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The short- and long-term effects of methotrexate on the rat skeleton

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Title:
The short- and long-term effects of methotrexate on the rat skeleton
Creator:
Wheeler, Donna L., 1962-
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English
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xii, 174 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Bone density ( jstor )
Bone formation ( jstor )
Bones ( jstor )
Femur ( jstor )
Minerals ( jstor )
Osteoblasts ( jstor )
Osteoclasts ( jstor )
Osteoporosis ( jstor )
Rats ( jstor )
Tibia ( jstor )
Bone resorption ( lcsh )
Cancer -- Chemotherapy -- Complications ( lcsh )
Methotrexate -- Side effects ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 165-172).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Donna L. Wheeler.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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THE SHORT- AND LONG-TERM EFFECTS
OF METHOTREXATE ON THE RAT SKELETON








BY

DONNA L. WHEELER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1993













ACKNOWLEDGMENTS

I would like to thank Dr. Robert E. Vander Griend for suggesting the area

of chemotherapy-induced osteopenia for study, and for his guidance and

encouragement throughout this work. I would like to thank Dr. R. William Petty and

the Department of Orthopaedics for generously supplying the funds for this project.

I am also thankful for the contributions of Dr. Thomas J. Wronski to my mastery of

histomorphometry and understanding of osteoporosis. I am also indebted to Dr.

Gary J. Miller, Dr. James E. Graves, Dr. Scott K. Powers, and Dr. David Lowenthal

for their guidance and instruction, enabling me to develop as a scientist.

I would like to acknowledge the loyal support of my friend and colleague,

Ernest E. Keith. His expertise, instruction, and assistance in the care of laboratory

animals were fundamental to the completion of this project. He also provided

valuable assistance in histomorphometric processing. Special thanks are extended to

Mia Park for her assistance in animal care, tissue processing, data acquisition, data

processing, and data entry.

I am indebted to Dr. Martha Campbell-Thompson and the Department of

Gastroenterology for the use of their microscope and Vidas imaging equipment. I

would also like to thank the Department of Exercise and Sport Sciences for the use

of their dual-energy x-ray absorptiometer and to Lunar Corporation for supplying the

software needed to use this machine.







Finally, I would like to acknowledge the support of Kris Billhardt. Her love,

friendship, inspiration, patience, and editing skills were instrumental in the

completion of this research. I would also like to thank my parents, Jack and Jane

Wheeler, for their undying support and encouragement.














TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... .................................. ii

LIST OF TABLES ...... ....................................... vi

LIST OF FIGURES ..... ......................................... vii

ABSTRACT ......... ....................................... xi

CHAPTERS

1 INTRODUCTION ................................... 1
Bone Remodeling ................ ........ ........ 3
Involutional Bone Loss .......................... 8
Mineral Regulating Mechanisms ................... 11
Mineral Regulating Hormones .. ............ 11
Growth Regulating Hormones ............... 15
Sex Hormones ........................... 16
Exercise ................................ 17
Types of Osteoporosis ........................... 18
Problem Statement ............................. 18
Research Objectives ....................... 19
Hypotheses ............................. 20
Delim stations ............................ 21
Lim stations ............................. 21

2 REVIEW OF THE LITERATURE ...................... 22
Clinical Research .............................. 23
Animal Research .............................. 28
Methods of Skeletal Assessment ................... 30

3 MATERIALS AND METHODOLOGY .................. 33
Anim al Care .................................. 33
Bone Histomorphometry ......................... 35
Cancellous Bone ......................... 35
Cortical Bone ............................ 38
Quantification of Bone Parameters ............ 39









Biomechanical Testing .........
Dual-Energy X-Ray Absorptiometry
Statistical Analysis .............

4 RESULTS .. .....................
Bone Histomorphometry ........
Cancellous Bone ........
Cortical Bone ..........
Biomechanics ................
Dual-Energy X-Ray Absorptiometry

5 DISCUSSION .....................
Summary ...................
Recommendations for Future Work


APPENDICES

A


B

C


D

E

F

G


S

CANCELLOUS BONE FIXATION, DEHYDRATION
AND METHYL METHACRYLATE EMBEDDING ......

MODIFIED VON KOSSA STAIN ....................

CORTICAL BONE FIXATION, DEHYDRATION
AND EMBEDDING IN BIOPLASTIC .................

COMPUTER CODE FOR IMAGE ANALYSIS ..........

DEXA REPEATABILITY STUDY ....................

SAS PROGRAMS FOR STATISTICAL ANALYSIS .......

QUICK REFERENCE FOR ABBREVIATIONS .........


REFERENCES ........ ..............................

BIOGRAPHICAL SKETCH .............................


140


.144


S148

S150

S159

S161

.164


. 41
. 46
. 47

. 48
. 50
. 50
. 66
. 102
. 119

. 124
. 136
. 138


....... 165

....... 173













LIST OF TABLES


Page


Table

1.1

1.2


4.1

4.2

4.3

4.4

4.5

4.6

E.1

G.1


Factors associated with osteoporosis .................... ....... 2

Effects of mineral regulating hormones on
serum calcium and phosphate ............................ 13

Cancellous bone parameters ............................. 52

Femoral cortical bone parameters ................ ...... 70

Tibial cortical bone parameters ........................... 71

Femoral torsional biomechanical parameters ............... 104

Tibial torsional biomechanical parameters .................. 105

Dual-energy x-ray absorptiometry values for BMD ........... 120

Results of DEXA reliability study ........................ 160

Standard Abbreviations ............................... 164














LIST OF FIGURES


Figure Page

1.1 Cancellous bone remodeling .............................. 4

1.2 Cortical bone remodeling ................................ 5

1.3 Involutional bone loss ................................. 10

2.1 Mechanism of action of Methotrexate ...................... 24

3.1 Photograph of femur and tibia with ends
embedded in low melting-point metal ...................... 42

3.2 Graphical depiction of biomechanical parameters ............. .45

4.1 Rat weight changes with time ........................... 49

4.2 Tibial cancellous bone volume ........................... 53

4.3 Tibial cancellous osteoclast surface ........................ 54

4.4 Tibial cancellous longitudinal bone growth .......... ...... .55

4.5 Tibial cancellous mineralizing surface ..................... ..56

4.6 Tibial cancellous mineral apposition rate ................... 57

4.7 Tibial cancellous bone formation rate ..................... .58

4.8 Photomicrograph of baseline cancellous bone volume .......... 59

4.9 Photomicrographs of cancellous bone volume at 30 days ........ 60

4.10 Photomicrographs of cancellous bone volume at 80 days ....... 61

4.11 Photomicrographs of cancellous bone volume at 170 days ....... 62








4.12


4.13


4.14


4.15

4.16

4.17

4.18

4.19

4.20

4.21

4.22

4.23

4.24

4.25

4.26

4.27

4.28

4.29

4.30

4.31


Photomicrographs of fluorescent labels on
cancellous bone surfaces at 30 days ....................... 63

Photomicrographs of fluorescent labels on
cancellous bone surfaces at 80 days ....................... 64

Photomicrographs of fluorescent labels on
cancellous bone surfaces at 170 days ........................ ..65

Femoral total bone tissue area ..... ..................... 72

Tibial total bone tissue area ............................ 73

Femoral marrow area .................................. 74

Tibial marrow area ................................... 75

Femoral cortical bone area .............................. 76

Tibial cortical bone area ............................... 77

Femoral mean cortical bone width ........................ 78

Tibial mean cortical bone width .......................... 79

Femoral polar moment of inertia ......................... 80

Tibial polar moment of inertia ........................... 81

Femoral periosteal mineralizing surface .................... 82

Tibial periosteal mineralizing surface ................... ... 83

Femoral periosteal mineral apposition rate ................. .84

Tibial periosteal mineral apposition rate .................... 85

Femoral periosteal bone formation rate ................... .86

Tibial periosteal bone formation rate ...................... 87

Photomicrograph of the femoral cross-section
of the baseline control animal ........................... 88








Photomicrograph of the tibial cross-section
of the baseline control animal ........................... 89


4.32


4.33

4.34

4.35

4.36

4.37

4.38

4.39

4.40

4.41

4.42

4.43

4.44

4.45

4.46

4.47

4.48

4.49

4.50

4.51

4.52

4.53


of femoral cross-sections at 30 days


of tibial cross-sections at

of femoral cross-sections

of tibial cross-sections at

of femoral cross-sections


30 days ...

at 80 days .

80 days .

at 170 days


Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs

Photomicrographs


of femoral periosteal surface

of tibial periosteal surface at

of femoral periosteal surface

of tibial periosteal surface at

of femoral periosteal surface


Photomicrographs of tibial periosteal surface at


at 30 day

30 days

at 80 day

80 days

at 170 da

170 days


........ 90

........ 91

........ 92

........ 93

........ 94

. ...... 95

's ...... 96

........ 97

,s ...... 98

........ 99

iys .... 100

...... 101


Photograph of a typical fracture pattern following torsional test

Femoral breaking torque ..............................

Tibial breaking torque ................................

Femoral twist angle at failure ...........................

Tibial twist angle at failure .............................

Femoral energy absorbed at failure ......................

Tibial energy absorbed at failure ........................

Femoral torsional stiffness .............................

Tibial torsional stiffness ...............................


106

107

108

109

110

111

112

113

114


of tibial cross-sections at 170 days








4.54 Femoral torsional strength ............................. 115

4.55 Tibial torsional strength ............................... 116

4.56 Femoral polar moment of inertia associated with torsional fracture 117

4.57 Tibial polar moment of inertia associated with torsional fracture 118

4.58 Femoral bone mineral density .......................... 121

4.59 Tibial bone mineral density ............................. 122

4.60 Vertebral bone mineral density ......................... 123













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

The Short- and Long-term Effects of Methotrexate on the Rat Skeleton

By

Donna L. Wheeler

December 1993



Chairman: James E. Graves, Ph.D.
Major Department: Health and Human Performance


The chemotherapy drug, Methotrexate (MTX), has been shown to decrease

bone mass and increase the incidence of bone fractures. Existing studies measure

bone parameters at one point in time following chemotherapy and do not monitor

the bone's recovery. This study's purpose was to use a rat model to determine the

long-term effects of MTX on bone volume, turnover, mineralization, density, and

strength.

Sprague-Dawley rats were randomly assigned to either control or MTX

groups. Daily MTX and saline injections were given for two five-day courses to drug

treatment and control groups, respectively. Fluorochrome compounds were injected

prior to sacrifice to monitor actively mineralizing bone surfaces. One control and

MTX group were sacrificed at 30, 80, and 170 days following treatment initiation.

xi







Both femurs, both tibias, and 2 lumbar vertebrae were harvested for cancellous and

cortical bone histomorphometry, biomechanics (torsion), and densitometry (DEXA).

Standard cancellous and cortical histomorphometric parameters were measured from

undecalcified, methyl methacrylate embedded sections from the right proximal tibia

and tibial and femoral diaphyses. The contralateral femur and tibia were torsionally

loaded to failure and standard mechanical parameters were measured. Excised

bones were scanned using DEXA to measure bone density. All bone responses were

statistically analyzed using a 2-way ANOVA followed by Duncan's multiple

comparison procedure (significance: p=0.05).

Cancellous bone volume was significantly lower in the MTX treated group at

80 and 170 days. Cancellous mineralizing surface and longitudinal bone growth were

significantly depressed at all time points yet mineral apposition rate was only

depressed at the 170 day point. Cancellous osteoclast surface was increased at all

time points for the MTX treated animals. Cortical cross-sectional area and

periosteal mineral apposition rate were significantly lower for both the femur and

tibia in the MTX groups at all time points. However, periosteal mineralizing surface

was significantly depressed in the MTX group only for the femur. MTX had minimal

effects on the biomechanical parameters and bone density measurements.

Methotrexate had long-term negative effects on both cortical and cancellous

bone. The drug decreased bone volume, decreased bone formation, and decreased

osteoblast activity.













CHAPTER 1
INTRODUCTION


The disease osteoporosis results from a decrease in bone mass and bone

strength leading to an increase in the incidence of bone fractures. Osteoporosis is

an enormous public health problem which is responsible for over a million bone

fractures in the U.S. each year (61). The most common sites of osteoporotic

fractures are the vertebrae, hip, and distal forearm. One-third of all women over 65

will have vertebral fractures and, by the eighth decade of life, one of every three

women and one of every six men will have had a hip fracture (61). The direct and

indirect costs of osteoporosis are estimated to be over $6.1 billion annually in the

United States.

Peak bone mass is achieved by the third decade of life and then slowly

declines from this point. The magnitude of peak bone mass as well as the rate at

which bone is lost contribute to the development of osteoporosis. Many factors have

been shown to be associated with osteoporosis with the most important being age,

gender, race, and hormonal status. Osteoporosis is most commonly seen in

postmenopausal white women, Asian women, and older men (60). Other factors

such as genetics, lifestyle, nutritional factors, medical disorders, or drugs have been

shown to significantly affect bone health. A summary of osteoporosis risk factors are

presented in Table 1.1.









Table 1.1 Factors Associated with Osteoporosis


Genetic


Lifestyle


White or Asian ethnicity
Female gender
Positive family history
Small body frame (<127 lbs)


Smoking
Inactivity
Nulliparity
Excessive exercise (producing amenorrhea)
Early natural menopause
Late menarche


Nutritional Factors Milk intolerance
Low calcium intake
Vegetarianism
Excessive alcohol intake
Caffeine
Consistently high protein intake

Medical Disorders Anorexia nervosa
Thyrotoxicosis
Parathyroid overactivity
Cushing's syndrome
Type I diabetes
Abnormal gastrointestinal function
Abnormal hepatobiliary function
Occult osteogenesis imperfecta
Mastocytosis
Rheumatoid arthritis
Prolactinoma
Hemolytic anemia
Drugs Thyroid replacement therapy
Glucocorticoid drugs
Anticoagulants
Chronic lithium therapy
Chemotherapy (Methotrexate)
GnRH agonist or antagonist therapy
Anticonvulsants







3

The following sections will discuss bone remodeling, involutional bone loss,

physiological mineral regulating mechanisms, types of osteoporosis, and will conclude

with the development of the problem being studied.



Bone Remodeling

Throughout the body, bone is continually being remodeled, where bone is

resorbed and replaced by new bone. In order to maintain bone mass it is essential

that this remodeling process remain balanced. The amount of bone resorbed should

be replaced by an equal or greater amount of new bone or there will be a net loss

of bone mass. If this imbalance remains uncorrected this condition can lead to

osteoporosis. This section will describe the normal bone remodeling sequence and

discuss problems which might affect the normal cycle.

Figure 1.1 and Figure 1.2 describe the normal sequence of cancellous and

cortical bone remodeling, respectively. Remodeling occurs in a programmed

sequence at discrete bone foci called bone remodeling units (BMU) (27). The steps

of bone remodeling can be described as ACTIVATION, RESORPTION, and

FORMATION and are similar for cancellous and cortical bone.

At the beginning of each cancellous remodeling cycle (Figure 1.1), activation

occurs at previously inactive bone surfaces. These surfaces are covered with bone

lining cells, presumably of osteoblastic lineage. These lining cells respond to bone-

resorbing hormones and expose the bone surface to osteoclast progenitor cells.

Osteoclast progenitors then replicate and differentiate into mature osteoclasts. The













(a)



I

I
~^ ^jT~
1-s


i


- p


Cancellous bone remodeling
Oc=osteoclast; Opc=osteoprogenitor cells;
Ob=osteoblast; BLC=bone lining cells
(a) Resorption is completed before formation
(b) Resorption followed closely by formation
(Figure adapted from W.S.S.Jee (37))


Ob

/,


BLC


Figure 1.1















C3


CO




U m


a 0



o0





a o1
U
E-





S0
IUJ



to
a


a




a II ||
C ciii
cB o.







M 0
E mewU 4




S ZII II








I






6

osteoclasts secrete lysosomal enzymes that degrade the collagen matrix and hydrogen

ions which dissolve the bone mineral. After the osteoclasts resorb the bone there is

a reversal phase in which macrophages may appear to either complete resorption or

initiate the next stage of remodeling, the formation phase. During formation,

osteoblasts replace the bone that was resorbed by the osteoclasts. For successful

bone formation the osteoblasts must be able to adequately replicate, differentiate,

and produce normal bone matrix. Since osteoblasts lay down bone on existing bone

spicules, it is important for there to be a template of unresorbed bone in the

resorption cavity on which the osteoblasts may build the new bone matrix.

Cortical bone remodeling is similar, in principle, to cancellous remodeling.

The osteoclasts first resorb a canal or tunnel which is then refilled centripetally by

osteoblastic apposition, forming a new osteon or secondary Haversian system (Figure

1.2).

The completion of the osteoclastic resorption phase in humans requires

approximately 21 days for cancellous bone and 24 days for cortical bone. The

osteoblasts then create a new structural bone unit in approximately 91 days and 124

days for cancellous and cortical bone, respectively. Therefore, the completion of a

cancellous BMU requires 112 days, and a cortical BMU requires 147 days (39).

In normal young adults, the resorption and formation phases are tightly

coupled and bone mass is maintained. However, there is a natural bone loss with

age which implies an uncoupling or imbalance in the phases of bone remodeling, with

an increase in resorption over formation. The slow age-dependent phase of bone








7

loss results mainly from impaired bone formation, where the osteoclasts create

resorption cavities of normal depth but the osteoblasts fail to refill them completely

(21,56). In other words, age-related bone loss occurs due to reduction in the rate of

bone formation with no change in the rate of bone resorption (39). Over time, the

uncoupled remodeling cycles can cause significant thinning of spicules of cancellous

bone until the osteoclastic resorption cavities penetrate the thickness of bone. These

resorptive perforations create discontinuities in the cancellous scaffold, resulting in

loss of mechanical integrity and strength of the bone.

Increased bone turnover (activation of new BMUs) can also result in net bone

loss. Postmenopausal accelerated bone loss is associated with a high rate of bone

turnover (80), where more osteoclasts are present and these cells create deeper

resorption cavities (21,56). Although there is more bone being played down due to

the increased bone turnover, there is even greater bone resorption (38).

Drug induced osteoporosis may affect the normal remodeling cycle in a variety

of ways depending on the cellular actions of the drug. The drug can either increase

or decrease the number of osteoblasts or osteoclasts or affect the quality and quantity

of bone being played down by osteoblasts. Cytotoxic drugs may also affect bone

metabolism indirectly by inducing gonadal damage, thus decreasing the levels of

circulating sex hormones. The mechanism of drug-induced bone loss is typically due

to diminished osteoblastic number, activity, or quality of bone.

The focus of this study is on the chemotherapy drug, Methotrexate, which

interferes with the replication of DNA within the cells. Faulty DNA replication








8

would decrease the number of osteoblasts and osteoclasts and would, therefore,

decrease the overall bone formation and resorption. Based on Methotrexate's

mechanism of action, this drug would decrease the surface of bone lined by

osteoblasts and osteoclasts and reduce the overall bone turnover.



Involutional Bone Loss

Both men and women experience age-related bone loss. Bone mass continues

to increase until approximately the age of 30, then after a transient period of

stability, age-related bone loss begins. Women lose approximately 35% of their

cortical bone mass and 50% of cancellous bone mass over their lifetime, whereas

men lose only 23% and 33% of their cortical and cancellous bone, respectively (50).

Bone loss occurs in a biphasic pattern for both cortical and cancellous bone in

women; with a sustained slow phase that occurs in both sexes and a transient

accelerated phase that occurs in women after menopause.

For cortical bone, bone is lost at approximately 0.3 to 0.5 %/year and

increases with aging until it levels out late in life (50). For women, an accelerated

postmenopausal phase of cortical bone loss is superimposed on the slow phase to

increase bone loss to a rate of 2 to 3 %/year immediately after menopause. This

accelerated bone loss decreases exponentially to baseline slow phase levels after

about 8 to 10 years (50).

Cancellous bone loss starts to occur earlier in life than cortical bone loss.

Controversy exists as to the rate of cancellous bone loss. Some researchers claim a








9

linear decrease of 0.6 %/year (61), while others predict a curvilinear decrease of

2.4 %/year in women (11) and a linear 1.2 %/year for men (51). Because cancellous

bone has a greater surface area than cortical bone, it is more metabolically active

and, therefore, more responsive to changes in external loading (exercise), mineral,

hormonal, and metabolic status. The effect of hormonal status is reflected in the

earlier onset of bone loss for cancellous bone and the more rapid rate of cancellous

bone loss following menopause compared to cortical bone. This accelerated

cancellous bone loss, however, is shorter in duration than the accelerated

postmenopausal phase of cortical bone loss (61). In addition to involutional (age-

related) bone loss, drugs and diseases can exacerbate the bone loss by either

increasing the rate of bone loss, diminishing peak bone mass or both. In general, the

development of osteoporosis is related to two factors: (1) level of peak bone mass,

and (2) the rate of loss of bone after achieving peak bone mass.

Figure 1.3 graphically represents natural bone loss in humans. As discussed

above, peak bone mass occurs at the approximate age of 30 and slowly declines at

a constant rate due to age-associated changes. In women, an accelerated bone loss

occurs at menopause where the contributions of age and hormonal status combine.

Following this accelerated phase, bone loss resumes a slow decline until late in life,

while bone mass appears to stabilize at an osteopenic level. If a disease requires

drug therapy such as glucocorticoids, anticonvulsants, or chemotherapy (4,7,24,30,61),

the age-related bone loss is confounded by drug induced bone loss. As can be seen

in Figure 1.3, the bone fracture threshold is also an important consideration in the



















T 1


5 35 45 55 65 75 85


Age (years)


-Males -- Females


Figure 1.3


Involutional bone loss
(curves based on 1.2%/year)


menopause


Fx threshold


1.1


0.9 -


m
t0.7



0.5



0.3
2








11

bone density spectrum. This threshold represents the level of bone density at which

a person's statistical risk for osteoporotic fractures increases. As depicted in Figure

1.3, the lower the peak bone mass and the greater the rate of bone loss the sooner

a person will reach this fracture threshold.



Mineral Regulating Mechanisms

Endocrine status, endogenous mineral balance, and mechanical loading

regulate both acquisition of peak bone mass and rates of bone loss and gain. The

following sections will discuss the effects of systemic mineral-regulating hormones,

growth-regulating hormones, sex hormones, and exercise on bone.



Mineral-Regulating Hormones

Bone is the reservoir for 99% of the body's calcium. Therefore, hormones

which regulate calcium homeostasis are extremely important for the regulation of

bone mass. In addition to the hormones responsible for calcium metabolism,

adequate dietary intake is essential for healthy bones. Only 30% of ingested calcium

is absorbed into the system; therefore, the consumption of at least 800 mg of calcium

a day to prevent the bone reserve of calcium from being depleted is very important.

