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@ Copyright by

DANIEL JOSEPH SELKE
1981

A STUDY OF SELECTED LIGAMENTS AND TENDONS
OF THE KNEES AND ANKLES
OF THE RHESUS MONKEY, BABOON
AND CHIMPANZEE

By

Daniel Joseph Selke

A THESIS

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

MASTER OF SCIENCE

Department of Metallurgy, Mechanics, and Material Science

1981

ABSTRACT
A STUDY OF SELECTED LIGAMENTS AND TENDONS
OF THE KNEES AND ANKLES

OF THE RHESUS MONKEY, BABOON
AND CHIMPANZEE

By
Daniel Joseph Selke

The purpose of this research was to study the mechanical properties
of the flexor hallucis longus and tendo-calcaneus tendons of the ankles,
and the medial collateral and patellar ligaments of the knees from the
rhesus monkey, baboon, and chimpanzee using a three hour experimental
protocol which included preconditioning, constant strain rate, cyclic,
and relaxation tests. The preconditioning tests, in most cases, indicated
a lack of stability of the preconditioning due to a long term relaxation
phenomena. Constant strain rate data were used to measure the value of
the tangent modulus which correlated well with the percent of elastin
found from histological observations. The maximum stress, tangent
modulus, load input energy, and hysteresis area all decreased with a
decrease in strain rate. Cyclic and relaxation tests showed a short term
viscoelastic response of comparable nature being linear with the natural

logarithm of time.

ACKNOWLEDGEMENTS

The author wishes to express his deepest appreciation and
gratitude to the following people for making this phase of his
graduate work possible:

To Dr. Robert Wm. Little, his major advisor, for his constant
encouragement and friendship, and especially, his valuable assistance
in the preparation of this thesis;

To Mr. David L. Hyler for his friendship and technical assistance
in this study;

To Mr. Robert Schaeffer for his technical assistance in the smooth
operation of the testing equipment used in this study;

To Ms. Jane Walsh for her friendship, and especially her technical
assistance in the histological preparations used in this study;

To the United States Air Force for the financial support required
for this study under contract number F336l5-79-C-0514.

To his family, especially his parents, Joseph and Libia, for their
understanding, encouragement, endless patience, and interest in his

graduate work and academic education.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

LIST OF FIGURES

LIST OF TABLES .
INTRODUCTION
MATERIALS AND METHODS

Sample Preparation . . .
Dissection of the Knee Region
Dissection of the Foot Region

Histological Observations
Tendo-calcaneus Tendon .
Flexor Hallucis Longus Tendon
Patellar Ligament . .

Medial Collateral Ligament

Gripping . .

Mechanical Testing Equipment

Mechanical Testing Protocol .
Geometric Properties

RESULTS AND DISCUSSIONS .
CONCLUSION
BIBLIOGRAPHY

iv

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LIST OF FIGURES

Figure Page
l. Typical Stress-Strain Curve for Connective Tissue ....... 4
2. Schematic Drawing of Testing Grips ............... 18
3. Preconditioning Stability of Flexor Hallucis Longus Tendon . . . 24
4. Preconditioning Stability of Tendo-calcaneus Tendon ....... 25
5. Preconditioning Stability of Medial Collateral Ligament ..... 26
6. Preconditioning Stability of Patellar Ligament ......... 27
7. Average Stress-Strain Curves at a Constant Strain Rate of

lOO%/sec for the Flexor Hallucis Longus Tendon ......... 30
8. Average Stress-Strain Curves at a ConStant Strain Rate of

100%lsec for the Tendo-calcaneus Tendon ............. 3l
9. Average Stress-Strain Curves at a Constant Strain Rate of

lOO%/sec for the Medial Collateral Ligament ........... 32
l0. Average Stress-Strain Curves at a Constant Strain Rate of

lOO%/sec for the Patellar Ligament ............... 33
ll. Average Cyclic Relaxation Curves for the Flexor Hallucis

Longus Tendon .......................... 37
l2. Average Cyclic Relaxation Curves for the Tendo-calcaneus Tendon . 38
l3. Average Cyclic Relaxation Curves for the Medial Collateral

Ligament ....... . ..... . . . ............ 39
14. Average Cyclic Relaxation Curves for the Patellar Ligament . . . 40
l5. Average Standard Relaxation Curves for the Flexor Hallucis

Longus Tendon . . . . . . . ........ . . . . . . ..... 43
16. Average Standard Relaxation Curves for the Tendo-calcaneus

Tendon ............................. 44

Figure

17.

18.

Page
Average Standard Relaxation Curves for the Medial Collateral
Ligament ............. - ............... 45
Average Standard Relaxation Curves for the Patellar Ligament 46

vi

LIST OF TABLES

Table Page

1. Cross-sectional Areas of the Flexor Hallucis Longus Tendon
from Histological Slides .................... l0

2. Cross—sectional Areas of the Tendo-calcaneus Tendon
from Histological Slides .................... ll

3. Cross-sectional Areas of the Medial Collateral Ligament
from Histological Slides .................... l2

4. Cross-sectional Areas of the Patellar Ligament
from Histdlogical Slides .................... l3

5. A Summary of Averages for Specific Mechanical Properties
at a Constant Strain Rate of lOO%/sec .............. 29

6. A Summary of Percentages for Specific Mechanical Properties
at Variable Strain Rates .................... 36

vii

INTRODUCTION

Tendons and ligaments play a major role in the body's support
system, and have stimulated much interest and many investigations to
determine their mechanical and histological properties. Tendons consist
of fibrous cords of connective tissue that join muscle fibers to bones.
Ligaments are also fibrous connective tissues that provide bone-to-bone
Junctions giving the necessary support and strength to the joints.

The basic study of the connective tissues, tendons and ligaments, is
fundamental to understanding the mechanical properties of collagenous
tissues. A brief survey of earlier work will be presented as the
various methods of testing, specimen choice and data analysis used by
different researchers have led to results which are hard to compare.

