to be

no more beneficial than a normal enforced exercise regime on the healing of ligament.

Noyes (55, 56) examined of the effects of immobilization with subsequent
remobilization on the mechanical and histological properties of knee ligaments. The

decreased mechanical properties of the bone-ligament-bone complex due to 8 weeks of

immobilization could only be reversed by a l2 month remobilization period. Thus. a
year of remobilization was required before the normal, healthy material properties of

the tissue returned.

Woo et al. (82) examined the effect of a similar protocol of immobilization
followed by remobilization with MCL—bone complexes. Following both 9 week and I2
week immobilization periods. the groups which were remobilized for 9 weeks showed a
rapid return to mechanical properties similar to controls. Larsen and coworkers (4|)
saw similar results when they studied the effects of 4 weeks of immobilization on the
elastic stiffness of rat anterior and posterior cruciate ligaments. followed by
subsequent remobilization for 6 weeks. The tensile stiffness of the ligaments was no
different than that of the controls. It is apparent that exercise does can reverse some

of the effects of immobilization.

Salter et al. (65) have conducted a series of studies in which there was
histological evidence that faster healing and regeneration of articular cartilage occurs
with CPM. It was also demonstrated that CPM is significantly superior to both
immobilization and intermittent active motion in stimulation of healing and regeneration
of articular tissues. However, Athanasiou et al. (8) found cast immobilization to result

in more favorable healing of articular cartilage than with CPM.

Moran, Kelley, and Salter (5|) found CPM to result in early repair of full-
thickness laceration of the patellar tendon: a better result than what was seen with
immobilization or intermittent activity. Loitz et al (44) found 3 weeks of CPM to result
in a tendon with mechanical properties intermediate to those of 3 week immobilized

tendons and control tendons. However, the elastic stiffness of the 3 week immobilized

group and the 3 week CPM group were comparable, at 79% and 76% of controls
respectively. Thus. CPM was seen to counterbalance the immobilization-related

biomechanical alterations in the tendon.

Woo et al (80) also showed positive effects of CPM on tendon repair. The
tendons which underwent varying periods of CPM following immobilization showed a
higher rate of repair than those tendons immobilized continuously for the same period

of time ranging from 3 weeks to l2 weeks.

Namba et al. (52) evaluated the effect of immobilization and CPM on joint
stiffness. After having induced intra-articular defects in rabbit ankles, CPM was able to

maintain joint stiffness similar to pre-injury levels.

It is apparent that the frequency of CPM may affect the rate of repair as well as
the tensile properties of the tissue being rehabilitated. In I990, Woo et al. (7 I). using
canine flexor tendons, demonstrated a higher frequency CPM regimen to exhibit higher
(values for linear sloPe, ultimate load, and energy absorption than that of lower
frequency CPM protocol. Thus, there is evidence that CPM, applied after certain
periods of stress-deprivation. results in the return of normal mechanical properties

despite the differences in time of immobilization and CPM times for the studies.
Common Experimental Models and Procedures
The research done in this thesis makes it is imperative to have an understanding

of the various models that have been utilized to study the effects of mechanical stimuli

on connective tissue. The choice of an in vitro tissue culture model should become clear

upon evaluation of the other immobilization and mobilization methods used in the

literature.

There are not many methods by which joints are immobilized for experimental
evaluation. The most common method of immobilization has been with extra-articular
pin-fixation of the joint. while it is in a flexed position I l. 20. 35. 43. 44’ 73. 83: 34. The
general procedure entails the insertion of a stainless steel pin through both of the
articulating bones of the joint, thus forcing the knee joint to be immobile in a hyper-
flexed position. However, such flexion can place extreme tension on portions of the
cruciate and collateral ligaments. While this method results in immobility of the knee
joint, portions of the soft tissues may be stressed position when the joint is flexed.
Thus. depending on which tissue is being evaluated. there is a distinct possibility that

some tissues are under tension.

Another manner by which tissue is immobilized is by the application of a cast 4"
55' 74. 30. Cast immobilization is not as prevalent as pin-immobilization. The cast can
be applied to the entire limb, the entire body, or just limited to the area of injury. If
the animal is allowed activity while being casted, the casts have a tendency of loosening.
Thus, midway through the treatment the cast needs to be changed. For both pin
immobilization and cast immobilization. there is little control of the amount of stress or

the lack of stress that the tissue experiences due to the gross nature of both methods.

Mobilization methods vary more with the type of study and the animal model
being used. Many of the current studies are utilizing CPM as a remobilization technique
23’ 43’ 44. 5" 52. 55. However, the cycles and duration of CPM are not standardized
nor well documented. Some studies have relied strictly on cage activity for mobilization
of the animals 55. 55» 32. This method of mobilization does not allow for evaluation of

IS

the loads being experienced by the tissues in any manner. The same predicament
occurs with running animals on a treadmill 73' 74. 34 , manual flexion of joints 55- 3° ,

and swimming protocols 4'.

Thus, upon evaluation of the possible methods by which to study the effects of
mechanical stimulus on connective tissue, none provided the type of experimental
control as the use of an in vitro tissue culture model. This model provides for the
complete lack of stress for the evaluation of stress-deprivation effects; it also allows for
the precise control of the amount of load and the method of loading for evaluation of

the effects of loading on the tissue.

This experimental model in also based on the concept that diffusion is the
effective process in providing nutrients to certain animal tissues allowing them to be
maintained in a tissue culture environment. Connective tissues have been successfully
maintained and evaluated in a tissue culture environment 9. 22' 25' 28' 3" 34’ 42. 47.
49- 57. 599 54’ 53. Previous work has shown that whole tissue segments can be
favorably maintained in a chemically defined. cell-free environment in the absence of a
circulating blood supply or a bathing synovial fluid. The principle behind tissue culture
lies in the ability to provide an in vitro nutritional environment for the explanted tissue
which approximates, as closely as possible, that which occurs in the intact animal '1: vivo.
The canine FDP tendon was selected as the tissue of choice for this study. Because a
portion of the FDP tendon derives its nourishment from diffusion of synovial fluid in the
tendon sheath. it was a perfect choice of tissues for the tissue culture model used in

this study.

The FDP tendon has avascular and vascular regions (24. 58). The avascular D-region
(S8) was the portion of the tendon used in this study. There has been considerable
controversy regarding the relative roles of vascular perfusion and synovial fluid diffusion
as nutritional sources for flexor tendons: many have investigated this route by obsewing
how the FDP tendon heals (22. 28. 46. 47. 49). The flexor retinaculum synovial bands.
or vinicula. provide blood supply to flexor tendons (67). Several studies have
demonstrated that synovial fluid diffusion provides sufficient nutrition for the FDP
tendon (22. 28. 3|. 38. 47-50) both with tissue culture models and with intact. 'n viva

studies.