With age, less calcium is absorbed, and the daily intake of calcium should be

increased in order to maintain calcium homeostatis and healthy bones. Calcium is

the most common nutritional supplement given to postmenopausal and osteoporotic

patients. Studies continue to show, however, that calcium is not effective by itself in








12

preventing postmenopausal bone loss or reversing osteoporosis. Calcium intake may

be more important during the period of growth and development to allow

maximization of peak bone mass (37).

The goal of mineral-regulating hormones is to maintain physiologic serum

calcium, magnesium, and phosphate levels. The three main mineral-regulating

hormones are parathyroid hormone (PTH), calcitonin (CT) and 1,25dihyroxyvitamin

D (calcitriol or 1,25D). These hormones act on three target tissues: bone, intestine,

and kidney. Table 1.2 supplies a summary of the actions of these mineral-regulating

hormones in the regulation of serum calcium (Ca) and phosphate (Pi) at the different

target tissues. Magnesium is not included in this table; its regulation by these

hormones is either nonexistent or inadequately defined. The role of calcitonin in

mineral regulation is modest compared to the effects of PTH and 1,25D.

Vitamin D is either obtained through dietary foodstuffs or absorbed from the

sun. In both cases this form of "raw"vitamin D is not active, and a series of enzymes

in the liver and kidney must manufacture the active form, 1,25D. PTH is secreted

by the parathyroid gland in response to low levels of serum calcium and 1,25D.

Calcitonin is secreted by the thyroid gland in response to hypercalcemia. The actions

and interaction of these hormones maintain mineral homeostasis.

Ca and Pi enter the blood through the intestine, leave it through the kidney,

and are stored in the bone. In order to maintain homeostasis, the net absorption of

Ca and Pi in the intestine must be equal to the net excretion of these ions by the

kidney. The absorption of Ca and Pi depends on the dietary intake and the










Table 1.2
Effects of Mineral-Regulating Hormones on Serum Calcium and Phosphate


Bone Gut Kidney NET
Ca Pi Ca Pi Ca Pi Ca Pi

PTH + 4' 4 ,
CT 4, 4, *, ,- 4 ,
1,25D t + +

KEY: PTH = parathyroid hormone
CT = calcitonin
1,25D = 1,25-dihydroxyvitamin D
Ca = calcium
Pi = phosphate
t = increase in serum mineral level when hormone acts on target tissue
= decrease in serum mineral level when hormone acts on target tissue
= no effect in serum mineral levels when hormone acts on target tissue
(Figure adapted from D.D. Bikle (6))








14

efficiency of absorption. Glomerular filtration of these ions in the kidney is relatively

constant, so the kidney reabsorbs Ca and Pi relative to the body's needs. Bone

provides the major buffer for maintaining constant levels of Ca and Pi in the blood.

This is achieved by balancing bone formation (which deposits these ions in bone)

with bone resorption (which releases these ions into the bloodstream). PTH, 1,25D,

and calcitonin act on the intestine, kidney, and skeleton and maintain mineral

balance. 1,25D has the positive effect of increasing serum Ca and Pi levels by

increasing absorption in the intestines and increasing reabsorption in the kidney.

However, 1,25D in combination with PTH mobilizes Ca and Pi from bone. The net

effect of 1,25D is to increase both serum Ca and Pi.

PTH regulates serum Ca and Pi by stimulating the release of these ions from

the skeletal reservior and by stimulating Ca reabsorption and inhibiting Pi

reabsorption in the kidney. PTH also affects 1,25D levels by stimulating its renal

synthesis which, in turn, leads to increased intestinal absorption of Ca and Pi. The

net effect of PTH is to increase serum Ca and decrease serum Pi. PTH levels have

been shown to increase with age, which may partly explain age-related bone loss (45).

Chronically high PTH level (hyperparathyroidism) increases the number and activity

of osteoclasts (6). However, animal studies have shown that intermittent (pulsatile)

doses of PTH have the opposite effect, stimulating bone formation and resulting in

significant increases in cancellous and cortical bone (85,86). Human studies have

also confirmed that pulsatile PTH administration increases bone mineral density in








15

postmenopausal women and proves to be a promising prevention and treatment for

osteoporosis (17,47).

Calcitonin, although of less importance to mineral homeostasis than PTH and

1,25D, is a potent inhibitor of bone resorption by decreasing the activity and number

of osteoclasts. The secretion of CT is regulated by serum calcium levels. High

serum Ca stimulates CT secretion, which decreases the release of Ca to the blood

by bone resorption. Calcitonin is currently used for the treatment of osteoporosis.

Treatment of high turnover osteoporosis (postmenopausal) with calcitonin has been

shown to increase bone mass but its effects are less consistent and sustained than

estrogen therapy (60).

1,25D, PTH, and calcitonin act on the skeleton, kidney, and intestines to

regulate blood levels of Ca and Pi. Regulation entails control of how much comes

into the body from the diet, how much leaves the body through the kidney, and how

much is stored and released from the bone. The different hormones, ions, and target

tissues involved communicate and interact to ensure the precise regulation of these

important minerals.



Growth-regulating Hormones

Several systemic hormones associated with growth are important in the

development of peak bone mass and strength. These hormones include growth

hormone, glucocorticoids, thyroid hormone, and insulin. Growth hormone is a very

important determinant of skeletal mass and acts through changes in the production








16

of insulin-like growth factor 1 (IGF-1), which is a potent stimulator of skeletal growth

(10,60). Glucocorticoids have complex effects on bone metabolism. Excess

glucocorticoids produce bone loss primarily through suppression of osteoblast

function. They also inhibit intestinal calcium absorption leading to secondary

hyperparathyroidism and increased bone resorption. Glucocorticoids also promote

bone loss by inhibiting renal calcium reabsorption which may contribute to increased

PTH secretion (34). Thyroid hormones increase bone turnover but tend to stimulate

resorption more than formation, resulting in net bone loss. Insulin has been shown

to stimulate osteoblastic collagen synthesis at physiological concentrations and may

also affect calcium transport and vitamin D metabolism (60).



Sex Hormones

It has been well established that estrogen deficiency leads to the development

of osteoporosis. Accelerated bone loss has been noted in postmenopausal women

or after oophorectomy (43,54,79-82,84,85), as well as in ammenorrheic women

(19,57). Estrogen replacement therapy has been helpful in preserving bone mass

under estrogen deficient conditions in humans (18,36) and animals (29,80). A

similar link to osteoporosis has been noted in androgen deficient men (22). Upon

withdrawal of estrogen or androgen, bone turnover is increased, where bone

resorption is greater than bone formation, resulting in a net loss of bone mass.









Exercise

Another important factor promoting bone health is the presence of

mechanical stimulation which provides impetus for modeling, remodeling, and

mineralization (12,26,63-65,75). Weightbearing exercise, therefore, becomes an

important modality to preserve or increase bone mass. Weight-bearing exercise has

been shown to augment bone mass in athletes (41,46,49,78)and maintain or improve

bone mass in aging individuals and postmenopausal women (1,14,32,72). In fact, men

and women subjected to high intensity physical training have been shown to increase

their bone mineral content (BMC) and bone mineral density (BMD) between 5 and

20% when compared to age-matched sedentary controls (46,49). Some studies have

shown that exercise tends to be site specific, augmenting bone mass only in the

specific bones loaded during the exercise routine (41,78). Other researchers (32)

have found an increase in BMC in both appendicular and axial skeleton with a

combined exercise program which incorporated both weight-bearing aerobic exercise

and strength training.



As described in the previous paragraphs, bone homeostasis is affected by many

factors including natural aging, nutritional state, hormonal status, and activity level.

When the equilibrium is upset by alterations in body function due to disease, surgery,

or drug therapy, drastic changes in bone mass can occur.








18

Types of Osteoporosis

Osteoporosis can be classified into three types: (1) Type I or Postmenopausal,

(2) Type II or Senile, and (3) Type III or Therapy Induced (5). Type I and type II

osteoporosis result from either lack of estrogen at menopause (women) or from

natural aging (men and women), respectively. Type I osteoporosis affects mainly

cancellous bone with the most common fracture sites being the vertebrae (crush

fractures) and the distal radius. Type II osteoporosis affects both cancellous and

cortical bone with the most common fracture sites being the vertebrae (multiple

wedge fractures) and hip. Type III osteoporosis can result from chronic

administration of pharmacological agents which affect circulating levels of

parathyroid hormone, vitamin D metabolites, osteocalcin, calcium absorption, and

renal conservation of calcium. Type III osteoporosis can also occur from short- or

long-term treatment of cytotoxic (chemotherapy) drugs such as Adriamycin or

Methotrexate (4,31,52,59,73).





Problem Statement

Drugs are given to cure disease or improve the function of organ systems. In

the case of cancer, chemotherapy drugs are used to kill actively growing cells. While

cancer cells are usually actively growing and, therefore, affected by the

chemotherapy, the drugs do not discriminate between neoplastic and normal rapidly

growing cells. Chemotherapy, therefore, can have deleterious effects on non-targeted








19

tissue such as epithelial tissue (i.e. hair follicles, lining of the gut), hematopoietic

marrow cells, and bone cells, resulting in hair loss, gastrointestinal distress,

immunosuppression, and osteoporosis.

Osteoporosis is a silent disease, since the health of the skeleton is not

outwardly apparent and the symptoms are minimal until catastrophic fracture.

Chemotherapy may attend to the more obvious disease, cancer, while creating other

diseases in the process. The focus of this work is on the secondary disease,

osteoporosis, resulting from administration of the chemotherapy drug, Methotrexate.

This project will explore the pathophysiology and severity of Methotrexate-induced

osteoporosis using an animal model. The recovery of bone after withdrawal of drug

treatment will also be investigated. Histomorphometry, biomechanics, and dual-

energy x-ray absorptiometry will be used to asess bone quantity and quality.



Research Objectives

MTX has been shown to have immediate adverse effects on bone turnover

and fracture healing using a rat model (23,24,35,74). However, administration of

more than one course of MTX and long-term bone recovery have not been

investigated. Therefore, the objectives of this study are outlined below:

(1) To study the short- and long-term effects of MTX on the skeleton by using 3

month old male Sprague-Dawley rats.








20

(2) To quantify the effects of MTX on bone characteristics at different times after

withdrawal of MTX treatment using histomorphometric, biomechanical, and

radiological methods.

(3) To verify the pathophysiology of MTX-associated osteoporosis proposed by

others.

(4) To propose changes in the existing chemotherapy protocols to prevent or treat

secondary osteoporosis.



Hypotheses

The Sprague Dawley rat will be used to test the following hypotheses:

(1) Two courses of Methotrexate will induce osteopenia and depress bone

formation 30 days following treatment as reported by others after one course

of Methotrexate (24).

(2) Osteoblast and osteoclast function will return to normal (age-matched control

levels) after the drug is cleared from the tissues at approximately 170 days.

(3) Although cellular function will be restored after 170 days, the osteopenia

resulting from Methotrexate influence will persist. Methotrexate will,

therefore, cause sustained loss of cancellous and cortical bone, bone mineral

density, and mechanical strength.









Delimitations

The following demarcations are recognized in this study:

(1) Only male Sprague-Dawley rats were used in this study.

(2) Two 5-day courses of Methotrexate chemotherapy were given to the rats.

(3) The quantity and quality of the skeleton was examined at three points in time

following completion of chemotherapy: 30, 80, and 170 days.



Limitations

The following weaknesses are acknowledged in this study:

(1) Animals were studied instead of humans.

(2) Longitudinal measures of bone quality and quantity were not made due to the

invasive nature of some of these measurements.

(3) Complete clearance of methotrexate from bone was not achieved at 170 days,

therefore, recovery was not fully recognized.













CHAPTER 2
REVIEW OF THE LITERATURE


Cancer is a far-reaching disease with often tragic outcomes. Cancer cells can

arise in any body tissue, at any age. Cancer cells can invade local tissues by direct

extension or they can spread throughout the body by way of lymphatic or vascular

channels. The size and scope of cancer disease is overwhelming. Approximately 56

million Americans will be diagnosed with cancer -- this is approximately 1 in 4.

These statistics make it extremely likely that each of us will face the disease at some

point in our life, either personally or through the care of a loved one. Although two

thirds of those diagnosed with cancer will die of their disease, the percentages of

survivors are increasing due to improved efficacy of adjuvant therapies such as

radiation therapy and chemotherapy.

Chemotherapy agents, although often effective in destroying and controlling

neoplastic cell growth, have many adverse side effects on normal tissue and body

function. Many drugs cause nausea, vomiting, alopecia, cardiotoxicity, anorexia,

myelosuppression, renal damage, liver toxicity, and osteopenia. The primary interest

of this investigation concerns the chemotherapy drug Methotrexate and the adverse

effects of this drug on the skeleton.

Methotrexate (MTX) is a common antineoplastic agent used to treat acute

lymphoblastic leukemia, choriocarcinoma in women, breast carcinomas, testicular








23

carcinomas, head and neck carcinomas, osteosarcomas, chondrosarcoma,

fibrosarcoma, liposarcoma, lymphosarcoma, Hodgkin's disease, lung cancer

(squamous and small cell types), and, at lower doses, severe psoriasis and rheumatoid

arthritis. MTX is classified as an antimetabolite and is a folic acid analog. It

competitively inhibits the enzyme dihydrofolate reductase. This enzyme, involved in

protein synthesis, catalyzes a reaction to convert nucleic acids to DNA (see Figure

2.1). Therefore, MTX inhibits DNA synthesis by depleting the cell of the DNA

building blocks. MTX is carried by the blood and about 90% is filtered out of the

bloodstream within 48 hours. However, a large percentage of the drug remains in

the tissues where it was captured and its effects are very long-lasting and often

considered irreversible, especially with chronic MTX therapy (35).

MTX has been shown to have an adverse effect on the skeleton in both

humans and animals. The following sections will detail existing research in this area,

both clinical and animal. Methods to quantify bone changes will be reviewed.



Clinical Research

Nesbit et al. (52) studied children with acute lymphocytic leukemia who were

treated with high doses of MTX. The most apparent toxicity with these children

were associated with pulmonary inflammation (pneumonia) as well as inflammation

of bladder, vaginal, and pleural epithelium. Ulceration of the gastrointestinal tract

was also noted. Chronic administration of MTX resulted in liver cirrhosis and

osseous changes. MTX associated hepatotoxicity (16) and osteoporosis (59) has also





























DIHYDROFOLIC ACID


FOLIC ACID


DIHYDROFOLATE
REDUCTASE


-I


TETRAHYDROFOLIC ACID


I

PURINE SYNTHESIS
THYMIDYLIC ACID SYNTHESIS


On


METHOTREXATE


Figure 2.1 Mechanism of action of Methotrexate


N N


OH N


CN,
CONN -CN
COON


N N




N* N
NH l N


C-n- CONH CH
Co
CH


DNA


I








25

been reported by others. Approximately 20% of Nesbit's patients receiving chronic

MTX treatment experienced bone pain and/or fractures associated with osteopenia;

the researchers attribute these osseous changes to MTX induced abnormalities in

calcium metabolism hypercalcemiaa).

Atkinson et al. (4) studied children with acute lymphoblastic leukemia in

attempts to elucidate the physiologic mechanism leading to osteoporosis. It was

uncertain if the cancer itself was affecting the bone mineral or if the chemotherapy

used to treat the disease was affecting the gastrointestinal and renal handling of

nutrients, causing alterations in mineral homeostasis and leading to abnormal

turnover of bone mineral and osteoporosis. The chemotherapy protocols in this study

used a combination of the following drugs over a 24 month period: prednisone,

vincristine, L-asparaginase, methotrexate, 6-mercaptopurine, and doxorubicin. This

study provided evidence that the chemotherapy protocol mentioned above caused an

imbalance in the mineral homeostasis including hypomagnesium, hypocalcemia, and

hypoparathyroidism. These abnormal calcium and magnesium levels, caused by

chemotherapy, indirectly altered bone turnover.

Ragab et al. (59) reported on 11 children with acute lymphoblastic leukemia

being treated with MTX therapy for more than 6 months who developed severe bone

pain and/or fractures in their lower extremities. These children were diagnosed

radiographically with osteopenia. Four of the patients were withdrawn from MTX

therapy. Serial radiographs indicated improvements in bone density after 6 drug-free

months. In contrast to Nesbit's study, these children did not experience MTX-







26

associated hypercalcemia. Ragab postulated that MTX alters either protein

metabolism and/or bone cell activity to induce osteopenia and speculated that these

changes are reversible.

Similar results were noted by Stanisavljevic and Babcock (73) in their review

of 37 children treated for leukemia with MTX. A high incidence of bone fractures

and bone pain were noted in these children. Fracture healing was delayed and non-

unions were common. Those children taken off of MTX went on to achieve normal

fracture repair. Stanisavljevic and Babcock (73) surmised MTX inhibits osteogenesis;

however, this effect appears to be reversible. MTX binds to dihydrofolate reductase

months after a single MTX dose, yet it has been postulated that the free unbound

intra-cellular MTX inhibits DNA synthesis (13). If this is true, the cells affected by

MTX therapy would be capable of resuming normal function after MTX withdrawal.

Gnudi et al. (31) studied the bone mineral content of 59 osteosarcoma

patients treated with different doses of MTX using single photon absorptiometry.

They analyzed the radius at the mid-shaft (primarily cortical bone) and the distal

metaphysis (rich in cancellous bone). These researchers found a dose-dependent

reduction in bone mineral content at the cancellous-rich distal radius with higher

doses of MTX having a more detrimental effect. They concluded that high doses of

MTX or low doses over long periods of time may severely compromise bone mass

and strength. Long-term follow-up bone mineral content measurements after

withdrawal of MTX treatment were not reported for these patients.








27

Clinical research (4,52,73) has verified the detrimental effects of

chemotherapy, specifically MTX, on bone through documentation of fracture

incidence. More quantitative evidence of MTX's effect on bone has been presented

in a recent study using single photon absorptiometry showing a reduction in bone

mineral content with MTX treatment (31). The mechanism of action of MTX on

bone and the permanancy on the osteoporotic effects, however, are unknown. Some

researchers (4,52) speculate the high incidence of fractures in MTX patients is due

to the drug's affect on mineral homeostasis through either alterations in protein

metabolism or cellular activity. Other possible explanations for the high fracture

incidence associated with cancer and MTX treatment include (1) the neoplastic

disease itself weakening the bone (not related to MTX treatment); (2) inflammation

reaction due to MTX-associated cell necrosis affecting the bone matrix; (3)

malnutrition associated with abnormal absorption of nutrients in the intestines caused

by MTX; (4) decreased physical activity due to illness; or (5) a direct relationship to

the cellular alterations in osteoblasts and osteoclasts caused by MTX treatment.

Most clinical researchers speculate the osteoporotic effects are transitory

(59,73); however, they base this theory soley on fracture incidence and observations

of fracture repair. No longitudinal quantitative clinical studies have been conducted

to investigate the bone's response over time to MTX treatment. It is uncertain if

bone recovers after chronic chemotherapy. If bone density remains depressed after

withdrawal of chemotherapy, this would decrease the peak bone mass the patient

would accrue in their lifetime, making them at risk for osteoporotic fracture







28

throughout their life. In order to further investigate the pathophysiology and the

time course of MTX-associated osteoporosis, animal models have been used to

facilitate quantification of bone characteristics through histomorphometry and

destructive biomechanical tests.



Animal Research

An abstract by Tross et al. (74) was the first to report the effects of the

chemotherapy drugs, Methotrexate (MTX) and Adriamycin (ADR), on bone turnover

and strength using a rat model. One 5-day course of daily chemotherapy injections

were given intraperitoneally, the animals were given fluorochrome label on day 7 and

13, and sacrificed on day 14 for histomorphometric and biomechanical assessment

of bone morphometry and strength. No changes in the torsional biomechanical

strength, stiffness, and energy absorbed at failure were noted for the drug treated

rats. However, significant decreases were noted in cancellous bone volume, osteoid

surface, and osteoblast surface in the drug treated animals compared to controls.

MTX and ADR treatment was not reported to affect osteoclast surface or mean

cortical thickness.

Freidlaender et al. (24) expanded Tross's previous work into manuscript form.

As outlined in the previous paragraph, the researchers administered one 5 day course

of MTX and ADR to rats and, using quantitative histomorphometric techniques,

measured the bone's response 2 weeks after initiation of the protocol. These

researchers found significant reductions in cancellous bone volume and bone








29

formation rate during this short-term study. This confirms the osteotoxic effects of

MTX and ADR immediately following acute administration, but does not determine

the ability of bone to recover from the drug insult.

Freidlaender's group (23) continued their research into the skeletal effects of

MTX by evaluating fracture healing. In this study Sprague Dawley rats received a

transverse fracture of the femur using a bone saw. The fracture was fixed internally

with an intramedullary K-wire. Rats were divided into 3 groups: (1) control rats

which received no treatment following fracture fixation; (2) MTX treated rats which

received one 5-day course of MTX injections; and (3) Radiation treated rats which

were irradiated with 250 rad fractions for 10 days following surgery. Groups of rats

in each of the 3 treatment groups were sacrificed at 1,2, 4, 8, and 12 weeks following

surgical fixation. Following sacrifice, bone harvest, and embedding procedures,

longitudinal sections were made through the callus. Callus formation was graded

based on the amount of repair present. Radiation and MTX treatments retarded

callus formation at all time intervals when compared to the control animals. MTX

treated animals also failed to regain femoral torsional strength following fracture

even at extended time periods (24 weeks). Similar results were also reported by

Hajj et al. (35) using a similar model where both bending strength and histological

grade of callus formation were significantly compromised in animals receiving weekly

injections of MTX compared to control animals. Burchardt et al. (8) also reported

suppressed bone/callus formation and reduced junction strength in chemotherapy

treated dogs in repair of segmental cortical non-unions.







30

Existing animal studies have elucidated the pathophysiology of MTX-

associated osteoporosis. MTX directly affects the cellular activity of osteoblasts as

observed by a significant decrease in bone volume, osteoblast surface, osteoid

surface, and bone formation rate (24,74). However, no study, animal or human, has

investigated the recovery of bone following completion of chemotherapy treatment.

All existing studies measure bone parameters at only one point in time after

treatment. It is unknown if the bone mass remains depressed indefinitely, slowly

recovers to normal levels, or recovers to osteopenic levels below the normal range.



Methods of Skeletal Assessment

Quantification of skeletal changes due to aging, menopause, exercise, or drug

treatment depends on the type of study (human or animal), resources available, and

desired accuracy of measurement. In vivo studies involving humans require

noninvasive means to assess bone changes unless skeletal biopsies are indicated.