One of the most comprehensive surveys of investigations on the
structure and function of mammalian tendon was conducted by D.H. Elliott
in 1965 [1]. He stated that besides primarily transmitting tensions,
tendons had important secondary functions. These were "the removal of
the bulk of a muscle from the joint over which it acts, the concentration
of muscular pull on to a small area of bone, the modification of muscle
action over several joints by retaining bands and the possible protection
of the muscle by the buffer action of its tendon during an unexpected
stress". In 1975, Evans and Barbenel [2] defined tendons as serving

mainly as force transmitters, but having a second mechanical function in

their ability to store energy elastically.

Elliott [1] also included a histological description of the
structure of tendons in his l965 paper. He described tendon as consisting
almost entirely of collagenous tissue whose structural unit is the
collagen fiber, or primary tendon bundle, and constitutes approximately
80% of the dry weight of the tendon. Primary tendon bundles are
organized into secondary tendon bundles, which are organized into
fasciculi, or tertiary tendon bundles. In l973, Minns et al [3]
described tendons as consisting "largely of parallel wavy bundles or
sheets of collagen fibers surrounded by the interfiber matrix and a
sparSe network of elastic fibers. The collagen fibers provide the great
strength of the tissue while the elastic fibers seem to provide
elastic recovery and draw the collagen fibers back to the wavy condition
in the relaxed state". They also described the structure of ligaments
which differs from that of tendons. While collagen is a major constituent
in most tendons and ligaments, elastic fibers were reported to be more
prevalent in ligaments. When in the relaxed state, the collagen
fibers in the ligaments were much less aligned than in tendons. A more
complete structural hierarchy of tendon was given by Kastelic et al [4]
in l978. To briefly summarize this hierarchy, they described tendon
as consisting of five units of tropocollagen that made up micro-fibrils,
which form together to make sub-fibrils that generate fibrils producing
fascicles, and finally constituting a tendon.

Rigby et al [5], in l959, determined-that the geometry of the
collagen fiber in wet rat-tail showed a macroscopic banded or crinkly
pattern when in its natural position. Their further observations
indicated that the apparent helical microstructure of the tendon was an

illusion resulting from the angle and type of illumination, and found the

wave-form disappeared with less stretching than the amount required to
eliminate the helix. The wet rat-tail tendon was also teased into
many hundreds of wavy "subfibers" which showed no evidence of inter-
twining. A very detailed study was also done in 1972 by Diamant et al
[6], who analyzed the periodic bands along the rat-tail tendons by
using polarizing optics. They discovered that the periodic pattern
along the tendon had a two-dimensional planar and sinusoidally shaped
arrangement. Evans and Barbenel [2], in 1975, studied the way fibers
were grouped into bundles or fasciculi and recognized that the dimensions
and arrangements of these bundles vary considerably from tendon to tendon.
Much has been written about the viscoelastic properties of biological
tissues. As long as the strain did not exceed approximately 4% for
strain rates between l - 20%/minute, Rigby et al [5] found that the
rat-tail tendon's mechanical behavior was reproducible, or reversible,
when the tendon was allowed to rest a few minutes after each elongation.
Partington and Wood [7] in l963, showed that the stress-strain
properties of rat-tail tendon fibers were reversible up to 2% strain.
If fibers were stretched more than 3%, the mechanical behavior was
irreversible, and the fibers did not return to their original length when
released.
In the reversible region, tendons and ligaments exhibited a non-
linear behavior. A typical stress—strain curve is shown in Figure l,
and can be divided into three ranges. In Region I, the response was
linear due to the elastin fibers resisting extension, while the wavy
collagen fibers were straightened or aligned, but do not carry the load.
Therefore, the waviness of collagen determined the extent of the Region

I response. The secondary range, Region II, showed gradually increasing

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slope due to the stiffening of the tissue by the loading or the variation
in length of the collagen fibers. The slope of Region II was determined
by the distribution of the original waviness for individual collagen
fibers. In the works of Rigby et al [5] in l959, Millington et al [8]

in l97l, and Diamant et al [6] in l972, it was concluded that Region III
exhibited a high, constant slope as all straightened collagen fibers
resisted further extension. The tangent modulus or slope was determined
by the quantity and structure of collagen in the tissue, and represented
a measure of the stiffness.

In l959, Rigby et al [5] computed an average maximum slope from the
stress-strain curves for wet rat-tail tendon, to be 8.0 i 2.0 x l09 dynes/
cm2 (800 z 200 MPa) at a strain rate of lO%/min. They also noticed that
strains up to 20% could be reached without collagen fiber bundles break-
ing if the strain rate was sufficiently slow, less than l%/min.

When strain rate was increased, the stress-strain curves were identical,
but shifted toward the stress axis. Elliott [l] in 1965, measured the
mechanical tensile strength of certain tendons from different mammalian
species. For example, the human tendo-calcaneus tendon had a tensile
strength of 4.7 kg/mm2 (0.48 MPa). Minns et al [3], in 1973, observed
complete elastic recovery in human Achilles tendon specimens that were
loaded up to 2% strain, and gave values for the failure stress for
certain tendons and ligaments. The tensile strength of human Achilles
tendon was given as 4950 - 8000 lbf/in2 (l.73 - 2.79 MPa), while for the
ligamentum nuchae from an adult cow was 200 - 2500 lbf/inz (0.07 - 0.87
MPa). Viidik [9] in 1980, reported a higher ultimate strength of tendons
as 50 - l00 Wm2 (50 - 100 MPa) with an ultimate strain in the range

of l5 - 30%.