Manske and associates first evaluated the nutrient pathways to flexor tendons
approximately fifteen years ago (50) and determined that hydrogen molecules were
transported to and across the synovium. and subsequently into the flexor tendon by
diffusion; this occurred in the absence of vascular attachments. In studies following this
one. Manske and Lesker and others (38. 47. 49) were able to demonstrate similar
results of synovial diffusion in vitro. via the assistance of flexor tendon healing in various
animal species. Cell proliferation and increased collagen content was the indicating

factor in progressive healing in culture.

Graham et al. (28) found flexor tendons to be fully capable of healing in a tissue
culture environment. Similarly. Gelberman and associates (47) were able to successfully
exclude any vascular contributions to the healing process of injured flexor tendons of
four animal species by demonstrating that tendons could heal on their own in tissue
culture. Hooper and his associates studied the nutrient pathways of flexor tendons by
evaluating clearance of a radionucleide in canine flexor tendons with an 'n viva study

(38). They saw similar results to Manske. Graham. and Gelberman. This study

I7

demonstrated that flexors tendon receive a majority of its nutrition from the
surrounding tissues via diffusion. The high content of radionucleide in the tissue
suggested that this was more than could possibly be transported by the surrounding

system of vessels.

From the literature it is apparent that the alterations occurring due to
immobilization following injury have deteriorating consequences for the uninjured soft
tissue present. As tissue culture models have been observed to be suitable for the
maintenance of whole connective tissue segments. it is feasible to use such a model to

study the alterations occurring due to stress-deprivation in vitro.

l8

III. MATERIALS AND METHODS

This chapter provides a general description of the experimental methods and
techniques that were utilized to perform the specified tests. collect and analyze the
data. and evaluate the histological results. All of the biomechanical testing and histology
was done in the Laboratory for Comparative Orthopaedic Research (LCOR) in the
College of Veterinary Medicine at Michigan State University. Methodology for all
three phases of the study is provided. with a emphasis on tissue culture technique and

biomechanical testing.

Tissue Evaluated - Flexor Digitorum Profundus Tendon

The canine flexor digitorum profundus tendon (FDP) was the connective tissue
chosen for this study. The FDP has been utilized in many previous studies and is well-
suited for this in vitra model because of it's nutrient pathways. The FDP tendon consists
of distinct avascular and vascular regions (Figure l). The avascular region has been

shown to receive a majority of its nutrition via diffusion of small solutes from the

surrounding synovial fluid 2" 22' 23- 3 I . 46, 479 49' 50.

The Okuda D-region (S8) of the FDP tendon was isolated for this study.
Okuda documented the biomechanical. histological, and biochemical properties of the
canine FDP tendon (58). The tendon was divided into five regions dependent upon

these

20

 

Figure l: A schematic depiction of the avascular and vascular regions of the canine
flexor digitorum profundus tendon (2|).

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characteristics (Figure 2). The Okuda D-region consists of dense. hypocellular collagen
bundles. located between two strictly fibrocartilaginous areas (Okuda B/C- and E-
regions). Being a predominantly tendinous area within a tendon sheath. the Okuda D-

region was well suited to maintain in culture and to apply tensile load in culture.

The tendons were obtained from dogs being utilized in other unrelated studies.
and from dogs being used in the College of Veterinary Medicine teaching laboratories.
The dogs were adult mongrels of mixed age and sex. The animals were euthanized with
an intravenous injection of sodium pentobarbital (Fatal Plus). Immediately following
euthanasia, the tissue was harvested and four FDP tendons were taken from each of the

four limbs in every animal.

Dissection Technique:

A lateral incision was made along the foot and the foot pad in order to exposed
the flexor digitorum superficialis (FDS) tendons. The FDS tendons were then
transected proximally and reflected distally in order to expose the underlying FDP
tendons. sheath. and pulley systems. The synovium and sheath was cleared from the
tendon before transection. The FDP tendons were then transected approximately 3
mm proximal to their distal attachment on the third phalanx and pulled gently in the
proximal direction. through the sheath. Subsequently. the proximal end of the FDP
tendons was transected and placed in Dulbecco's modified Eagle medium solution
containing a IO % concentration of an antibiotic/antimycotic solution
(penicillinlstreptomycin): this solution is the antibacterial wash solution. The tendons
were then taken to the tissue culture facility of the LCOR where the remainder of the
tissue preparation was carried out in a tissue culture hood. The common proximal

attachment of the FDP tendons

22

was trimmed off in order to separate the four individual tendons. prior to the
antibacterial wash sequence. The Okuda A-region and the distal-most portion of the

Okuda E-region were also trimmed (Figure 3).

 

 

 

 

 

 

 

”\
M/

l<- A ->l<- BIC —>l<— D ->l<-— E —>I

 

Figure 3: Schematic depiction of the canine flexor digitomm profundus tendon. the
vertical lines indicating the regions at which trimming was done. with reference to the
Okuda regions (26).

Antibacterial Wash Sequence:

After harvest. the individual tendons were rinsed in a wash solution of
Dulbecco's modified Eagle medium (DMEM) containing IO % antibiotic/antimycotic
solution (Gibco Laboratories. Grand Island. New York). All tendons that were to be
utilized for zero time controls were rinsed once in the wash solution and then
immediately frozen at - 80 ° C until time of tensile testing. The experimental tendons

were taken through the washes prior to tissue culture protocol. The tendons were

23

taken through a series of three '19 hour washes in I00 mm x 20 mm petri dishes
(Corning Glassworks. Corning. NY). with approximately 40 ml of wash media. This
procedure was done to ensure the removal of external contamination prior to tissue
culture. The entire wash procedure was conducted in an aseptic environment under
the tissue culture hood. Tendons were placed into tissue culture immediately following

this wash sequence.

Tissue Culture Techniques

The in vitro model for stress-deprivation and subsequent loading entailed the
maintenance of the FDP tendons in a tissue culture environment. The process is
technically referred to as ”organ culture” as the culture is of a three-dimensional.
undisaggregated tissue which has all of the histological features of the tissue in viva. In
this study a complex, defined media. M-l99 (Gibco Laboratories. Grand Island. New
York). was utilized. Previous in vitro studies done with flexor tendons have utilized
medium M-l99 with supplements 22. 32' 47. Thus. M-I99 medium has been shown to
successfully maintain tendons in a tissue culture environment. The media was stored at
4 ° C until the tissue culture media was to be made. The culture media was prepared
by adding vitamin and other nutrient supplements to the M-l99 media. Fetal bovine
serum (FBS) and ascorbate were the nutritional supplements utilized. FBS (Gibco
Laboratories. Grand Island. NY) provides growth factors and cytokines to the media in
proportions similar to what the tissue would normally get in viva. Ascorbate (Sigma. St.
Louis, MO) is a vitamin C supplement which is necessary in providing a substrate for
collagen synthesis in tissue culture. Antibiotic/antimycotic (ab/am) solution (Gibco
Laboratories. Grand Island. NY) was added to the media solution as an antibacterialand
anti fungal supplement. The culture media was prepared under the tissue culture hood
in sterile

