THE EFFECTS or EXERCISE AND DEIRAINING ON THE
STIFFNESS OF BONE

Dissertation for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
ANNE ELIZABETH IRWIN
1976

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thesis entitled

THE EFFECTS OF EXERCISE AND DETRAINING
ON THE STIFFNESS OF BONE

presented by
Anne Elizabeth Irwin

has been accepted towards fulfillment
of the requirements for

Ph.D. Biomechanics

& Engineering Mechanics

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ABSTRACT

THE EFFECTS OF EXERCISE AND DETRAINING
ON THE STIFFNESS OF BONE

By

Anne Elizabeth Irwin

The purpose of this study was to investigate the
effects of two chronic exercise programs and a period
of detraining on the stiffness of rat femurs.

Eighty normal, 72-day-old, male, albino rats of
the Sprague-Dawley strain were randomly assigned to one
of three treatment groups and to one of five duration
periods. Prior to the start of the treatments, all
animals were allowed a twelve-day adjustment period to
adapt to laboratory conditions. The treatments were:
sedentary-control (CON); short-duration, high-intensity
interval running (SHT); and long-duration, low-intensity
endurance running (LON). The animals had access to a
commercial animal diet and water ad libitum. The treat-
nents were administered under controlled environmental
conditions once a day, five days per week (Monday
through Friday), for either eight or sixteen weeks.

Eight- and sixteen-week periods of detraining followed

Anne Elizabeth Irwin

during which all animals remained in their cages and no
exercise was administered.

Animals were sacrificed after zero, eight, six-
teen, twenty-four, and thirty-two weeks of treatments.
The animals chosen for sacrifice were selected on the
bases of their apparent health and their training per-
formances. The final sample included sixty animals.
Each animal was sacrificed by an intraperitoneal injec-
tion of a 6.48 percent sodium pentobarbital (Halatal)
solution. The right and left femurs were surgically
removed while they were continuously moistened with
Ringer's saline solution.

Bone stiffness (modulus of elasticity) was
determined by bending tests. These tests were designed
to determine the load-deformation relationship for bone.
All bones were tested within a temperature range of
22-260C and within one week of sacrifice. Double point
loading was used. The distance to thickness ratios
ranged from 1:1 to 2:1. The average strain rate was
6.01 X 10-4/sec. A cross-section of the bone, at the
point of load application, was obtained by using a
jeweler's saw. The crossésection was placed in a micro-
scopic projector; its image was projected onto graph
paper; and the image was traced. The elastic modulus
and the moment of inertia of the bone cross—section were

calculated.

Anne Elizabeth Irwin

After dependence of stiffness on strain rate was
eliminated through a regression technique, a univariate-
repeated measures technique was used to test differences
at the .10 level of significance between elastic moduli
of the three training groups, the five duration groups,
and the right and left bones. The Newman-Keuls method
was used to find the source of each significant univariate
F-ratio. Means and standard deviations were computed
for the elastic modulus for all groups.

Over all treatments and durations there was no
significant difference between the right and left bones.
The only significant difference among treatment groups
occurred after the sixteen-week training period at which
time the bones of the LON group were less stiff than
those of either the SHT group or the CON group. The SHT
treatment did not significantly affect bone stiffness.
The bones of the CON and SHT groups significantly
increased in stiffness after sixteen weeks of training
and then became less stiff after sixteen weeks of de-
training. Therefore, some factor, such as the aging
process, may cause rat femurs to gain stiffness before
and lose stiffness after 196 days of age. At no dura-
tion did the LON treatment cause a significant increase

in bone stiffness above the original level.

THE EFFECTS OF EXERCISE AND DETRAINING
ON THE STIFFNESS OF BONE

By

Anne Elizabeth Irwin

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Departments of:
Health, Physical Education and Recreation;
Metallurgy, Mechanics and Materials Science

1976

ACKNOWLEDGMENTS

The author wishes to thank Dr. William W.
Heusner (Department of Health, Physical Education and
Recreation), for acting as my thesis director and for
guidance in the development of this dissertation. In
addition, appreciation is also extended to Dr. Robert W.
Little (Departments of Mechanical Engineering and of
Metallurgy, Mechanics and Materials Science), for
guidance in my engineering courses of study and for
assistance in the development of this dissertation.
Thanks are also given to the other members of the
Guidance Committee: to Dr. wayne D. Van Huss (Depart-
ment of Health, Physical Education and Recreation), to
Dr. wade O. Brinker (Department of Small Animal Surgery
and Medicine), and to Dr. William N. Sharpe, Jr.
(Department of Metallurgy, Mechanics and Materials
Science) for their guidance and support. In addition,
gratitude is expressed to Dr. Gary L. Cloud (Department
of Metallurgy, Mechanics and Materials Science), for
guidance during the early stages of organizing and
calibrating the experimental apparatus; to Mr. Edgar
Conley for assistance in designing and building the

testing apparatus; to Mr. Robert L. Wells for assistance

ii

with the electrical and electronic aspects of the
experiment; to Mr. David Anderson for assistance in
drawing the figures; and to Dr. George Piotrowski and
the Florida State University System for their assistance

in processing data through their computer.

iii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

Chapter

I.

II.

III.

INTRODUCTION

Bone Stiffness .

Need for the Study
Purpose of the Study
Scope of the Study .
Limitations of the Study
Definitions .

REVIEW OF LITERATURE

Mechanical Properties of Bone
Bending Tests
Rates of Loading . .
Elastic Modulus Values
Experimental Effects on Bone .
Storage .
Temperature .
Delayed Testing .
Effects of Exercise and Detraining on Bone
Exercise .
Detraining
Paired Bones

RESEARCH METHODS .

Experimental Animals
Treatment Groups

Control (CON)

Short (SHT)

Long (LON) .
Duration Periods
Sample Size
Experimental Procedures
Animal Care

iv

Page
vi

vii

\IN \I \J'IU'IUJNNN H

Chapter Page

Sacrifice Procedures . . . . . . . 22
Bone Stiffness Analysis . . . . . . 23
Mechanical Testing Techniques . . . . 23
Elastic Modulus Equation . . . . . 34
Elastic MOdulus of the Bone . . 39

Equation for the MOment of Inertia of a
Cross- section . . . 4O

Moment of Inertia of the Bone Cross-

section . . . . . . . . 43
Strain Rate Equation . . . . . . . 44
Strain Rate of the Bone . . . . . . 45
Treatment of the Data . . . . . . . 46
Design . . . . . . . 46
Significance Level . . 47

Eliminating the Dependence of Stiffness
on Strain Rate . . . . . . . . 47
Statistical Procedures . . . . . . 48
IV. RESULTS AND DISCUSSION . . . . . . . 50
Results . . . . . . . . . . . . 50
Question I . . . . . . . . . . 50
Question II . . . . . . . . . . 55
Question III . . . . . . . . . 57
Question IV . . . . . . . . . . 58
Discussion . . . . . . . . . . . 60
V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. 63
Conclusions . . . . . . . . . . 64
Recommendations . . . . . . . . . 65
REFERENCES CITED . . . . . . . . . . . 67
APPENDICES . . . . . . . . . . . . . 77
A. TRAINING PROGRAMS . . . . . . . . 78
B. FEMUR LENGTHS . . . . . . . . . . 83
c. FEMUR WEIGHTS . . . . ' . . . . . . 85

Table

10.

LIST OF TABLES

A Review of the Literature of Bending Tests

A Review of the Literature of the Mechanical
Properties of Bone Subjected to Bending
Tests . . . . . . . . . .

A Review of the Literature of the Experi-
mental Properties of Bone Subjected to
Bending Tests .

Final Cell Frequencies

A 3X5X2 Factorial Design with Cell Fre-
quencies . . . . .

Means and Standard Deviations for the
Stiffness of Rat Femurs Presented by
Treatment, Duration, and Paired Bones
(km /cm2

Analysis of Variance for Bone Stiffness with
a Repeated-measures Option for the Right
and Left Bones of the Animal . .

Means and Standard Deviations for the
Stiffness of Rat Femurs Presented by
Treatment, Duration, and Animal

Differences Between the Mean Elastic Moduli
(R + L) of the Treatment Groups within
each Treatment Duration . . .

Differences Between the Mean Elastic Moduli

(R + L) of the Duration Groups within each
Treatment Group . . . . .

vi

Page

ll

14
20

46

51

55

S6

58

59

Figure

\OCDNmU'IJ-‘UJN

10.

ll.

12.
13.
14.

LIST OF FIGURES

A Rat Femur (left side)

Micrometer and Jeweler's Saw

Bending Text Fixture

Testing Apparatus

Specimen Grips

Recording Apparatus

Load, Deformation, and Time Strip Chart
Load Versus Deformation Plot
Microscopic Projector

Tracing of the Cross-Sectional Area of
the Bone Projection

Free-body Diagram of a Beam in Pure
Bending

Bending Moments of a Cross-section
Distance (Y) from the Centroid

Average Elastic Moduli with Standard Error
of the Means . . . . . . .

vii

Page
24
25
27
28
29
31
32
33
35

36

37
40
45

53

CHAPTER I

INTRODUCTION

Physicians and physical educators have recommended
exercise for strengthening the human body. However, very
little is known about the effects of exercise and de-
training on bone.