Non-invasive methods commonly used to measure bone density include: (1) single-

photon absorptiometry (SPA), (2) dual-photon absorptiometry (DPA), (3) dual-

energy x-ray absorptiometry (DEXA), and (4) quantitative computed tomography

(QCT). DEXA, with it's high accuracy, quick scan times, and low radiation dose is

the most popular method to assess bone density in humans (15,28,40,55,68). DEXA

has also proven to be accurate for either in vivo or ex vivo animal studies measuring

either whole skeleton or appendicular density (2,3,33,48,67). These radiographic

methods measure the amount of mineral in the skeleton but do not offer the








31

capability of evaluating bone architecture or structure, assessing actively mineralizing

bone surfaces, or measuring bone quality or strength. Invasive measurements supply

more information but require painful biopsies for human studies or animal sacrifice.

Methods commonly used to assess bone quantity and quality following animal

sacrifice are histomorphometry and mechanical testing. Histomorphometric

techniques enable quantification of cancellous bone volume, cortical bone area,

cortical thickness and bone surface lined with osteoblasts or osteoclasts (42). When

fluorescent compounds are used to label the actively mineralizing bone,

histomorphometry can also quantify mineralizing surface, mineral apposition rate,

bone formation rate, and longitudinal bone growth (25).

Whole bones, cortical sections, or cancellous bone blocks can be mechanically

tested to determine the failure load, ultimate strength and stiffness of the specimen.

Weight bearing activities apply a complex loading environment to the skeleton

consisting of bending, torsion, and compressive loads. Mechanical tests of long bones

are typically loaded either in torsion or bending. Compressive tests are also used for

testing cancellous blocks or vertebral segments.

Bone mass, assessed either through DEXA or histomorphometry, provides

important information concerning bone health. However, the quality or strength of

the bone is also very important. The amount of cancellous or cortical bone mass can

be depressed or the amount of bone mineral compromised and not affect the

structural strength of the bone due to compensatory changes in bone geometry.







32

Similarly, adverse changes in bone geometry can decrease the structural strength of

bone without changes in bone mass or mineral content.













CHAPTER 3
MATERIALS AND METHODOLOGY

The following sections detail the experimental protocol concerning animal

care, bone histomorphometry, biomechanical testing, dual-energy x-ray

absorptiometry (DEXA), and statistical analysis.



Animal Care

The protocol for this experiment was approved by the University Animal Use

Committee to assure humane treatment of animals and prevent undo suffering.

Sixty-nine male Sprague-Dawley rats (120 days old) with an average body weight of

415 g were randomly assigned to 4 control groups and three drug treatment groups.

Six rats were selected for the baseline control group and nine rats were chosen for

three control groups to be sacrificed at 30, 80, and 170 days. Similarly, three drug

treatment groups were also randomly chosen with 12, 10, and 14 rats in the 30, 80,

and 170 day groups, respectively. More animals were used in the 170 day group in

anticipation of death from MTX toxicity. Following a 2 week acclimation period,

treatment was initiated and baseline control rats were euthanized. Methotrexate

(MTX) was administered intraperitoneally (i.p.) in a 0.5 ml bolus injection using a

dose of 0.75 mg/kg/day. This dose is comparable to approximately one-third the

daily dose used for humans (69,70). Two courses of MTX were administered to the







34

treated rats in a cycle involving 5 consecutive days of drug injections, followed by 9

drug-free days, followed by another 5 days of injections (5-ON/9-OFF/5-ON). Two

courses of chemotherapy were used in this protocol to provide consistency with

typical clinical multiple-course sequelae used with human patients. Control rats

received a 0.5 ml i.p. bolus injection of saline using the same injection cycle (5-

ON/9-OFF/5-ON) as the MTX-treated rats.

All rats were injected with fluorochrome compounds on 2 separate occasions,

15 mg/kg of Demeclocycline (Lederle Laboratories, Pearl River, NY) on the 14th

day prior to sacrifice and 15 mg/kg of Calcein (Sigma Chemical Co., St. Louis, MO)

on the 7th day prior to sacrifice in order to monitor actively mineralizing bone

(dynamic histomorphometric parameters).

The rats were housed individually, with an ambient temperature of 24o-26 C

and a 12hr/12hr light/dark cycle. The activity of the rats was not monitored but was

limited to the confines of relatively small cages. The rats were fed Purina Rat

Laboratory Chow (St. Louis, MO) with 1.0% calcium and 0.9% phosphorus and

water ad libitum.

The rats were euthanized by i.p. injections of sodium pentobarbital

(100mg/kg). Bilateral femurs and tibiae as well as the 2nd and 3rd lumbar vertebrae

were harvested. The left tibia and femur and the vertebral segments were stripped

of all soft tissue. The tibia was sawed into 3 segments to allow infiltration of fixative:

the proximal third, middle diaphysis including the tibiofibular junction, and the small

distal segment which was discarded. The cortical bone of the anterior aspect of the








35

proximal tibia was shaved to expose cancellous bone tissue to the fixative solution.

The proximal and distal metaphyses of the femur were removed by cross sectional

saw cuts and discarded leaving the femoral diaphysis for analysis. All bone segments

were immediately fixed in 10% formalin-alcohol for future histomorphometric

analysis (see Appendix A). The right limb (tibia and femur) was disarticulated at the

hip, wrapped in saline soaked gauze and frozen at -700 C for future biomechanical

analysis. DEXA evaluation was performed on all excised bones prior to histological

and biomechanical testing.



Bone Histomorphometry

The right tibia and femur segments were transferred from 10% formalin-

alcohol to 70% ethyl alcohol (ETOH) two days after harvest. The proximal tibia was

chosen over the femur for cancellous histomorphometry evaluation due to the ability

to standardize the sampling area relative to the growth plate. The tibial diaphysis

proximal to the tibiofibular junction and the mid-diaphysis of the femur were used

for analysis of cortical bone.



Cancellous Bone

The proximal tibia was prepared for undecalcified cancellous bone

histomorphometry through dehydration in graded solutions of ETOH (70%, 95% and

100% for at least two days at each concentration) and xylene (for one day). The

sample was then infiltrated with a series of solutions containing methyl methacrylate,







36

dibutyl phthalate, and benzoyl peroxide. The recipes and detailed procedures for

methyl methacrylate embedding are included in Appendix A. The methyl

methacrylate solution was polymerized and the anterior aspect of the embedded

proximal tibia specimen was ground flat using a dental grinding wheel (Buffalo

Dental Manufacturing Co. Inc., Syosset, NY) to approximately one-third the depth

of the metaphysis. Thin longitudinal sections, 4 and 8 micrometers thick, were then

cut using an AO Autocut/Jung 1150 microtome (Cambridge Instruments, West

Germany). The thinner sections were placed on slides and stained according to the

Von Kossa method with a tetrachrome counterstain (Polysciences, Inc., Warrington,

PA). The recipe and procedure for the modified Von Kossa stain is included in

Appendix B. The 8 lim sections were left unstained and illuminated under ultraviolet

light to analyze the fluorescent labels for cancellous dynamic histomorphometry.

Two bone sections of the proximal tibia from each animal were analyzed for

both static and dynamic bone parameters. The standardized sample site (3mm

square window) was taken approximately 1 mm distal to the growth

plate/metaphyseal junction. The following static histomorphometric measurements

(25) were made on the 4 Am sections:

(1) Cancellous Bone Volume Cn.BV/TV (%) Cn.BV/TV is the percentage of

cancellous bone tissue (bone, marrow, and unmineralized osteoid) composed

of mineralized bone matrix.








37

(2) Osteoclast Surface Cn.Oc.S./BS (%) Cn.Oc.S./BS is the percentage of

cancellous bone surface with osteoclasts (bone resorbing cells) present and is

considered an index of bone resorption.

The following dynamic bone measurements (25) were made on the unstained, 8 pm-

thick sections under ultraviolet illumination to enable observation of the

fluorochrome labeling:

(3) Longitudinal Bone Growth Cn.LBG (tm/day) Cn.LGB is the mean

distance between final fluorochrome label (Calcein) and the growth

plate/metaphyseal junction divided by the time between last label injection

and sacrifice.

(4) Mineralizing Surface Cn.MS (%) Cn.MS is the percentage of cancellous

bone surface with double fluorochrome labels and is an index of bone

formation.

(5) Mineral Apposition Rate Cn.MAR (pm/day) Cn.MAR is the mean

distance between the two fluorochrome markers divided by the time interval

between administration of the labels and is an index of osteoblast activity.

(6) Bone Formation Rate Cn.BFR/BS (m3/nm2/day) Cn.BFR/BS is

calculated by multiplying mineralizing surface by mineral apposition rate and

is an index of bone turnover.









Cortical Bone

Cortical bone segments of the tibia and femur were dehydrated in serial

solutions of 70%, 95%, and 100% ETOH and acetone. The bone segments were

then embedded in bioplastic (TAP Plastics, Inc., Dublin, CA) as described in

Appendix D. The bone blocks were then sectioned to 100 J/m using a Bueler Isomet

low-speed bone saw (Lake Bluff, IL) with a diamond chip blade (#801-137 LECO

Corp., St. Joseph, MI). The sections were mounted on slides for analysis. The

following static bone measurements were made:

(1) Total Bone Tissue Area Ct.T.Ar (mm2) Ct.T.Ar is the area within

periosteal perimeter which includes cortical bone and marrow.

(2) Marrow Area Ct.Ma.Ar (mm2) Ct.Ma.Ar is the area within the

endocortical perimeter which contains marrow.

(3) Cortical Bone Area Ct.Ar (mm2) Ct.Ar is the area of only cortical bone

(marrow area subtracted from the total bone tissue area).

(4) Mean Cortical Width Ct.Wi(mm) Ct.Wi is the average width or thickness

of the cortical bone sampled at 12 positions around the circumference.

(5) Polar Moment of Inertia J (mm4) J is the geometric property which is

calculated by modeling the cross-section of the bone as a hollow ellipse and

is represented by the following equation:

J = [r.(ab3 +a3b-(a-t)(b-t)3-(a-t)3(b-t))]/4

where a is the minor axis of the ellipse, b is the major axis of the ellipse, and

t is the mean cortical thickness (Ct.Wi) of the cross-section.








39

Under ultraviolet illumination the following dynamic bone parameters were

measured:

(6) Periosteal Mineralizing Surface Ps.Ms (%) Ps.MS is the percentage of the

cortical periosteal surface with double fluorochrome labels and is an index of

bone formation.

(7) Periosteal Mineral Apposition Rate Ps.MAR (im/day) Ps.MAR is the

average distance between the two fluorochrome markers divided by the time

interval between administration of the labels and is an index of osteoblast

activity on the periosteal surface.

(8) Periosteal Bone Formation Rate Ps.BFR (Jm3l/Im2/day) Ps.BFR/BS is

calculated by multiplying periosteal mineralizing surface by the periosteal

mineral apposition rate.



Quantification of Bone Parameters

Two methods were used to quantify the cancellous and cortical bone

parameters listed in the previous section. The static bone parameters were measured

using a Vidas imaging system (Kontron Electronics, West Germany) and customized

software programs. The dynamic bone parameters were measured using a bone

histomorphometry package by Bioquant (R & M Biometrics Corp., Nashville, TN).

Previous work has shown the Vidas imaging system to be more consistent and

reliable than the Bioquant hand digitizing system for static histomorphometric

measurements (76). The Vidas system was not used for dynamic histomorphometric







40

measurements due to the absence of proper fluorescent filters to enable viewing of

the demeclomycin and calcein labels.

The Vidas imaging system consisted of a Zeiss Axiophot (Zeiss, West

Germany) microscope, Hamamatsu C2400 high resolution black and white CCTV

video camera (Hamamatsu City, Japan), and 386 IBM-based computer with expanded

memory capabilities. The cancellous and cortical bone sections were imaged with the

video camera and the pixel information of these images were stored on the 386

microcomputer. The density contrast between the dark stained bone spicules and the

lighter blue stained marrow allowed semi-automatic differentiation between the bone

and marrow for calculation of cancellous bone volume. The cortical cross-sections,

however, were not stained and the periosteal and endocortical perimeters were

digitized to calculate bone areas. Cortical thickness and polar moment of inertia

were also calculated by a special program written to identify the geometry of the

cross section and sample, at 300 increments, the cortical width to calculate mean

cortical width and major and minor diameters for the elliptical model. The computer

codes written to make all cancellous and cortical calculations are presented in

Appendix D.

Dynamic parameters were measured with the Bioquant Bone Morphometry

System. Surfaces of interest on the bone were viewed with a Nikon Labophot

microscope (Nikon, Inc., Garden City, NY) and digitized with a Hipad digitizing

tablet (R & M Biometrics Corp., Nashville, TN). A camera lucida enabled the

digitizing cursor to be visible in the microscopic field of view. An interface between








41

the digitizing tablet and an Apple HIe microcomputer (Cupertino, CA) allowed the

transfer of bone surface lengths to the Bioquant software where calculations were

performed to obtain the desired histomorphometric parameters.





Biomechanical Testing

The right tibia and femur, complete with surrounding soft tissue, were stored

at -70 C following harvest. Bones were thawed slowly to 25oC, cleaned of soft

tissue, and saturated in saline solution and kept chilled until testing. DEXA scans

were made of these bones immediately after removing the soft tissue and were

biomechanically tested the next morning.

The distal and proximal metaphyses were embedded in low temperature metal

potting material in a specially designed mold. When the metal was poured into the

mold, it produced a metal block surrounding the bone ends which fit into the torsion

tester's grip. The distance between the proximal and distal ends were standardized

to provide tibia and femur gauge lengths of 23.6 mm and 20.75 mm, respectively.

See Figure 3.1 for a photograph of a femur and tibia with bone ends embedded in

low melting point metal.

The right tibia and femur from each animal were tested to failure in external

torsion about the long axis of the bone on a rapid loading torsional testing machine

(A.H. Burstein, Cleveland, OH) at a loading rate of 12 rad/sec (9). Torsional tests

were selected for mechanical assessment because these tests apply uniform torque























































Figure 3.1 Photograph of femur and tibia with ends
embedded in low melting point metal








43

along the bone length, allowing failure to occur at the weakest part of the test

segment.

Raw data, including torque applied (N m) and angular displacement (degrees)

at failure, were collected at 1000 Hz per channel and subsequently stored on a 386-

IBM microcomputer through an analog to digital interface with the torsional testing

machine. The cross-sectional geometry of the bone was analyzed in order to

calculate the polar moment of inertia and the torsional strength. The cross-sections,

therefore, were taken immediately adjacent to the torsional fracture. Cross-sections,

approximately 1000 pm in thickness, were prepared from undecalcified bone using

a Unimat-SL circular saw (model #DB200, American Edelstaal, Inc., NY, NY). Two

cross-sections were cut from each bone and glued with cyanoacrylate to glass slides.

These sections were then ground flat to approximately 300 Am using a Dremel Moto-

tool (model 285, Emerson Electric Co., Racine, WI). These cross sections were then

analyzed with a Vidas imaging system, as mentioned in the previous

histomorphometry section, to obtain the following parameters:

(1) Total Bone Tissue Area (mm2) = A

(2) Marrow Area (mm2) = A.

(3) Cortical Bone Area (mm2) = AC

(4) Mean Cortical Thickness (mm) = t

(5) Polar Moment of Inertia (mm4) = J







44

The following biomechanical parameters were either measured or calculated

using custom designed algorithms created for digital processing software (DADisp,

DSP Development Corporation, Cambridge, MA):

(1) Torque at failure (N m) = Tu

(2) Twist angle at failure (degrees) = en

(3) Energy absorbed at failure (N degrees) = Eu

(4) Stiffness (N/degrees) = S

(5) Torsional strength at failure (N/m2) = 7,



To and eu were measured directly during testing. Tu was plotted against eu to

calculate Eu and S which represent the area under the torque-angle curve and the

slope of the elastic region of the torque-angle curve, respectively. The biomechanical

parameters are represented in graphical form in Figure 3.2. Due to the lamellar

nature of cortical bone in the rat, there is minimal plastic deformation, therefore, a

linear regression from the minimum torque and angle to the maximum torque and

angle was used to determine stiffness. r, was then calculated using the hollow

ellipse model (20,62), where

r7 = Tu-b/J

and the polar moment of inertia,

J = [Tr.(ab3 +a3b-(a-t)(b-t)-(a-t)(b-t))]/4




































Twist Angle (e)


Figure 3.2 Graphical depiction of biomechanical parameters







46

where T. is the torque at failure, a is the minor axis of the ellipse, b is the major axis

of the ellipse, and t is the mean thickness of the cross-section calculated from 12

positions around the circumference of the ellipse.



Dual Energy X-Ray Absorptiometrv

Ex-vivo measurements of bone mineral density were made for the excised

femurs, tibiae, and vertebrae using a dual energy x-ray absorptiometer (DEXA)

(Lunar Radiation, Madison, WI). The DEXA, with special high resolution software

supplied by the company, is capable of accurately scanning small bone samples (3,48).

The DEXA machine was calibrated prior to each use with a standard calibration

block consisting of known density materials. Repeatability tests were run using

excised rat tibiae and femurs to determine the coefficient of variation for this

procedure. The results from these repeatability tests are presented in Appendix E.

The cortical bone segments of the left tibial and femoral diaphyses, which

were stored in 70% ETOH for histomorphometric analyses at the time of scan, were

positioned on a 2 cm thick piece of acrylic with the anterior aspects of the bone

facing the scan sensor. The acrylic material was required in the scan area to provide

a contrast density, similar to tissue density, to the excised bones. The bones were

scanned side-by-side in the scanning area. The vertebral segments were scanned in

a manner similar to the left tibia and femur.

The intact right tibia and femur (which were kept moist and chilled in saline

baths and ice in order to prevent material degradation prior to mechanical testing)








47

were scanned in a saline filled petri dish on top of the acrylic contrast medium. The

bones were positioned and scanned in the same manner as the left tibia and femur.

The scans were analyzed by software provided by Lunar. The density scans

produced a pixel map of density gradients. The edges of the bone were detected by

an automatic algorithm. The edges were often misplaced by this algorithm and were

corrected by manual intervention. The bone mineral density (g/cm2) was calculated

for each excised bone.



Statistical Analysis

A two-way analysis of variance (ANOVA) was used to determine if drug

treatment (MTX or CTL) or time following treatment (30, 80, and 170 days) or the

interaction between drug treatment and time had significant effects on the response

variables which include the histomorphometric, DEXA, and biomechanical

measurements detailed in the previous sections. If significant interactions were

noted, a one-way ANOVA was run to determine the effects of drug treatment at

each time interval and time for each treatment group, separately. If differences

among groups were noted with the ANOVA, a Duncan's multiple comparison

procedure was used to determine which groups were different from one another. A

significance level was set for all tests at the p = 0.05 level. PC-SAS version 6.03

(SAS, Inc., Cary, NC) was used for all the statistical analyses. A copy of the code

used for these analyses is presented in Appendix F.












CHAPTER 4
RESULTS


The weights of the animals were monitored every week throughout the study.

A plot of the weight changes with time are presented in Figure 4.1. Although the

methotrexate treated rats had lower body weights throughout the study, this

difference was not significant (p> 0.05). There was a significant increase in weight

for both the MTX and CTL rats with time (p<0.05). Animals experienced no

noticeable side effects from the MTX treatment other than the slight decrease in

body mass.

The following sections present the results of the histomorphometric analysis

cancellouss and cortical bone), biomechanical testing, and dual-energy x-ray

absorptiometry on the bones of control and methotrexate treated rats. Abbreviations

of bone parameters are used when presenting the results and in the graphical

presentation. In order to make these abbreviations more readable, a glossary of

abbreviations is provided in Appendix G for quick reference.






















0.56

0.54

0.52

S 0.5

0.48

. 0.46
G)
S0.44

0.42

0.4

0.38


Figure 4.1 Rat weight changes with time


0 50 100 150

Time (days)


SCTL MTX







50

Bone Histomorphometry

Cancellous Bone

The means and standard deviations for the cancellous bone parameters are

presented in Table 4.1. A graph of tibial cancellous bone volume (Cn.BV/TV) with

time in shown in Figure 4.2. The statistical results of the 2-way and 1-way ANOVAs

are summarized on the right-hand side of the graph. The results for all parameters

are presented in a similar manner in subsequent graphs. The 2-way ANOVA

indicated a significant interaction between treatment and time (p=0.003), therefore,

separate 1-way ANOVAs were run for treatment by time and time by treatment. As

indicated in the graph, CTL rats had significantly higher CN.BV/TV at 80 and 170

days. Peak Cn.BV/TV for MTX rats occurred at 30 days with significant decreases

after this point, whereas, the CTL rats' peak Cn.BV/TV occurred at 80 days.

Figure 4.3 presents a graph of cancellous osteoclast surface (Cn.Oc.S), an

indicator of bone resorption. The MTX treated rats had significantly higher Cn.Oc.S

at 30, 80, and 170 days. Cn.Oc.S declined with age in both MTX and CTL rats.

Cancellous longitudinal bone growth (Cn.LBG) is presented in Figure 4.4. As

indicated in the statistical summary, there is not a significant interaction between

treatment and time. Therefore, the general effects of treatment (CTL or MTX) and

time (30, 80, and 170) are evaluated. In this case both treatment and time do have

a significant effect on Cn.LBG. The CTL rats had generally higher rates of

longitudinal growth than MTX and there was a general decline in longitudinal bone

growth with age in both groups.








51

The dynamic parameter of cancellous mineralizing surface (Cn.MS), indicative

of active bone forming surface, is shown in Figure 4.5. CTL rats had significantly

higher Cn.MS at all measurement intervals. The Cn.MS was highest at 30 days and

decreased to lower levels at 80 and 170 days for both CTL and MTX rats.

Osteoblastic activity was quantified by cancellous mineral apposition rate

(Cn.MAR) in Figure 4.6. The osteoblastic activity between CTL and MTX rats was

significantly different only at 170 days, with CTL rat's Cn.MAR exceeding MTX rats.

Again, there was an age-associated decline in Cn.MAR with highest values at 30 days

and decreasing to the lowest levels at 170 days for both CTL and MTX rats.

Cancellous bone formation rate (Cn.BFR), an index of bone turnover, is

presented in Figure 4.7. CTL rats had higher bone turnover at all time intervals (30,

80, and 170 days). Bone turnover was also age-dependent with highest levels

occurring at 30 days followed by decrease and plateau at 80 days for both CTL and

MTX animals.