24

conditions. using warm (37°C) solutions. At the time of culture media preparation. the
solutions and M-l99 media were filtered through a 0.20 p. tissue culture filter (Nalgene.
Rochester. New York). The protocol for making 500 ml of M-l99 tissue culture media
is given below:

435 ml of M-l99 media (87 %)

5 ml of antibiotic/antimycotic solution (I %)

l0 ml of ascorbate (2 %)

50 ml of fetal bovine serum (|0%)

Once the media was made. it was stored in the 37 ° C water bath until the time
of its use. Multi-well (six l0 ml wells per dish) plastic culture dishes (Falcon. Becton
Dickinson. Lincoln Park. NJ) were used to maintain the tendon while in tissue culture.
Approximately 9 ml of tissue culture media. at 37 ° C. was pipetted into each well in
the dish. The tendons were placed into the wells with sterile forceps under the tissue
culture hood (Figure 4). Following this step. the dishes were placed in a tri-gas,
humidified incubator (Forma Scientific. Inc.. Marietta. Ohio) which was maintained at
37.0 ° C with a I0 % C02. The culture media was changed every other day under

sterile conditions.
Tissue Preparation

The examination of the first hypothesis involves the evaluation of material
properties of tendons that have been deprived of stress or immobilized for different

time periods. In vitro stress-deprivation was achieved by maintaining the tendons in

tissue culture for specified periods of time. The experimental animals were divided into

25

groups based on the duration of tissue culture immobilization. The tendons were
separated into the following experimental groups:

(i) I week immobilized in tissue culture

(ii) 2 weeks immobilized in tissue culture

(iii) 3 weeks immobilized in tissue culture

(iv) 4 weeks immobilized in tissue culture

(v) 5 weeks immobilized in tissue culture

(vi) 6 weeks immobilized in tissue culture

(vii) 8 weeks immobilized in tissue culture

The time zero controls for each animal in this phase of this study were frozen
immediately following harvest, as noted previously. A summary of the tendons utilized
for each experimental group in this stress-deprivation portion of the study is provided
in Table I. At the end of each experimental time period. the respective tendons were
removed from tissue culture and frozen at - 80 ° C in individual freezer tubes until the
time of tensile testing. In order to maintain tissue integrity the tissue must be deep

frozen at temperatures below - 39 ° C (I 3).

For each animal used in each experimental group. a time zero control group was
established. A control group was required in order to attain a normalized data set.
Thus. each experimental tendon has it's own set of control tendons. This procedure
serves to minimize the effects of variations in tendon size. age. sex. and breed between
the animals used. In this manner. the parameters evaluated in the ”experimental”
tendon group are normalized when compared to the same parameters in the respective
control group: both groups having similar tendon size and being from one animal. thus

eliminating any changes occurring due to age. sex. and breed.

26

27

 

 

Figure 4: Photograph showing the freshly-trimmedcanine FDP tendons in the tissue
culture dish with culture media, prior to incubation.

28

 

 

WQWMRS
STRESS DEPRIVATION CONTROL (To) EXPERIMENTAL‘
PERIOD
I WEEK n = l8 n = 25
2WEEKS n=27 n=3l
3 WEEKS n =l3 n = ID
4 WEEKS n = I5 n = IS
5 WEEKS n = l2 n = 8
6 WEEKS n = l8 n = 8
8 WEEKS n = l9 n = 9

 

3 The disparity in the number of tendons used per experimental group is a result of

the pooling of data from all three phases of the study.

Freeze-Thaw Procedure:

It has been hypothesized that the deterioration of material properties in tendon
due to varying periods of stress-deprivation is probably a cell-mediated response. In
order to explore this theory. a subset of tendons in which the cells had been killed

were examined.

The cells were killed using a freeze/thaw technique. The tendons were taken
through a series of freeze and thaw cycles immediately after harvest. The tendons were
frozen in liquid nitrogen (- I96 ° C) for approximately l0 seconds and subsequently
"thawed” in warm (37 ° C) wash media for approximately 30 seconds. This cycle was
repeated five times in order to ensure complete cell death. This freeze/thaw procedure
was shown to result in complete cell death due to destruction of the cell membrane
due to ice crystal formation (23. 24). Following the freeze/thaw cycles. the tendons
were taken through the same wash procedure as the tendons from previous
experimental groups. After the washes the tendons were placed in tissue culture for
the specified stress-deprivation periods. The time zero control tendons were frozen in

the - 80 ° C freezer immediately after harvest.

Four animal groups were utilized for a freeze/thaw protocol to kill the cells
before immobilization in tissue culture. The four animals were each divided into the
following experimental groups:

(i) zero time controls.

(ii) 2 weeks stress-deprivation (freeze/thaw).

(iii) 4 weeks stress-deprivation (freeze/thaw)
Once the tendons in the experimental groups had been in tissue culture for their
specified time periods. they were removed and frozen in the - 80 ° C freezer until the
time of tensile testing.

29

The second and third phases of this study involve mobilization and re-
mobilization of the respective tendons. Mobilization was the cyclic loading of the
tendon immediately after harvest. Re-mobilization entailed the cyclic loading of a
tendon previously immobilized for a specific time period. The loading of the tendons in
tissue culture was accomplished by susPending a specified weight from the tendon in a
test tube of culture media. Loading was done by securing the suspended tendon to a
specially designed cyclic passive motion machine. The tendon hanging and loading

procedure is described in the following section.

Tendon Hanging Procedure:

The tendons are clipped on both the fibrocartilaginous Okuda BIC and E-
regions. both adjacent to the Okuda D-region test area. Small (9 mm) metal clips
(Autoclips. Becton Dickinson, Parsippany. NJ) were used for this purpose. The distal
metal clip was attached to a 50 gram (0.49 N) stainless steel calibration weight via nylon
suture thread (Surgilon. American Cyanimide Company, Pearl River. NY). The proximal
metal clip was used to secure the loaded tendon to the motion device. with nylon
thread. This entire procedure was done in the hood using sterilized equipment. Sterile
50 ml conical test tubes (Fisherbrand. Fisher Scientific. Pittsburgh. PA) were filled with
47.5 ml of M-l99 culture media. 5 mm holes were made in the center of the test tube
caps (sterilized prior to loading). allowing for full excursion of the tendon during the
cyclic loading regime. The loaded tendons remain in the media-filled test tube both

during loading and during rest.

A load of 50 grams was chosen after evaluating the effects of both lower and

higher loads on the tissue (32). This load has been seen to result in tissue strains

30

approximately 0.5 % (as determined by stress-strain curves of tensile loading of the

canine FDP tendon). This strain falls well within slow. passive physiological strain levels.