Many studies have been conducted over the last
hundred years to investigate the physical and mechanical
behavior of bone (21,34,49,71). During the past twenty
years, engineering techniques have been employed to study
the mechanical preperties of bone. Through the analysis
of stress and strain in materials, researchers know that
bone behaves as a transversely isotrOpic material and
displays elastic, plastic, and viscoelastic properties.
The elastic pr0perties of bone have been studied more
extensibly than either the plastic or viscoelastic
properties.

Only within the last five years have there been
studies to evaluate the effects of chronic exercise on
the behavior of bone (58,75). None of these have

included the effects of detraining.

Bone Stiffness

 

Bone stiffness is the resistence of bone to
deformation. It is dependent on the rate of loading, the
temperature, and the moisture content of the bone.

The elastic modulus has most frequently been used
as a measure of bone stiffness. It can be determined by
using one of four methods of testing: bending, compression,
tension, or torsion testing. A bending test is recommended

because there is less chance of grip slippage (7).

Need for the Study
Not only is little known about the effects of
exercise and detraining on bone, but there also is no
information on different exercise training programs and
their effects on bone stiffness. In addition, more
evidence concerning the effects of exercise on paired
bones is needed to verify whether changes are uniform

between the right and the left bone.

Purpose of the Study
It was the purpose of this study to investigate
the effects of two exercise training programs and a
subsequent detraining program on bone stiffness. More
specifically, an answer was sought to the following

questions:

Question I

 

What is the stiffness of rat bones, as measured
by the elastic modulus, after a planned program of

exercise and detraining?

Question II

 

Does the elastic modulus vary between paired

femurs?

Question III

 

Does the elastic modulus vary as a function of

the various exercise treatments?

Question IV
Does the elastic modulus vary as a function of

duration of the various exercise treatments?

Scope of the Study

 

The purpose of this study was to determine if
bone stiffness is affected by a chronic exercise program
and/or by a subsequent detraining program. The effects
of exercise and detraining on the stiffness of paired
bones also was examined.

Eighty normal, male, albino rats were used in
this study for several reasons. The rat is easily
trained to run on a wheel. The running wheels were

already constructed for use in a previous study. Also,

the rat femur has been used in earlier bone stiffness
studies.

The design of this study included three treat-
ments and five durations. The treatments were:
sedentary-control (CON); short-duration, high-intensity
interval running (SHT); and long-duration, low-intensity
endurance running (LON). The animals within each group
were sacrificed after five preselected treatment dura-
tions. The durations were: prior to the start of the
treatments, at eight weeks and sixteen weeks during the
exercise program, and at twenty-four weeks and thirty-
two weeks during the detraining programs

The SHT and LON exercise training programs were
implemented using electronically controlled running
wheels (89). The wheels regulated the length and
intensity of the running which in specific combination
created programs of exercise designed to stimulate
anaerobic and aerobic metabolic processes. Following
common practice, these programs will hereinafter be
referred to as "anaerobic" and "aerobic" types of
exercise. The exercise programs increased in length and
intensity for eight weeks. The exercise then was main-
tained at this level for eight more weeks. At that time,
no exercise was given for sixteen weeks of detraining.

The sixteen weeks of training and the sixteen weeks of

detraining were estimated to be of adequate length to

cause a change in the stiffness of bone.

Limitations of the Study

1. The design of this study, based on data from
a previous study on muscle, included four animals per
cell. This may not have been a large enough sample.

2. Shock was the stimulus used to motivate the
animals to run. There was no control in this study for
shock. In previous unpublished studies, the amount of
shock used has been shown to be insufficient to produce a
change in either skeletal or cardiac muscle. However, no
information is available on the effects of the shock
stimulus on bone tissue.

3. The sixteen-week periods of exercise and
detraining may not have been long enough to produce
changes in bone stiffness.

4. The results of this study are specific to the
femur bones of male albino rats that begin a program of
exercise at eighty-four days of age. The data apply only
to the specific training and detraining regimens studied

in this investigation.

Definitions

 

Stiffness

 

A measure of the resistence of a material to

deformation.

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HI

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Pb -

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RAH

Flt-

Stress
The internal force per unit area within a
material resisting the deforming action of an outside

force.

Strain

A measure of deformation per unit length within

a material.

Elastic Modulus

 

The amount of stress theoretically required to

produce a unit strain in a linear elastic material.

Load

 

A force or weight applied to an object.

Deformation

 

The amount an object changes in length or

shape as a result of a load placed on it.

CHAPTER II

REVIEW OF LITERATURE

This review of literature focuses on the mechani-
cal properties of bone, the possible effects of testing
conditions on bone, the effects of exercise and detraining
on bone, and the comparative characteristics of paired
bones. A discussion of the mechanical properties of bone
includes bending tests, rates of loading, and the
elastic modulus values determined in previous studies.
Testing conditions which may effect the stiffness of bone

are storage, temperature, and the delayed testing of bone.

Mechanical Properties of Bone

Bending Tests
‘ In the bones of living animals, the tension
induced by bending is the most dangerous stress (23,69).
Resistance to tension is more critical than resistance
to compression. In research, the bending test is used
because slippage of the testing grips is minimized (7).
Also, a slightly curved test piece will not alter the
value of the elastic modulus (21).

However, in bending tests there are two assump-

tions which are usually made (7): plane sections in bone

7

will remain plane, and bone material is both homogeneous
and isotropic.

In studies of bone strength and bone stiffness,
bending tests have been performed by a number of investi-
gators. Both whole bones and machined specimens were
used. A ten-year review of the literature of bending
tests is presented in Table 1.

All of the studies of bending tests that were
reviewed used a single point of loading. With a single
loading point, normal strain occurs along with shearing
strain at the maximum bending point. With double point
loading, only normal strain occurs at the point of
maximum bending. The central region is subjected to a
constant bending moment, called pure bending (82).

All investigators that have reported breaking
stress values have used a stress formula for linearly
elastic isotropic materials. The use of this formula
should be restricted to isotropic substances and then is
correct only when used within the linear range of stress
values before breakage. However, bone shows some plastic
yielding and is an anisotropic material (21). Burstein
and co-workers found that, if a bending test is carried
beyond the yield point, an elastic-plastic analysis must
be performed (16).

In isotropic materials bent as end-supported

beams, strength and the deformations were found to vary

 

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with the distance between supports. Distance to specimen
thickness ratios of 15, 20, and 25:1 were found to pro-
duce no differences in the elastic modulus, but to
increase or decrease the ratio out of this range
decreases the value of the elastic modulus (77). Some

researchers did not report the distance between supports.

Rates of Loading

 

The time-dependent viscoelastic property of bone
makes the strain rate used in bending tests an important
variable. The elastic modulus increases with a more
rapid deformation rate (49,63,77).

Pope and Outwater (68) were the only researchers
investigated that reported the strain rate used in their
bending testing (Table 2). Several other researchers
reported the rate of movement of the testing machine,

but this alone does not define strain rate.

Elastic Modulus Values

 

In previous studies, the elastic modulus values

5

were reported as ranging from .0235 X 10 kgm/cm2 to

2.00 X 105 kgm/cmz. But, the elastic modulus is dependent
on the following experimental conditions: the type of
storage, the temperature, and the rate of loading of the
bone during testing. Table 2 summarizes a review of the

literature reporting the elastic modulus and how it was

obtained.

11

 

 

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12

As Table 2 reveals, many studies either lack
important information regarding the elastic modulus or
are inconsistent in regard to the data reported.
Temperature and the type of storage will be discussed
in a later section of this chapter.

The moment of inertia is dependent on the shape
of the cross-section of the material being tested. It

2dA, which is

is represented by the equation Iyy = fy
known as the second moment of the cross-sectional area.
Bone is an asymmetrical object and each cross-section of
bone takes a different shape. Therefore, the equation
for the moment of inertia of each bone cross-section is
different. As Table 2 indicates, many researchers have
studied a machined bone specimen of solid, regular-shaped,
cross-sectional areas. Mather (55,56,57) studied whole

bones and used Mohr's method of determining a hollow,

irregular, cross-sectional area (5).

Experimental Effects on Bone
Storage
Researchers have found that the method of storage
prior to testing has a direct effect on the stiffness and
strength of bone. The values of stiffness and strength
are lower in bones that have been stored in a wet solution,
such as Ringer's or saline, than in bones that have been

stored dry (49). Wet storage is considered to be closer

 

1.

:4

LI

13

to 12.2133 conditions. Therefore, if inferences are to
be drawn for living bone, wet storage should be the
method used.

Sedlin and Hirsch discovered that within ten
minutes a bone exposed to air will dry sufficiently to
cause an increase in the stiffness of the bone (77).
Table 3 shows that frequently bones have been exposed to
air for as long as two hours prior to testing. Therefore,
many investigators have reported inflated values for the
strength and stiffness of bones that have not been
stored in solutions.

Some researchers have frozen bone for storage.
Sedlin and Hirsch found that freezing does not signifi-

cantly effect the stiffness or strength of bone (77).

Temperature

 

A viscoelastic material such as bone is tempera-
ture dependent. Table 3 indicates that few authors have
reported the temperature at which their experiments were
conducted. One recent study has tried to test bones at
a temperature comparable to the normal body temperature

of the animal (77).