Comparative photomicrographs of cancellous bone volume are presented in

Figures 4.8, 4.9, 4.10, and 4.11 for the 0, 30, 80, and 170 day time intervals,

respectively. Each figure presents the control animal's bone in part "a"and the MTX

treated animal in part "b" for ease of comparison. Photomicrographs capturing the

fluorescent labels on the cancellous surface are shown in Figures 4.12,4.13,and 4.14

for the 30, 80, and 170 time intervals, respectively. Again, these figures are

composite photographs showing both the control and drug treated animal.

















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Statistics
trt x time: p=0.003
trt:
CTL>MTX @80,170 (*)

time:
CTL: 80>0,30,170
MTX: 30>80,170


Time (days)


CTL
- MTX


Figure 4.2 Tibial cancellous bone volume


14-


10 -

























Statistics

trt x time: p=0.05

trt:
MTX>CTL
@30,80,170 (*)
time:

CTL: 0>30>80,170
MTX: 30>80>170


0 100
0 50 100 150


-CTL

+- MTX


Time (days)


Figure 4.3 Tibial cancellous osteoclast surface


2.5 -


1.5 h


s-








55











35


30
Statistics

- 25 trt x time: p=0.643
* trt: p=0.0001
E 20- CTL>MTX (*)

time: p=0.0001
15 30>80>170


015






0 so loo 15o CTL

Time (days) MTX


Figure 4.4 Tibial cancellous longitudinal bone growth





























Statistics


*\





*


trt x time: p=0.0003

trt:
CTL>MTX
@30,80,170 (*)
time:

CTL: 30>80,170
MTX: 30>80,170


(V '


0 50


100

Time (days)


CTL

- MTX


Figure 4.5 Tibial cancellous mineralizing surface


30-


25




































*r


50 100 150


Time (days)


Statistics

trt x time: p=0.045


CTL> MTX
@170 (*)


time:
CTL: 30>80,170
MTX: 30>80>170







S CTL

-- MTX


Figure 4.6 Tibial cancellous mineral apposition rate


2.5 H


C2

E

" .5
n-


, 1
0


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0


ni '














































I Ik


0 50


100

Time (days)


Statistics

trt x time: p=0.0001

trt:
CTL>MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80,170








S CTL

+ MTX


Figure 4.7 Tibial cancellous bone formation rate


80 -


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Cortical Bone

The means and standard deviations for the cortical bone parameters are

presented in Table 4.2 and 4.3 for the femur and tibia, respectively. Figures 4.15 and

4.16 present the cortical total tissue area (Ct.T.Ar) as a function of time for the

femur and tibia, respectively. For both bones, CTL had significantly higher Ct.T.Ar

than MTX at 170 days but equivalent bone areas at prior measuring times. The

Ct.T.Ar steadily increased with time in the femur for both CTL and MTX animals.

The Ct.T.Ar of the CTL rats' tibia also followed a similar steady increase, however,

the MTX rats increased only until the 80 day time point and then experienced a

drastic decrease in Ct.T.Ar to baseline levels.

The marrow area (Ct.Ma.Ar) of the femur and tibia are presented in Figures

4.17 and 4.18, respectively. For both bones the MTX animals had larger marrow

areas than the CTL animals when taken over all time intervals. There was a general

trend for increased marrow areas with time in the femur. However, in the tibia, the

marrow area indicated a significant increase over baseline levels only at 170 days.

The cortical bone area (Ct.Ar) is the portion of the total bone tissue area

comprised of cortical bone. Figure 4.19 and 4.20 show the Ct.Ar for the femur and

tibia, respectively. The CTL animals had significantly greater femoral and tibial

cross-sectional cortical areas than the MTX rats at 30, 80, and 170 days. The femurs

of the CTL rats showed increases in Ct.Ar with time, whereas the MTX rats showed

no significant changes in Ct.Ar from baseline levels. The Ct.Ar of the tibia increased







67

with time for the CTL rats but only increased up to 80 days for the MTX rats at

which point a significant decrease in bone area was noted to below baseline levels.

The mean cortical width (Ct.Wi) of the femoral and tibial cross-section are

shown in Figures 4.21 and 4.22,respectively. Femoral Ct.Wi was significantly greater

for the CTL rats compared to the MTX rats at all time intervals. Femoral Ct.Wi

increased with age for the CTL rats yet didn't change for the MTX rats. Tibial

Ct.Wi was also significantly greater for the CTL compared to MTX rats at 30, 80,

and 170 days. Peak Ct.Wi occurred in the tibia at 80 days for both MTX and CTL

rats. The tibial Ct.Wi of CTL rats then stabilized, whereas, the Ct.Wi of the MTX

rats decreased to below baseline levels.

The polar moment of inertia (J) is presented in Figures 4.23 and 4.24 for the

femur and tibia, respectively. The CTL rats had significantly higher J-values than the

MTX rats at 170 days for both the femur and tibia. The J-value for CTL rats

experienced an age-related increase in the femur and tibia. The MTX rats had peak

femoral and tibial J-values at 80 days.

The dynamic cortical bone parameter, periosteal mineralizing surface (Ps.MS),

is presented in Figures 4.25 and 4.26for the femur and tibia, respectively. There was

a significant difference in femoral Ps.MS between CTL and MTX rats with CTL

exhibiting higher Ps.MS values at 30, 80, and 170 days. There was an age-related

decline in femoral Ps.MS for both CTL and MTX rats with the MTX rats exhibiting

a greater rate of Ps.MS loss with time. The tibia demonstrated a significant







68

decrease in Ps.MS with time with the CTL and MTX rats exhibiting similar rates of

Ps.MS change.

Periosteal mineral apposition rate (Ps.MAR), an index of osteoblast activity,

is presented in Figures 4.27 and 4.28 for the femur and tibia, respectively. Although

there was not a significant interaction between treatment and time for the femur, the

CTL had significantly greater Ps.MAR than MTX treated animals and a decrease in

Ps.MAR with time. The statistical results from the tibia indicated that CTL animals

had higher Ps.MAR than MTX rats at all time intervals and a significant decrease

in Ps.MAR with time for both CTL and MTX animals.

Graphs for femoral and tibial periosteal bone formation (Ps.BFR) rate are

presented in Figures 4.29 and 4.30, respectively. Femoral Ps.BFR was significantly

lower for the MTX rats at all time intervals than the CTL animals and exhibited a

steady decline with time for both CTL and MTX animals. Tibial Ps.BFR was only

significantly lower for the MTX at the 30 day time interval; after that point both

CTL and MTX exhibited similar periosteal bone formation. Tibial Ps.BFR was

higher at 30 days for both MTX and CTL animals than at 80 and 170 days with a

rapid age-related decrease between 30 and 80 days.

Representative photomicrographs of cortical bone area for the femur and tibia

are presented in Figures 4.31 and 4.32,respectively, for the baseline control animals.

Photographs of the femoral and tibial cortical bone areas for the 30 day time interval

are presented in Figures 4.33 and 4.34, 80 day time interval in Figures 4.35 and 4.36,

and the 170 day time interval in Figures 4.37 and 4.38. Each figure presents the








69

control animal's bone in part (a) and the MTX treated animal in part (b) for ease

of comparison. Photomicrographs capturing the fluorescent labels on the periosteal

cortical surface of the femur and tibia are shown in Figures 4.39 and 4.40 for the 30

day time interval, Figures 4.41 and 4.42 for the 80 day time interval, and Figures 4.43

and 4.44 for the 170 day time interval. Again, these figures are composite

photographs showing both the control and drug treated animal.












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Statistics

trt x time: p=0.002
trt:
CTL>MTX
@170 (*)
time:

CTL: 170>80,30,0
MTX: 170,80>30


10'
0 50 100 150


-CTL

+ MTX


Time (days)


Figure 4.15 Femoral total bone tissue area


N
E

































1


0 50 100 150


Time (days)


Statistics
trt x time: p=0.0005
trt:
CTL> MTX
@170 (*)
time:

CTL: 170,80>30,0
MTX: 80>30,170






S CTL

-+ MTX


Figure 4.16 Tibial total bone tissue area


E
E
S6.5 -


6 6


0 5.5-


E






















K I
--"c


50 100 150


Time (days)


Statistics
trt x time: p=0.283
trt:
p=0.0012
MTX>CTL (*)
time:
p=0.0001
170>80>30,0







CTL

I MTX


Figure 4.17 Femoral marrow area


5.5 -


N 5
E
E

.5
03

54
O


3.5 [


0


K .








75














1.5- Statistics
o trt x time: p=0.349

SE p=0.0001
E MTX>CTL (*)
A- time:

0 C p=0.021
1170>80,30,0







0.5
0 50 100 150 CTL

Time (days) MTX


Figure 4.18 Tibial marrow area





















10


9.5


9


N 8.5
E

E8
<
S7.5


7


6.5


6


100

Time (days)


Statistics

trt x time: p=0.0001
trt:
CTL> MTX
@30,80,170 (*)

time:

CTL: 170>80,30>0
MTX: none









S CTL

+ MTX


Figure 4.19 Femoral cortical bone area


0 50


- -...
























Statistics

trt x time: p=0.0001
trt:
CTL>MTX
@30,80,170 (*)
time:
CTL: 170,80>30>0
MTX: 80>30>170


0 50 100 150


Time (days)


Figure 4.20 Tibial cortical bone area


5.5 -


N
E 5
E


S4.5



4


/*


- IT`


CTL

-+ MTX




























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S *

*.

PT- -,


1j


Statistics
trt x time: p=0.0001
trt:
CTL>MTX
@30,80,170 (*)
time:
CTL: 170>80,30>0
MTX: none


0.6 --
0 50


100
Time (days)


CTL

-- MTX


Figure 4.21 Femoral mean cortical bone width


0.9 H


0.7 H




























*`


0 50 100 150


Time (days)


Statistics
trt x time: p=0.0001


CTL>MTX
@30,80,170 (*)


time:
CTL: 170,80>30>0
MTX: 80>30>170







CTL

-- MTX


Figure 4.22 Tibial mean cortical bone width


0.9 H


0.8


5-










































0 50 100 150


Time (days)


Statistics

trt x time: p= 0.0003
trt:
CTL> MTX
@170 (*)
time:
CTL: 170>80,30,0
MTX: 170,80>30







CTL

-+ MTX


Figure 4.23 Femoral polar moment of inertia





























*r



5T


Ar


Statistics

trt x time: p=0.019


CTL> MTX
@170 (*)


time:
CTL: 170,80>30,0
MTX: 80>170,30


0 50 100 150


Time (days)


CTL

-- MTX


Figure 4.24 Tibial polar moment of inertia






















100-



90 Statistics

trt x time: p=0.0035
80 trt:
CTL>MTX
*s @30,80,170 (*)
70 \
70 time:

"V CTL: 30>80,170
60- MTX: 30>80>170



50 -



40
0 50 100 150 -- CTL

Time (days) + MTX


Figure 4.25 Femoral periosteal mineralizing surface




















100

90-

80 \Statistics

70- trt x time: p=0.704

60 -trt: p=0.337
60\

50 -
time: p=0.0001
40- 30>80>170

30 -

20 -

10-

0
0 50 100 150 -"- CTL

Time (days) + MTX


Figure 4.26 Tibial periosteal mineralizing surface
























3-


S2.5-


E 2-


1.5


i 1 -


*k


0.5 -


0 50 100 150


Time (days)


Statistics

trt x time: p=0.07

trt: p=0.0001
CTL>MTX (*)

time: p=0.0001
30>80>170









CTL

+ MTX


Figure 4.27 Femoral periosteal mineral apposition rate























2.5 -


*k


% *

r------- __ *


Statistics

trt x time: p=0.015
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80>170


0 50 100 150


Time (days)


CTL

- MTX


Figure 4.28 Tibial periosteal mineral apposition rate































*T


Y *


- *-


Statistics

trt x time: p=0.039

trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80>170
MTX: 30>80>170


0 50


100

Time (days)


CTL

- MTX


Figure 4.29 Femoral bone formation rate


250



200



150-



100-



50-


"0

N
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E


E


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u0
I.
nL


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250 1


C 200

cm
E
3
S150
E


O 100


50 H


0 50 100 150

Time (days)


Statistics

trt x time: p=0.0024
trt:
CTL> MTX
@30 (*)
time:
CTL: 30>80,170
MTX: 30>80,170








SCTL

+ MTX


Figure 4.30 Tibial periosteal bone formation rate










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INGEST IEID E5R9T1BFE_J1DB93 INGEST_TIME 2017-07-13T21:25:18Z PACKAGE AA00003685_00001
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FILES



THE SHORT- AND LONG-TERM EFFECTS
OF METHOTREXATE ON THE RAT SKELETON
BY
DONNA L. WHEELER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993

ACKNOWLEDGMENTS
I would like to thank Dr. Robert E. Vander Griend for suggesting the area
of chemotherapy-induced osteopenia for study, and for his guidance and
encouragement throughout this work. I would like to thank Dr. R. William Petty and
the Department of Orthopaedics for generously supplying the funds for this project.
I am also thankful for the contributions of Dr. Thomas J. Wronski to my mastery of
histomorphometry and understanding of osteoporosis. I am also indebted to Dr.
Gary J. Miller, Dr. James E. Graves, Dr. Scott K. Powers, and Dr. David Lowenthal
for their guidance and instruction, enabling me to develop as a scientist.
I would like to acknowledge the loyal support of my friend and colleague,
Ernest E. Keith. His expertise, instruction, and assistance in the care of laboratory
animals were fundamental to the completion of this project. He also provided
valuable assistance in histomorphometric processing. Special thanks are extended to
Mia Park for her assistance in animal care, tissue processing, data acquisition, data
processing, and data entry.
I am indebted to Dr. Martha Campbell-Thompson and the Department of
Gastroenterology for the use of their microscope and Vidas imaging equipment. I
would also like to thank the Department of Exercise and Sport Sciences for the use
of their dual-energy x-ray absorptiometer and to Lunar Corporation for supplying the
software needed to use this machine.
ii

Finally, I would like to acknowledge the support of Kris Billhardt. Her love,
friendship, inspiration, patience, and editing skills were instrumental in the
completion of this research. I would also like to thank my parents. Jack and Jane
Wheeler, for their undying support and encouragement.

TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
Bone Remodeling 3
Involutional Bone Loss 8
Mineral Regulating Mechanisms 11
Mineral Regulating Hormones 11
Growth Regulating Hormones 15
Sex Hormones 16
Exercise 17
Types of Osteoporosis 18
Problem Statement 18
Research Objectives 19
Hypotheses 20
Delimitations 21
Limitations 21
2 REVIEW OF THE LITERATURE 22
Clinical Research 23
Animal Research 28
Methods of Skeletal Assessment 30
3 MATERIALS AND METHODOLOGY 33
Animal Care 33
Bone Histomorphometry 35
Cancellous Bone 35
Cortical Bone 38
Quantification of Bone Parameters 39
iv

Biomechanical Testing 41
Dual-Energy X-Ray Absorptiometry 46
Statistical Analysis 47
4 RESULTS 48
Bone Histomorphometry 50
Cancellous Bone 50
Cortical Bone 66
Biomechanics 102
Dual-Energy X-Ray Absorptiometry 119
5 DISCUSSION 124
Summary 136
Recommendations for Future Work 138
APPENDICES
A CANCELLOUS BONE FIXATION, DEHYDRATION
AND METHYL METHACRYLATE EMBEDDING 140
B MODIFIED VON KOSSA STAIN 144
C CORTICAL BONE FIXATION, DEHYDRATION
AND EMBEDDING IN BIOPLASTIC 148
D COMPUTER CODE FOR IMAGE ANALYSIS 150
E DEXA REPEATABILITY STUDY 159
F SAS PROGRAMS FOR STATISTICAL ANALYSIS 161
G QUICK REFERENCE FOR ABBREVIATIONS 164
REFERENCES 165
BIOGRAPHICAL SKETCH 173
v

LIST OF TABLES
Table Page
1.1 Factors associated with osteoporosis 2
1.2 Effects of mineral regulating hormones on
serum calcium and phosphate 13
4.1 Cancellous bone parameters 52
4.2 Femoral cortical bone parameters 70
4.3 Tibial cortical bone parameters 71
4.4 Femoral torsional biomechanical parameters 104
4.5 Tibial torsional biomechanical parameters 105
4.6 Dual-energy x-ray absorptiometry values for BMD 120
E.l Results of DEXA reliability study 160
G.l Standard Abbreviations 164
vi

LIST OF FIGURES
Figure Page
1.1 Cancellous bone remodeling 4
1.2 Cortical bone remodeling 5
1.3 Involutional bone loss 10
2.1 Mechanism of action of Methotrexate 24
3.1 Photograph of femur and tibia with ends
embedded in low melting-point metal 42
3.2 Graphical depiction of biomechanical parameters 45
4.1 Rat weight changes with time 49
4.2 Tibial cancellous bone volume 53
4.3 Tibial cancellous osteoclast surface 54
4.4 Tibial cancellous longitudinal bone growth 55
4.5 Tibial cancellous mineralizing surface 56
4.6 Tibial cancellous mineral apposition rate 57
4.7 Tibial cancellous bone formation rate 58
4.8 Photomicrograph of baseline cancellous bone volume 59
4.9 Photomicrographs of cancellous bone volume at 30 days 60
4.10 Photomicrographs of cancellous bone volume at 80 days 61
4.11 Photomicrographs of cancellous bone volume at 170 days 62
vii

4.12 Photomicrographs of fluorescent labels on
cancellous bone surfaces at 30 days 63
4.13 Photomicrographs of fluorescent labels on
cancellous bone surfaces at 80 days 64
4.14 Photomicrographs of fluorescent labels on
cancellous bone surfaces at 170 days 65
4.15 Femoral total bone tissue area 72
4.16 Tibial total bone tissue area 73
4.17 Femoral marrow area 74
4.18 Tibial marrow area 75
4.19 Femoral cortical bone area 76
4.20 Tibial cortical bone area 77
4.21 Femoral mean cortical bone width 78
4.22 Tibial mean cortical bone width 79
4.23 Femoral polar moment of inertia 80
4.24 Tibial polar moment of inertia 81
4.25 Femoral periosteal mineralizing surface 82
4.26 Tibial periosteal mineralizing surface 83
4.27 Femoral periosteal mineral apposition rate 84
4.28 Tibial periosteal mineral apposition rate 85
4.29 Femoral periosteal bone formation rate 86
4.30 Tibial periosteal bone formation rate 87
4.31 Photomicrograph of the femoral cross-section
of the baseline control animal 88
viii

4.32 Photomicrograph of the tibial cross-section
of the baseline control animal 89
4.33 Photomicrographs of femoral cross-sections at 30 days 90
4.34 Photomicrographs of tibial cross-sections at 30 days 91
4.35 Photomicrographs of femoral cross-sections at 80 days 92
4.36 Photomicrographs of tibial cross-sections at 80 days 93
4.37 Photomicrographs of femoral cross-sections at 170 days 94
4.38 Photomicrographs of tibial cross-sections at 170 days 95
4.39 Photomicrographs of femoral periosteal surface at 30 days 96
4.40 Photomicrographs of tibial periosteal surface at 30 days 97
4.41 Photomicrographs of femoral periosteal surface at 80 days 98
4.42 Photomicrographs of tibial periosteal surface at 80 days 99
4.43 Photomicrographs of femoral periosteal surface at 170 days .... 100
4.44 Photomicrographs of tibial periosteal surface at 170 days 101
4.45 Photograph of a typical fracture pattern following torsional test . 106
4.46 Femoral breaking torque 107
4.47 Tibial breaking torque 108
4.48 Femoral twist angle at failure 109
4.49 Tibial twist angle at failure 110
4.50 Femoral energy absorbed at failure Ill
4.51 Tibial energy absorbed at failure 112
4.52 Femoral torsional stiffness 113
4.53 Tibial torsional stiffness 114
IX

4.54 Femoral torsional strength 115
4.55 Tibial torsional strength 116
4.56 Femoral polar moment of inertia associated with torsional fracture 117
4.57 Tibial polar moment of inertia associated with torsional fracture . 118
4.58 Femoral bone mineral density 121
4.59 Tibial bone mineral density 122
4.60 Vertebral bone mineral density 123
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
The Short- and Long-term Effects of Methotrexate on the Rat Skeleton
By
Donna L. Wheeler
December 1993
Chairman: James E. Graves, Ph.D.
Major Department: Health and Human Performance
The chemotherapy drug, Methotrexate (MTX), has been shown to decrease
bone mass and increase the incidence of bone fractures. Existing studies measure
bone parameters at one point in time following chemotherapy and do not monitor
the bone’s recovery. This study’s purpose was to use a rat model to determine the
long-term effects of MTX on bone volume, turnover, mineralization, density, and
strength.
Sprague-Dawley rats were randomly assigned to either control or MTX
groups. Daily MTX and saline injections were given for two five-day courses to drug
treatment and control groups, respectively. Fluorochrome compounds were injected
prior to sacrifice to monitor actively mineralizing bone surfaces. One control and
MTX group were sacrificed at 30, 80, and 170 days following treatment initiation.
xi

Both femurs, both tibias, and 2 lumbar vertebrae were harvested for cancellous and
cortical bone histomorphometry, biomechanics (torsion), and densitometry (DEXA).
Standard cancellous and cortical histomorphometric parameters were measured from
undecalcified, methyl methacrylate embedded sections from the right proximal tibia
and tibial and femoral diaphyses. The contralateral femur and tibia were torsionally
loaded to failure and standard mechanical parameters were measured. Excised
bones were scanned using DEXA to measure bone density. All bone responses were
statistically analyzed using a 2-way ANOVA followed by Duncan’s multiple
comparison procedure (significance: p=0.05).
Cancellous bone volume was significantly lower in the MTX treated group at
80 and 170 days. Cancellous mineralizing surface and longitudinal bone growth were
significantly depressed at all time points yet mineral apposition rate was only
depressed at the 170 day point. Cancellous osteoclast surface was increased at all
time points for the MTX treated animals. Cortical cross-sectional area and
periosteal mineral apposition rate were significantly lower for both the femur and
tibia in the MTX groups at all time points. However, periosteal mineralizing surface
was significantly depressed in the MTX group only for the femur. MTX had minimal
effects on the biomechanical parameters and bone density measurements.
Methotrexate had long-term negative effects on both cortical and cancellous
bone. The drug decreased bone volume, decreased bone formation, and decreased
xii
osteoblast activity.