Cyclic Loading Procedure:

Tendons were loaded using a Specially designed cyclic loading devices (Figure 5).
The device consists of an arm attached to a rotating cam which was motorized by a
small. slow speed electric motor (Edmund Scientific Co.. Barrington. NJ) which had a
set frequency of l rotation per minute. Tendons were loaded for 2 hours a day at 5
days a week This frequency and duration of loading was gleaned from previous CPM

studies.

The loaded tendon was suspended in the 50 ml test tube of culture media from
the arm of the device. As gleaned mentioned above. the device allowed for a
loading/unloading cycle of l rotation per minute via the rotating cam which raised and
lowered the suspended tendon at this frequency. This frequency was equivalent to 30
seconds of rest (when the arm traveled through the lower hemisphere of the rotation
area) and 30 seconds of loading (when the arm traveled through the upper hemisphere
of the rotation area). A schematic depicting this set-up is shown in Figure 5. This
loading/unloading frequency was achieved by adjusting the length of the suture thread
before loading. All cyclic loading periods began with the tendon having a small pre-load
(i.e. the suture thread was not slack. as when it was at rest. yet it was not fully
tensioned) with the device arm at a ”3 o'clock" or "9 o'clock" position. This set-up
ensured proper 30 second loading and 30 second unloading of the tendons. At the end
of each loading period, the tendons were brought to a rest position with the arms at a
"6 o'clock” stance (Figure 6). Similar to the previous phases of this study. the zero time

controls from these groups were rinsed with I0 % ablam solution of DMEM and

3l

immediately frozen at - 80 ° C. Four experimental groups were established to evaluate

the second and third hypotheses of this study. the groups were as follows:

(i) 2 weeks stress-deprived (immobilized).

(ii) 2 weeks loaded (mobilized)

(iii) 2 weeks stress-deprived (immobilized) l 2 weeks
loaded (re-mobilized).

(iv) 4 weeks stress-deprived (immobilized)

Each experimental group consisted of tendons from more than one animal. A
summary of the tendons in each animal group is provided in Table 2. The tendons
involved in the second phase of the study were mobilized or loaded for a period for 2
weeks in a tissue culture environment . The tendons were taken through the wash
procedure and then immediately started on the loading regimen previously described.
After the loading period. the tendons were removed from tissue culture and frozen in

the - 80 ° C freezer.

The third phase of the study entailed stress-deprivation of the tendons for a 2
week period immediately followed by a loading (re-mobilization) period for the same
time. Following this 4 week program. the tendons were removed from tissue culture

and frozen in the - 80 ° C freezer.

32

33

 

 

2 1 1 Ann-.1 0 0:0." M'PI '-i-u°9 0 '1"I
Emu:
EXPERIMENTAL CONTROL (T 0) EXPERIMENTAL
GROUP
2 WEEK LOADED n = 22 n = l9
2 WEEK
ST RESS-DEPRIVED n = 27 n = 3|
2 WEEK STRESS-
DEPRIVJ 2 WEEK
LOADED n= I8 n= l9
4 WEEK

STRESS-DEPRIVED n = IS n = l5

 

34

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Ii:

Biomechanical Testing of Tendons

The structural properties of every tendon were determined from tensile testing.
Acquisition of tendon mechanical data was accomplished with a servo hydraulic
mechanical testing machine (lnstron 850l M) aided by a micro-computer (Commodore

Amiga 2500).

Tissue Preparation:

The individual tendons were removed from the - 80 °C freezer and allowed to
thaw at room temperature (for ~ l0 minutes). The tendons were kept in M-l99 media
to maintain hydration before testing. Once the tendon had thawed. it was trimmed at
the Okuda BIC and E-regions to provide a sufficient gripping area for testing purposes.

All measurements were taken after the tissue was secured in the grips.

Once the grips were fastened to the lnstron attachments. the initial gauge length
(lo). width. and thickness measurements were taken. These measurements were taken
with hand-held digital calipers (Mitutoyo Corporation, Tokyo. Japan). All measurements
were taken with an approximate load of 0.098 N (l0 g) on the tendon. This load was
required to eliminate any slack in the tendon. The tendon was kept moist throughout
the measurement procedure. This was done by dousing the tendon with the M-l99
media. A schematic diagram depicting all points at which measurements were taken is
shown in Figure 7. The tendon was kept in a saline (0.9%) bath. at room temperature.
while testing (Figure 8). Because no changes have been seen in the mechanical
properties of collagenous tissue over the temperature range of 0 to 37 ° C (I3. l5).

this temperature was thought to be sufficient during the testing period.

36

Tensile Test Parameters and Procedure:

As noted above. all tensile tests were performed using an lnstron 850l-M
mechanical testing machine. A 25 lb. load cell (Data Instruments. Acton. MA) was
utilized for testing. All tendons were tested to a 9.8l N (I000 g) load at strain rate
equivalent to 0.5 % of the tendon gauge length per second. This rate was derived from
previous studies (60) and from testing done prior to this experiment (unpublished data).
In preliminary tests, this rate resulted in the best response of the tendon upon
observing the load-deformation graph - i.e. it allowed for sufficient linear region prior to
reaching the 9.8l N final load limit. All tendons were tested with no pre-conditioning.
A data acquisition program was provided with the required parameters prior to testing
in order to sample data at a rate of 80 hertz. Upon completion of the test. the tendon
was removed from the grips. the gripped portion removed. and the test-region of the

tendon fixed in ID % buffered formalin for histology.

The tensile testing data was saved directly to fl0ppy disk in order for subsequent
manipulation on a computer spread-sheet program. Three columns of data were
obtained: time (in seconds), an amplified position of the actuator (in volts). and load
readout through the load cell (in volts). This data was later manipulated, with the aid of
scaling factors. to obtain values that would provide the required load-deformation plots

for data analysis.

37

38

 

 

 

 

 

 

 

 

 

ol

 

 

T01
‘LIO’
I

 

 

 

 

 

 

 

 

 

 

0 0
___,1,.__
__.§ q._.
__.J {—

0 0

 

 

 

 

MW

 

 

 

 

 

 

 

 

Wm

Figure 7: Schematic depiction of all points at which measurements were taken on the
FDP tendon prior to mechanical testing. All measurements were taken with
an approximate load of 0.098 N (l0 g) on the tendon.

39

 

 

Figure 8: Photograph of the FDP tendon fixed in the grips within the bath apparatus
prior to tensile testing.

Histology

Following the mechanical testing each tendon was trimmed (leaving only test
region - the Okuda D-region) and put in l0% buffered formalin for fixing of the tendon.
The tendons were fixed for at least 24 hours before any tissue processing could be
done. The standard tissue processing procedure of dehydration. and infiltration was
done prior to embedding the tendon samples in paraffin. The paraffin blocks were
oriented such that longitudinal sections of the FDP tendon could be cut. 5.0 u thick
sections were cut on a rotary microtome and mounted on glass slides. These sections
were then stained with a standard Hematoxylin and Eosin (H 8: E) staining procedure.
This protocol resulted in the cell nuclei retaining blue stain, while the cytoplasm and

collagen fibers retaining a pink stain.