Delayed Testing

 

Properties in isolated bone change over a period
of time (14). In some studies, bone has been tested as

late as four weeks after it was removed from the animal

14

 

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15

(16). In the available literature, the effects of the
delayed testing were not significant for a period of
four days (24). More information must be gathered in
this area.

Effects of Exercise and
IDetréinIhgionTBone

 

 

Exercise

Several researchers have studied the effects of
various types of exercise or overload on bone. These
include: voluntary running, artificially increased
gravity, walking, voluntary exercising, and mechanical
vibration (3,47,32,52,58).

Only one published article could be found which
reported the effects of controlled exercise on bone.
Saville and Whyte forced rats to run in electrically
controlled revolving drums (75). Running and resting
were alternated in fifteen-minute intervals of time.
Each rat ran about 2000 m/day during each five-day exer-
cise week. How the rats were motivated to run, however,
was not noted. The rats were sacrificed after 2, 4, 6,
8, and 10 weeks of exercise. The results of the study
revealed that, between the exercised and the control
groups, there were no significant differences in breaking
force or in other breaking stress measurements. The
author's report indicates that the breaking force and

the cortical thickness measurements may not have been

l6

precise enough to demonstrate differences. However, they
did find that muscular hypertrophy was associated with a
proportional bone hypertrophy. The converse has been
shown to be true: disuse causes proportional atrophy of
both bone and muscle (2,34).

Voluntary exercise has been shown toproduce
slight reductions in the percent of water in bone (32).
In the same study, Donaldson and Meeser were able to show
that changes in the weight of the bone were due to changes

in cell size and not to the number of cells in the bone.

Detraining_

 

No studies were found which dealt with the effects

of detraining on the stiffness of bone.

Paired Bones

 

Paired bone differences have been studied by
several researchers (18,56,57,58,64,70,74). In a study
of dog ulnas, Puhl g§_§l. found no significant differences
between right and left bones which were subjected to
torque tests (70). Other researchers have used right and
left bones interchangeably when studying bone strength
(56,58,64,74). No studies were found which evaluated
differences between right and left bones due to exercise

and detraining.

CHAPTER III
RESEARCH METHODS
The purpose of this study was to investigate the

effects of two exercise programs and a period of detrain-

ing on the stiffness of rat femurs.

Experimental Animals

 

Eighty normal, male, albino rats of the Sprague-
Dawley strain1 were received in the laboratory when they
were 72 days of age. At this time, each animal was
assigned randomly to one of three treatment groups and to
one of five duration periods. Prior to the start of the
treatments, all animals were allowed a twelve-day adjust-

ment period to adapt to laboratory conditions.

Treatment Groups

 

The three treatment groups in this study were:

Control (CON)

 

The control animals did not receive any special

treatment .

 

1
Illinois.

Obtained from Hormone Assay Laboratory, Chicago,

17

18

Short (SHT)

 

These animals were subjected to a short-duration,
high-intensity interval running program which was
administered once a day, five days per week (Monday
through Friday), for either 8 or 16 weeks. The 16-week
training period was followed by 8-week and 16-week periods
of detraining during which the animals were not allowed to
exercise. The intensity of the running program was
increased gradually up to the 37th day of training (Tuesday
of the 8th week) and then was held constant through the
80th day (Friday of the 16th week). Between the 37th and
80th days of training, the program consisted of eight
bouts of exercise with 2.5 minutes of inactivity between
bouts. Each bout contained six repetitions of a 10-
second work interval alternated with five 40-second rest
intervals. During the work intervals, the animals were
forced to run at the relatively fast speed of 99 m/min.
See Appendix A for a complete description of the first

37 days of this training program.

Long»(LON)

 

These animals were subjected to a long-duration,
low-intensity endurance running program. The treatment
schedule for this exercise regimen was the same as that
for the SHT program. Between the 37th and 80th days of

training, the LON program consisted of four bouts of

(D

19

exercise with 2.5 minutes of inactivity between bouts.
Each bout was a continuous 12.5-minute run at 36 m/min.
Appendix A includes a complete description of the first

37 days of the LON program.

Duration Periods

Animals were sacrificed after zero, eight, six—
teen, twenty-four, and thirty-two weeks of treatments.
These duration groups are referred to here as 0-wk,
8-wk, l6-wk, 24-wk, and 32-wk animals, respectively. The
animals were 84, 140, 196, 252, and 308 days of age at
sacrifice.

The 0-wk animals were sacrificed following the
initial adjustment period. The 8-wk animals were sacri-
ficed three training days (six calendar days) after the
SHT and LON programs reached maximum intensity. The 16-
wk animals were sacrificed three calendar days after the
training programs were terminated. 'The 24-wk and 32-wk
animals were sacrificed after eight and sixteen weeks of

the detraining phase of the study had elapsed.

Sample Size

 

A total of four animals per treatment group were
sacrificed at the end of each duration period. These
animals were selected on the basis of their apparent
health and their training performances. The final sample

included sixty animals (Table 4).

20

TABLE 4.--Final Cell Frequencies.

 

 

 

Duration
Treatment
O-wk 8-wk 16-wk 24-wk 32-wk
CON 4 4 4 4 4
SHT 4 4 4 4 4
LON 4 4 4 4 4

 

Experimental Procedures

 

The treatments began, following the twelve-day
adjustment period, when all animals were eighty-four days
of age. The SHT and LON treatments were administered in
the Human Energy Research Laboratory, Michigan State
University, East Lansing, Michigan. Body weights of the
exercised animals were recorded before and after each
training period. The CON animals were weighed weekly.

Each SHT and LON animal was trained in an indi-
vidual controlled-running wheel (CRW) (89). Prior to
the start of training and during all rest periods, the
wheel was braked to restrict voluntary activity. At the
beginning of each work interval, the brake automatically
was released allowing the wheel to rotate freely. At
the same time, a light above the wheel was illuminated
as a signal for the animal to start running. A programmed
acceleration period of 1 to 3 seconds was allowed during

which the animal was supposed to achieve a predetermined

21

running speed. If the animal failed to run at or faster
than that speed during the acceleration period, the light
remained on and a mild electric shock (0.8, 1.0, or 1.2
ma) was administered through the running surface. In
this case, the light and the shock remained on until the
animal reached the preset speed at which time they both
were extinguished. If the animal responded to the light
and attained the desired speed during the acceleration
period, the light was extinguished immediately and no
shock was applied. This light-shock sequence was repeated
each time the animal dropped below the desired running
speed during a work interval. After a three-day learning
period, most of the animals had learned to run by this
avoidance-response operant conditioning technique and
thus were exposed to a minimum of shock.

After each training period, cumulative duration
of shock (ODS) and total revolutions run (TRR) were
indicated on each CRW. Percent expected revolutions
(PER) and percent shock free time (PSF) were calculated
from CD5 and TRR along with the total expected revolu-
tions run (TER) and total work time (TWT): PER = 100 X
TRR/TER and PSF = 100 - (100 X CDS/TWT). PER and PSF

values were used to evaluate animal performance (45).

Animal Care

 

All animals were maintained under relatively

constant conditions which included daily handling,

 

H

 

22

humidity and temperature control, and regular cage
cleaning. The animals were housed in standard, indi-
vidual sedentary cages (24 cm X 18 cm.X 18 cm) through-
out the training and detraining programs. The animals
had access to a commercial animal diet2 and water ad
libitum.

The animals were trained in their normal active
period during the first few hours of darkness. For the
convenience of the laboratory staff, the light sequence
in the animal quarters was automatically timed to alter
the animal's active period by having the lights off

between 1:00 pm and 1:00 am.

Sacrifice‘Procedures

 

Five sacrifices of twelve animals each were con-
ducted eight weeks apart (see Table 4). Each sacrifice
took place on a Monday morning and included four animals
from each of the three treatment groups: CON, SHT, and
LON. The 8-wk and l6-wk animals were sacrificed seventy-
two hours after the last treatments of the SHT and LON
animals assigned to those duration periods. Only those
animals which were healthy and those animals of the SHT
and LON groups whose mean PER and PSF values were 75 or

higher were selected for sacrifice (45).

 

2Wayne Laboratory-Blox, Allied Mills, Inc.,
Chicago, Illinois.

23

Final body weights were recorded. Each animal
then was sacrificed by an intraperitoneal injection
(4 mg/lOOg body weight) of a 6. 48 percent sodium pento-
barbital (Halata13) solution.

The right and left femurs were removed from the
animal (Figure l). The joint capsules, tendons, and
ligaments were trimmed from the bones with scissors.

The muscles, nerves, and arteries were carefully scraped
away from the bones with a surgical knife. The bones
were continuously moistened with Ringer's saline solution.
Using a micrometer, graduated in .001 millimeter units
(Figure 2), femur lengths were measured along a line
from the most proximal point of the greater trochanter
to the most distal point of the lateral condyle. See
Appendix B for the femur lengths. The bones were
weighed wet and then stored in Ringer's saline solution

at 4 to 6°C. See Appendix C for the femur weights.