Biological materials exhibit viscoelastic behavior in any time-
dependent test. This was revealed in rate dependency for stress-strain
tests at different constant strain rates, and for hysteresis under cyclic
loading, cyclic stress relaxation, and stress relaxation under constant
strain. In l972, Fung [l0] noted that the hysteresis loops of canine
artery decreased with succeeding cycles to a steady state after a number
of cycles. Torp et al [ll] in l974, recorded decreases in hysteresis
loop area and maximum stress at a constant maximum strain of 2% for
rat-tail tendon. They also showed that the decay in maximum stress
versus the log of the number of cycles is almost a linearly decreasing
function. This decay rate was similar to stress relaxation at constant
strain. However, the strain level was constant for the stress relaxa-
tion, while in the cyclic tests, the deformation was cyclically applied
to a constant strain level.

The researchers referenced used different methods of testing and
data analysis, but all found that tendons and ligaments exhibited visco-
elastic properties that could be defined from stress-strain curves,
hysteresis, cyclic, and relaxation data. With these parameters a
mathematical model could be developed to describe this tissue.

This thesis will present the mechanical properties and histological
characteristics of the medial collateral and patellar ligaments of the
knee and the flexor hallucis longus and tendo-calcaneus tendons of the
ankle of the rhesus monkey, baboon, and chimpanzee, to determine
parameters that could be used in the development of a mathematical
model. ‘These viscoelastic properties will be defined using data from

different stress-strain rates, hysteresis, cyclic, and relaxation tests.

MATERIALS and METHODS

Special techniques in sample preparation, gripping, and testing
protocol were adopted from a previous study [l2]. These aspects
will be discussed in detail, including a concise description of the

testing equipment.
Sample Preparation

Tendo-calcaneus and flexor hallucis longus tendons and patellar
and medial collateral ligaments were dissected from rhesus monkey,
baboon and chimpanzee carcasses which were supplied by the Air Force
Aerospace Medical Research Laboratories. These animals were medically
healthy and not previously used in experimentation. After death, the
primates were chilled in dry ice immediately, and transported to the
testing laboratory. The lower limbs were removed from the hip socket
and stored at -5°C until further dissections could be performed.

While frozen, the lower limbs were sectioned into knee and foot regions
and stored separately in identifiable plastic bags. Prior to dissection
and testing, the tissue was allowed to thaw at room temperature.
Dissection was performed in a humid environment using a humidifier

blowing air over the tissue.
Dissection of the Knee Region:

Skin and extraneous tissues were removed to expose the ligaments of

interest. The patellar ligament was removed first due to its accessibil-
7

ity. The test specimen ran from the apex of the patella bone to the
anterior tibia below the condyle. A surgical chisel was used with
moderate blows from a rubber mallet against the condyle of the anterior
tibia to insure a bone-ligament—bone specimen. Next, the medial
collateral ligament was dissected from the medial epicondyle of the femur
to the medial tibial shaft, again yielding a bone-ligament-bone

subject with use of a chisel and mallet. The bony attachments of both

ligaments were sanded smooth on a sander to facilitate mechanical

gripping.
Dissection of the Foot Region:

Skin and superficial tissue of the plantar aspect of the foot
along with the posterior side of the leg was removed to expose the inner
structures. First, the tendo-calcaneus tendon was dissected after
being freed of fascia from the gastrocnemius muscle to the calcaneum.
The tendon was severed below the muscle and separated from underlying
tissue all the way to the calcaneum where the bony attachment was
preserved. Next, the flexor hallucis longus muscle and tendon were
exposed by removing the soleus muscle, and extraneous muscles and ten-
dons. The part of the tendon used for testing was severed before the
osseo-fibrous tunnel and at the midpoint of the plantar exposure.

After the dissections were completed, all tissues were wrapped in
moist paper towels and stored at 4°C for not more than 24 hours, at
which time mechanical testing was performed.

After all mechanical testing was completed, the tissues were fixed

in l0% buffered formalin for several days. A post-fixation in Bouin's

fluid for 24 hours followed. Samples were then sectioned in transverse
and longitudinal planes and processed according to standard paraffin
embedding methods. The tissue blocks were cut at7u thickness on a
rotary microtome and stained. The stains routinely used were
Hematoxylin-Eosin for general morphology [l3], Davenport's modification
of Halmi's for collagen and elastin [l4], and Frankel's 0rcein for
elastic fibers [15]. Histological samples were taken from ligaments and
tendons of both the right and left limbs of the three primates.
Cross-sectional areas were measured by placing metric graph paper
underneath the slides and "counting squares" using a microscope.

The areas of different ligaments and tendons are given in Tables 1 to 4.
Details of area measurements are described under "Geometric Properties"

(See page 21).
Histological Observations
Tendo-calcaneus Tendon:

The tendo-calcaneus is a large broad tendon located on the
posterior aspect of the lower limb. It attaches the gastrbcnemius
muscle, which composes a large portion of the back of the leg, to the
calcaneum (heel bone). Where the gastrocnemius fibers unite with the
tendo-calcaneus, the soleus muscle tendon integrates into the main body
of the tendo-calcaneus, producing a "two—tendon“ structure in the
middle and proximal areas. In the distal area, there is a twisting and
fanning out of the fibers providing a broad area of insertion as the

tendon attaches to the calcaneum.

'10

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14

Microscopically, the tendo-calcaneus is composed predominantly of
collagen with varying amounts of loose connective tissue and vascula-
tion. Two distinct segments are usually seen in the middle and proxi-
mal regions, but are not evident in the most distal sections.

Loose connective tissue and small blood vessels are seen between these
segments. The collagen fiber bundles are parallel and slightly to
moderately wavy in nature and elastic fibers are evident in all areas.
Elastin content from histological slides ranged from approximately

5 - l0% for the chimpaniee, 20% for the rhesus monkey, and up to a
value of 30% for the baboon. In areas of collagen-muscle attachment,
the muscle fibers joined the collagen fibers at angles up to 30°.