The stained tendon sections were observed with a light microsc0pe (Olympus
BH-2, New Jersey Scientific. Middlebush. NJ). Cell shape. relative cell number
(cellularity). and the status of cell nuclei were observed with standard light microsc0py.
Collagen fiber alignment was evaluated using polarized light microscopy. The
birefringent nature of collagen allowed for viewing of the sinusoidal crimp pattern in the

individual collagen fibers. as well as the relative alignment of the fibers.

Data Analysis

The raw data that was obtained from each tensile test was evaluated with the
assistance of a computer spread-sheet program. This allowed for manipulation of the
raw data into the functional. scaled data required for a load-deformation plot. The
scaling factors were utilized. specifically. to convert the load and deformation values (in
volts) to the metric units required for proper interpretation and analysis of the data.

The load was recorded in grams. while the deformation was recorded in millimeters.

Load-deformation curves were developed directly from the computer spread-
sheet program. The load-deformation plots allowed for the calculation of the stiffness
values (K) for each tendon tested. The stiffness values for all tendons were computed
manually directly from the load-deformation output graph. A sample load-deformation
graph is shown in Figure 9. This graph is not an actual plot of a canine FDP tendon

tensile test. it is a schematic depiction.

Calculation of Stiffness (K):

The stiffness of the tendon was calculated directly from the load-deformation
curve. The slope of the load-deformation curve designates the stiffness of the tendon.
i.e.:

stiffness = change in load I change in deformation
This is shown in Figure 9. A tangent to the load-deformation curve was drawn with in
the linear region. The tangent was drawn in a manner such that the largest portion of
linearity in the curve's linear region was utilized. The stiffness values were recorded in

units of N/mm.

4|

42

 

 

 

 

 

 

 

LOAD
(‘0 8) K : A Load
7 A Deformation
l ,
H Athbrrmtbn DEFORHATION
(5 mm)

 

 

Figure 9: Sample load-deformation curve indicating regions at which stiffness (K)
measurements would be taken for further calculations and analysis.

The stiffness represents a structural prOperty. The modulus of elasticity or
tensile modulus is based on a linear relationship between stress (force per unit area)
and strain. which translates into the stress being directly proportional to the strain for
elastic materials. For biological materials. which are viscoelastic in nature. the tensile
modulus is used to represent the ”elasticity" of the material. Structural properties of a
tissue can be converted to material properties by utilizing individual tissue
characteristics. In this manner the stiffness (K) of a tendon can be converted to a
material property. the tensile modulus (E). by the use of the initial gauge length (lo) and
the cross-sectional area (A) of the respective tendon. A brief derivation of the
equations used for the calculation of the tensile modulus in this study is provided

below. Detailed calculations are provided in the Appendix.

W

A summary of the symbols:
F = force or load in N
5 = deformation in mm
IO = initial gauge length of tendon in mm
A = cross-sectional area of tendon in mm2

K = stiffness coefficient of tendon in N I mm

E = tensile modulus of tendon in mega Pascal
0' = stress (= force/unit area) in mega Pascal
8 = % strain

The load-deformation curve the coefficient of stiffness. K. is obtained:

K=Fl5 ...inNI mm2(l)

= To obtain the tensile modulus. E. of the tendon

E=ol§
where o=FIA
e= Silo
E= (FlA)(5llo) ...inNImm2

=> The tensile modulus (E) value is obtained in N I mm2 . it is converted to Pascals.
I NIm2= I Pascal

=9 Following conversion of N I mm2 to | N l m2 Pascals can be used as the
functional unit.

Once the value for the elastic modulus (E) for each experimental tendon was calculated.

it was expressed as a percentage of it's respective control tensile modulus value. The

time zero control tensile modulus was expressed as a mean value of all zero time

control tensile modulus in the respective experimental group.

= if Econtrol = tensile modulus of the control group (in MPa)

if Eexperimental = tensile modulus of the experimental group (in MPa)

=> Then the reported values for each experimental group were expressed as:

Eexperimental (%) = Eexperimental (in MPa) I Econtrol (in MPa)

Eexperimental = E as a % of control

43

In this manner. each experimental tendon was normalized by a control from the same
animal. Thus. all results are expressed with the experimental tensile modulus as a

percentage of its individual control.

Statistical Analysis

Following the collection and processing of the mechanical data, statistical analysis was
done for all of the data. To evaluate the changes in tensile modulus with the increasing
periods of stress-deprivation. a one-way analysis of variance (ANOVA) was done.
Multiple confidence intervals were utilized to determine statistical significance in this
method. In this manner. the effect of time (corresponding to increased periods of
stress-deprivation) on the material properties of the respective tendon groups can be
observed. This test was used only to evaluate the data from the stress-deprivation

group.

The Student's t-Test was utilized to determine if any change was occurring
between the control groups and the respective experimental groups. as well as between
the different experimental groups. For this evaluation. the mean elastic modulus for a
particular experimental group was expressed as a percentage of the respective control
tensile modulus. In this manner. a mean value for the tensile modulus a percentage of
it's control as well as the standard deviation was obtained. Statistical analysis was done
both with each raw data (percentage of control tensile modulus values) value. as well as
with each mean and standard deviation within the group value. The t-Test was done
using 99th percentile confidence interval. with "p" values below 0.05 indicating

validation of the hypothesis - or statistical significance.

IV. RESULTS

The results listed in this chapter are written in direct relation to the stated

hypotheses of this study.

H I: Stress-Deprivation Study

Tensile testing of all tendons in this portion of the study resulted in a classic
load-deformation curve (Figure ID). The tensile modulus (E) was calculated from the
linear region of the load-deformation curve using the method described in the materials
and methods chapter. The tensile moduli for each control and stress-deprived tendon
group is shown in Table 3. Because of the variation in size. age. sex. and breed of the
animals utilized. the values for the tensile modulus were normalized by their respective
control tensile moduli. This allows for valid comparisons between each of the
experimental groups. deSpite being from different animals. All values for the
experimental tensile modulus were expressed as percentages of individual zero time

control tensile modulus:

E as % Of contrOl = Eexperi’nental I Econtrol

The mean tensile moduli of each group. expressed as a percent of control results are
presented in Figure II. The effect of stress-deprivation over time on the tensile

modulus was determined by performing a one way analysis of variance (ANOVA)

45

statistical test. The effect of time on stress-deprivation was shown to be statistically
significant with a p value of 0.000. However. statistical significance between certain
groups was not significantly different as determined by the Student's t-Test. The
modulus of elasticity for the 2 weeks and 3 weeks stress-deprived groups were
statistically the same, as were the tensile modulus of the 5 weeks. 6 week. and 8 weeks
stress-deprived groups. However. the tensile modulus for the I week stress-deprived
group was statistically different from the 2 week stress-deprived group; similarly the 3
week stress-deprived group showed statistical significance with the 4 week stress-
deprived group. Finally. the 4 week stress-deprived group was statistically different

from the 5 week stress-deprived group.