CHAPTER 1
INTRODUCTION
The disease osteoporosis results from a decrease in bone mass and bone
strength leading to an increase in the incidence of bone fractures. Osteoporosis is
an enormous public health problem which is responsible for over a million bone
fractures in the U.S. each year (61). The most common sites of osteoporotic
fractures are the vertebrae, hip, and distal forearm. One-third of all women over 65
will have vertebral fractures and, by the eighth decade of life, one of every three
women and one of every six men will have had a hip fracture (61). The direct and
indirect costs of osteoporosis are estimated to be over $6.1 billion annually in the
United States.
Peak bone mass is achieved by the third decade of life and then slowly
declines from this point. The magnitude of peak bone mass as well as the rate at
which bone is lost contribute to the development of osteoporosis. Many factors have
been shown to be associated with osteoporosis with the most important being age,
gender, race, and hormonal status. Osteoporosis is most commonly seen in
postmenopausal white women, Asian women, and older men (60). Other factors
such as genetics, lifestyle, nutritional factors, medical disorders, or drugs have been
shown to significantly affect bone health. A summary of osteoporosis risk factors are
presented in Table 1.1.
1

2
Table 1.1 Factors Associated with Osteoporosis
Genetic
White or Asian ethnicity
Female gender
Positive family history
Small body frame (< 127 lbs)
Lifestyle
Smoking
Inactivity
Nulliparity
Excessive exercise (producing amenorrhea)
Early natural menopause
Late menarche
Nutritional Factors
Milk intolerance
Low calcium intake
Vegetarianism
Excessive alcohol intake
Caffeine
Consistently high protein intake
Medical Disorders
Anorexia nervosa
Thyrotoxicosis
Parathyroid overactivity
Cushing’s syndrome
Type I diabetes
Abnormal gastrointestinal function
Abnormal hepatobiliary function
Occult osteogenesis imperfecta
Mastocytosis
Rheumatoid arthritis
Prolactinoma
Hemolytic anemia
Drugs
Thyroid replacement therapy
Glucocorticoid drugs
Anticoagulants
Chronic lithium therapy
Chemotherapy (Methotrexate)
GnRH agonist or antagonist therapy
Anticonvulsants

3
The following sections will discuss bone remodeling, involutional bone loss,
physiological mineral regulating mechanisms, types of osteoporosis, and will conclude
with the development of the problem being studied.
Bone Remodeling
Throughout the body, bone is continually being remodeled, where bone is
resorbed and replaced by new bone. In order to maintain bone mass it is essential
that this remodeling process remain balanced. The amount of bone resorbed should
be replaced by an equal or greater amount of new bone or there will be a net loss
of bone mass. If this imbalance remains uncorrected this condition can lead to
osteoporosis. This section will describe the normal bone remodeling sequence and
discuss problems which might affect the normal cycle.
Figure 1.1 and Figure 1.2 describe the normal sequence of cancellous and
cortical bone remodeling, respectively. Remodeling occurs in a programmed
sequence at discrete bone foci called bone remodeling units (BMU) (27). The steps
of bone remodeling can be described as ACTIVATION, RESORPTION, and
FORMATION and are similar for cancellous and cortical bone.
At the beginning of each cancellous remodeling cycle (Figure 1.1), activation
occurs at previously inactive bone surfaces. These surfaces are covered with bone
lining cells, presumably of osteoblastic lineage. These lining cells respond to bone-
resorbing hormones and expose the bone surface to osteoclast progenitor cells.
Osteoclast progenitors then replicate and differentiate into mature osteoclasts. The

4
Figure 1.1 Cancellous bone remodeling
Oc=osteoclast; Opc=osteoprogenitor cells;
Ob=osteoblast; BLC=bone lining cells
(a) Resorption is completed before formation
(b) Resorption followed closely by formation
(Figure adapted from W.S.S.Jee (37))

Cavity
Resorption
Cavity
Early Haversian
System
Completed Haversian
System
Figure 1.2 Cortical remodeling unit. Longitunidal and cross sections through remodeling tunnel
A=osteoclasts; B=precursor cells; C= capillary loop; D=mononuclear cells
E=osteoblasts; F=flattened cells; G=cement lines (Figure adapted from W.S.S.Jee (39))

6
osteoclasts secrete lysosomal enzymes that degrade the collagen matrix and hydrogen
ions which dissolve the bone mineral. After the osteoclasts resorb the bone there is
a reversal phase in which macrophages may appear to either complete resorption or
initiate the next stage of remodeling, the formation phase. During formation,
osteoblasts replace the bone that was resorbed by the osteoclasts. For successful
bone formation the osteoblasts must be able to adequately replicate, differentiate,
and produce normal bone matrix. Since osteoblasts lay down bone on existing bone
spicules, it is important for there to be a template of unresorbed bone in the
resorption cavity on which the osteoblasts may build the new bone matrix.
Cortical bone remodeling is similar, in principle, to cancellous remodeling.
The osteoclasts first resorb a canal or tunnel which is then refilled centripetally by
osteoblastic apposition, forming a new osteon or secondary Haversian system (Figure
1.2).
The completion of the osteoclastic resorption phase in humans requires
approximately 21 days for cancellous bone and 24 days for cortical bone. The
osteoblasts then create a new structural bone unit in approximately 91 days and 124
days for cancellous and cortical bone, respectively. Therefore, the completion of a
cancellous BMU requires 112 days, and a cortical BMU requires 147 days (39).
In normal young adults, the resorption and formation phases are tightly
coupled and bone mass is maintained. However, there is a natural bone loss with
age which implies an uncoupling or imbalance in the phases of bone remodeling, with
an increase in resorption over formation. The slow age-dependent phase of bone

7
loss results mainly from impaired bone formation, where the osteoclasts create
resorption cavities of normal depth but the osteoblasts fail to refill them completely
(21.56). In other words, age-related bone loss occurs due to reduction in the rate of
bone formation with no change in the rate of bone resorption (39). Over time, the
uncoupled remodeling cycles can cause significant thinning of spicules of cancellous
bone until the osteoclastic resorption cavities penetrate the thickness of bone. These
resorptive perforations create discontinuities in the cancellous scaffold, resulting in
loss of mechanical integrity and strength of the bone.
Increased bone turnover (activation of new BMUs) can also result in net bone
loss. Postmenopausal accelerated bone loss is associated with a high rate of bone
turnover (80), where more osteoclasts are present and these cells create deeper
resorption cavities (21,56). Although there is more bone being layed down due to
the increased bone turnover, there is even greater bone resorption (38).
Drug induced osteoporosis may affect the normal remodeling cycle in a variety
of ways depending on the cellular actions of the drug. The drug can either increase
or decrease the number of osteoblasts or osteoclasts or affect the quality and quantity
of bone being layed down by osteoblasts. Cytotoxic drugs may also affect bone
metabolism indirectly by inducing gonadal damage, thus decreasing the levels of
circulating sex hormones. The mechanism of drug-induced bone loss is typically due
to diminished osteoblastic number, activity, or quality of bone.
The focus of this study is on the chemotherapy drug, Methotrexate, which
interferes with the replication of DNA within the cells. Faulty DNA replication

8
would decrease the number of osteoblasts and osteoclasts and would, therefore,
decrease the overall bone formation and resorption. Based on Methotrexate’s
mechanism of action, this drug would decrease the surface of bone lined by
osteoblasts and osteoclasts and reduce the overall bone turnover.
Involutional Bone Loss
Both men and women experience age-related bone loss. Bone mass continues
to increase until approximately the age of 30, then after a transient period of
stability, age-related bone loss begins. Women lose approximately 35% of their
cortical bone mass and 50% of cancellous bone mass over their lifetime, whereas
men lose only 23% and 33% of their cortical and cancellous bone, respectively (50).
Bone loss occurs in a biphasic pattern for both cortical and cancellous bone in
women; with a sustained slow phase that occurs in both sexes and a transient
accelerated phase that occurs in women after menopause.
For cortical bone, bone is lost at approximately 0.3 to 0.5 %/year and
increases with aging until it levels out late in life (50). For women, an accelerated
postmenopausal phase of cortical bone loss is superimposed on the slow phase to
increase bone loss to a rate of 2 to 3 %/year immediately after menopause. This
accelerated bone loss decreases exponentially to baseline slow phase levels after
about 8 to 10 years (50).
Cancellous bone loss starts to occur earlier in life than cortical bone loss.
Controversy exists as to the rate of cancellous bone loss. Some researchers claim a

9
linear decrease of 0.6 %/year (61), while others predict a curvilinear decrease of
2.4 % /year in women (11) and a linear 1.2 %/year formen (51). Because cancellous
bone has a greater surface area than conical bone, it is more metabolically active
and, therefore, more responsive to changes in external loading (exercise), mineral,
hormonal, and metabolic status. The effect of hormonal status is reflected in the
earlier onset of bone loss for cancellous bone and the more rapid rate of cancellous
bone loss following menopause compared to cortical bone. This accelerated
cancellous bone loss, however, is shorter in duration than the accelerated
postmenopausal phase of cortical bone loss (61). In addition to involutional (age-
related) bone loss, drugs and diseases can exacerbate the bone loss by either
increasing the rate of bone loss, diminishing peak bone mass or both. In general, the
development of osteoporosis is related to two factors: (1) level of peak bone mass,
and (2) the rate of loss of bone after achieving peak bone mass.
Figure 1.3 graphically represents natural bone loss in humans. As discussed
above, peak bone mass occurs at the approximate age of 30 and slowly declines at
a constant rate due to age-associated changes. In women, an accelerated bone loss
occurs at menopause where the contributions of age and hormonal status combine.
Following this accelerated phase, bone loss resumes a slow decline until late in life,
while bone mass appears to stabilize at an osteopenic level. If a disease requires
drug therapy such as glucocorticoids, anticonvulsants, or chemotherapy (4,7,24,30,61),
the age-related bone loss is confounded by drug induced bone loss. As can be seen
in Figure 1.3, the bone fracture threshold is also an important consideration in the

BMD (g/cm
10
25 35 45 55 65 75 85 95
Age (years)
— Males ~~ Females
Figure 1.3 Involutional bone loss
(curves based on 1.2%/year)

11
bone density spectrum. This threshold represents the level of bone density at which
a person’s statistical risk for osteoporotic fractures increases. As depicted in Figure
1.3, the lower the peak bone mass and the greater the rate of bone loss the sooner
a person will reach this fracture threshold.
Mineral Regulating Mechanisms
Endocrine status, endogenous mineral balance, and mechanical loading
regulate both acquisition of peak bone mass and rates of bone loss and gain. The
following sections will discuss the effects of systemic mineral-regulating hormones,
growth-regulating hormones, sex hormones, and exercise on bone.
Mineral-Regulating Hormones
Bone is the reservoir for 99% of the body’s calcium. Therefore, hormones
which regulate calcium homeostasis are extremely important for the regulation of
bone mass. In addition to the hormones responsible for calcium metabolism,
adequate dietary intake is essential for healthy bones. Only 30% of ingested calcium
is absorbed into the system; therefore, the consumption of at least 800 mg of calcium
a day to prevent the bone reserve of calcium from being depleted is very important.
With age, less calcium is absorbed, and the daily intake of calcium should be
increased in order to maintain calcium homeostatis and healthy bones. Calcium is
the most common nutritional supplement given to postmenopausal and osteoporotic
patients. Studies continue to show, however, that calcium is not effective by itself in

12
preventing postmenopausal bone loss or reversing osteoporosis. Calcium intake may
be more important during the period of growth and development to allow
maximization of peak bone mass (37).
The goal of mineral-regulating hormones is to maintain physiologic serum
calcium, magnesium, and phosphate levels. The three main mineral-regulating
hormones are parathyroid hormone (PTH), calcitonin (CT) and 1,25 dihyroxyvitamin
D (calcitriol or 1,25D). These hormones act on three target tissues: bone, intestine,
and kidney. Table 1.2 supplies a summary of the actions of these mineral-regulating
hormones in the regulation of serum calcium (Ca) and phosphate (Pi) at the different
target tissues. Magnesium is not included in this table; its regulation by these
hormones is either nonexistent or inadequately defined. The role of calcitonin in
mineral regulation is modest compared to the effects of PTH and 1,25D.
Vitamin D is either obtained through dietary foodstuffs or absorbed from the
sun. In both cases this form of "raw"vitamin D is not active, and a series of enzymes
in the liver and kidney must manufacture the active form, 1,25D. PTH is secreted
by the parathyroid gland in response to low levels of serum calcium and 1,25D.
Calcitonin is secreted by the thyroid gland in response to hypercalcemia. The actions
and interaction of these hormones maintain mineral homeostasis.
Ca and Pi enter the blood through the intestine, leave it through the kidney,
and are stored in the bone. In order to maintain homeostasis, the net absorption of
Ca and Pi in the intestine must be equal to the net excretion of these ions by the
kidney. The absorption of Ca and Pi depends on the dietary intake and the

13
Table 1.2
Effects of Mineral-Regulating Hormones on Serum Calcium and Phosphate
Bone
Gut
Kidney
NET
Ca
Pi
Ca
Pi
Ca
Pi
Ca
Pi
PTH
t
t
t
Jr
t
i
CT
i
i
i
i
i
1.25D
T
t
t
t
t
t
t
KEY: PTH = parathyroid hormone
CT = calcitonin
1,25D = 1,25-dihydroxyvitamin D
Ca = calcium
Pi = phosphate
t = increase in serum mineral level when hormone acts on target tissue
i = decrease in serum mineral level when hormone acts on target tissue
«-* = no effect in serum mineral levels when hormone acts on target tissue
(Figure adapted from D.D. Bikle (6))

14
efficiency of absorption. Glomerular filtration of these ions in the kidney is relatively
constant, so the kidney reabsorbs Ca and Pi relative to the body’s needs. Bone
provides the major buffer for maintaining constant levels of Ca and Pi in the blood.
This is achieved by balancing bone formation (which deposits these ions in bone)
with bone resorption (which releases these ions into the bloodstream). PTH, 1,25D,
and calcitonin act on the intestine, kidney, and skeleton and maintain mineral
balance. 1,25D has the positive effect of increasing serum Ca and Pi levels by
increasing absorption in the intestines and increasing reabsorption in the kidney.
However, 1,25D in combination with PTH mobilizes Ca and Pi from bone. The net
effect of 1,25D is to increase both serum Ca and Pi.
PTH regulates serum Ca and Pi by stimulating the release of these ions from
the skeletal reservior and by stimulating Ca reabsorption and inhibiting Pi
reabsorption in the kidney. PTH also affects 1,25D levels by stimulating its renal
synthesis which, in turn, leads to increased intestinal absorption of Ca and Pi. The
net effect of PTH is to increase serum Ca and decrease serum Pi. PTH levels have
been shown to increase with age, which may partly explain age-related bone loss (45).
Chronically high PTH level (hyperparathyroidism) increases the number and activity
of osteoclasts (6). However, animal studies have shown that intermittent (pulsatile)
doses of PTH have the opposite effect, stimulating bone formation and resulting in
significant increases in cancellous and cortical bone (85,86). Human studies have
also confirmed that pulsatile PTH administration increases bone mineral density in

15
postmenopausal women and proves to be a promising prevention and treatment for
osteoporosis (17,47).
Calcitonin, although of less importance to mineral homeostasis than PTH and
1,25D, is a potent inhibitor of bone resorption by decreasing the activity and number
of osteoclasts. The secretion of CT is regulated by serum calcium levels. High
serum Ca stimulates CT secretion, which decreases the release of Ca to the blood
by bone resorption. Calcitonin is currently used for the treatment of osteoporosis.
Treatment of high turnover osteoporosis (postmenopausal) with calcitonin has been
shown to increase bone mass but its effects are less consistent and sustained than
estrogen therapy (60).
1,25D, PTH, and calcitonin act on the skeleton, kidney, and intestines to
regulate blood levels of Ca and Pi. Regulation entails control of how much comes
into the body from the diet, how much leaves the body through the kidney, and how
much is stored and released from the bone. The different hormones, ions, and target
tissues involved communicate and interact to ensure the precise regulation of these
important minerals.
Growth-regulating Hormones
Several systemic hormones associated with growth are important in the
development of peak bone mass and strength. These hormones include growth
hormone, glucocorticoids, thyroid hormone, and insulin. Growth hormone is a very
important determinant of skeletal mass and acts through changes in the production

16
of insulin-like growth factor 1 (IGF-1), which is a potent stimulator of skeletal growth
(10,60). Glucocorticoids have complex effects on bone metabolism. Excess
glucocorticoids produce bone loss primarily through suppression of osteoblast
function. They also inhibit intestinal calcium absorption leading to secondary
hyperparathyroidism and increased bone resorption. Glucocorticoids also promote
bone loss by inhibiting renal calcium reabsorption which may contribute to increased
PTH secretion (34). Thyroid hormones increase bone turnover but tend to stimulate
resorption more than formation, resulting in net bone loss. Insulin has been shown
to stimulate osteoblastic collagen synthesis at physiological concentrations and may
also affect calcium transport and vitamin D metabolism (60).
Sex Hormones
It has been well established that estrogen deficiency leads to the development
of osteoporosis. Accelerated bone loss has been noted in postmenopausal women
or after oophorectomy (43,54,79-82,84,85), as well as in ammenorrheic women
(19,57). Estrogen replacement therapy has been helpful in preserving bone mass
under estrogen deficient conditions in humans (18,36) and animals (29,80). A
similar link to osteoporosis has been noted in androgen deficient men (22). Upon
withdrawal of estrogen or androgen, bone turnover is increased, where bone
resorption is greater than bone formation, resulting in a net loss of bone mass.

17
Exercise
Another important factor promoting bone health is the presence of
mechanical stimulation which provides impetus for modeling, remodeling, and
mineralization (12,26,63-65,75). Weightbearing exercise, therefore, becomes an
important modality to preserve or increase bone mass. Weight-bearing exercise has
been shown to augment bone mass in athletes (41,46,49,78)and maintain or improve
bone mass in aging individuals and postmenopausal women (1,14,32,72). In fact, men
and women subjected to high intensity physical training have been shown to increase
their bone mineral content (BMC) and bone mineral density (BMD) between 5 and
20% when compared to age-matched sedentary controls (46.49). Some studies have
shown that exercise tends to be site specific, augmenting bone mass only in the
specific bones loaded during the exercise routine (41,78). Other researchers (32)
have found an increase in BMC in both appendicular and axial skeleton with a
combined exercise program which incorporated both weight-bearing aerobic exercise
and strength training.
As described in the previous paragraphs, bone homeostasis is affected by many
factors including natural aging, nutritional state, hormonal status, and activity level.
When the equilibrium is upset by alterations in body function due to disease, surgery,
or drug therapy, drastic changes in bone mass can occur.

18
Types of Osteoporosis
Osteoporosis can be classified into three types: (1) Type I or Postmenopausal,
(2) Type II or Senile, and (3) Type III or Therapy Induced (5). Type I and type II
osteoporosis result from either lack of estrogen at menopause (women) or from
natural aging (men and women), respectively. Type I osteoporosis affects mainly
cancellous bone with the most common fracture sites being the vertebrae (crush
fractures) and the distal radius. Type II osteoporosis affects both cancellous and
cortical bone with the most common fracture sites being the vertebrae (multiple
wedge fractures) and hip. Type III osteoporosis can result from chronic
administration of pharmacological agents which affect circulating levels of
parathyroid hormone, vitamin D metabolites, osteocalcin, calcium absorption, and
renal conservation of calcium. Type III osteoporosis can also occur from short- or
long-term treatment of cytotoxic (chemotherapy) drugs such as Adriamycin or
Methotrexate (4,31,52,59,73).
Problem Statement
Drugs are given to cure disease or improve the function of organ systems. In
the case of cancer, chemotherapy drugs are used to kill actively growing cells. While
cancer cells are usually actively growing and, therefore, affected by the
chemotherapy, the drugs do not discriminate between neoplastic and normal rapidly
growing cells. Chemotherapy, therefore, can have deleterious effects on non-targeted

19
tissue such as epithelial tissue (i.e. hair follicles, lining of the gut), hematopoietic
marrow cells, and bone cells, resulting in hair loss, gastrointestinal distress,
immunosuppression, and osteoporosis.
Osteoporosis is a silent disease, since the health of the skeleton is not
outwardly apparent and the symptoms are minimal until catastrophic fracture.
Chemotherapy may attend to the more obvious disease, cancer, while creating other
diseases in the process. The focus of this work is on the secondary disease,
osteoporosis, resulting from administration of the chemotherapy drug, Methotrexate.
This project will explore the pathophysiology and severity of Methotrexate-induced
osteoporosis using an animal model. The recovery of bone after withdrawal of drug
treatment will also be investigated. Histomorphometry, biomechanics, and dual¬
energy x-ray absorptiometry will be used to asess bone quantity and quality.
Research Objectives
MTX has been shown to have immediate adverse effects on bone turnover
and fracture healing using a rat model (23,24,35,74). However, administration of
more than one course of MTX and long-term bone recovery have not been
investigated. Therefore, the objectives of this study are outlined below:
(1) To study the short- and long-term effects of MTX on the skeleton by using 3
month old male Sprague-Dawley rats.

20
(2) To quantify the effects of MTX on bone characteristics at different times after
withdrawal of MTX treatment using histomorphometric, biomechanical, and
radiological methods.
(3) To verify the pathophysiology of MTX-associated osteoporosis proposed by
others.
(4) To propose changes in the existing chemotherapy protocols to prevent or treat
secondary osteoporosis.
Hypotheses
The Sprague Dawley rat will be used to test the following hypotheses:
(1) Two courses of Methotrexate will induce osteopenia and depress bone
formation 30 days following treatment as reported by others after one course
of Methotrexate (24).
(2) Osteoblast and osteoclast function will return to normal (age-matched control
levels) after the drug is cleared from the tissues at approximately 170 days.
(3) Although cellular function will be restored after 170 days, the osteopenia
resulting from Methotrexate influence will persist. Methotrexate will,
therefore, cause sustained loss of cancellous and cortical bone, bone mineral
density, and mechanical strength.

21
Delimitations
The following demarcations are recognized in this study:
(1) Only male Sprague-Dawley rats were used in this study.
(2) Two 5-day courses of Methotrexate chemotherapy were given to the rats.
(3) The quantity and quality of the skeleton was examined at three points in time
following completion of chemotherapy: 30, 80, and 170 days.
Limitations
The following weaknesses are acknowledged in this study:
(1) Animals were studied instead of humans.
(2) Longitudinal measures of bone quality and quantity were not made due to the
invasive nature of some of these measurements.
(3) Complete clearance of methotrexate from bone was not achieved at 170 days,
therefore, recovery was not fully recognized.