The tendon capsule is composed of loose connective tissue with muscle
fibers often seen on the anterior side. ‘This connective tissue varies
from a compact layer at the posterior surface to a broader area of

loose connective tissue on the anterior surface.
Flexor Hallucis Longus Tendon:

The flexor hallucis longus tendon has an elliptical cross-
section and extends distally from the flexor hallucis longus muscle in
the posterior of the lower leg, through the osseo-fibrous tunnel
along the plantar aspect of the foot, through the fibrous distal
sheath and attaches to the bottom of the distal phalange of the
first toe. At the microscopic level, it is an ovoid structure made up
of compact collagen fiber bundles with small amounts of loose connec-
tive tissue between the fascicles. Areas of fasculation are also

found among the groups of bundles, while muscle can be seen along the

15

capsule surface on the anterior aspects. Loose connective tissue

and blood vessels are found on the anterior surface, while the
capsule on the posterior surface is very compact, showing very little
loose connective tissue or vasculation. The parallel fiber bundles
are slightly to moderately wavy, and elastic fibers are evident at all
levels as well as in the surrounding loose connective tissue.

Elastin content ranged from approximately 5 - l0% for the rhesus
monkey, and approximately 5% for the baboon, to a value of less than

1% for the chimpanzee.
Patellar Ligament:

The patellar ligament is a broad, thick ligament situated on the
anterior surface of the knee. It attaches from the tibia below the
condyles to the non-articulating area of the apex of the patella bone.
The ligament is composed of compact collagen bundles with a broad area
of loose connective tissue surrounding the joint capsule. Extensions of
the Capsular connective tissue invade the structure dividing the
fascicles into segments. This is particularly noted at the proximal and
distal ends with less marked segmentation in the middle region.

The fiber bundles are broad, parallel, and slightly wavy. Large areas
of loose connective tissue and vasculation are seen between groups of
bundles. Elastic fibers are also seen in all areas sampled, with a
high content of approximately 25 - 30% for the baboon and approximately
10 - 15% for the chimpanzee. No value for elastin content was estimated
for the rhesus monkey, since the samples used for testing were so

small that once histological preparations were made for determining

16

ligament cross-section, insufficient material was available for
longitudinal sections. It is worthy to note that in the middle region
on the posterior side of a longitudinal section of the patella, there
exists a narrow band of parallel fibers that runs perpendicular to the
fiber bundles of the main ligament. These fibers are moderately

wavy and are separated from the rest of the ligament by a zone of loose

connective tissue and blood vessels.
Medial Collateral Ligament:

The medial collateral ligament (also known as the tibial collat-
eral) is a long, thin, flattened ligament. It attaches at the top of
the medial epicondyle of the femur, transverses the medial aspect of
the knee, and attaches to the medial tibial shaft.

Microscopically, it has an elongated, elliptic cross-sectional
structure composed of round fascicles with small amounts of loose
connective tissue between them. A thick capsule surrounds the liga-
mentous structure. Longitudinally, the fiber bundles are compact,
parallel, relatively acellular and moderately wavy. Loose connective
tissue with some vasculation is observed in the capsular composition.
Elastic fibers are found approximately 25 - 30% for all species within

the fiber bundles, and in greater amounts in the ligament capsule.
Gripping

The concept that ligaments constrain the motion of the knee was

the basis of the grip design. The bone attachments for the ligaments

17

were held by the grips, and the axial displacements were imposed by
the testing machine. By tightening the screws to secure the bony
attachments within the grips, slippage of the tissue was prevented

or kept at a minimum. The function of tendons is to transmit forces
from muscle to bone. To eliminate slippage for all ligaments and
tendons, especially at the muscle end of the tendon, a waterproof
abrasive mesh (silicon carbide 120 grit "sand screen" by 3M) was
epoxied to the surface of the grips. This increased the effectiveness
of the grips and at the same time helped in preventing any damage in
cutting or tearing of the tissue during testing. The grips were
designed to be much stiffer than the samples tested so that the motion
of the testing machine would be equal to the sample displacement.

The same grips were used for all specimens and are shown schematically
in Figure 2. The upper gripping plate was attached to the actuator of
the testing machine and the lower plate to the load cell. The sample

was held against these plates by stainless steel bars.
Mechanical Testing Equipment

Mechanical testing of the tissue specimens was conducted with an
Instron (Model 1331) machine that is hydraulically powered and
electronically controlled to produce uniaxial extensions at rates up
to 1 m/s. Sample extension (grip motion) and load were recorded with
a digital storage oscilloscope (Nicolet Model 201) and stored for
subsequent analysis on flexible, magnetic diskettes (Verbatim mini-
disksTM). The data was finally transferred to a micro-computer

(Digital pdp 11/03) for analysis.

18

attached to actuator

sand-screen mesh

specimen

stainless steel
bars

 

 

 

attached to load cell

Figure 2 Schematic Drawing of Testing Grips

19

All ligaments and tendons were tested at room temperature
(27°C) in a chamber which is supplied with water-saturated air.
An additional physiological saline (0.9%) drip was used over the
samples to sustain tissue moisture. A stereo-microscope (WILD MSA)
and camera (WILD MPS Sl) were attached to the chamber base and used to
observe samples during testing. The chamber and microscope could be

rotated to view the specimen at different angles.
Mechanical Testing Protocol

Preliminary testing was used for grip design refinement and
the confirmation of the general viscoelastic nature of the tissues.
Current literature does not establish any definitive physiological
limit on load and deflection of these tissues. Each tissue was
ramped slowly to the point where the load deflection response appeared
linear. This maximum extension at which the specimen was ramped
established the maximum strain, E*. Initial testing was used to
establish the following protocol for examination of the response to
successive extensions, the relaxation of load, and the load response

to haversine extensions at various frequencies.

A. Preconditioning

l) Thaw test sample, wet with normal saline.

2) Mount sample and tighten grips.

3) Ramp slowly to establish a strain level E* well into linear
region III, which will be the maximum non-destructive strain.

4) Hold at E* for 2 minutes and tighten grips.