Bone Stiffness Analysis
Mechanical Testing Techniques
In order to measure the stiffness of bone, the
modulus of elasticity was determined through bending
tests. These tests were designed to determine the load—
deformation relationship for bone, which in turn was

used to calculate the elastic modulus.

3From Jensen-Salsberg Laboratories, Division of
Richardson-Merrel, Inc., Kansas City, Missouri.

 

24

 

 

Figure l.--A Rat Femur (left side).

25

 

 

 

Figure 2.--Micrometer and Jeweler's Saw.

26

All bones were tested within one week of sacri-
fice. A concurrent study showed that there was no
significant difference within the sixteen-week duration
group either when tests were conducted on bone immediately
after sacrifice from animals that were sacrificed
immediately or 72 hours after completing the exercise
program or when tests were conducted on bone one week
after sacrifice. Furthermore, there was no significant
difference, at the .10 level, between the elastic moduli
of the right and left bones.

The specimens for testing were removed from wet
storage and were kept in Ringer's solution until they
reached a temperature of 22 to 26°C. The specimens were
maintained within this temperature range throughout the
testing procedure. The bones were removed individually
from.the storage solution and were placed in a 3-inch
diameter plexiglass chamber containing a test bath of
Ringer's solution (Figure 3). The temperature of the
test solution was kept constant by circulating water
from a constant temperature source through a copper heat-
exchange coil in the bottom of the bath. The temperature
of the Ringer's bath was measured with a thermometer,
graduated in .1°C units. The source temperature was
checked by a Bronwill Model 20 constant temperature

circulator (Figure 4). A linear relationship was shown

27

 

 

Figure 3.--Bending Test Fixture.

28

 

 

Figure 4.--Testing Apparatus.

29

to exist between the temperature of the source and the
temperature of the Ringer's bath over a large temperature
range.

The grips used to hold the specimens at both ends
in a horizontal position were C-shaped minimizing slip-
page (Figure 5). A two-point loading hook made contact
on the linea aspera in the middle of the posterior side
of the bone. The distance between each loading point and
each outside support was .221 inches. The span between
the two loading points was .101 inches (Figure 5). The
distancezthickness ratios ranged from 1:1 to 2:1. These

ratios were lower than those reported in other studies.

 

 

=.543:
(FLZZIH
L-20=.IOI
O
\z

 

 

Figure 5.--Specimen Grips.

30

The machine used to conduct the tests was operated
by driving a crosshead with two finely threaded screws
mounted in stainless steel ball bearings (Figure 3). A
Statham UL4—50 load cell accessory was inserted into the
stationary upper plate of the test fixture. This cell
was powered by a Statham SClOOl Universal Transducer
Readout unit.

The displacement of the crosshead was measured
with a steel clip gauge inserted between the crosshead
and the base (Figure 3). The clip gauge was constructed
from steel shim stock .01 inches in thickness, .20 inches
in width, and about 6.00 inches in length. Budd Metal—
film Strain Gauges, Type 015-624, were attached to both
the convex and concave sides of the clip gauge. The
strain gauges were balanced in a bridge circuit by a
Heath Decade Box Resistance. The clip gauge was used
with an Ellis Associates Model BAM-l Bridge Amplifier
Meter.

The testing fixtures were powered by a 1/70
horsepower Bodine Model NSR-12R shunt wound motor. The
speed of the motor was kept constant by a Minarik Model
W-l4 speed control unit (Figure 4).

The loads, displacements, and loading rates of
all specimens were recorded on both a Sanborn 322 Dual
Channel DC Amplifier Recorder and a Varian F-80 X-Y

Recorder (Figures 6, 7 and 8). A voltage regulator and

31

 

Figure 6.--Recording Apparatus

32

 

I___

 

Figure 7.--Load, Deformation, and Time.
Strip Chart.

33

 

 

Figure 8.--Load Versus Deformation Plot.

34

two low pass filters (a 10K resistor and a 50-150 uf DC
capacitor) were used to hold the line voltage constant
and to filter out irrelevant noise.

A cross-section of the bone, at the point of
load application, was obtained by using a jeweler's saw
(Figure 2). The distal end of the bone was held in a
vice between two soft pads. While the bone was being
sawed, Ringer's solution was applied to the bone to
avoid drying. A cross-section, 1/32 to 1/16 inches in
thickness, was placed on a glass slide. The slide was
placed in a microscopic projector (Figure 9). The cross-
sectional image was projected onto graph paper and the
outline was traced. The point of load application was

marked (Figure 10).

Elastic Modulus Equation
The free-body diagram (Figure 11) for a linearly
elastic, simple beam in pure bending is described by a

load-intensity function (q(x)) (20):

q(x) = -EIvIV = RA<x-0>_1- P<x-a>_l- P<x-(L-a) >_1 + RB<x-L>__1
3.1
where: fn(x) = <x-a>n is a family of singularity
functions
when n30 and x<a, then fn(x) = 0
when n30 and x>a, then fn(x) = (x-a)n.

35

ea :~_,"—’f“fiI
. -‘ ‘ .l
t. "°
‘1

 

Figure 9.--Microscopic Projector.

36

 

 

Figure 10.--Tracing of the Cross-Sectional
Area of the Bone Projection.

32

when n<0 and xia, then fn(x) = 0, and

when n<0 and x=a, then fn(x) is infinite; and
where: at x=a, <x-a>O is the unit step function,

at x=a, <x-a>1 is the unit ramp function,

<x-a>_1 is the unit concentrated load function,

and <x-a>_2 is the unit concentrated moment function.

2!
D'
N

I)
G!

 

 

 

 

 

 

 

 

Figure ll.--Free—body Diagram of a Beam
in Pure Bending.

38

Integration of equation 3.1 four times results in
equation 3.2 for the deflection of the beam at any

position on the x-axis.

 

 

 

 

~ RAfx20>3 P<x.-a>3 P<x.-L+a>3 Clx3 szz
+C4 3.2

The boundary conditions at x = 0 are

III

-V(x) = "EIV = 0 :9 C1 = 0’
Mb = -EivII = o =>c = o and
(x) 2 ’

The boundary conditions at x = L+ are

_ ~ III _ _ _ _ =
Mb = -EIvII = o s>R L - P(L-a) - P(a) = o and
(x) A ’

~ RA(L)3 P(L-a)3 P(a)3
v<x> = -EIv = o =>——3——— - —_—6___ - _—6—— + C3L==0.

3.3

Solving equations 3.3 simultaneously yields

RB = P,
RA = P,
PL2 + P L-a)3 Pa3

C3 = ”’6‘“ L ‘+ 6L ' 3.4

39

substituting the values of equation 3.4 and x = % into
the deflection equation (3.2) results in an equation for

the center deflection (maximum deflection 6) of the beam:

6 = -v = Pa L2 - 82 3 5
Vmax if 7? 7? ' '

Substituting the values a = .221 inches and L = .543
inches into equation 3.5 and solving for the elastic

modulus yields the equation:

E

—3— (.01420) kgm/cmz, 3,6
612

load placed on the bone and measured by

the load cell,

where P

6 = center deflection measured by the strain
clip gauge, and
I = an equivalent moment of inertia of the

cross—sectional area of bone.

Elastic Modulus of the Bone

Each bone to be analyzed was loaded until plastic
yielding occurred. Load (P) versus deformation (6) was
recorded on a Varian F-80 X—Y recorder. Load and
deformation values along only the linear portion of the
recording were used. Within this segment of the curve,
the bone responded as a linearly elastic material. The
obtained load and deformation values were used in equation

3.6.

40

Equation for the Moment of
Inertia ofia Gross-section

 

 

For the moment of inertia of the irregular
cross-sectional area of a bone, a general case will be
considered where the y- and z-axes are centroidal axes,
not principal axes, and where bending will occur in the
xy and xz planes as a result of the bending couples -My

and -M2 (Figure 12).

 

 

 

Figure 12.--Bending Moments of a
Cross-section.

 

41

The positive curvature in the xy plane can be denoted

by ky = 5L3 where Dy is the radius of curvature along
Y

the y-axis. The positive curvature in the xz plane can

be denoted by k2 = iLy where oz is the radius of curva-

pz

ture along the z-axis. The stress at point A with

coordinates y,z is

o = + k EY + k EZ. 3.7
x y 2

Since the origin of the axes is at the centroid of the
cross-section, the resultant force in the x-direction

is zero. That is,
fodi = 0 and kyEdeA + szdeA = 0.

The moment about the y-axis is

_ _ 2
-My - fondA — kyEfYZdA + szfZ dA 3.8

where Iy = IZZdA and Iyz = fYZdA. Therefore, equation

3.8 can be rewritten as

M = -k EI — k EI . 3.9
Y Y yz Z Y

The moment about the z-axis is

-M = - fo YdA = -k EszdA - k EfYZdA, 3.10
z x y z

where Iz = szdA and Iyz = fYZdA. Therefore, equation

3.10 can be rewritten as

 

42

M = k EI + k EI . 3.11
z y z z yz

Solving equations 3.9 and 3.11 simultaneously yields

M I z + MZI M IZ + MZI z
k = '.I _ and k2 = - E(I_I——:_I_XZ).
y y z yz y z yz
3.12

Substituting equations 3.12 into equation 3.7 yields the
total bending stress at point A for the general case

-(M I + M I )z + (M I + M I )Y
y,z z yz 4y yz z y; .