46

47

LOAD vs. DEFORMATION

 

 

 

 

-2003-

ggggaasgsaas°°SSS°s

83328§§2E3§§ §§§ §

q'c'o'oooo'o'o'oo'o' q'qsqs 9'
DEFORMATIONOnmm)

Figure I0: Load-deformation curve of time zero control canine FDP tendon. Depicts
the classic load-deformation response of tendon.

48

TENSILE MODULUS (expressed as a X of control)

IN.” 1 92.33%

  

57-9IX 56.89% 56.46%

        

 

l 2 3 4 5 6 8
WEEK WEEK WEEK WEEK WEEK WEEK WEEK
(n=24) (n=3 I) (n= I0) (n= l5) (n=8) (n=8) (n=9)

Figure l I: Histogram depicting the time-dependent change in the tensile modulus (E)
with increased periods of stress-deprivation.

49

:3 : z' 0 T EELATI u'D L a. 0L
W

 

 

 

 

 

 

 

 

 

 

STRESS DEPRIVATION ELASTIC MODULUS OF
PERIOD STRESS-DEPRIVED n
GROUP (in MPa)

To control 87.37 :I: 7.04 83
l week 80.7l :l: l.03 24
2 weeks 67.05 :I: 5.70 30
3 weeks 69.88 :I: l.25 '0
4 weeks 55.49 i 0.97 '6
5 weeks 50.44 :I: L66 3
6 weeks 50.40 :I: L86 3
8 weeks 50. I 8 :l: 5.77 9

 

 

 

 

Histological assessment of the stress-deprived tendons demonstrated
progressive alteration in tissue morphology compared to normal tendon (Figure l2).
These changes included changes in cell shape and number. Following 2 weeks of stress-
deprivation. the cells were morphologically similar to those seen in the control tendons
(Figure l3). With increased time of stress-deprivation a decrease in cellularity became
more evident. By 4 weeks cell nuclei were more rounded and showed occasional
fragmentation (Figure I4). After 6 weeks of stress-deprivation a marked decrease in
cellularity was observed (Figure l5). In addition. a majority of the collagen fibers
demonstrated a random alignment differing markedly from the typical parallel
orientation observed in the control tendons. Following 8 weeks of stress-deprivation.
very few cells were observed. The few cells which remained showed evidence of
degradation (Figure l6). and collagen alignment varied with some areas depicting normal

crimp. other areas showed erratic crimp patterns.

Freeze-Thaw Tendon Group:

The tendons which were taken through the freeze-thaw procedure prior to the
immobilization in vitro were evaluated in a similar manner. Tensile testing again resulted
in classic load-deformation curves. with no deviation seen in either groups. The
tendons that were freeze-thawed and subsequently maintained in tissue culture for 2
weeks had an tensile modulus of 99.3% of the respective time zero controls. Similarly
those tendons freeze-thawed and stress-deprived for 4 weeks had an tensile modulus of
l00.2 % of their respective controls (Figure l7). Both the 2 week and 4 week freeze-

thawedlstress-deprived groups were shown to be

50

5|

 

 

Figure l2: Histological appearance of H&E stained time zero control canine FDP
tendon. as seen with light microscope (at 40X magnification).

52

 

 

 

Figure I3: Histological appearance of H&E stained canine FDP tendon after 2 weeks
of stress-deprivation. Observed with light microscope at 40X magnification.

53

 

I

Figure l4: Histological appearance of H&E stained canine FDP tendon after 4 weeks
of stress-deprivation. Observed with light microscope at 40X magnification.

54

 

 

 

Figure l5: Histological appearance of H&E stained canine FDP tendon after 6 weeks
of stress-deprivation. Observed with light microscope at 40X magnification.

55

 

l
l
l I
Figure l6: Histological appearance of H&E stained canine FDP tendon after 8 weeks
of stress-deprivation. Observed with light microscope at 40X magnification.

56

TENSILE MODULUS (expressed as e X of control)

    

 

loo.oox ( , 98x92 . loo.st
80.00% -»
60.00% --
40.00:; ‘-
2o.oox +-
0.00% e- . .
2 WEEK 4WEEK
STRESS- STRESS-
DEPRIVED , DEPRIVED
(n=l9) (n=2l)

Figure I1: Histogram depicting the effect of freeze-thaw technique with subsequent
stress-deprivation on the tensile modulus of the canine FDP tendon.

statistically the same (p>0.05) when compared to their respective time zero controls.
by the Student t-Test. Similarly. the 2 week and 4 week values were statistically the

same (p>0.05)

Histological evaluation of the 2 week and 4 week freeze-thawed. stress-deprived
tendons demonstrated a complete absence of cells (Figure I8). However collagen

orientation and alignment did not appear to be adversely affected.

H2: Mobilization Study

This portion of the investigation examined the effects of a controlled program of

cyclic loading. on the material properties of the tendons maintained in vitro.

Using the previously described cyclic loading regimen. the tensile modulus for
the tendons mobilized for 2 weeks was 84 % of their individual controls. While this
elastic modulus value was higher than that shown by the 2 week stress-deprived tendon

group it was not statistically different (Figure I9).

Histological appearance of the tendons cyclically loaded for 2 weeks was
comparable to that seen in the zero time control tendons (Figure 20). However. the
cellular morphology and collagen crimp pattern in the 2 week mobilized specimens was
better than that observed with the 2 weeks immobilized group. The tendon showed
high cellularity. with elongated spindle-shaped within normal. parallel oriented collagen
fibers. Thus. maintenance of cellular morphology was seen despite the slight diminishing

of mechanical prOperties.

57

58

 

 

1

Figure I8: Histology (H&E stained) of canine FDP tendon following freeze-thaw
technique and 2 weeks stress-deprivation. Complete absence of cellularity is seen.
(Observed with light microscopy, 40X magnification).

2C

Fl}
dej

59

TENSILE MODULUS (expressed as e % of control)

 

 

I00.00% 1'
80.00% --
60.00% --
40.00% <-
20.00% I
0.00% - ’ ‘

2 WEEK 2 WEEK

LOADED STRESS-

(n=l9) DEPRIVED

(n=3l)

Figure I9: Histogram depicting the variations seen between tendon groups stress-
deprived for 2 weeks and those cyclically loaded for 2 weeks.

60

 

Figure 20: Histological section of canine FDP tendon cyclically loaded (with 50 gram
load) for 2 weeks in tissue culture. Viewed with light microscopy at 40 X magnification.