5) Unload and wait 10 minutes.

6) Ten constant rate cycles of 1% per second to E*.

7) Wait 5 minues.

8) Determine initial unloaded length 2.

9) Three tests to E* at 1% per second with 5 minute wait after
each test.

20

Constant Strain Rate Loading and Unloading

One test at 100% per second to E* followed by 5 minute wait.

One test at 1% per second to E* followed by 5 minute wait.

Dne test at 0.01% per second to E* followed by 5 minute wait.

Two tests to E* at 1% per second with 5 minute wait after each
to check preconditioning stability.

C. Cyclic Tests

Examination of the data from the last test in 8.4 will establish the

strain,

£11, at the transition from the non-linear toe region,

region II, to the linear region III.

1)
2)
3)
4)
5)
5)
7)
8)

II at 10 Hertz for 40 seconds

Cycle strain from 0.4EII to E
followed by a 5 minute wait.

Using the same minimum and maximum strains as test C.1, cycle
40 seconds at 1 Hertz followed by a 5 minute wait.

Using same minimum and maximum strains as test C.1, cycle

40 seconds at 0.1 Hertz followed by a 5 minute wait.

Check preconditioning stability by test 814.

Using a strain equal to E + 0.2 (E* - E ) as a minimum and
E* as the maximum level, cycle 40 seconds at 10 Hertz
followed by a 5 minute wait.

Using the minimum and maximum strains from C.5, cycle

40 seconds at l Hertz followed by a 5 minute wait.

Using the minimum and maximum strains from C.5, cycle
40 seconds at 0.1 Hertz followed by a 5 minute wait.

Check preconditioning stability by test 8.4. '

D. Relaxation

From the second test in C.8, determine new E

II transition strain or

confirm EII from test series C.

1)
2)
3)
4)
5)

Ramp at 100% per second to 0.7EII and hold until relaxation
approaches zero (approximately 10 minutes).

Return to zero strain and wait an equal time as relaxation time
in 0.1.

Ramp at 100% per second to E* and hold until relaxation
approaches zero (approximately 25 minutes).

Return to zero strain and wait an equal time as relaxation time
in D.3.

Check preconditioning stability by test 8.4.

 

21
Geometric Properties:

The initial length of a tendon or ligament was defined as the
distance between "grip-to-gripf and was measured with the unloaded
tissue in place in the testing machine. Crude cross-sectional areas
were taken at this time, width times thickness, but the areas used
in data analysis were obtained by measurements from histological slides
made after testing. The cross-sectional areas calculated did not include
the surrounding connective tissue having a large elastin content in
the peripheral area. Any discrepancy of the cross-sectional areas of the
tendons and ligaments recorded between right and left leg can be
explained by the fact that the primates tested were of different sex
and body weight. Thus, the chimpanzee's left and right patellar ligament
and flexor hallucis longus tendon were taken from two physically
different specimens, one weighing nearly three times more than the

other.

RESULTS and DISCUSSION

Constant strain rate loading and unloading data were collected in
digital form with the Nicolet digital oscilloscope, and then trans-
ferred to the pdp 11/03 computer for analysis. A three-degree
polynomial least square technique was employed to smooth this constant
strain rate data for noise elimination. These polynomial curves were
used to calculate smooth stress-strain curves, hysteresis and energy
areas, and the tangent moduli from data obtained in the experimental
program. I

The experimental protocol was constructed to collect data that
would allow for the development of a mathematical model using a
hereditary integral and/or structural analysis. This thesis is concerned
with finding those parameters needed for the hereditary integral
analysis. A qualitative discussion of these physical parameters is
included, but no attempt was made to put them back into the hereditary
model so as to discern whether or not this analysis could serve as a
predictive model. Although this predictive model will not be discussed
here, it is the subject of future Ph.D. work.

The concept of an initial material adjustment of biological
tissues undergoing a series of mechanical tests was termed "precondition-
ing" by Fung [5]. The precise definition of preconditioning is not
possible since it is not exactly known nor fully understood what has

happened in the tissue during this initial period. Therefore a rigid,

22

 

23

experimental protocol (See page 19) was established to help stabilize
and record any short or long time dependency effects of the tissue's
viscoelastic nature.

In order to help stabilize the material response, the tissue was
initially exposed to a preconditioning process that focused on the
repeatability of tests at a certain level of strain equal to or greater
than those used in the test program. It is not known whether or not
the initial or preconditioning changes which occur are independent of the
other observed viscoelastic effects. As suggested in the current
literature, these preconditioning effects may be due to changes in the
state of cross-linking, alteration in the state of hydration, or
realignment of the fiber matrix. Constant strain rate loading and
unloading, cyclic, and relaxation tests were performed sequentially to
record the tissue's mechanical properties. Overall preconditioning
stability was measured by conducting a constant strain rate test at 1%
per second after each testing sequence (See Figures 3 to 6). As seen
from these graphs, the maximum stress levels of subsequent check loops
dropped during this test program to approximately 75 to 95% of the value
attained on the initial cycle for the tendons (flexor hallucis longus
and tendo-calcaneus), and approximately 70 to 95% for the ligaments
(medial collateral and patellar). There was no preconditioning data
recorded for the patellar ligament of the baboon and the tendo-calcaneus
tendon of the chimpanzee due to the accidental erasure of data. The
check loops throughout the protocol showed the lack of stability of the
preconditioning response due to a long term relaxation phenomena during