0x = I'I - I 2 3'13
y z z

 

Y

In this study, the bending moment about the
y-axis is equal to zero (My = 0). Therefore, equation
3.13 takes the form

_ Mz (IyY - Iyzz).

Ox - I I - I 2
Y z yz

 

The curvature of bending is only in the xy plane, or

k = z _ .
y EZIyIz Iyz2)

If the axes were principal axes, then Iyz = 0 and

k = -%—u Since the axes are centroidal axes, the

43

equivalent moment of inertia of the irregular cross—

section is

I=Yz YZ- 3.14

 

Moment of Inertia of the
Bone‘Cfoss-section

 

 

Each cross-section to be analyzed was projected
onto graph paper so that the outer contour would lie
within a Cartesian field of 30 units by 30 units with a
scale factor of 50 units/cm. Both the inner border and
the outer border of the bone were described by a series
of coordinate points which were selected arbitrarily.

The coordinates were recorded on computer cards. The
S.C.A.D.S.4 computer program developed by G. Piotrowski
and G. I. Kellman (67) was used to compute all geometric
properties of the cross-section. The program fills in a
smooth curve between the input points to establish the
cross-sectional geometry. The y-axis was the axis of
load application. The line of load was determined as
continuous from the point of load application through the
cross-section bisecting the inner elliptical cavity. The
cross-sectional area (A), the location of the centroid
of the cross-section (§,§), and the various area moments

of inertia (1y, Iz, Iyz) were calculated using the IBM

 

4
Sections.

A Stress Calculator for Arbitrarily Drawn

44

5

System/370-165 computer of the N.E.R.D.C. The values

calculated were used as the parameters for equation 3.13.

Strain Rate Equation

 

For the strain rate of each bone analyzed, the

standard stress-strain relationship was used:
0 = Eex. 3.15

A stress equation can be formulated using bending
moments and the moment of inertia of the cross-section

as follows:

MZY PaY
g = _,_.= _,_3 3.1
x Iz 'Iz 6

substituting equation 3.16 into 3.15 yields

PaY _
1E: - Eex. 3.17

Strain rate was calculated by differentiating both

sides of equation 3.17 with respect to time:

dP aY Edex
HE:=-Ti't_

Rewritten, this formula yields the following equation

for strain rate:

 

5North East Regional Data Computing Center of
the Florida State University System.

45

94%

aY
._f_. 3.18
z

[‘11

Strain Rate of the Bone

 

Each bone to be analyzed was loaded until plastic
yielding occurred. Lead versus time was recorded on a
Sanborn 322 Dual Channel DC Amplifier recorder. Load
and time values along the linear portion of the
recording were used. The (Y) from the centroid of the
cross-section to the outer surface of the bone along the
y-axis was measured in millimeters (Figure 13). Values
calculated and measured in this manner were used in
equation 3.18. The average strain rate was 6.01 x
10-4/sec. This value was lower than the strain rate

values used in other studies.

 

 

 

Figure l3.--Distance (Y) from the Centroid.

 

46

Treatment of the Data

 

Desigp

The study was designed as a three-way factorial
experiment with repeated measures on only the third
factor. Factor A, training, had three levels: CON,
SHT, and LON. Factor B, duration, had five levels:
O-wk, 8-wk, l6-wk, 24-wk, and 32-wk. These two factors
have been described previously. Factor C, right versus
left, was included to permit repeated measures to be
taken on paired bones of each animal. A tabular repre-
sentation of the experimental design, with cell fre-

quencies, can be seen in Table 5.

TABLE 5.--A 3X5X2 Factorial Design with Cell Frequencies.

 

Factor C: Right vs Left

 

 

Factor A: Factor B:
Training Durat1on Right Bone Left Bone
(CON) (Oewk) 4 4
(8-wk) 4 4
(16 -wk) 4 4
(24-wk) 4 4
(32-wk) 4 4
(SHT) (O-wk) 4 4
(8—wk) 4 4
(16-wk) 4 4
(24-wk) 4 4
(32 -wk) 4 4
(LON) (O-wk) 4 4
(8-wk) 4 4
(l6-wk) 4 4
(24-wk) 4 4
(32-wk) 4 4

 

47

Significance Level

 

The .10 level of significance was selected for
all inferential statistical analyses. This choice
reflected the fact that the sample size was limited to
four observations per cell by practical considerations.
At the present state of knowledge, the consequences of
failing to detect a potentially important treatment
effect seemed to be nearly as great as those associated
with claiming a significant effect when none exists.
Differences observed at the .10 level (more easily
detected than at the usual .01 or .05 levels) might
provide direction for future research. Therefore, the
probability of making a Type II error was reduced even
though the risk of committing a Type I error was
increased.

Eliminating the Dgpendence of
Stiffness on Strain Rate

Strain rate was not controlled during data
collection. It was not randomized across treatments,
durations, the right and left femur, or subjects. Thus,
the effect of strain rate had to be eliminated between
groups as well as within groups. A regression technique
was used for this purpose. The assumptions of linearity
of regression and homoscedasticity were met. The raw
values of the elastic modulus were corrected to esti-

mated values corresponding to the mean strain rate of

 

48

6.01 X 10-4/sec. This procedure did not alter the

overall mean value of the elastic modulus.

Statistical Procedures

 

The statistical analyses used in this study,
both inferential and descriptive, are indicated for each

of the following research questions that were asked:

Question I.--What is the stiffness of rat bones,

 

as measured by the elastic modulus, after selected
programs of exercise and detraining?

Descriptive statistics, including means and
standard deviations, were computed for the elastic
modulus by treatment, duration, and right versus left

sides of the body.

Question II.-—Does the elastic modulus vary as

 

a function of paired femurs?

The MANOVA program (41) was used, with the
univariate-repeated measures option, to test the sig-
nificance of the difference between the elastic moduli

of the right and the left femurs.

Qpestion III.--Does the elastic modulus vary

 

as a function of exercise?
The MANOVA program (41) was used, with the

univariate-repeated measures option, to test the

49

significance of the differences between the elastic

moduli of the three training groups.

Qpestion IV.--Does the elastic modulus vary as a

 

‘function of duration of exercise and detraining?
The MANOVA program (41) was used, with the

univariate-repeated measures option, to test the

significance of the differences between the elastic

moduli when the data were grouped by duration.

CHAPTER IV

RESULTS AND DISCUSSION

The purpose of this study was to investigate the
effects of two training programs and a subsequent
detraining period on bone stiffness in adult male albino
rats. More specifically, the study sought to determine
the stiffness of the femur after planned programs of
exercise and detraining as well as the individual effects
of the exercise treatments and various treatment dura-
tions. Bones from.the right and left sides of the body

were compared.

Results
The results of both inferential and descriptive
statistical procedures are reported here in an attempt

to answer the four major questions posed earlier.

Question I

 

What is the stiffness of rat femurs, as measured
by the elastic modulus, after planned programs of exer-
cise and detraining?

Means and standard deviations of the elastic

modulus were computed separately for the right and left

50

51

 

 

 

 

 

 

 

 

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52

femurs of each treatment group at the conclusion of each
duration period. These values are presented in Table 6.
The overall low stiffness values found in this study may
have been due either to the wet storage technique used,
to the low strain rate used, or to the small distance:
thickness ratios used during testing.

The elastic modulus decreased during each time
interval except one in all treatment groups (Figure 14).
A significant increase in all treatment groups was noted
for the interval between the 8-wk and the 16-wk training
periods. There was a trivial increase in the mean
elastic modulus of the left femur of the LON treatment
group during the interval between the l6-wk training
period and the 24-wk detraining period. At the conclu-
sion of each duration period, the average values of the
elastic modulus for the LON and SHT groups were less
than the average value for the CON group with two
exceptions: (a) at 32 weeks, the right femur of the LON
group had a greater average value than did the right
femur of the CON group; and (b) at 8 weeks, the left
femur of the SHT group had a greater average value than
did the left femur of the CON group. The average values
of the elastic modulus for the SHT group were greater
than those for the LON group after both 8 and 16 weeks

of training. However, at 24 weeks and 32 weeks, the

53

  

 

O CONTROL

0 SHORT
X LONG

 

 

 

 

 

 

 

l
24
252
OETRAINING

 

 

O x

l_ l

I 1

\O x
. D
O A O
l l I l l l l l l 1 1%]

ID 0 ID 0 ID 0 0
¢ 0' [0 to N N —

(son I awe/won) smnoow DIISV‘IB

Figure l4.--Average Elastic Moduli with
Standard Error of the Means.

TRAIN. WK.

|96

I40
TRAINING

AGE (days)

 

 

 

I
O
84

a

AVERAE ELASTIC MODLLI WITH STANDARD ERROR (f TI-E LEANS

54

average values for the SHT group drOpped below those for
the LON group.

The differences between the average stiffness
values for the right and the left femurs were no greater
than the within group variability. Therefore, bone
stiffness appears to have little to do with which side
of the body is being studied.