CHAPTER 2
REVIEW OF THE LITERATURE
Cancer is a far-reaching disease with often tragic outcomes. Cancer cells can
arise in any body tissue, at any age. Cancer cells can invade local tissues by direct
extension or they can spread throughout the body by way of lymphatic or vascular
channels. The size and scope of cancer disease is overwhelming. Approximately 56
million Americans will be diagnosed with cancer — this is approximately 1 in 4.
These statistics make it extremely likely that each of us will face the disease at some
point in our life, either personally or through the care of a loved one. Although two
thirds of those diagnosed with cancer will die of their disease, the percentages of
survivors are increasing due to improved efficacy of adjuvant therapies such as
radiation therapy and chemotherapy.
Chemotherapy agents, although often effective in destroying and controlling
neoplastic cell growth, have many adverse side effects on normal tissue and body
function. Many drugs cause nausea, vomiting, alopecia, cardiotoxicity, anorexia,
myelosuppression, renal damage, liver toxicity, and osteopenia. The primary interest
of this investigation concerns the chemotherapy drug Methotrexate and the adverse
effects of this drug on the skeleton.
Methotrexate (MTX) is a common antineoplastic agent used to treat acute
lymphoblastic leukemia, choriocarcinoma in women, breast carcinomas, testicular
22

23
carcinomas, head and neck carcinomas, osteosarcomas, chondrosarcoma,
fibrosarcoma, liposarcoma. lymphosarcoma, Hodgkin's disease, lung cancer
(squamous and small cell types), and, at lower doses, severe psoriasis and rheumatoid
arthritis. MTX is classified as an antimetabolite and is a folic acid analog. It
competitively inhibits the enzyme dihydrofolate reductase. This enzyme, involved in
protein synthesis, catalyzes a reaction to convert nucleic acids to DNA (see Figure
2.1). Therefore, MTX inhibits DNA synthesis by depleting the cell of the DNA
building blocks. MTX is carried by the blood and about 90% is filtered out of the
bloodstream within 48 hours. However, a large percentage of the drug remains in
the tissues where it was captured and its effects are very long-lasting and often
considered irreversible, especially with chronic MTX therapy (35).
MTX has been shown to have an adverse effect on the skeleton in both
humans and animals. The following sections will detail existing research in this area,
both clinical and animal. Methods to quantify bone changes will be reviewed.
Clinical Research
Nesbit et al. (52) studied children with acute lymphocytic leukemia who were
treated with high doses of MTX. The most apparent toxicity with these children
were associated with pulmonary inflammation (pneumonia) as well as inflammation
of bladder, vaginal, and pleural epithelium. Ulceration of the gastrointestinal tract
was also noted. Chronic administration of MTX resulted in liver cirrhosis and
osseous changes. MTX associated hepatotoxicity (16) and osteoporosis (59) has also

24
Nv^ycM>',:vv ^
COOM
I
CM»
C H,
I
CONM - CM
FOLIC ACID
DIHYDROFOLATE
REDUCTASE
N "
-"•//Nj/nX
NH > n CM a
COOM
I
CM i
I
CM ,
I
COMM — CM
I
COOM
METHOTREXATE
DIHYDROFOLIC ACID
TETRAHYDROFOLIC ACID
I
PURINE SYNTHESIS
THYMIDYLIC ACID SYNTHESIS
I
DNA
Figure 2.1 Mechanism of action of Methotrexate

25
been reported by others. Approximately 20% of Nesbit’s patients receiving chronic
MTX treatment experienced bone pain and/or fractures associated with osteopenia;
the researchers attribute these osseous changes to MTX induced abnormalities in
calcium metabolism (hypercalcemia).
Atkinson et al. (4) studied children with acute lymphoblastic leukemia in
attempts to elucidate the physiologic mechanism leading to osteoporosis. It was
uncertain if the cancer itself was affecting the bone mineral or if the chemotherapy
used to treat the disease was affecting the gastrointestinal and renal handling of
nutrients, causing alterations in mineral homeostasis and leading to abnormal
turnover of bone mineral and osteoporosis. The chemotherapy protocols in this study
used a combination of the following drugs over a 24 month period; prednisone,
vincristine, L-asparaginase, methotrexate, 6-mercaptopurine, and doxorubicin. This
study provided evidence that the chemotherapy protocol mentioned above caused an
imbalance in the mineral homeostasis including hypomagnesium, hypocalcemia, and
hypoparathyroidism. These abnormal calcium and magnesium levels, caused by
chemotherapy, indirectly altered bone turnover.
Ragab et al. (59) reported on 11 children with acute lymphoblastic leukemia
being treated with MTX therapy for more than 6 months who developed severe bone
pain and/or fractures in their lower extremities. These children were diagnosed
radiographically with osteopenia. Four of the patients were withdrawn from MTX
therapy. Serial radiographs indicated improvements in bone density after 6 drug-free
months. In contrast to Nesbit’s study, these children did not experience MTX-

26
associated hypercalcemia. Ragab postulated that MTX alters either protein
metabolism and/or bone cell activity to induce osteopenia and speculated that these
changes are reversible.
Similar results were noted by Stanisavljevic and Babcock (73) in their review
of 37 children treated for leukemia with MTX. A high incidence of bone fractures
and bone pain were noted in these children. Fracture healing was delayed and non¬
unions were common. Those children taken off of MTX went on to achieve normal
fracture repair. Stanisavljevic and Babcock (73) surmised MTX inhibits osteogenesis;
however, this effect appears to be reversible. MTX binds to dihydrofolate reductase
months after a single MTX dose, yet it has been postulated that the free unbound
intra-cellular MTX inhibits DNA synthesis (13). If this is true, the cells affected by
MTX therapy would be capable of resuming normal function after MTX withdrawal.
Gnudi et al. (31) studied the bone mineral content of 59 osteosarcoma
patients treated with different doses of MTX using single photon absorptiometry.
They analyzed the radius at the mid-shaft (primarily cortical bone) and the distal
metaphysis (rich in cancellous bone). These researchers found a dose-dependent
reduction in bone mineral content at the cancellous-rich distal radius with higher
doses of MTX having a more detrimental effect. They concluded that high doses of
MTX or low doses over long periods of time may severely compromise bone mass
and strength. Long-term follow-up bone mineral content measurements after
withdrawal of MTX treatment were not reported for these patients.

27
Clinical research (4,52,73) has verified the detrimental effects of
chemotherapy, specifically MTX, on bone through documentation of fracture
incidence. More quantitative evidence of MTX’s effect on bone has been presented
in a recent study using single photon absorptiometry showing a reduction in bone
mineral content with MTX treatment (31). The mechanism of action of MTX on
bone and the permanancy on the osteoporotic effects, however, are unknown. Some
researchers (4,52) speculate the high incidence of fractures in MTX patients is due
to the drug’s affect on mineral homeostasis through either alterations in protein
metabolism or cellular activity. Other possible explanations for the high fracture
incidence associated with cancer and MTX treatment include (1) the neoplastic
disease itself weakening the bone (not related to MTX treatment); (2) inflammation
reaction due to MTX-associated cell necrosis affecting the bone matrix; (3)
malnutrition associated with abnormal absorption of nutrients in the intestines caused
by MTX; (4) decreased physical activity due to illness; or (5) a direct relationship to
the cellular alterations in osteoblasts and osteoclasts caused by MTX treatment.
Most clinical researchers speculate the osteoporotic effects are transitory
(59,73); however, they base this theory soley on fracture incidence and observations
of fracture repair. No longitudinal quantitative clinical studies have been conducted
to investigate the bone’s response over time to MTX treatment. It is uncertain if
bone recovers after chronic chemotherapy. If bone density remains depressed after
withdrawal of chemotherapy, this would decrease the peak bone mass the patient
would accrue in their lifetime, making them at risk for osteoporotic fracture

28
throughout their life. In order to further investigate the pathophysiology and the
time course of MTX-associated osteoporosis, animal models have been used to
facilitate quantification of bone characteristics through histomorphometry and
destructive biomechanical tests.
Animal Research
An abstract by Tross et al. (74) was the first to report the effects of the
chemotherapy drugs, Methotrexate (MTX) and Adriamycin (ADR), on bone turnover
and strength using a rat model. One 5-day course of daily chemotherapy injections
were given intraperitoneally, the animals were given fluorochrome label on day 7 and
13, and sacrificed on day 14 for histomorphometric and biomechanical assessment
of bone morphometry and strength. No changes in the torsional biomechanical
strength, stiffness, and energy absorbed at failure were noted for the drug treated
rats. However, significant decreases were noted in cancellous bone volume, osteoid
surface, and osteoblast surface in the drug treated animals compared to controls.
MTX and ADR treatment was not reported to affect osteoclast surface or mean
cortical thickness.
Freidlaender et al. (24) expanded Tross’s previous work into manuscript form.
As outlined in the previous paragraph, the researchers administered one 5 day course
of MTX and ADR to rats and, using quantitative histomorphometric techniques,
measured the bone’s response 2 weeks after initiation of the protocol. These
researchers found significant reductions in cancellous bone volume and bone

29
formation rate during this short-term study. This confirms the osteotoxic effects of
MTX and ADR immediately following acute administration, but does not determine
the ability of bone to recover from the drug insult.
Freidlaender’s group (23) continued their research into the skeletal effects of
MTX by evaluating fracture healing. In this study Sprague Dawley rats received a
transverse fracture of the femur using a bone saw. The fracture was fixed internally
with an intramedullary K-wire. Rats were divided into 3 groups: (1) control rats
which received no treatment following fracture fixation; (2) MTX treated rats which
received one 5-day course of MTX injections; and (3) Radiation treated rats which
were irradiated with 250 rad fractions for 10 days following surgery. Groups of rats
in each of the 3 treatment groups were sacrificed at 1,2,4, 8, and 12 weeks following
surgical fixation. Following sacrifice, bone harvest, and embedding procedures,
longitudinal sections were made through the callus. Callus formation was graded
based on the amount of repair present. Radiation and MTX treatments retarded
callus formation at all time intervals when compared to the control animals. MTX
treated animals also failed to regain femoral torsional strength following fracture
even at extended time periods (24 weeks). Similar results were also reported by
Hajj et al. (35) using a similar model where both bending strength and histological
grade of callus formation were significantly compromised in animals receiving weekly
injections of MTX compared to control animals. Burchardt et al. (8) also reported
suppressed bone/callus formation and reduced junction strength in chemotherapy
treated dogs in repair of segmental cortical non-unions.

30
Existing animal studies have elucidated the pathophysiology of MTX-
associated osteoporosis. MTX directly affects the cellular activity of osteoblasts as
observed by a significant decrease in bone volume, osteoblast surface, osteoid
surface, and bone formation rate (24,74). However, no study, animal or human, has
investigated the recovery of bone following completion of chemotherapy treatment.
All existing studies measure bone parameters at only one point in time after
treatment. It is unknown if the bone mass remains depressed indefinitely, slowly
recovers to normal levels, or recovers to osteopenic levels below the normal range.
Methods of Skeletal Assessment
Quantification of skeletal changes due to aging, menopause, exercise, or drug
treatment depends on the type of study (human or animal), resources available, and
desired accuracy of measurement. In vivo studies involving humans require
noninvasive means to assess bone changes unless skeletal biopsies are indicated.
Non-invasive methods commonly used to measure bone density include: (1) single¬
photon absorptiometry (SPA), (2) dual-photon absorptiometry (DPA), (3) dual¬
energy x-ray absorptiometry (DEXA), and (4) quantitative computed tomography
(QCT). DEXA, with it’s high accuracy, quick scan times, and low radiation dose is
the most popular method to assess bone density in humans (15,28,40,55,68). DEXA
has also proven to be accurate for either in vivo or ex vivo animal studies measuring
either whole skeleton or appendicular density (2,3,33,48,67). These radiographic
methods measure the amount of mineral in the skeleton but do not offer the

31
capability of evaluating bone architecture or structure, assessing actively mineralizing
bone surfaces, or measuring bone quality or strength. Invasive measurements supply
more information but require painful biopsies for human studies or animal sacrifice.
Methods commonly used to assess bone quantity and quality following animal
sacrifice are histomorphometry and mechanical testing. Histomorphometric
techniques enable quantification of cancellous bone volume, cortical bone area,
cortical thickness and bone surface lined with osteoblasts or osteoclasts (42). When
fluorescent compounds are used to label the actively mineralizing bone,
histomorphometry can also quantify mineralizing surface, mineral apposition rate,
bone formation rate, and longitudinal bone growth (25).
Whole bones, cortical sections, or cancellous bone blocks can be mechanically
tested to determine the failure load, ultimate strength and stiffness of the specimen.
Weight bearing activities apply a complex loading environment to the skeleton
consisting of bending, torsion, and compressive loads. Mechanical tests of long bones
are typically loaded either in torsion or bending. Compressive tests are also used for
testing cancellous blocks or vertebral segments.
Bone mass, assessed either through DEXA or histomorphometry, provides
important information concerning bone health. However, the quality or strength of
the bone is also very important. The amount of cancellous or cortical bone mass can
be depressed or the amount of bone mineral compromised and not affect the
structural strength of the bone due to compensatory changes in bone geometry.

32
Similarly, adverse changes in bone geometry can decrease the structureal strength of
bone without changes in bone mass or mineral content.

CHAPTER 3
MATERIALS AND METHODOLOGY
The following sections detail the experimental protocol concerning animal
care, bone histomorphometry, biomechanical testing, dual-energy x-ray
absorptiometry (DEXA), and statistical analysis.
Animal Care
The protocol for this experiment was approved by the University Animal Use
Committee to assure humane treatment of animals and prevent undo suffering.
Sixty-nine male Sprague-Dawley rats (120 days old) with an average body weight of
415 g were randomly assigned to 4 control groups and three drug treatment groups.
Six rats were selected for the baseline control group and nine rats were chosen for
three control groups to be sacrificed at 30, 80, and 170 days. Similarly, three drug
treatment groups were also randomly chosen with 12, 10, and 14 rats in the 30, 80,
and 170 day groups, respectively. More animals were used in the 170 day group in
anticipation of death from MTX toxicity. Following a 2 week acclimation period,
treatment was initiated and baseline control rats were euthanized. Methotrexate
(MTX) was administered intraperitoneally (i.p.) in a 0.5 ml bolus injection using a
dose of 0.75 mg/kg/day. This dose is comparable to approximately one-third the
daily dose used for humans (69,70). Two courses of MTX were administered to the
33

34
treated rats in a cycle involving 5 consecutive days of drug injections, followed by 9
drug-free days, followed by another 5 days of injections (5-ON/9-OFF/5-ON). Two
courses of chemotherapy were used in this protocol to provide consistency with
typical clinical multiple-course sequelae used with human patients. Control rats
received a 0.5 ml i.p. bolus injection of saline using the same injection cycle (5-
ON/9-OFF/5-ON) as the MTX-treated rats.
All rats were injected with fluorochrome compounds on 2 separate occasions,
15 mg/kg of Demeclocycline (Lederle Laboratories, Pearl River, NY) on the 14th
day prior to sacrifice and 15 mg/kg of Calcein (Sigma Chemical Co.,St. Louis, MO)
on the 7th day prior to sacrifice in order to monitor actively mineralizing bone
(dynamic histomorphometric parameters).
The rats were housed individually, with an ambient temperature of 24°-26°C
and a 12hr/12hr light/dark cycle. The activity of the rats was not monitored but was
limited to the confines of relatively small cages. The rats were fed Purina Rat
Laboratory Chow (St. Louis, MO) with 1.0% calcium and 0.9% phosphorus and
water ad libitum.
The rats were euthanized by i.p. injections of sodium pentobarbital
(100mg/kg). Bilateral femurs and tibiae as well as the 2nd and 3rd lumbar vertebrae
were harvested. The left tibia and femur and the vertebral segments were stripped
of all soft tissue. The tibia was sawed into 3 segments to allow infiltration of fixative:
the proximal third, middle diaphysis including the tibiofibular junction, and the small
distal segment which was discarded. The cortical bone of the anterior aspect of the

35
proximal tibia was shaved to expose cancellous bone tissue to the fixative solution.
The proximal and distal metaphyses of the femur were removed by cross sectional
saw cuts and discarded leaving the femoral diaphysis for analysis. All bone segments
were immediately fixed in 10% formalin-alcohol for future histomorphometric
analysis (see Appendix A). The right limb (tibia and femur) was disarticulated at the
hip, wrapped in saline soaked gauze and frozen at -70° C for future biomechanical
analysis. DEXA evaluation was performed on all excised bones prior to histological
and biomechanical testing.
Bone Histomorphometrv
The right tibia and femur segments were transferred from 10% formalin-
alcohol to 70% ethyl alcohol (ETOH) two days after harvest. The proximal tibia was
chosen over the femur for cancellous histomorphometry evaluation due to the ability
to standardize the sampling area relative to the growth plate. The tibial diaphysis
proximal to the tibiofibular junction and the mid-diaphysis of the femur were used
for analysis of cortical bone.
Cancellous Bone
The proximal tibia was prepared for undecalcified cancellous bone
histomorphometry through dehydration in graded solutions of ETOH (70%, 95% and
100% for at least two days at each concentration) and xylene (for one day). The
sample was then infiltrated with a series of solutions containing methyl methacrylate,

36
dibutyl phthalate, and benzoyl peroxide. The recipes and detailed procedures for
methyl methacrylate embedding are included in Appendix A. The methyl
methacrylate solution was polymerized and the anterior aspect of the embedded
proximal tibia specimen was ground flat using a dental grinding wheel (Buffalo
Dental Manufacturing Co. Inc., Syosset, NY) to approximately one-third the depth
of the metaphysis. Thin longitudinal sections, 4 and 8 micrometers thick, were then
cut using an AO Autocut/Jung 1150 microtome (Cambridge Instruments, West
Germany). The thinner sections were placed on slides and stained according to the
Von Kossa method with a tetrachrome counterstain (Polysciences, Inc., Warrington,
PA). The recipe and procedure for the modified Von Kossa stain is included in
Appendix B. The 8 /xm sections were left unstained and illuminated under ultraviolet
light to analyze the fluorescent labels for cancellous dynamic histomorphometry.
Two bone sections of the proximal tibia from each animal were analyzed for
both static and dynamic bone parameters. The standardized sample site (3mm
square window) was taken approximately 1 mm distal to the growth
plate/metaphyseal junction. The following static histomorphometric measurements
(25) were made on the 4 /xm sections:
(1) Cancellous Bone Volume -Cn.BV/TV (%) -Cn.BV/TV is the percentage of
cancellous bone tissue (bone, marrow, and unmineralized osteoid) composed
of mineralized bone matrix.

37
(2) Osteoclast Surface - Cn.Oc.S./BS (%) - Cn.Oc.S./BS is the percentage of
cancellous bone surface with osteoclasts (bone resorbing cells) present and is
considered an index of bone resorption.
The following dynamic bone measurements (25) were made on the unstained, 8 ¿zm-
thick sections under ultraviolet illumination to enable observation of the
fluorochrome labeling:
(3) Longitudinal Bone Growth - Cn.LBG (fim/day) - Cn.LGB is the mean
distance between final fluorochrome label (Calcein) and the growth
plate/metaphyseal junction divided by the time between last label injection
and sacrifice.
(4) Mineralizing Surface - Cn.MS (%) - Cn.MS is the percentage of cancellous
bone surface with double fluorochrome labels and is an index of bone
formation.
(5) Mineral Apposition Rate - Cn.MAR (¿xm/day) - Cn.MAR is the mean
distance between the two fluorochrome markers divided by the time interval
between administration of the labels and is an index of osteoblast activity.
(6) Bone Formation Rate - Cn.BFR/BS (/xm3/¿xnr/day) - Cn.BFR/BS is
calculated by multiplying mineralizing surface by mineral apposition rate and
is an index of bone turnover.

38
Cortical Bone
Cortical bone segments of the tibia and femur were dehydrated in serial
solutions of 70%, 95%, and 100% ETOH and acetone. The bone segments were
then embedded in bioplastic (TAP Plastics, Inc., Dublin, CA) as described in
Appendix D. The bone blocks were then sectioned to 100 ¿mi using a Bueler Isomet
low-speed bone saw (Lake Bluff, IL) with a diamond chip blade (#801-137 LECO
Corp., St. Joseph, MI). The sections were mounted on slides for analysis. The
following static bone measurements were made:
(1) Total Bone Tissue Area - Ct.T.Ar (mm2) - Ct.T.Ar is the area within
periosteal perimeter which includes cortical bone and marrow.
(2) Marrow Area - Ct.Ma.Ar (mm2) - Ct.Ma.Ar is the area within the
endocortical perimeter which contains marrow.
(3) Cortical Bone Area - Ct.Ar (mm2) - Ct.Ar is the area of only cortical bone
(marrow area subtracted from the total bone tissue area).
(4) Mean Cortical Width - Ct.Wi(mm) - Ct.Wi is the average width or thickness
of the cortical bone sampled at 12 positions around the circumference.
(5) Polar Moment of Inertia - J (mm4) - J is the geometric property which is
calculated by modeling the cross-section of the bone as a hollow ellipse and
is represented by the following equation:
J = [7T(ab3+a3b-(a-t)(b-t)3-(a-t)3(b-t))]/4
where a is the minor axis of the ellipse, b is the major axis of the ellipse, and
t is the mean cortical thickness (Ct.Wi) of the cross-section.