the testing sequence, and therefore comparisons of one test with another

24

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28

in the testing sequence cannot be made, with exception of the
chimpanzee, which showed a relatively stable response. The scatter of
the data did not allow for significant variation between species.
Constant strain rate data were also used to determine the relative
stiffness, or tangent modulus, of the tendons and ligaments for the
three primates (See Table 5). The results recorded in Table 5 were
all tabulated for a strain rate of 100% per second. Figures 7 to 10
illustrate for the specimens tested average constant strain rate curves,
which were used to compute the tangent moduli, defined as the slope in
the linear region of these curves. Comparisons were made between the
primates for each tissue. The chimpanzee had the largest value of
tangent moduli of 1060 MPa for the tendo-calcaneus tendon, while for the
same tissue, the rhesus monkey had a value of 670 MPa and the baboon a
value of 280 MPa. Except for the rhesus monkey's patellar ligament,
which indicated a low value of 75 MPa, the tangent moduli values
recorded in Table 5 were within the range of 300-1000 MPa as reported by
Swanson [16] in 1971 for tendon. As seen from the histology and
morphology for all four tissues (See pages 9 to 16), elastin was more
prominent in the peripheral connective tissues than in the area with
dense collagen fiber bundles. An estimated value of the amount of
elastin was given for each primate tissue from the stained longitudinal
sections used in histological studies. No precise measurement could be
made due to the elastic fiber waviness exhibiting eccentric length and
amplitude. The collagen fiber bundles which carry the load are oriented
in the direction of the tensile load, while the function of the sur-

rounding matrix was assumed by Torp et a1 [13] in 1974, to be that of

 

29

 

 

 

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STRESS (N/mleOG)

30

40 _.

C>—-<> Rhesus monkey
35 - c1——CJ Baboon
Ar-A Chimpanzee

30‘

25..

20 _

15 _

10.. .

 

 

 

 

I If If I l l l l
4 5 6 7 8 9 10

STRAIN %

Figure 7. Average Stress-Strain Curves at a Constant Strain Rate of
100%/Sec. for the Flexor Hallucis Longus Tendon

STRESS (N/mleO‘)

31

 

 

 

40 _-
1
0—0 Rhesus monkey
35 .
III—Cl Baboon ,
H Chimpanzee
30__
25..) ,
20_‘ .
A
15..1 .
A
10.. . ‘
I
A ‘ -
5" I
A I
o I
‘ 1
0__ .4:
I I I I l I VI I II I
O 1 2 3 4 5 6 7 8 9 10

STRAIN %

Figure 8. Average Stress-Strain Curves at a Constant Strain Rate of
100%/Sec.for the Tendo-calcaneus Tendon

STRESS (N/m2x106)

32
40._

0—0 Rhesus monkey

[lriZI Baboon
(Sr-{A Chimpanzee

35..

30

25

8
J

In"
4

10— A

 

0 ,.,£ -
-J I 41' I I I l7 I I I

0 l 2 3 4 5 6 7 8 9 10

STRAIN %

Figure 9. Average Stress-Strain Curves at a Constant Strain Rate of
100%/Sec. for the Medial Collateral Ligament

 

33

 

 

40..
C>-<3 Rhesus monkey
35- .Cl-‘CI Baboon
lfirfifll Chimpanzee
30—-
25—.
”O
‘5; 20..m
E A
\
5
V)
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E 15-
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A
5.4. '
‘ C
L ' ' . .
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0 I I I F I I r
0 1 2 3 4 5 6 7 8 9 IO

STRAIN %

Figure 10. Average Stress-Strain Curves at a Constant Strain Rate of
100%/Sec. for the Patellar Ligament

34

holding the fibers together. Therefore, the larger the amounts of
elastin present among the collagen fiber bundles, the smaller the tangent
modulus. A variation in elastin content was observed between species

and may represent a functional adaptation reflecting different lower

limb usage by the various species. For example, the elastin content in

the tendo-calcaneus tendon was 5 - 10% for the chimpanzee with a

tangent modulus of 1060 MPa, while the rhesus and baboon had correspond-
ing values of 20% and 670 MPa, and 30% and 280 MPa, respectively.

The differences seen in Table 5 in maximum test strains among
primate tissues were due to the protocol in establishing'E*, defined as
the maximum test extension of the linear region on the stress-strain
curve. The maximum test strain values were directly dependent upon the
initial test length of the ligament or tendon which varied between
species and tests. Individual fibers had different points of attachment
to specific bones or muscle bodies, but the initial length was defined
as the "grip-to-grip" distance and was measured with the unloaded tissue
in place in the testing machine. The average maximum test strain for
the tendons was between 6.80 - 8.63% for all the primates, while for
the ligaments the values were between 4.28 - 9.64%. No correlatiOn
between tendons and ligaments can be made at maximum test strains, so
comparisons for the tissues were made at a strain level of 5.0%, using
extrapolated data from the average constant stress-strain curves in
some cases (See Figures 7 to 10). The average stress at a strain level
of 5.0% was 4.80 - 28.50 MPa for the tendons, and 1.00 - 19.20 MPa for
the ligaments, for all three primates. A pattern was noticed for the

tendons between the average maximum stress and the % hysteresis energy

35

to loading energy. When the average maximum stress increased, the

% hysteresis energy to loading energy also increaSed. No pattern was
found for the ligaments, suggesting a larger number of tissue samples
shoUld be tested before conclusions can be drawn about hysteresis
areas. Since few viscoelastic tests of this nature have been conducted
on soft connective tissue, hysteresis data cannot be compared to other
investigators' works.

Strain rate effects, hysteresis and energy areas were also determined
from constant strain rate data (See Table 6). Each primate tissue was
tested consecutively at three different strain rates; 100, l, and 0.01%
per second. For each specimen, the maximum stress, tangent modulus,
load input energy, and hysteresis area all decreased with decrease in
strain rate implying a strain rate dependency. An attempt was made to
select a parameter which reflected the energy expended in the deformation
of the tissue. This parameter was determined by taking the percentage
of the hysteresis area at a particular strain rate relative to the
hysteresis area at a strain rate of 100% per second. Nhen strain rate
decreases, the greatest drop in hysteresis area occurred for the
ligaments of the baboon. The medial collateral recorded a decrease to
28.9% at a strain rate of 0.01% per second, while the patellar ligament
had a value of 44.7% at a strain rate of 0.01% per second. The largest
drop in hysteresis area for the tendons occurred for the chimpanzee to a
value of 52.9% at a strain rate of 0.01% per second for the flexor hallu-
cis longus, and a value of 58.7% at a strain rate of 0.01% per second
for the tendo-calcaneus.