In summary, the computed means and standard
deviations of the elastic modulus suggest that:

(a) over the 32 weeks of training and detraining, the
rat femurs in all treatment groups (including the CON
group) initially decreased in stiffness, then increased,
and finally decreased below the original level;

(b) during the training period, the SHT group had a
greater average stiffness than did the LON group, but
both experimental groups had values below that of the
CON group; (c) during the detraining period, the SHT
group had a lower average stiffness than did the LON
group, but both experimental groups had values below
that of the CON group; (d) there were no apparent dif-
ferences between the right and left femurs over either
treatment durations or treatment groups; and (e) the
intragroup variabilities suggest that there was a wide
range of individual values for the elastic modulus

within most of the treatment-duration groups.

55

Qpestion II

 

Does the elastic modulus vary as a function of
paired femurs?

A multiple analysis of variance (MANOVA) pro-
cedure was used, with the repeatedemeasures option, to
test the significance of the difference between the
elastic moduli of the right and the left femurs. This
technique was considered appropriate since the paired
femurs that were obtained from each animal provided a
set of bivariate dependent data. A summary of the

analysis of variance results is presented in Table 7.

TABLE 7.--Analysis of Variance for Bone Stiffness with
a Repeated-measures Option for the Right and
Left Bones of the Animal.

 

 

Source Variablea F Pb
Treatment 1 2.69 0.079
2 0.71 0.495
Duration 1 16.54 0.0001
2 0.73 0.577
Treatment by Duration 1 0.95 0.484
2 0.94 0.497

 

a1 = The right and left femur considered
collectively;
2 = The difference between the right and
left femur.

bProbability values were rounded to the nearest
.001 unless otherwise listed.

56

Examination of the univariate F—ratios indicated that
there were no significant differences between the right
and left femurs over treatment, over duration, or over
interaction at the .10 level. Therefore, in answering
Questions III and IV, the stiffness values for the
right and left femurs were totaled and considered

collectively (see Table 8).

TABLE 8.—-Means and Standard Deviations for the Stiff-
ness of Rat Femurs Presented by Treatment,
Duration, and Animal (kgm/cm2 X 103).

 

 

 

 

 

 

 

 

 

Short Long Control Total
Duration Mean Mean Mean Mean
(S.D.) (S.D.) (S.D.) ‘
O-wk Control 26.5 27.8 29.7 28.0
(3.4) (1.8) (4.8)
8-wk Training 25.5 18.9 23.4 22.6
(5.1) (4.4) (8.9)
l6-wk Training 36.2 29.6 40.5 35.4
(4.4) (7.4) (13.0)
24-wk Detraining 24.1 26.2 29.5 26.6
(2.6) (3.2) (4.3)
32-wk Detraining 16.8 18.0 18.0 17.6
(2.2) (4.3) (2.4)
TOTAL 25.8 24.1 28.2

(3.5) (4.2) (6.7)

 

57

Question III

 

Does the elastic modulus vary as a function of
exercise?

A repeated-measures (MANOVA) procedure was used
to test the significance of the differences between the
elastic moduli of the three treatment groups. A summary
of the analysis of variance results is presented in
Table 7. Examination of the univariate F—ratios indi-
cated that the interaction effect was not significant
at the .10 level (F = 0.95, P = 0.484). However, a
significant univariate F-ratio for the main effect of
treatment was obtained (F = 2.69, P = 0.079).

The source of the significant treatment effect
was determined by using the Newman-Keuls test (90). The
differences between the treatment means that were sig-
nificant at the .10 level are indicated by an asterisk
in Table 9. Significant contrasts were found only at
the conclusion of the l6-wk training period. At that
time, both the SHT group and the CON group had signifi-

cantly greater average stiffness values than did the LON

group; however, the CON - SHT contrast was not significant.
These results would suggest that femur stiffness is not
influenced significantly by the SHT running program and
that it is decreased by the LON program only after 16

weeks of training.

58

TABLE 9.--Differences Between the Mean Elastic Moduli
(R + L) of the Treatment Groups within each
Treatment Duration (kgm/cm2 X 103).

 

Contrast

 

Treatment Duration
CON - SHT CON - LON LON - SHT

 

O-wk Control + 6.3 + 3.7 + 2.5
8-wk Training - 4.2 + 8.8 -13.0
16-wk Training + 8.6 +21.7* -l3.0*
24-wk Detraining +10.8 + 6.6 + 4.1
32-wk Detraining + 2.4 + .0 + 2.3

 

*
Significant at the .10 level.

Question IV

 

Does the elastic modulus vary as a function of
duration of exercise and detraining?

A repeated-measures (MANOVA) procedure was used
to test the significance of the differences between the
elastic moduli of the various duration groups. A
summary of the analysis of variance results may be
found in Table 7. A significant univariate F-ratio for
the main effect of duration was obtained (F = 16.54,

P = 0.0001).

The Newman-Keuls method was used to locate the
source of significance in the duration effect. The
differences between means are shown in Table 10. The

asterisks indicate significant contrasts (P < 0.10).

59

TABLE 10.--Differences Between the Mean Elastic Moduli
(R + L) of the Duration Groupg within each
Treatment Group (kgm/cm2 X 10 ).

 

Treatment Group

 

 

C°ntraSt CON SHT LON
l6-wk - 32-wk +44.9* +38.6* +23.2*
l6-wk - 24-wk +21.9* +24.0* + 6.8
l6-wk - 8-wk +34.2* +21.3* +21.3*
l6-wk - 0-wk +21.6* +19.3* + 3.6
O-wk - 32-wk +23.2* +19.3* +19.5*
O-wk - 24-wk + .2 + 4.7 + 3.1
O-wk - 8-wk +12.6 + 2.0 +19.5*
24-wk - 32-wk +23 . 0* +14 . 5* +16 . 4*
24-wk - 8-wk +46.8 - 2.6 +14.5*
8-wk - 32-wk _ +10.6* +17.2* + 1.8

 

*
Significant at the .10 level.

Within the CON group, the highest stiffness
value was observed at 16 weeks. This value differed
significantly from those found at all other treatment
durations. The O—wk control, the 8-wk training, and the
24-wk detraining means differed significantly from the
32-wk detraining mean, but they did not differ signifi-
cantly from each other. Since the CON animals remained
in sedentary cages throughout the experiment, there

appears to have been some factor other than training

60

(perhaps aging) which caused the bones to gain stiffness
before and lose stiffness after the conclusion of the
l6-wk treatment period when the animals were 196 days

of age.

The pattern of contrasts within the SHT group was
the same as that seen in the CON group except that the
24-wk detraining mean was less than the 8-wk training
mean. The reverse was true in the CON group. These

results suggest that the SHT running program had no sig-

_—- I 37.33::-

nificant effect on the stiffness of rat femurs over time.
In the LON group, the 0-wk, l6-wk, and 24-wk

' means were not significantly different from each other
but were significantly greater than the 8-wk and 32-wk
means. The difference between the 8—wk and the 32-wk
means was not significant. These results suggest that
the bone stiffness decreased shortly after the beginning
of training, increased after 8 weeks, and then decreased
again after 8 weeks of detraining. At no time did the
LON program cause a significant increase in bone stiff-

ness above the initial level.

Discussion

 

The results of this study show that a decrease in
rat femur stiffness occurs during 16 weeks of long-
duration low-intensity endurance training and that some

factor, such as the aging process, causes rat femurs to

61

gain stiffness before and lose stiffness after 196 days
of age. Stiffness of bone, which is the resistence to
deformation, should not be confused with the breaking
strength of bone or the stress at which failure occurs.
A significant correlation (r = 0.4105, P < 0.01) has
been found in humans between stiffness and breaking load
of the femur (57). Data are not available to indicate
whether a loss or a gain in bone stiffness is desirable
at a given age.

The current results appear to be in conflict with
those of Jankovich (47) which showed an increase in the
elastic modulus due to vibration of bone and those of
Saville and Whyte (75) which showed no significant dif-
ferences in breaking force between exercised and control
animals. However, both the intensity and the duration
of the exercise programs were different in those studies
than in the present investigation.

Beary (9) observed that any change in the crystal-
line structure of the bone significantly alters the
elastic modulus. Currey (28) showed that the elastic
modulus of bone rises very rapidly with an increase in
the mineral content. He also found that a marked
increase in the stiffness of bone, with a corresponding
change in ash content, is due to an elongation of the
apatite crystals without a corresponding increase in

their thickness (22). Bartley and co-workers (8)

62

studied the relationship of aging to bone strength in
humans and observed that the ash content per unit volume
of bone increases up to 20 or 30 years of age and then
decreases until it eventually reaches a value below the
birth level. The results of the present study are in
keeping with the findings of Bartley (8), salomon and
Volpin (73), and Mather (55) in that there was an
increase in stiffness and then a decrease as aging took
place. However, based on the assumption that the life
span of man is thirty times that of the albino rat (31),
the drop in bone stiffness in this study occurred at a
relatively earlier age than it does in the human. Hirsch
and Evans (46) observed that the elastic modulus
increases from infancy to 14 years of age, but they
did not study the phenomenon beyond this age.
Insufficient data was obtained to calculate the
yield strength values of all the femurs. The data that
are available (not reported in this study) suggest that
there is an increase in yield strength after eight weeks
of detraining of the experimental animals. Further

research needs to be done in this area.