39
Under ultraviolet illumination the following dynamic bone parameters were
measured:
(6) Periosteal Mineralizing Surface -Ps.Ms(%) - Ps.MS is the percentage of the
cortical periosteal surface with double fluorochrome labels and is an index of
bone formation.
(7) Periosteal Mineral Apposition Rate - Ps.MAR (pim/day) - Ps.MAR is the
average distance between the two fluorochrome markers divided by the time
interval between administration of the labels and is an index of osteoblast
activity on the periosteal surface.
(8) Periosteal Bone Formation Rate - Ps.BFR (/zm3/^m:/day) - Ps.BFR/BS is
calculated by multiplying periosteal mineralizing surface by the periosteal
mineral apposition rate.
Quantification of Bone Parameters
Two methods were used to quantify the cancellous and cortical bone
parameters listed in the previous section. The static bone parameters were measured
using a Vidas imaging system (Kontron Electronics, West Germany) and customized
software programs. The dynamic bone parameters were measured using a bone
histomorphometry package by Bioquant (R & M Biometrics Corp., Nashville, TN).
Previous work has shown the Vidas imaging system to be more consistent and
reliable than the Bioquant hand digitizing system for static histomorphometric
measurements (76). The Vidas system was not used for dynamic histomorphometric

40
measurements due to the absence of proper fluorescent filters to enable viewing of
the demeclomycin and calcein labels.
The Vidas imaging system consisted of a Zeiss Axiophot (Zeiss, West
Germany) microscope, Hamamatsu C2400 high resolution black and white CCTV
video camera (Hamamatsu City, Japan), and 386 IBM-based computer with expanded
memory capabilities. The cancellous and cortical bone sections were imaged with the
video camera and the pixel information of these images were stored on the 386
microcomputer. The density contrast between the dark stained bone spicules and the
lighter blue stained marrow allowed semi-automatic differentiation between the bone
and marrow for calculation of cancellous bone volume. The cortical cross-sections,
however, were not stained and the periosteal and endocortical perimeters were
digitized to calculate bone areas. Cortical thickness and polar moment of inertia
were also calculated by a special program written to identify the geometry of the
cross section and sample, at 30° increments, the cortical width to calculate mean
cortical width and major and minor diameters for the elliptical model. The computer
codes written to make all cancellous and cortical calculations are presented in
Appendix D.
Dynamic parameters were measured with the Bioquant Bone Morphometry
System. Surfaces of interest on the bone were viewed with a Nikon Labophot
microscope (Nikon, Inc., Garden City, NY) and digitized with a Hipad digitizing
tablet (R & M Biometrics Corp., Nashville, TN). A camera lucida enabled the
digitizing cursor to be visible in the microscopic field of view. An interface between

41
the digitizing tablet and an Apple He microcomputer (Cupertino, CA) allowed the
transfer of bone surface lengths to the Bioquant software where calculations were
performed to obtain the desired histomorphometric parameters.
Biomechanical Testing
The right tibia and femur, complete with surrounding soft tissue, were stored
at -70°C following harvest. Bones were thawed slowly to 25°C, cleaned of soft
tissue, and saturated in saline solution and kept chilled until testing. DEXA scans
were made of these bones immediately after removing the soft tissue and were
biomechanically tested the next morning.
The distal and proximal metaphyses were embedded in low temperature metal
potting material in a specially designed mold. When the metal was poured into the
mold, it produced a metal block surrounding the bone ends which fit into the torsion
tester’s grip. The distance between the proximal and distal ends were standardized
to provide tibia and femur gauge lengths of 23.6 mm and 20.75 mm, respectively.
See Figure 3.1 for a photograph of a femur and tibia with bone ends embedded in
low melting point metal.
The right tibia and femur from each animal were tested to failure in external
torsion about the long axis of the bone on a rapid loading torsional testing machine
(A.H. Burstein, Cleveland, OH) at a loading rate of 12 rad/sec (9). Torsional tests
were selected for mechanical assessment because these tests apply uniform torque

42
Figure 3.1 Photograph of femur and tibia with ends
embedded in low melting point metal

43
along the bone length, allowing failure to occur at the weakest part of the test
segment.
Raw data, including torque applied (N m) and angular displacement (degrees)
at failure, were collected at 1000 Hz per channel and subsequently stored on a 386-
IBM microcomputer through an analog to digital interface with the torsional testing
machine. The cross-sectional geometry of the bone was analyzed in order to
calculate the polar moment of inertia and the torsional strength. The cross-sections,
therefore, were taken immediately adjacent to the torsional fracture. Cross-sections,
approximately 1000 pan in thickness, were prepared from undecalcified bone using
a Unimat-SL circular saw (model #DB200, American Edelstaal, Inc.,NY, NY). Two
cross-sections were cut from each bone and glued with cyanoacrylate to glass slides.
These sections were then ground flat to approximately 300 pan using a Dremel Moto-
tool (model 285, Emerson Electric Co.,Racine, WI). These cross sections were then
analyzed with a Vidas imaging system, as mentioned in the previous
histomorphometry section, to obtain the following parameters:
(1) Total Bone Tissue Area (mm2) = At
(2) Marrow Area (mm2) = Am
(3) Cortical Bone Area (mm2) = Ac
(4) Mean Cortical Thickness (mm) = t
(5) Polar Moment of Inertia (mm4) = J

44
The following biomechanical parameters were either measured or calculated
using custom designed algorithms created for digital processing software (DADisp,
DSP Development Corporation, Cambridge, MA):
(1) Torque at failure (N m) = Tu
(2) Twist angle at failure (degrees) = 0U
(3) Energy absorbed at failure (N degrees) = Eu
(4) Stiffness (N/degrees) = S
(5) Torsional strength at failure (N/m2) = ru
Tu and eu were measured directly during testing. Tu was plotted against 0U to
calculate Eu and S which represent the area under the torque-angle curve and the
slope of the elastic region of the torque-angle curve, respectively. The biomechanical
parameters are represented in graphical form in Figure 3.2. Due to the lamellar
nature of cortical bone in the rat, there is minimal plastic deformation, therefore, a
linear regression from the minimum torque and angle to the maximum torque and
angle was used to determine stiffness. tu was then calculated using the hollow
ellipse model (20,62), where
ru = Tu b/J
and the polar moment of inertia,
J = [7r(ab3+a3b-(a-t)(b-t)3-(a-t)3(b-t))]/4

Torque (N*m)
45
Figure 3.2 Graphical depiction of biomechanical parameters

46
where Tu is the torque at failure, a is the minor axis of the ellipse, b is the major axis
of the ellipse, and t is the mean thickness of the cross-section calculated from 12
positions around the circumference of the ellipse.
Dual Energy X-Ray Absorptiometry
Ex-vivo measurements of bone mineral density were made for the excised
femurs, tibiae, and vertebrae using a dual energy x-ray absorptiometer (DEXA)
(Lunar Radiation, Madison, WI). The DEXA, with special high resolution software
supplied by the company, is capable of accurately scanning small bone samples (3,48).
The DEXA machine was calibrated prior to each use with a standard calibration
block consisting of known density materials. Repeatability tests were run using
excised rat tibiae and femurs to determine the coefficient of variation for this
procedure. The results from these repeatability tests are presented in Appendix E.
The cortical bone segments of the left tibial and femoral diaphyses, which
were stored in 70% ETOH for histomorphometric analyses at the time of scan, were
positioned on a 2 cm thick piece of acrylic with the anterior aspects of the bone
facing the scan sensor. The acrylic material was required in the scan area to provide
a contrast density, similar to tissue density, to the excised bones. The bones were
scanned side-by-side in the scanning area. The vertebral segments were scanned in
a manner similar to the left tibia and femur.
The intact right tibia and femur (which were kept moist and chilled in saline
baths and ice in order to prevent material degradation prior to mechanical testing)

47
were scanned in a saline filled petri dish on top of the acrylic contrast medium. The
bones were positioned and scanned in the same manner as the left tibia and femur.
The scans were analyzed by software provided by Lunar. The density scans
produced a pixel map of density gradients. The edges of the bone were detected by
an automatic algorithm. The edges were often misplaced by this algorithm and were
corrected by manual intervention. The bone mineral density (g/cnr) was calculated
for each excised bone.
Statistical Analysis
A two-way analysis of variance (ANOVA) was used to determine if drug
treatment (MTX or CTL) or time following treatment (30, 80, and 170 days) or the
interaction between drug treatment and time had significant effects on the response
variables which include the histomorphometric, DEXA, and biomechanical
measurements detailed in the previous sections. If significant interactions were
noted, a one-way ANOVA was run to determine the effects of drug treatment at
each time interval and time for each treatment group, separately. If differences
among groups were noted with the ANOVA, a Duncan’s multiple comparison
procedure was used to determine which groups were different from one another. A
significance level was set for all tests at the p = 0.05 level. PC-SAS version 6.03
(SAS, Inc., Cary, NC) was used for all the statistical analyses. A copy of the code
used for these analyses is presented in Appendix F.

CHAPTER 4
RESULTS
The weights of the animals were monitored every week throughout the study.
A plot of the weight changes with time are presented in Figure 4.1. Although the
methotrexate treated rats had lower body weights throughout the study, this
difference was not significant (p>0.05). There was a significant increase in weight
for both the MTX and CTL rats with time (p<0.05). Animals experienced no
noticeable side effects from the MTX treatment other than the slight decrease in
body mass.
The following sections present the results of the histomorphometric analysis
(cancellous and cortical bone), biomechanical testing, and dual-energy x-ray
absorptiometry on the bones of control and methotrexate treated rats. Abbreviations
of bone parameters are used when presenting the results and in the graphical
presentation. In order to make these abbreviations more readable, a glossary of
abbreviations is provided in Appendix G for quick reference.
48

Weight (kg)
49
CTL + MTX
Figure 4.1 Rat weight changes with time

50
Bone Histomorphometrv
Cancellous Bone
The means and standard deviations for the cancellous bone parameters are
presented in Table 4.1. A graph of tibial cancellous bone volume (Cn.BV/TV) with
time in shown in Figure 4.2. The statistical results of the 2-way and 1-way ANOVAs
are summarized on the right-hand side of the graph. The results for all parameters
are presented in a similar manner in subsequent graphs. The 2-way ANOVA
indicated a significant interaction between treatment and time (p=0.003), therefore,
separate 1-way ANOVAs were run for treatment by time and time by treatment. As
indicated in the graph, CTL rats had significantly higher CN.BV/TV at 80 and 170
days. Peak Cn.BV/TV for MTX rats occurred at 30 days with significant decreases
after this point, whereas, the CTL rats’ peak Cn.BV/TV occurred at 80 days.
Figure 4.3 presents a graph of cancellous osteoclast surface (Cn.Oc.S), an
indicator of bone resorption. The MTX treated rats had significantly higher Cn.Oc.S
at 30, 80, and 170 days. Cn.Oc.S declined with age in both MTX and CTL rats.
Cancellous longitudinal bone growth (Cn.LBG) is presented in Figure 4.4. As
indicated in the statistical summary, there is not a significant interaction between
treatment and time. Therefore, the general effects of treatment (CTL or MTX) and
time (30, 80, and 170) are evaluated. In this case both treatment and time do have
a significant effect on Cn.LBG. The CTL rats had generally higher rates of
longitudinal growth than MTX and there was a general decline in longitudinal bone
growth with age in both groups.

51
The dynamic parameter of cancellous mineralizing surface (Cn.MS), indicative
of active bone forming surface, is shown in Figure 4.5. CTL rats had significantly
higher Cn.MS at all measurement intervals. The Cn.MS was highest at 30 days and
decreased to lower levels at 80 and 170 days for both CTL and MTX rats.
Osteoblastic activity was quantified by cancellous mineral apposition rate
(Cn.MAR) in Figure 4.6. The osteoblastic activity between CTL and MTX rats was
significantly different only at 170 days, with CTL rat’s Cn.MAR exceeding MTX rats.
Again, there was an age-associated decline in Cn.MAR with highest values at 30 days
and decreasing to the lowest levels at 170 days for both CTL and MTX rats.
Cancellous bone formation rate (Cn.BFR), an index of bone turnover, is
presented in Figure 4.7. CTL rats had higher bone turnover at all time intervals (30,
80, and 170 days). Bone turnover was also age-dependent with highest levels
occurring at 30 days followed by decrease and plateau at 80 days for both CTL and
MTX animals.
Comparative photomicrographs of cancellous bone volume are presented in
Figures 4.8, 4.9, 4.10, and 4.11 for the 0, 30, 80, and 170 day time intervals,
respectively. Each figure presents the control animal’s bone in part "a"and the MTX
treated animal in part "b" for ease of comparison. Photomicrographs capturing the
fluorescent labels on the cancellous surface are shown in Figures 4.12,4.13,and 4.14
for the 30, 80, and 170 time intervals, respectively. Again, these figures are
composite photographs showing both the control and drug treated animal.

Table 4.1. Cancellous Histomorphometry
Treatment
Group
Cn.BV/TV
(%)
Cn.Oc.S
(%)
Cn.LBG
{¡x m/day)
Cn.MS
(%)
Cn.MAR
in m/day)
Cn.BFR/BS*10'2
(^m3//inr/day)
CO (n=6)
7.42±2.63
2.29±0.61
-
-
-
-
C30 (n=9)
8.94±2.49
1.43±0.29
26.29±4.52
26.89±6.86
2.37±0.51
64.42+25.1
C80 (n=9)
12.16±5.92
0.71±0.22
15.22±1.87
14.08±5.43
1.64±0.23
22.99±8.71
C170 (n=9)
10.10±3.11
0.39±0.15
8.36±1.89
9.99±4.39
1.57 ±0.46
15.70±8.18
M30 (n = 12)
9.17±3.62
2.08±0.60*
21,85±3.16*
8.9U3.86*
2.17±0.37
19.25±8.15*
M80 (n= 10)
5.61+3.21*
1,56±0.41*
12.23±1.79*
4.99+1.64*
1,44±0.38
7.16±2.64*
M170 (n=14)
4.10±1.62*
0.65±0.24*
5.19±0.65*
3.94±1.92*
0.85±0.09*
3.40±1.72*
NOTE: All values are mean ± standard deviation
* = significantly different from age-matched controls (p<0.05)
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
C170 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
Cn.BV/TV = cancellous bone volume
Cn.Oc.S. = cancellous osteoclast surface
Cn.LBG = cancellous longitudinal bone growth
Cn.MS = cancellous mineralizing surface
Cn.MAR = cancellous mineral apposition rate
Cn.BFR/BS = cancellous bone formation rate

Cn.BV/TV (%)
53
Time (days)
Statistics
trt x time: p = 0.003
trt:
CTL> MTX @80,170 (*)
time:
CTL: 80> 0,30,1 70
MTX: 30>80,170
Figure 4.2 Tibial cancellous bone volume

54
Time (days)
Statistics
trt x time: p = 0.05
trt:
MTX>CTL
@30,80,170 (*)
time:
CTL: 0>30>80,170
MTX: 30>80> 170
CTL
MTX
Figure 4.3 Tibial cancellous osteoclast surface

Cn.LBG (um/day)
55
Time (days)
Statistics
trt x time: p = 0.643
LUl p = 0.0001
CTL> MTX (*)
time: p = 0.0001
30> 80>170
CTL
MTX
Figure 4.4 Tibial cancellous longitudinal bone growth

Cn.MS (%)
56
Time (days)
Statistics
trt x time: p = 0.0003
trt:
CTL>MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80,170
CTL
MTX
Figure 4.5 Tibial cancellous mineralizing surface

Cn.MAR^um/day)
57
Time (days)
Statistics
trt x time: p = 0.045
trt:
CTL> MTX
@170 (*)
time:
CTL: 30>80,170
MTX: 30>80> 1 70
— CTL
♦ MTX
Figure 4.6 Tibial cancellous mineral apposition rate

58
Statistics
trt x time: p = 0.0001
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80,170
— CTL
MTX
Figure 4.7 Tibial cancellous bone formation rate

Figure 4.8 Photomicrograph of baseline cancellous bone volume

(a) control
Figure 4.9 Photomicrographs
(b) methotrexate
of cancellous bone volume at 30 days

(a) control
(b) methotrexate
ON
Figure 4.10 Photomicrographs of cancellous bone volume at 80 days

ON
to
(a) control
(b) methotrexate
Figure 4.11 Photomicrographs of cancellous bone volume at 170 days

Figure 4.12 Photomicrographs of fluorescent labels on cancellous bone surfaces at 30 days

(a) control (b) methotrexate
Figure 4.13 Photomicrographs of fluorescent labels on cancellous bone surfaces at 80 days

(a) control
(b) methotrexate
o\
Figure 4.14 Photomicrographs of fluorescent labels on cancellous bone surfaces at 170 days

66
Cortical Bone
The means and standard deviations for the cortical bone parameters are
presented in Table 4.2 and 4.3 for the femur and tibia, respectively. Figures 4.15 and
4.16 present the cortical total tissue area (Ct.T.Ar) as a function of time for the
femur and tibia, respectively. For both bones, CTL had significantly higher Ct.T.Ar
than MTX at 170 days but equivalent bone areas at prior measuring times. The
Ct.T.Ar steadily increased with time in the femur for both CTL and MTX animals.
The Ct.T.Ar of the CTL rats’ tibia also followed a similar steady increase, however,
the MTX rats increased only until the 80 day time point and then experienced a
drastic decrease in Ct.T.Ar to baseline levels.
The marrow area (Ct.Ma.Ar) of the femur and tibia are presented in Figures
4.17 and 4.18, respectively. For both bones the MTX animals had larger marrow
areas than the CTL animals when taken over all time intervals. There was a general
trend for increased marrow areas with time in the femur. However, in the tibia, the
marrow area indicated a significant increase over baseline levels only at 170 days.
The cortical bone area (Ct.Ar) is the portion of the total bone tissue area
comprised of cortical bone. Figure 4.19 and 4.20 show the Ct.Ar for the femur and
tibia, respectively. The CTL animals had significantly greater femoral and tibial
cross-sectional cortical areas than the MTX rats at 30, 80, and 170 days. The femurs
of the CTL rats showed increases in Ct.Ar with time, whereas the MTX rats showed
no significant changes in Ct.Ar from baseline levels. The Ct.Ar of the tibia increased

67
with time for the CTL rats but only increased up to 80 days for the MTX rats at
which point a significant decrease in bone area was noted to below baseline levels.
The mean cortical width (Ct.Wi) of the femoral and tibial cross-section are
shown in Figures 4.21 and 4.22.respectively. Femoral Ct.Wi was significantly greater
for the CTL rats compared to the MTX rats at all time intervals. Femoral Ct.Wi
increased with age for the CTL rats yet didn't change for the MTX rats. Tibial
Ct.Wi was also significantly greater for the CTL compared to MTX rats at 30, 80,
and 170 days. Peak Ct.Wi occurred in the tibia at 80 days for both MTX and CTL
rats. The tibial Ct.Wi of CTL rats then stabilized, whereas, the Ct.Wi of the MTX
rats decreased to below baseline levels.
The polar moment of inertia (J) is presented in Figures 4.23 and 4.24 for the
femur and tibia, respectively. The CTL rats had significantly higher J-values than the
MTX rats at 170 days for both the femur and tibia. The J-value for CTL rats
experienced an age-related increase in the femur and tibia. The MTX rats had peak
femoral and tibial J-values at 80 days.
The dynamic cortical bone parameter, periosteal mineralizing surface (Ps.MS),
is presented in Figures 4.25 and 4.26 for the femur and tibia, respectively. There was
a significant difference in femoral Ps.MS between CTL and MTX rats with CTL
exhibiting higher Ps.MS values at 30, 80, and 170 days. There was an age-related
decline in femoral Ps.MS for both CTL and MTX rats with the MTX rats exhibiting
a greater rate of Ps.MS loss with time. The tibia demonstrated a significant

68
decrease in Ps.MS with time with the CTL and MTX rats exhibiting similar rates of
Ps.MS change.
Periosteal mineral apposition rate (Ps.MAR), an index of osteoblast activity,
is presented in Figures 4.27 and 4.28 for the femur and tibia, respectively. Although
there was not a significant interaction between treatment and time for the femur, the
CTL had significantly greater Ps.MAR than MTX treated animals and a decrease in
Ps.MAR with time. The statistical results from the tibia indicated that CTL animals
had higher Ps.MAR than MTX rats at all time intervals and a significant decrease
in Ps.MAR with time for both CTL and MTX animals.
Graphs for femoral and tibial periosteal bone formation (Ps.BFR) rate are
presented in Figures 4.29 and 4.30, respectively. Femoral Ps.BFR was significantly
lower for the MTX rats at all time intervals than the CTL animals and exhibited a
steady decline with time for both CTL and MTX animals. Tibial Ps.BFR was only
significantly lower for the MTX at the 30 day time interval; after that point both
CTL and MTX exhibited similar periosteal bone formation. Tibial Ps.BFR was
higher at 30 days for both MTX and CTL animals than at 80 and 170 days with a
rapid age-related decrease between 30 and 80 days.
Representative photomicrographs of cortical bone area for the femur and tibia
are presented in Figures 4.31 and 4.32.respectively, for the baseline control animals.
Photographs of the femoral and tibial cortical bone areas for the 30 day time interval
are presented in Figures 4.33 and 4.34. 80 day time interval in Figures 4.35 and 4.36.
and the 170 day time interval in Figures 4.37 and 4.38. Each figure presents the

69
control animal’s bone in part (a) and the MTX treated animal in part (b) for ease
of comparison. Photomicrographs capturing the fluorescent labels on the periosteal
cortical surface of the femur and tibia are shown in Figures 4.39 and 4.40 for the 30
day time interval, Figures 4.41 and 4.42 for the 80 day time interval, and Figures 4.43
and 4.44 for the 170 day time interval. Again, these figures are composite
photographs showing both the control and drug treated animal.