Graphs of cyclic relaxation data can be found in Figures 11 to 14.

36

Table 6. A Summary of Percentages for
Specific Mechanical Properties at Variable Strain Rates

Percent of 100%/Sec. Strain Rate Values

Ligament Species Strain Maximum* Tangent+ Loadede Hysteresis#
or Rate Stress Modulus ' Input Area
Tendon %/Sec. % % Energy,% %
Flexor Rhesus 100.00 100.0 100.0 100.0 130.g
1.00 97.1 91.0 87.5 .
"al‘ucls 0.01 83.0 79.0 73.0 87.7
L°"9"5 Baboon 100.00 100.0 100.0 100.0 100.0
Tendon 1.00 98.2 93.5 87.4 85.7
0.01 86.3 82.0 74.8 79.5
Chimp 100.00 100.0 100.0 100.0 100.0
1.00 93.0 91.5 85.5 64.9
0.01 83.6 77.1 78.9 52.9
Tendo- Rhesus 100.00 100.0 100.0 100.0 100.0
1.00 97.3 92.4 83.5 68.7
Ca‘ca"°“5 0.01 80.2 77.7 67.1 66.9
T°"d°“ Baboon 100.00 100.0 100.0 100.0 100.0
1.00 98.4 94.5 84.3 72.8
0.01 89.5 79.9 69.8 69.1
Chimp 100.00 100.0 100.0 100.0 100.0
1.00 88.9 85.3 82.5 80.3
0.01 83.0 65.1 79.8 58.7
Medial Rhesus 100.00 100.0 100.0 100.0 100.0
1.00 91.4 91.7 86.5 59.9
°°“ate’a‘ 0.01 80.3 81.3 75.6 55.5
Ligam°"t Baboon 100.00 100.0 100.0 100.0 100.0
1.00 91.2 89.9 73.8 30.7
0.01 78.0 82.0 61.6 28.9
Chimp 100.00 100.0 100.0 100.0 100.0
1.00 90.4 90.9 96.4 31.1
0.01 79.6 81.6 85.6 30.4
Patellar Rhesus 100.00 100.0 100.0 100.0 100.0
1.00 91.4 88.6 82.5 53.9
L‘game"t 0.01 65.4 64.2 57.4 48.9
Baboon 100.00 100.0 100.0 100.0 100.0
1.00 94.1 93.0 80.9 52.9
0.01 73.1 76.7 61.0 44.7
Chimp 100.00 100.0 100.0 100.0 100.0
1.00 97.1 93.4 92.1 86.1
0.01 92.5 78.2 82.3 82.4
* Average % of Maximum Stress for All Samples.
+ Average % of Tangent Modulus for All Samples.
@ Average % of Loaded Energy of Hysteresis Area for All Samples.
# Average % of Total Hysteresis Area for All Samples.

37

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41

Each primate tissue was tested at three different frequency levels;
10, l, and 0.1 cycles per second, and data was then plotted with all
three frequencies on the same graph. The cyclic data curves represent
the normalized stress at maximum amplitude versus the natural
logarithm of time, instead of versus the number of cycles as frequently
done in the literature. All stress values were normalized by the
initial peak stress which occurred at maximum amplitude during the first
cycle. A smooth transition between the different frequencies, with tests
separated by a five minute wait, was observed on all tests. The scatter
of the cyclic data did not allow for significant variation between spe-
cies. Comparisons between low to high frequencies cannot be made
because the three different frequencies tests were conducted at
different times in the testing protocol. The only way such a comparison
could be made would be to either (1) vary the order of test frequencies
or (2) run single frequency tests on many different specimens.
The smooth transition between frequencies may not indicate lack of
frequency dependence, but instead represented a simple time response,
dependent upon the total time of cyclic testing. The negative slope of
the linear region for the tendons ranged between 0.021 to 0.043, while
for the ligaments the slope ranged between 0.012 to 0.037. The initial
drop in cyclic relaxation showed a short term relaxation phenomena,
Similar to that observed in standard relaxation tests.

Two types of short term viscoelastic effects were examined and
compared. The first type of viscoelastic effect was the short term
relaxation phenomena represented by the change of the percent of peak

stress at maximum strain amplitude from cyclic relaxation data.

42

A second examination of short term viscoelastic effects used standard
relaxation tests (See Figures 15 to 18), to measure short time
(10 - 30 minutes) response. The normalized relaxation function, G(t),

can be approximated as a linear function of the logarithm of time.
G(t) = 1 - uln(t+l)

G(t) is a reduced relaxation function as defined by Fung [5], in a
hereditary integral formulation. A measure of the tissue's viscoelas-
ticity is given by the relaxation coefficient, u, and is calculated by ;-
finding the slope of the linear region of these curves. The coefficient
varied from 0.031 to 0.045 for the tendons, and from 0.022 to 0.038

for the ligaments. The scatter of the relaxation data did not allow

for significant variation between species. When the p values from the
relaxation data were compared to the slope values from the cyclic data
for each primate tissue, it was seen that the values were approximately
the same. This would indicate that the tissues seem to relax at the
same rate. However, it is difficult to make a comparison of these two
tests, since these tests were performed at two different time periods in

the test sequence.

43

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CONCLUSIONS

The results presented emphasize the importance of a controlled
test protocol. The check loops throughout the test program showed a
lack of stability of the preconditioning response due to a long term
relaxation phenomenon, except for the chimpanzee, which showed a
relatively stable response. Therefore, comparisons of one test with
another in the testing sequence cannOt be made. The scatter in the data
did not allow for significant variation between species.