CHAPTER V

SUMMARY, CONCLUSIONS,
AND RECOMMENDATIONS

The purpose of this study was to investigate
the effects on two exercise programs and a subsequent
detraining period of femur stiffness. Sixty adult
male rats of the Sprague-Dawley strain were assigned
randomly to three treatment groups: sedentary control
(CON), high-intensity running (SHT), and low-intensity
running (LON). Animals in the two experimental groups
were trained for 16 weeks and then confined to seden-
tary cages for 16 weeks of detraining. Four animels in
each treatment group were sacrificed after 0, 8, 16,
24, and 32 weeks of treatments.

A bending test was used to determine the load-
deformation relationship for the right and left femurs
of each animal. Values of the elastic modulus then
were calculated to indicate bone stiffness.

Means and standard deviations were computed
for the elastic modulus; and comparisons were made by
treatment group, treatment duration, and right versus

left femurs. A repeated-measures MANOVA technique was

63

64

used to test for the significance of differences between
the means. The source of each significant univariate F-
ratio was identified by the Newman-Keuls method.

The results of the study suggest that the femurs
of adult male albino rats vary in stiffness over a 32-wk
time period. There is a marked increase between 140 and
196 days of age. This initial increase is followed by a
decrease to values below the original level at 308 days
of age. The causal factor for these observed changes
may be simply the aging process. Great variability
exists between the stiffness values of individual
animals.

There appears to be no significant difference
between the stiffness values of the right and left
femurs. Although the SHT training program used in this
study does not alter femur stiffness, the LON program
gradually decreases bone stiffness. The LON treatment
effect becomes statistically significant after sixteen

weeks of training.

Conclusions

 

The following conclusions are drawn from the
results of this study:

1. Paired femurs of adult male rats do not
differ in stiffness over a 32-wk period of training and

detraining.

65

2. The short-duration, high-intensity exercise
program that was used in this study has no significant
effect on femur stiffness.

3. The long-duration, low-intensity exercise
program that was used significantly decreases femur
stiffness during 16 weeks of training.

4. Some factor, such as the aging process,
causes rat femurs to gain stiffness before and lose
stiffness after 196 days of age.

5. The relatively low stiffness values that
were found in this study may have been due either to
wet storage or to the low strain rate used, or to the

small distancezthickness ratios used during testing.

Recommendations

The following suggestions are made for future
research concerning bone stiffness:

1. A study of bone stiffness should be con-
ducted which includes the investigation of any
histological (structural) or biochemical changes that
may occur in bone due to exercise and detraining.

2. A study of the effects of exercise on bone
stiffness should be conducted in which the intensity of
exercise is increased gradually throughout an experi-

mental period of at least 16 weeks.

66

3. A study, including ultimate strength, yield
strength, and bone stiffness, should be conducted which
investigates the effects of exercise and detraining on
the plastic deformation and the viscoelastic properties

of bone.

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67

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Schemmel, Rachel, Mickelsen, Olaf & Mostosky, Ulreh,

"Skeletal size in obese and normal-weight litter-

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APPENDICES

77

APPENDIX A

78

APPENDIX A.--Standard Eight-week, Short-duration, High-

 

 

intensity Interval Training Program for
Postpubertal and Adult Male Rats in
Controlled-running Wheels.
N A
I: s 3
,l 0g g 3 m 3 5A a: BA
3 :3 as q... s S 8 :5 a 1: ES». '8 fig
3 H «so So 5 Ha a: as a en" 44:40 Ra o
.. .. :3 2:: a :3: a 3V V a: SS: ““3 W
.. ° ° 2:. 1;: .. em ° .3 :5 "’2 as: case so
a: a s 3.: SE a e: .- 53 .2. 5:: 3...: 35:: s:
3 Q G <64 3" a: MD. 2 E-dm U! adv E-‘OV E-‘MV l-‘V
0 4-T -2 3.0 40 00 10 1 1 5.0 0.0 1.5 40:00 --- ---
S=F -l 3.0 40 00 10 1 5.0 0.0 1 5 40:00 --- ---
1 18M 1 3.0 00 10 10 4O 3 5.0 1.2 1.5 49:30 450 1200
2=T 2 3.0 00 10 10 4O 3 5.0 1.2 1.5 49:30 450 1200
3=W 3 3.0 00 10 10 40 3 5.0 1.2 1.5 49:30 450 1200
4=T 4 2.5 00 10 10 40 3 5.0 1.2 2.0 49:30 600 1200
5=F 5 2.0 00 10 10 40 3 5.0 1.2 2.0 49:30 600 1200
2 1=M 6 1.5 00 10 10 28 4 5.0 1.2 2.5 51:40 700 1120
2=T 7 1.5 00 10 15 27 4 5.0 1.2 3.0 59:00 810 1080
3=W 8 1.5 00 10 15 27 4 5.0 1.2 3.0 59.00 810 1080
4=T 9 1.5 00 10 15 27 4 5.0 1.2 3.0 59:00 810 1080
5=F 10 1.5 00 10 15 27 4 5.0 1.2 3.0 59:00 810 1080
3 1=M 11 1.5 00 10 15 27 4 5.0 1.2 3.0 59:00 810 1080
2=T 12 1.5 00 10 20 23 4 5.0 1.2 3.5 59:40 805 920
3=W 13 1.5 00 10 20 23 4 5.0 1.2 3.5 59:40 805 920
4=T 14 1.5 00 10 20 23 4 5.0 1.2 3.5 59:40 805 920
5=F 15 1.5 00 10 20 23 4 5.0 1.2 3.5 59:40 805 920
4 1=M 16 1.5 00 10 20 23 4 5.0 1.2 3.5 59:40 805 920
2=T 17 1.5 00 10 25 20 4 5.0 1.0 4.0 60:00 800 800
3=W 18 1.5 00 10 25 20 4 5.0 1.0 4.0 60:00 800 800
4=T 19 1.5 00 10 25 20 4 5.0 1.0 4.0 60:00 800 800
5=F 20 1.5 00 10 25 20 4 5.0 1.0 4.0 60:00 800 800
5 1=M 21 1.5 00 10 25 20 4 5.0 1.0 4.0 60:00 800 800
2=T 22 1.5 00 10 30 16 4 5.0 1.0 4.5 55:40 720 640
3=W 23 1.5 00 10 30 16 4 5.0 1.0 4.5 55:40 720 640
4=T 24 1.5 00 10 30 16 4 5.0 1.0 4.5 55:40 720 640
5=F 25 1.5 00 10 30 16 4 5.0 1.0 4.5 55:40 720 640
6 1=M 26 1.5 00 10 30 16 4 5.0 1.0 4.5 55:40 720 640
2=T 27 2.0 00 10 35 10 5 5.0 1.0 5.0 54.35 625 500
3=W 28 2.0 00 10 35 10 5 5.0 1.0 5.0 54'35 625 500
4=T 29 2.0 00 10 35 10 5 5.0 1.0 5.0 54 35 625 500
5=F 30 2.0 00 10 35 10 5 5.0 1.0 5.0 54 35 625 500
7 1=M 31 2.0 00:10 35 10 5 5.0 1.0 5.0 54:35 625 500
2=T 32 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560
3=W 33 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560
4=T 34 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560
5=F 35 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560
8 1=M 36 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560
2=T 37 2.0 00'10 4O 6 8 2.5 1.0 5.5 52:10 660 480
3=W 38 2.0 00 10 4O 6 8 2.5 1.0 5.5 52'10 660 480
4=T 39 2.0 00 10 40 6 8 2.5 1.0 5.5 52:10 660 480
5=F 40 2.0 00 10 40 6 8 2.5 1.0 5.5 52:10 660 480

 

79

80

This standard program was designed using male rats
of the Sprague-Dawley Strain. All animals were between 70
and 170 days-of-age at the beginning of the program. The
duration and intensity of the program were established so
that 75 per cent of all such animals should have PSF and
PER scores of 75 or higher during the final two weeks.
Alterations in the rest time, repetitions per bout, number
of bouts, or time between bouts can be used to affect
changes in these values. Other strains or ages of animals
could be expected to respond differently to the program.

All animals should be exposed to a minimum of one
week of voluntary running in a wheel prior to the start of
the training program. Failure to provide this adjustment
period will impose a double learning situation on the
animals and will seriously impair the effectiveness of the
training program"

STANDARD SHORT-DURATION, HIGH-INTENSITY INTERVAL
MAINTENANCE PROGRAM FOR POSTPUBERTAL AND ADULT
MALE RATS IN CONTROLLED-RUNNING WHEELS

 

Total Total

Acc- Repe- Time Time Exp. Total
eler- Work ti- Bet- Run of Revo- Work
ation Time Rest tions No. ween Speed Prog. lu- Time

Time (min: Time per of Bouts Shock (ft/ (min: tions (sec)
(sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TWT

 

2.0 00:10 40 4 6 2.5 1.0 5.5 28:30 330 240

 

81

APPENDIX A.--Standard Eight-week, Long—duration, Low-
intensity Endurance Training Program for
Postpubertal and Adult Male Rats in
Controlled-running Wheels.