H3: Stress-Deprivation I Remobilization Study

The third hypothesis explored in this thesis was done to determine the ability of
cyclic loading to reverse or arrest the degenerative changes (decreased tensile modulus.

change in cell morphology) associated with stress-deprivation.

The tensile modulus of tendons stress-deprived for 2 weeks then cyclically
loaded for 2 weeks was found to be 80.8% of their individual controls. While this value
is higher than tendons stress-deprived for 2 weeks it was not statistically different.
However the tensile modulus for the remobilized tendons was significantly higher than

for the group stress-deprived for 4 weeks (Figure 2|)

Histological evaluation of the remobilized tendons showed a slightly different
cellularity as compared to the control tendons. However. the majority of the cells in
the tendon were Spindle-shaped and appeared healthy (Figure 22). This is in contrast to
the tendons deprived of stress for 4 weeks which demonstrated a distinct decrease in

cellularity and alterations in cellular morphology.

6l

62

TENSILE MODULUS (expressed as % of control)

l00.00% 1'

79.l9% 8033"
80.00% -~ -;; A .

   
           

 

e.

2 WEEK LOADED 2 WEEK STRESS- 2 WEEKSSTRESS— 4 WEEKS STRESS-
(n= l9) DEPRIVED DEPRIVEDIREMOB. DEPRIVED
(n=3l) (n= l9) (n= IS)

Figure 2|: Histogram comparing the effects of 2 weeks cyclic loading. 2 weeks
stress-deprivation. 2 weeks stress-deprivation/cyclic loading. and 4 weeks stress-
deprivation on the tensile modulus of canine FDP tendon.

63

 

 

Figure 22: Histological section (H&E staining) of canine FDP tendon deprived of stress
for 2 weeks and then cyclically loaded (with 50 gram load) for 2 weeks in tissue
culture. Viewed with light microscopy at 40 X magnification.

IV. DISCUSSION

The results of this stress-deprivation study are indicative of the same trend in
decreased material properties as seen in previous investigationszv ll. 32- 4" 43. 44' 54'
56. 52' 75. 82. The time-dependent decrease in the tensile modulus of the tendons
deprived of stress for 0 - 8 weeks is a clear indication of the injurious effect of
immobilization. A sharp decrease in tensile modulus (~ I3%) was observed as early as
after 2 weeks of stress-deprivation. This indicates that alterations secondary to lack of
stress occur soon after the procedure is commenced. From the onset of stress-
deprivation. the material properties of the tendon were seen to decrease significantly.
finally leveling off at approximately 6 weeks. The interesting circumstance of this level

of stress-deprivation was seen upon histological evaluation.

Histological assessment indicated that the fibrocytes began to deteriorate after 4
weeks of stress-deprivation. The change in cell shape observed can be interpreted in
two ways: either the previously spindle-shaped cells have now changed their shape due
to a lack of mechanical stimuli to provide them with a direction for alignment (parallel
to the direction of the mechanical stimuli). or new cells have arisen which are not
morphologically the same as the previous fibrocytes. It is probably more likely for the
cells to be changing their shape in response to their mechanical environment. It was also
observed that cellularity decreases with increased times of stress-deprivation becoming
significant after 4 weeks. This reduction in cellularity correlated well with the significant

decreases in tensile modulus at 4 weeks and 5 - 8 weeks.

64

To determine if the above noted immobilization-related changes were cell
mediated. the tendon cells were destroyed by freezing and then maintained in tissue
culture. The results revealed no significant change in the material properties had
occurred. This suggests that the cells do indeed play an active role in tissue
homeostasis. This is especially apparent when comparing the tensile moduli of the
tendons stress-deprived for 2 weeks and those freeze-thawed with subsequent stress-
deprivation for 2 weeks: similar results were seen upon comparison of the 4 week
groups (Figure 23). The 2 week stress-deprived group was significantly different than
the 2 week stress-deprived (freeze/thaw) group; similarly the 4 week stress-deprived
group was significantly different from the 4 week stress-deprived (freeze/thaw) group.
This disparity in the tensile modulus appears to be due to the elimination of any cellular
effect. This result would correlate well with the decreased elastic modulus observed at

4 - 8 weeks immobilization. when cellularity appears to diminish at a quick rate.

 

2 2 4 4
weeks weeks weeks weeks
(In) (in)

Figure 23: The tensile modulus of the 2 week stress-deprived and 4 week stress-
deprived groups as a % of their respective controls. Column graph depicts the effects
of absence of cells (freeze/thaw grOUp) on the tensile modulus of stress-deprived
tendon.

65

The lack of change in material properties with the absence of cellularity leads to the
conclusion that stress-deprivation causes specific changes to occur at the cellular level.
It is evident that the application of mechanical stress to connective tissue has a
beneficial effect. However. the distinct manner by which stress works is not well
understood. Few have postulated that stretch-activated ion channels on the surface
of cells are responsible for this benefit derived from mechanical stimulus. The channels
allow for a flux of ions to proceed through the cell membrane. The ion flux may be
responsible for stimulating cell metabolism. which is required for tissue health. The
stretch-activated ion channels are closely associated with the cellular cytoskeleton: thus
upon application of mechanical stress it would appear that cells would deform. This
translates to the direct deformation of the cytoskeleton. It is perhaps this deformation
that causes the activation of these ion channels. Thus. the basis of the time-dependent.
immobilization-related degradation may be explained by studying what occurs at the
cellular level of the tissue. Previous studies done with cyclic straining of connective
tissue in vitr026- 42- 68: 59 have shown that mechanical strain activates cellular
responses which result in increased cell proliferation and changes in protein synthesis.
By studying strains at the cellular level. correlation between what is occurring at the
global. tissue level and at the cell surface may help in gaining insight into the biological

basis of connective tissue injury and healing.

Following 6 weeks of immobilization - the typical immobilization period for
connective tissue deformities and/or injuries - a 45 % decrease in the tensile modulus
was seen in this study. As previously mentioned. the cellularity of the tendon was also
low. This may imply that similar alterations in morphology may occur in viva after 6

weeks of immobilization. The fact that clinical physiotherapy. following injury and

66

immobilization. can sometimes take up to l2 months before resulting in any increase in

strength or ease in mobility shows the damaging impact of a long immobilization period.

Gamble et al. (20) found chemical alterations to occur after 2 weeks of
immobility when they evaluated the enzymatic changes in the ligament of rabbit.
Morphological changes in the tendon after 4 weeks of immobilization were a result of a
decrease in GAG content. due to enzymatic degradation. This result is consistent with
the first great decrease in material prOperties and cellularity observed in the first
portion of this thesis investigation. This study has shown the deleterious effects of
stress-deprivation on the material properties of tendon: Loitz (44) found similar results
with short-term immobilization periods. noting a marked decrease in linear tensile

stress. Similar results were seen in other studies by Akeson3, Amiels. and Woo”.