Constant strain rate data were used to determine the relative
stiffness measured by the tangent modulus, for all three primates'
tendons and ligaments. The tangent moduli values recorded were
within the range of 300 - l000 MPa reported in the literature for
collagenous tissue. The value of the tangent modulus for each tissue
correlated well with the percent of elastin. The higher the elastin
content, the lower the value of the tangent modulus. Large variations
in the percent of elastin were observed histologically between the
species for the same tissue.

Differences in maximum test strains, due to the definition of the
maximum test extension in the linear region on the stress-strain curve,
made comparisons of properties at maximum test strains impossible, so
comparisons for the tissues were made at a strain level of 5.0%.

A pattern was noticed for the tendons between the average maximum stress

47

48

and the % hysteresis energy to loading energy. When average maximum
stress increased, the % hysteresis energy to loading energy also
increased. No pattern was found for the ligaments, but a larger number
of tissue samples should be tested before conclusions can be drawn about
hysteresis areas.

Strain rate effects, hysteresis and energy areas were also
determined from constant strain rate data. The maximum stress, tangent
modulus, load input energy, and hysteresis area all decreased with
decreasing strain rate. Since few viscoelastic tests of this nature
have been conducted on soft connective tissue, hysteresis data could
not be compared to other investigators' works.

Cyclic strain data showed a short term viscoelastic response of
comparable nature to that observed for standard relaxation tests.

No frequency dependency could be observed and the data showed a contin-
ued smooth decay through frequency change. The long term precondition-
ing instability prevented comparison of one frequency response to
another.

A second examination of viscoelastic effects used the standard
relaxation tests to measure short time effects. The normalized
relaxation function was approximated as a linear function of the
logarithm of time. The p value from the relaxation data was approxi-
mately the same as the slope value from the cyclic data, indicating
the relaxation of the tissue was the same.

The tissue tested in this thesis were the ligaments (medial
collateral and patellar) from the knees, and tendons (flexor hallucis

longus and tendo-calcaneus) from the ankles of the rhesus monkey,

49

baboon, and chimpanzee. The physical parameters needed for the
hereditary integral analysis were found from the experimental
protocol. No attempt was made to put these parameters back into the
hereditary analysis, but is the subject of future Ph.D. work to see

if this analysis could be used as a predictive model.

BIBLIOGRAPHY

10.

BIBLIOGRAPHY

Elliott, D.H., "Structure and Function of Mammalian Tendon,"
Biol. Rev., Vol. 40, l965, pp. 392-441.

 

Evans, J.H., and J.C. Barbenel, "Structural and Mechanical
Properties of Tendon Related to Function," Equine Veterinary J.,
Vol. 7, No. 1, January 1975, pp. l-B.

Minns, R.J., P.D. Soden, and 0.5. Jackson, "The Role of the
Fibrous Components and Ground Substance in the Mechanical
Properties of Biological Tissues: A Preliminary Investigation,"
J. Biomechanics, Vol. 6, l973, pp. l53-165.

 

Kastelic, J., A. Galeski, and E. Baer, “The Multicomposite
Structure of Tendon," Connective Tissue Res., Vol. 6, l978,
pp. ll-23.

Rigby, B.J., N. Hirai, J.D. Spikes, and H. Eyring, "The Mechanical
Properties of Rat Tail Tendon," J. Gen. Phys., Vol. 43, 1959,
pp. 265-283.

Diamant, J., A. Keller, E. Baer, M. Litt, and R.G.C. Arridge,
"Collagen: Ultrastructure and Its Relation to Mechanical
Properties as a Function of Ageing," Proc. R. Soc. Lond. B.,
Vol. 180, 1972, pp. 293-3l5.

 

Partington, F.R., and G.C. Wood, "The Role of Non-collagen
Components in the Mechanical Behaviour of Tendon Fibres,"
Biochim. Biophys. Acta., Vol. 69, 1963, pp. 485-495.

 

Millington, P.F., T.G. Gibson, J.H. Evans, and T.C. Barbenel,
"Structural and Mechanical Aspects of Connective Tissue,"
Advanced Biomed. Eng,, Vol. l, 1971, pp. 189-248.

 

Viidik, A., "Mechanical Properties of Parallel-fibred Collagenous
Tissues," Biolo of Collagen, Ed. by Viidik and Vuust, New York,
1980, pp. 3 - 5.

 

Fung, Y.C., "Stress-Strain-History Relations of Soft Tissues in
Simple Elongations," Biomechanics--Its Foundations and Objectives,
Eds. Fung, Perrone, Anliker, Englewood Cliffs, N.J., pp. lBl-ZOB.

50

ll.

12.

13.

14.

15.

16.

51

Torp, 5., R.G.C. Arridge, C.D. Armeniades, and E.Baer,
"Structure-Property Relationships in Tendon as a Function of
Age," Proceedings of 1974 Colston Conference, Department of
Physicis, University of Bristol, U.K., pp. 197-221.

Little, R.N., R.P. Hubbard, D.L. Hyler, and A.R. Slonim,
"Mechanical Properties of Spinal Ligaments for Rhesus Monkey,
Baboon, and Chimpanzee," Air Force Aerospace Medical Research
Laboratory-TR-81-40.

Manual of Histologic and Special Staining Technics, Armed Forces

 

IiStitute of Pathology. 2nd Edition, McGraw-Hill, 1960.

Davenport, N.D., "A Rapid Trichrome Staining Procedure for the
Identification of Tissue Types," Histochem. J., Vol. 11, 1979,
pp. 367-372. ‘

Lillie, R.D., and H.M. Fullmer, Histopatholo ic Technic and
Practical Histochemistry, 4th Edition, McGraw-Hill, 1976.
Swanson, S.A.V., "Biomechanical Characteristics of Bone,"

Advances in Biomedical Engineering, Vol. 1, Ed. by R.M. Kenedi,
New York, 1971, pp. 137-187.

 

 

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