 

 

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a I: :3 «(in 3~v' m g a. :z hug In aflsw Eco~v th‘v en~I
0 4-T -2 3.0 40:00 10 l 1 5.0 0.0 1.5 40:00 --- ---
5'? -1 3.0 40:00 10 l 5.0 0.0 1.5 40:00 --- ---
1 1-M 1 3.0 00:10 10 40 3 5.0 1.2 1.5 49:30 450 1200
2-T 2 3.0 00:10 10 40 3 5.0 1.2 1.5 49:30 450 1200
3'" 3 3.0 00:10 10 40 3 5.0 1.2 1.5 49.30 450 1200
4-T 4 2.5 00:20 10 30 2 5.0 1.2 1.5 34:40 450 1200
S-F 5 2.5 00:30 15 20 2 5.0 1.2 1.5 34:30 450 1200
2 l-M 6 2.0 00:40 20 15 2 5.0 1.2 2.0 34:20 600 1200
2-T 7 2.0 00:50 25 12 2 5.0 1.2 2.0 34:10 600 1200
3-W 8 1.5 01:00 30 10 2 5.0 1.2 2.0 34.00 600 1200
4=T 9 1.5 02:30 60 4 2 5.0 1.2 2.0 31.00 600 1200
5'? 10 1.0 02:30 60 4 2 5.0 1.2 2.0 31:00 600 1200
3 1-M 11 1.0 02:30 60 4 2 5.0 1.2 2.0 31:00 600 1200
2-T 12 1.0 05:00 0 1 5 2.5 1.2 2.0 35:00 750 1500
3-W 13 1.0 05:00 0 1 5 2.5 1.2 2.0 35:00 750 1500
4-T 14 1.0 05:00 0 l 5 2.5 1.2 2.0 35:00 750 1500
S-F 15 1.0 05:00 0 l 5 2.5 1.2 2.0 35:00 750 1500
4 1-M 16 1.0 05:00 O 1 5 2.5 1.2 2.0 35:00 750 1500
2=T 17 1.0 07:30 O 1 4 2.5 1.0 2.0 37:30 900 1800
3-W 18 1.0 07:30 0 1 4 2.5 1.0 2.0 37:30 900 1800
4= 19 1.0 07:30 0 1 4 2.5 1.0 2.0 37:30 900 1800
S-F 20 1.0 07:30 0 1 4 2.5 ‘ 1.0 2.0 37:30 900 1800
S l-M 21 1.0 07:30 0 l 4 2.5 1.0 2.0 37:30 900 1800
2-T 22 1.0 07:30 0 1 5 2.5 1.0 2.0 47:30 1125 2250
3-W 23 1.0 07:30 0 1 5 2.5 1.0 2.0 47.30 1125 2250
4=T 24 1.0 07:30 0 1 5 2.5 1.0 2.0 47:30 1125 2250
5=F 25 1.0 07:30 0 1 5 2.5 1.0 2.0 47:30 1125 2250
6 1=M 26 1.0 07:30 0 1 5 2.5 1.0 2.0 47:30 1125 2250
2=T 27 1.0 10:00 0 1 4 2.5 1.0 2.0 47:30 1200 2400
3=W 28 1.0 10:00 0 1 4 2.5 1.0 2.0 47 30 1200 2400
4=T 29 1.0 10:00 0 1 4 2.5 1.0 2.0 47:30 1200 2400
S-F 30 1.0 10:00 0 l 4 2.5 1.0 2.0 47:30 1200 2400
7 l-M 31 1.0 10:00 0 1 4 2.5 1.0 2.0 47:30 1200 2400
Z-T 32 1.0 10:00 0 l 5 2.5 1.0 2.0 60:00 1500 3000
3-W 33 1.0 10:00 0 1 5 2.5 1.0 2.0 60:00 1500 3000
4-T 34 1.0 10:00 0 1 5 2.5 1.0 2.0 60:00 1500 3000
S-F 35 1.0 10:00 0 1 5 2.5 1.0 2.0 60:00 1500 3000
8 l-M 36 1.0 10:00 0 l 5 2.5 1.0 2.0 60:00 1500 3000
2=T 37 1.0 12:30 0 l 4 2.5 1.0 2.0 57:30 1500 3000
3-W 38 1.0 12:30 0 1 4 2.5 1.0 2.0 57:30 1500 3000
4-T 39 1.0 12:30 0 1 4 2.5 1.0 2.0 57:30 1500 3000
S-F 40 1.0 12:30 0 1 4 2.5 1.0 2.0 57:30 1500 3000

 

82

This standard program was designed using male rats
of the Sprague-Dawley strain. All animals were between
70 and 170 days-of-age at the beginning of the program.
The duration and intensity of the program were estab-
lished so that 75 per cent of all such animals should
have PSF and PER scores of 75 or higher during the
final two weeks. Alterations in the work time, number
of bouts, or time between bouts can be used to affect
changes in these values. Other strains or ages of
animals could be expected to respond differently to the
program.

All animals should be exposed to a minimum of one
week of voluntary running in a wheel prior to the start
of the program. Failure to provide this adjustment
period will impose a double learning situation on the
animals and will seriously impair the effectiveness of
the training programs.

STANDARD LONG-DURATION, LOW-INTENSITY ENDURANCE
MAINTENANCE PROGRAM FOR POSTPUBERTAL AND ADULT
MALE RATS IN CONTROLLED RUNNING WHEELS

 

Total Total

Acc- Repe- Time Time Exp. Total
eler- WOrk ti- Bet- Run of Revo- Work
ation Time Rest tions No. ween Speed Prog. lu- Time

Time (min: Time per of Bouts Shock (ft/ (min: tions (sec)
(sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TWT

 

1.0 12:30 0 l 2 2.5 1.0 2.0 27:30 750 1500

 

APPENDIX B

83

APPENDIX B.--Femur Lengths (r .002 inches).

84

 

 

 

 

 

 

 

 

 

 

Control Short Long
Duran” Right Left Right Left Right Left
Mean Me an Mean Mean Mean Mean
Odwk Control 1.408 1.411 1.427 1.423 1.423 1.420
1.429 1.418 1.371 1.379 1.435 1.438
1.350 1.360 1.429 1.424 1.361 1.375
1.425 1.429 1.395 1.404 1.450 1.460
1.403 1.404 1.405 1.407 1.417 1.423
8-wk Training 1.556 1.561 1.516 1.520 1.540 1.550
1.567 1.575 1.473 1.475 1.555 1.547
1.575 1.575 1.491 1.495 1.505 1.502
1.535 1.524 1.523 1.525 1.515 1.529
1.558 1.558 1.500 1.503 1.528 1.532
16-wk Training 1.557 1.575 1.557 1.565 1.508 1.502
1.584 1.581 1.590 1.586 1.585 1.575
1.553 1.565 1.607 1.599 1.585 1.590
1.587 1.595 1.559 1.555 1.575 1.582
1.570 1.579 1.578 1.576 1.563 1.562
24-wk Detraining 1.644 1.650 1.621 1.620 1.573 1.575
1.613 1.605 1.653 1.655 1.590 1.583
1.653 1.650 1.665 1.650 1.610 1.617
1.631 1.630 1.611 1.613 1.623 1.637
1.635 1.633 1.637 1.634 1.599 1.603
32-wk Detraining 1.655 1.653 1.627 1.619 1.630 1.618
1.695 1.693 1.616 1.619 1.607 1.610
1.610 1.650 1.660 1.665 1.684 1.690
1.689 1.693 1.503 1.625 1.612 1.630
1.662 1.672 1.601 1.632 1.633 1.637

 

APPENDIX C

85

APPENDIX C.--Femur Weights (i .05 grams).

86

 

 

 

 

 

Control Short Long
Duran” Right Left Right Left Right Left
Mean Mean Mean Me an Mean Mean
O-wk Control 0.96 1.00 1.07 1.04 1.00 1.03
0.99 1.05 1.06 1.06 0.99 1.02
0.98 0 95 1 09 1 10 0 95 0 98
1.03 1.07 1.00 1.00 1.14 1.09
0.99 1.01 1.05 1.05 1.02 1.03
8-wk Training 1.37 1.30 1.23 1.22 1.33 1.37
1.47 1.44 1.21 1.22 1.27 1.34
1 30 1 30 1 14 1 18 1 20 1 20
1.30 1.31 1.28 1.40 1.38 1.30
1.36 1.33 1.21 1.25 1.29 1.30
16-wk Training 1.32 1.33 1.30 1.29 1.38 1.32
1.24 1.25 1.38 1.45 1.52 1.49
l 34 1 42 1 33 1 32 1 35 1.40
1.28 1.29 1.29 1.29 1.29 1.28
1.29 1.32 1.32 1.33 1.38 1.37
24~wk Detraining 1.51 1.53 1.48 1.51 1.39 1.33
1.35 1.35 1.48 1.52 1.37 1.42
1.35 1.46 1.56 1.53 1.34 1.38
1.42 1.42 1.41 1.39 1.46 1.46
1.40 1.44 1.48 1.48 1.39 1.39
32dwk Detraining 1.59 1.54 1.40 1.42 1.46 1.39
1.56 1.58 1.47 1.51 1.52 1.49
1 69 1.67 1.66 1.65 1 62 1.58
1.62 1.62 1.60 1.56 1.46 1.48
1.61 1.60 1.53 1.53 1.51 1.48

 

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