The evaluation of the effect of mechanical stimuli (cyclic loading) on the material
properties was studied in the second and third hypotheses of this study. The effect of
immediate mobilization (H2) on the material properties of tendon was evaluated in
order to gain insight as to what occurs in the healthy tissue when the injured joint is
mobilized immediately. Tissue morphology resembled that of untreated. healthy
tendon. whereas the material properties were less than the controls. Other studies
have seen a protocol of CPM to result in a tissue that is mechanically superior to an
immobilized tissue (44). While the cyclic loading regime utilized in this study resulted in
an increase in material properties. it was not a significant increase. The reason for this

is unclear but may be due to the limited number of specimens examine.

67

The effect of mobilization of tendons previously deprived of stress
(remobilization) was the focus of the final hypothesis of this study (H3). The aim was
to arrest the immobilization-related decreases in tensile modulus and cellularity by
application of controlled motion. The cyclic loading regime utilized in this study
resulted in a mechanically superior tendon following remobilization for 2 weeks. The
significance of this result is that remobilization was able to stop the immobilization-
related alterations. while retaining cellularity similar to that of the 2 weeks stress-
deprived group. after 4 weeks in a tissue culture environment. From this study it was
concluded that the controlled cyclic loading regime had a counter-balancing effect on
the harmful consequences of immobilization for 2 weeks. The results of this part of the
study indicate that the deteriorating effects of immobilization can be stopped and.

perhaps reversed with increased remobilized periods.

Thus. in regards to mobilization studies. this research was able to repeat. to a
certain extent. with an in vitro tissue culture technique. the results of previous studies in

which immobilized tissue was found to recover with the application of cyclic loading 23.
4|. SI. 52. 56. 65. 73. 74. 80. 82. 84.

Exercise. in many forms, has been shown to result in tissue than that tissues
which are mechanically superior to immobilized tissue. Tipton and his associates were
among the first to document the benefits of exercise 73-74. A majority of the studies
done examining the benefits of exercise or controlled motion on connective tissue have
either been in vivo animal model studies. Thus there is not any substantial prior data to
base the results of this in vitro study. except via inferences to the above mentioned

studies. CPM has been seen to cause an increase in tissue stiffness with respect to an

68

immobilized control tissue (44). The CPM tissues in Loitz's study (44) showed tensile
modulus values between those of the immobilized and the control groups. Similar

results were seen in this study.

It may be possible that controlled mobilization is more effective in rehabilitating
immobilized tissue. when applied for longer periods of time. Larsen (4|) found it
required 6 weeks of remobilization to successfully remove the harmful effects of a 4
week immobilization period. Others have found longer immobilization periods
followed by remobilization for the same time to result in superior tissue strength.
Woo (82) found a 9 week immobilization period. followed by a 9 week remobilization
period to result in increased tendon strength. as compared to controls. Further
investigation is required to examine the effects of a longer remobilization period.

utilizing an in vitro tissue culture model.

Because the tendons examined in this study had never been damaged or
surgically repaired. the results are pertinent for the assessment of immobilization and
remobilization effects on healthy tissue. Also. from a clinical viewpoint. the duration of
a rehabilitative program required to overcome the adverse effects of immobilization
alone would be much shorter than that required to overcome the combined effects of
surgery and subsequent immobilization. Therefore. realistically. the remobilization

portion of the rehabilitation may need to be longer than the immobilization period.

It is probable that varying levels of mechanical stimuli are needed for the
maintenance of tissue homeostasis. for the repair of injured tissue. as well as for tissue
that is remodeling. The in vitro model utilized in this study allowed for the precise

control of the loading parameters. such as frequency and duration. in exploring the

69

effect of mobilization on the material properties of connective tissue. This may allow
for the designing of more proficient rehabilitation procedures upon further

investigation.

Future Research Directions

Now that it has been established that the decrease in tensile modulus in a time-
dependent phenomenon. the next logical step is to evaluate how this process can be
stopped or reversed utilizing controlled passive motion. It is evident that 2 weeks of
remobilization is able to stop. if not fully reverse the detrimental changes occurring
after 2 weeks of immobilization. The increasing of the remobilization periods by
increments of weeks may be the next manner in which to sequentially document the

effects of an immobilization-remobilization protocol.

Another parameter to be examined is the effect of different frequencies of the
cyclic loading regimens. The frequency used for the mobilization studies in this research
was arbitrarily chosen: in order to commence the study a frequency was required and l
rpm was randomly chosen. Upon comparing slow and fast frequencies of CPM. faster
frequency has recently been documented to result in more favorable tissue tensile
properties (7|). This result is worth exploring. as the I rpm frequency utilized in this
study may well be a factor in the decrease/regaining of material properties of the
cyclically loaded tendons. Similarly it would be worthwhile to explore different

durations of cyclic loading of the tissue.

70

Conclusions

l. Stress-deprivation results in a progressive degradation of the material properties

of tendon. This is based on evaluation of the tensile modulus of the tissue.

2. For short time periods. cyclic loading of healthy tissue results in tissue
morphology similar to that of the non-treated. control tissue. However. normal

material properties were not maintained with short-term cyclic loading.
3. Cyclic loading of previously immobilized tissue appears to arrest the harmful

effects associated with short-term immobilization. However. significant reversal

of these alterations was not seen in this study.

7|

V. APPENDIX

APPENDIX

W
A summary of the symbols:

F = force or load in N
5 = deformation in mm
ID = initial gauge length of tendon in mm
A = cross-sectional area of tendon in mm2
K = stiffness coefficient of tendon in N I mm
E = elastic modulus of tendon in mega Pascal
0' = stress (= force/unit area) in mega Pascal
e = % strain

From the load-deformation curve:

K=AFI A8 ...inglmm2 (I)

E = A6 IAe in g I mm2 (2)

o = F I A = A6 = (AF)(I IA) ...in g I m2 (3)
IS constant

and A8 = change in length of tendon I original length of tendon
= deformation I original length

= Ae=5llo ...inmmImm(4)
IS constant

Upon substitution of equations (3) and (4) into equation (2):

=> E=AoIAe= (AF/A)(8llo)
= (AFXIO) I (5)(A)
= [(AF)/(5)I[('o)/(A)]

K = (AH/(5)

= E = (K)*[(|o)l(A)] = (K)(lo)l(A) inglmmz

72

Once the value for "E” was obtained In the units g I mm2 . the conversion was done
into the units of Pascals. as follows:

E = (K)(lo) I (A) = (glmm)(mmlmm2) = glmmz
E = I x I06 kg I m2
...conversion to Newtons:

g=9.8lmIs2

= E = (I x I06 kg I m2)*(9.8l mIs2)
= 9.8I x I06 (kg-mlsz) 1 (m2)

l kg-mls2 = l Newton
= E = 9.8l x I06 N/m2

I Nlmz = I Pascal

=> E = 9.8l x l06 Pascals = 9.8l mega Pascals (m-Pa)

73

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l0.

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