Trt
Grp
Limb
Ct.T.Ar
(mm2)
Ct.Ma.Ar
(mm2)
Ct.Ar
(mm2)
Ct.Wi
(mm)
J
(mm4)
Ps.Ms
(%)
Ps.MAR
(¿¿m/day)
Ps.BFR*10'2
(^m3//nn2/day)
CO
(n=6)
F
10.95
±0.49
4.22
±0.52
6.73
±0.21
0.718
±0.041
29.66
±2.71
-
-
-
C30
F
11.63
3.96
7.67
0.812
33.52
95.88
2.69
259.02
(n=9)
±0.735
±0.66
±0.52
±0.067
±4.46
±5.33
±0.36
±43.03
C80
F
12.12
4.24
7.87
0.812
36.29
86.99
1.99
173.77
(n=9)
±0.65
±0.50
±0.30
±0.036
±4.14
±8.57
±0.20
±26.88
C170
F
13.84
4.80
9.04
0.872
47.56
83.30
1.27
105.52
(n=9)
±0.91
±0.67
±0.34
±0.032
±6.48
±7.31
±0.16
±14.51
M30
F
11.09
4.08
7.02*
0.750*
30.73
89.80*
1.71*
154.17*
(n= 12)
±0.59
±0.49
±0.42
±0.056
±3.36
±5.51
±0.16
±19.84
M80
F
12.04
4.93*
7.10*
0.715*
37.10
66.96*
1.05*
70.34*
(n= 10)
±1.04
±0.64
±0.49
±0.029
±6.17
±8.87
±0.04
±9.29
M170
F
12.15*
5.28*
6.87*
0.675*
36.65*
55.77*
0.64*
35.82*
(n= 14)
±0.49
±0.48
±0.34
±0.040
—s .
±2.87
±14.40
±0.123
±9.71
NO
* -
= significantly different from age-matched controls (p<0.05)
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
Cl70 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
Ct.T.Ar = total cortical bone tissue area
Ct.Am.Ar = cortical marrow area
Ct.Ar = cortical bone area
Ct.Wi = cortical bone width
J = polar moment of inertia
Ps.Ms = periosteal mineralizing surface
Ps.MAR = periosteal mineral apposition rate
Ps.BFR = periosteal bone formation rate

Table 4.3 Tibial Cortical Morphometry
Trt
Grp
Limb
Ct.T.Ar
(mm2)
Ct.Ma.Ar
(mm2)
Ct.Ar
(mm2)
Ct.Wi
(mm)
J
(mm4)
Ps.Ms
(%)
Ps.MAR
(/im/day)
Ps.BFR*10'2
(/im3//mi2/day)
CO
(n=6)
T
5.41
±0.28
1.04
±0.19
4.37
±0.22
0.745
±0.041
6.63
±0.80
-
-
-
C30
T
5.79
1.01
4.78
0.793
7.62
79.63
2.15
173.95
(n=9)
±0.33
±0.14
±0.28
±0.041
±1.00
±10.35
±0.50
±52.19
C80
T
6.25
0.93
5.33
0.883
8.86
34.34
1.23
44.10
(n=9)
±0.73
±0.38
±0.44
±0.053
±2.62
±16.34
±0.29
±26.02
C170
T
6.60
1.12
5.47
0.858
9.64
15.05
0.92
15.23
(n=9)
±0.35
±0.23
±0.20
±0.039
±1.20
±7.93
±0.28
±10.34
M30
T
5.58
1.15
4.43*
0.732*
7.19
71.92
1.41*
103.59*
(n= 12)
±0.33
±0.19
±0.25
±0.040
±0.99
±13.16
±0.16
±33.23
M80
T
6.12
1.26*
4.86*
0.769*
8.56
33.25
0.83*
27.51
(n= 10)
±0.39
±0.15
±0.33
±0.043
±1.14
±14.72
±0.15
±12.08
M170
T
5.50*
1.41*
4.09*
0.658*
7.19*
13.20
0.60*
8.78
(n= 14)
±0.36
±0.18
±0.29
±0.042
±0.94
±7.62
±0.20
±6.71
NOTE: All values are mean ± standard deviation
* = significantly different from age-matched controls (p<0.05)
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
Cl70 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
Ct.T.Ar = total cortical bone tissue area
Ct.Am.Ar = cortical marrow area
Ct.Ar = cortical bone area
Ct.Wi = cortical bone width
J = polar moment of inertia
Ps.Ms = periosteal mineralizing surface
Ps.MAR = periosteal mineral apposition rate
Ps.BFR = periosteal bone formation rate

Ct.T.Ar (mm
72
Statistics
trt x time: p = 0.002
trt:
CTL>MTX
@170 (*)
time:
CTL: 170>80,30,0
MTX: 170,80 >30
CTL
MTX
Figure 4.15 Femoral total bone tissue area

Ct.T.Ar (mm
73
7.5
7
6
5.5
5
4.5
0 50 100 150
Time (days)
Statistics
trt x time: p = 0.0005
trt:
CTL> MTX
@170 (*)
time:
CTL: 1 70,80>30,0
MTX: 80>30,170
— CTL
♦- MTX
Figure 4.16 Tibial total bone tissue area

UlUJ) JV'BIARO
74
Time (days)
Statistics
trt x time: p = 0.283
trt:
p = 0.0012
MTX>CTL (*)
time:
p = 0.0001
170>80>30,0
CTL
♦ MTX
Figure 4.17 Femoral marrow area

Ct.Ma.Ar (mm
75
Time (days)
Statistics
trt x time: p = 0.349
trt:
p = 0.0001
MTX>CTL (*)
time:
p = 0.021
170>80,30,0
— CTL
MTX
Figure 4.18 Tibial marrow area

Ct.Ar (mm
76
0
50
100
150
Time (days)
Statistics
trt x time: p = 0.0001
trt:
CTL>MTX
@30,80,170 (*)
time:
CTL: 170> 80,30> 0
MTX: none
CTL
MTX
Figure 4.19 Femoral cortical bone area

Ct.Ar (mm
77
Statistics
trt x time: p = 0.0001
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 1 70,80>30>0
MTX: 80>30> 170
Time (days)
Figure 4.20 Tibial cortical bone area

Ct.Wi (mm)
78
Time (days)
Statistics
trt x time: p = 0.0001
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 1 70> 80,30>0
MTX:none
— CTL
♦ MTX
Figure 4.21 Femoral mean cortical bone width

Ct.Wi (mm)
79
0 50 100 150
Time (days)
Statistics
trt x time: p = 0.0001
trt:
CTL>MTX
@30.80,170 (*)
time:
CTL: 170,80>30>0
MTX: 80>30> 170
CTL
MTX
Figure 4.22 Tibial mean cortical bone width

J (mm
80
0 50 100 150
Time (days)
Statistics
trt x time: p = 0.0003
CTL>MTX
@170 (*)
time:
CTL: 170>80,30,0
MTX: 170.80>30
Figure 4.23 Femoral polar moment of inertia

J (mm
81
0 50 100 150
Time (days)
Statistics
trt x time: p = 0.019
trt:
CTL>MTX
@170 (*)
time:
CTL: 170,80> 30,0
MTX: 80> 170,30
CTL
MTX
Figure 4.24 Tibial polar moment of inertia

82
Statistics
trt x time: p = 0.0035
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80> 170
— CTL
♦ MTX
Figure 4.25 Femoral periosteal mineralizing surface

Ps.MS (%)
83
Statistics
trt x time: p = 0.704
trt: p = 0.337
time: p = 0.0001
30>80>170
CTL
MTX
Figure 4.26 Tibial periosteal mineralizing surface

Ps.MAR (um/day)
84
Statistics
trt x time: p = 0.07
ÃœH p = 0.0001
CTL>MTX (*)
time:
p = 0.0001
30>80> 170
CTL
MTX
Figure 4.27 Femoral periosteal mineral apposition rate

Ps.MAR ^um/day)
85
50 100
Time (days)
150
Statistics
trt x time: p = 0.015
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80,170
MTX: 30>80> 170
Figure 4.28 Tibial periosteal mineral apposition rate

Ps.BFR*10" (um /um /day)
86
Statistics
trt x time: p = 0.039
trt:
CTL> MTX
@30,80,170 (*)
time:
CTL: 30>80> 170
MTX: 30>80>170
Figure 4.29 Femoral bone formation rate

Ps.BFR*10 (um /urn /day)
87
Statistics
trt x time: p = 0.0024
CTL> MTX
@30 (*)
time:
CTL: 30>80,170
MTX: 30>80,170
— CTL
♦ MTX
Figure 4.30 Tibial periosteal bone formation rate

Figure 4.31 Photomicrograph of a femoral cross-section of a baseline control animal

Figure 4.32 Photomicrograph of a tibial cross-section of a baseline control animal

(a) control
(b) methotrexate
Figure 4.33 Photomicrographs of femoral cross-sections at 30 days

(a) control
(b) methotrexate
Figure 4.34 Photomicrographs of tibial cross-sections at 30 days

(a) control
(b) methotrexate
Figure 4.35 Photomicrographs of femoral cross-sections at 80 days

(a) control (b) methotrexate
Figure 4.36 Photomicrographs of tibial cross-sections at 80 days

(a) control
(b) methotrexate
Figure 4.37 Photomicrographs of femoral cross-sections at 170 days

VO
(a) control
(b) methotrexate
Figure 4.38 Photomicrographs of tibial cross-sections at 170 days

(a) control
(b) methotrexate
Figure 4.39 Photomicrographs of femoral periosteal mineralizing surfaces at 30 days

(a) control (b) methotrexate
Figure 4.40 Photomicrographs of tibial periosteal mineralizing surfaces at 30 days

(a) control
(b) methotrexate
Figure 4.41 Photomicrographs of femoral periosteal mineralizing surfaces at 80 days

(a) control (b) methotrexate
Figure 4.42 Photomicrographs of tibial periosteal mineralizing surfaces at 80 days

(a) control
(b) methotrexate
Figure 4.43 Photomicrographs of femoral periosteal mineralizing surfaces at 170 days

(a) control (b) methotrexate
Figure 4.44 Photomicrographs of tibial periosteal mineralizing surfaces at 170 days

102
Biomechanics
Tables 4.4 and 4.5 provide the means and standard deviations for the femoral
and tibial torsional biomechanical parameters at all time intervals. A photograph
displaying the spiral fracture typical of torsional failure is presented in Figure 4.45.
The graphs for breaking torque for the femur and tibia are presented in Figures 4.46
and 4.47, respectively. The statistical results for the femur indicate that there isa
general trend for increased breaking torque with time with the older rats (170 days)
having higher breaking torques than the younger baseline control rats (0 days). The
results from the tibial breaking torque, however, indicates a general increase in
breaking torque for the CTL rats over MTX rats.
The femoral and tibial twist angle (Figures 4.48 and 4.49) was significantly
increased at the 30 day point and decreased thereafter. There were no differences
between treatment groups.
The energy absorbed at failure for the femur and the tibia are presented in
Figures 4.50 and 4.51, respectively. The energy absorbed for the femur showed
variable results with the energy significantly higher at the 30 and 170 day intervals
(both CTL and MTX). The energy absorbed for the tibia was significantly greater
for the CTL animals than the MTX animals. Also there was significantly higher
energy at the 30 day interval than all other time points.
The graphs for femoral and tibial torsional stiffness are presented in Figures
4.52 and 4.53. There was no difference in stiffness between CTL and MTX animals

103
for either the femur or tibia. There was, however, higher stiffness recorded at the
80 day time point for the femur and the 80 and 170 day time point for the tibia.
Torsional strength, a parameter which combines the cross-section geometry
with the breaking torque, is presented in Figures 4.54 and 4.55 for the femur and
tibia, respectively. There were no significant treatment or time effects for either
bone.
The polar moment of inertia (J), measured adjacent to the torsional fracture,
in presented for the femur and tibia in Figures 4.56 and 4.57, respectively. The J-
value for the femur for the MTX animals was lower than the CTL animals at the 170
day point. There was also an increase in femoral J with time for both the CTL and
MTX animals. For the tibia, there was no significant difference between CTL and
MTX, however, there was a significant increase in J with time.

iaoie 4.4 remur biomechanics
Trt
Grp
Limb
(Nm)
9U
(degree)
Eu
(N deg)
S
(N/deg)
ru 102
(N/m2)
J
(mm4)
° 'll1
us
F
3.41
±0.47
3.17
±1.39
5.64
±3.10
1.29
±0.42
27.60
±2.98
29.78
±2.63
C30
(n=9)
F
3.93
±0.69
5.72
±3.09
10.79
±5.24
0.932
±0.51
30.72
±4.89
31.08
±4.15
C80
(n=9)
F
4.17
±0.51
3.38
±1.59
7.19
±3.75
1.48
±0.56
31.25
±4.95
34.26
±5.92
C170
(n=9)
F
4.91
±0.66
4.61
±1.07
11.30
±3.14
1.16
±0.37
31.72
±6.87
42.06
±5.47
M30
(n=12)
F
4.18
±0.94
5.71
±2.35
12.39
±7.41
0.83
±0.38
33.64
±7.29
30.59
±4.20
M80
(n= 10)
F
4.30
±0.82
3.80
±1.10
8.09
±2.40
1.27
±0.55
29.88
±5.83
36.23
±3.84
M170
(n= 14)
F
4.87
±1.21
4.36
±1.74
11.21
±6.65
1.23
±0.39
34.41
±10.13
36.79*
±5.07
* -
= significantly different from age-match controls
o
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
Cl70 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
Tu = torque at failure
0U = twist angle at failure
Eu = energy absorbed at failure
S = stiffness
ru = torsional strength at failure
J = polar moment of inertia

Table 4.5 Tibia Biomechanics
Trt
Grp
Limb
(NTm)
9U
(degree)
Eu
(N deg)
S
(N/deg)
T„ 102
(N/m2)
J
(mm4)
CO
(n=6)
T
2.62
±0.88
12.74
±3.77
16.09
±4.95
0.23
±0.10
60.69
±20.43
7.03
±0.51
C30
(n=9)
T
3.61
±0.52
16.19
±3.30
29.03
±6.81
0.23
±0.06
82.60
±9.86
7.33
±1.48
C80
(n=9)
T
3.85
±0.96
.10.29
±4.02
19.04
±6.30
0.45
±0.25
82.36
±20.60
8.05
±0.83
C170
(n=9)
T
3.41
±1.21
8.33
±5.51
16.62
±5.7
0.49
±0.15
68.61
±24.89
9.346
±2.39
M30
(n=12)
T
2.94
±0.59
13.02
±2.22
19.12
±5.09
0.23
±0.07
73.19
±13.94
6.50
±1.34
M80
(n= 10)
T
3.29
±0.57
8.47
±5.13
13.98
±7.95
0.51
±0.24
77.98
±16.99
7.17
±1.44
M170
(n= 14)
T
2.95
±0.74
8.84
±5.14
14.06
±10.38
0.42
±0.17
65.80
±18.07
8.09
±2.42
o
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
Cl70 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
Tu = torque at failure
6U = twist angle at failure
Eu = energy absorbed at failure
S = stiffness
ru = torsional strength at failure
J = polar moment of inertia

106
Figure 4.45 Photograph of typical fracture pattern following torsional test

Tu (N)
107
Time (days)
Statistics
trt x time: p = 0.863
trtl p = 0.632
time: p = 0.0019
170> 0
CTL
MTX
Figure 4.46 Femoral breaking torque

108
O 50 100 150
Time (days)
Statistics
trt x time: p = 0.937
trL p = 0.0213
CTL>MTX
time: p = 0.084
CTL
MTX
Figure 4.47 Tibial breaking torque

Ou (degrees)
109
Time (days)
Statistics
trt x time: p = 0.863
LEL p = 0.909
time: p = 0.005
30>0,80
— CTL
MTX
Figure 4.48 Femoral twist angle at failure

0U (degrees)
no
Time (days)
Statistics
trt x time: p = 0.489
tÚl p = 0.255
time: p = o.0026
30> 80,170
CTL
MTX
Figure 4.49 Tibial twist angle at failure

(N*deg)
in
Time (days)
Statistics
trt x time: p = 0.875
trtl p = 0.560
time: p = 0.0316
30,170>0
— CTL
♦ MTX
Figure 4.50 Femoral energy absorbed at failure

(N*deg)
112
Statistics
trt x time: p = 0.5515
trt: p = 0.0393
CTL> MTX (*)
time: p = 0.052
30>0,80,170
— CTL
♦ MTX
Figure 4.51 Tibial energy absorbed at failure

S (N/deg)
113
Time (days)
Statistics
trt x time: p = 0.602
trt: p = 0.514
time: p = 0.0133
80>30
CTL
MTX
Figure 4.52 Femoral torsional stiffness

S (N/deg)
114
Time (days)
Statistics
trt x time: p = 0.544
trt: p = 0.935
time: p = 0.0003
80,170>0,30
— CTL
♦ MTX
Figure 4.53 Tibial torsional stiffness

OUjW/n) "J,
115
O 50 100 150
Time (days)
Statistics
trt x time: p = 0.581
trt: p = 0.444
time: p = 0.473
- CTL
♦ MTX
Figure 4.54 Femoral torsional strength

T, (N/m2*10
116
Time (days)
Statistics
trt x time: p = 0.876
trt: p = 0.306
time: p = 0.069
— CTL
♦ MTX
Figure 4.55 Tibial torsional strength

J (mm
117
Time (days)
Statistics
trt x time: p = 0.05
tHi CTL> MTX
@170 (*)
time:
CTL: 170>0,30,80
MTX: 1 70,80>30
— CTL
♦ MTX
Figure 4.56 Femoral polar moment of inertia associated with torsional fracture

J (mm
118
Statistics
trt x time: p = 0.929
LfL p = 0.061
time: p = 0.021
170,80>30,0
— CTL
♦ MTX
Figure 4.57 Tibial polar moment of inertia associated with torsional fracture

119
Dual Energy X-rav Absorptiometry
The means and standard deviations for the DEXA obtained bone mineral
density (BMD) for the femur, tibia, and vertebrae are displayed in Table 4.6. The
BMD for the femur is presented in Figure 4.58 in graphical form. There is a gradual
increase of BMD with time for both the CTL and MTX groups. The statistical tests
indicate that the CTL rats have significantly higher BMD at 80 days. However, by
170 days the BMD of CTL and MTX rats are equivalent.
The BMD for the tibia is presented in Figure 4.59. There is not a significant
difference between CTL and MTX at any time interval. However, there is a
significant age-related increase in BMD for both CTL and MTX animals.
The vertebral BMD in presented in Figure 4.60. There is neither a significant
treatment nor time effect.

Table 4.6 DEXA Bone Mineral Density
Trt
Grp
Limb
BMD
(g/cm2)
Limb
BMD
(g/cm2)
Limb
BMD
(g/cm2)
CO
(n=6)
F
0.234
±0.012
T
0.193
±0.0082
V
0.255
±0.026
C30
(n=9)
F
0.225
±0.019
T
0.182
±0.019
V
0.259
±0.017
C80
(n=9)
F
0.249
±0.007
T
0.207
±0.008
V
0.260
±0.020
C170
(n=9)
F
0.263
±0.012
T
0.213
±0.011
V
0.253
±0.031
M30
(n= 12)
F
0.234
±0.017
T
0.187
±0.019
V
0.260
±0.026
M80
(n= 10)
F
0.243*
±0.009
T
0.203
±0.009
V
0.262
±0.020
M170
(n= 14)
F
0.264
±0.010
T
0.214
±0.015
V
0.239
±0.044
'JOTE: All values are mean ± standard deviation
* = significantly different than age-matched controls (p<0.05)
K>
o
KEY: CO = baseline control group
C30 = control group sacrificed at 30 days
C80 = control group sacrificed at 80 days
Cl70 = control group sacrificed at 170 days
M30 = methotrexate treated group sacrificed at 30 days
M80 = methotrexate treated group sacrificed at 80 days
Ml70 = methotrexate treated group sacrificed at 170 days
BMD = bone mineral density
F = femur
T = tibia
V = vertebrae

BMD (g/cm
121
Time (days)
Statistics
trt x time: p = 0.028
trt:
CTL> MTX
@80 (*)
time:
CTL: 170> 80>30,0
MTX: 170>80>30
CTL
MTX
Figure 4.58 Femoral bone mineral density

BMD (g/cm
122
Time (days)
Statistics
trt x time: p = 0.319
tjU p = 0.826
time: p = 0.0001
170>80>30,0
— CTL
♦- MTX
Figure 4.59 Tibial bone mineral density

BMD (g/cm
123
Time (days)
Statistics
trt x time: P = 0.611
till p = 0.621
time: p = 0.377
Figure 4.60 Vertebral bone mineral density

CHAPTER 5
DISCUSSION
As hypothesized in this study, Methotrexate had short- and long-term
detrimental effects on the rat skeleton. All cancellous bone parameters were
severely affected by the methotrexate treatment. Cancellous bone volume in treated
rats (Figure 4.2) was equivalent to levels in non-treated control rats at 30 days, but
experienced a 39% reduction between 30 and 80 days. Between 80 days and 170
days, the cancellous bone loss in the treated rats was probably due to age-related
mechanisms, as the rate of loss was similar to the rate in control rats. Reductions
in cancellous mineralizing surface (Figure 4.5) and bone formation rate (Figure 4.7)
at 30 days may have instigated the observed reductions in cancellous bone volume
(Figure 4.2) at 80 days. Friedlaender’s study (24) also reported significant reductions
(27%) in cancellous bone volume after one course of Methotrexate. However,
Friedlaender noted these changes at 30 days, whereas the effect ofMTX was delayed
in the present study, with significant reduction not seen until 80 days following
treatment. The cumulative MTX dose for both studies was the same. MTX
treatment was given over 10 days in the present study; in Friedlaeder’s study the
doses were given over 5 days. The differences in daily dose potency may possibly
account for the delay in cancellous bone loss observed in the present study.
124

125
Cancellous osteoblast function is measured histomorphometrically by
mineralizing surface and mineral apposition rate, which reflect the number and
activity of osteoblasts, respectively. A measure of net osteoblastic function, which
takes into account both mineralizing surface and mineral apposition rate, is
represented by bone formation rate. The number of osteoblasts was reduced by 67%
(mineralizing surface, Figure 4.5), yet their activity was close to normal, at least at
30 and 80 days (mineral apposition rate. Figure 4.6). The effect of MTX on mineral
apposition rate was not apparent until the 170 day point, when a 45% reduction was
noted. Bone formation rate was significantly reduced by 70% at 30 days in the MTX
treated animals (Figure 4.7). Bone formation in the MTX rats remained significantly
depressed at 80 and 170 days compared to their age-matched controls, however, the
rate of decline in bone formation was similar to that of the control animals. This
work confirms Friedlaeder’s (24) findings, which noted significant reductions in
cancellous mineralizing surface (49% reduction) and bone formation rate (58%
reduction) at 30 days.
The MTX treatment affected both the osteoblast and osteoclast cell lines.
The osteoblast function was significantly depressed by MTX (mineralizing surface,
mineral apposition rate, and bone formation rate), yet osteoclast population was
increased as reflected by the 31% to 54% increase in cancellous osteoclast surface
in the MTX treated rats (Figure 4.3). This work corresponds with Friedlaeder’s
findings (24), where these researchers found a slight (28%), but insignificant, increase
in osteoclast surface. Based on the mechanism of action of MTX, depletion ofDNA

126
building blocks, both osteoblast and osteoclast replication would theoretically be
reduced. Without osteoblasts, bone formation would diminish and without
osteoclasts, bone resorption would also be reduced. The cancellous bone
histomorphometric analysis confirmed the reduction in osteoblasts yet revealed a
slight but significant increase in osteoclast surface in the MTX treated rats (Figure
4.3). This may indicate that MTX affects the progenitor cells of the osteoblast and
osteoclast cell lines in different ways. Osteoblast precursors come from the
mesenchymal cell line, whereas osteoclast precursors are derived from hematopoietic
cells (39). The osteoblast mesenchymal precursor cells are located locally in the
bone marrow, whereas the osteoclast hematopoietic precursor cells are systemic and
migrate throughout the body via the bloodstream. This difference in osteoblast and
osteoclast progenitor cells may explain the different responses of these cells to MTX.
Nilsson et al. (53), through the radioactive labeling of calcium and proline,
discovered that MTX has a specific inhibiting effect on osteoblastic differentiation.
This finding would help to explain why MTX decreased osteoblast numbers and
function while not decreasing osteoclast numbers in the present study. The combined
effects of reduced cancellous bone formation and increased cancellous bone
resorption culminated in rapid bone loss and osteopenia.
Although cancellous bone is more metabolically active than cortical bone,
dramatic changes were also seen in cortical bone as a result of MTX treatment. Due
to the sluggish response of the less metabolically active cortical bone, the greatest
discrepancy in cortical bone between the MTX and control animals was not apparent

127
until the 170 day time interval (Figures 4.19 and 4.20). A 24% and 25% reduction
in cortical bone area was noted at 170 days for the femur and tibia, respectively.
Cortical bone loss occurred mainly from the endocortical bone surface but small
losses were also noted on the periosteal surfaces as reflected in the increases in
marrow area (Figures 4.17and 4.18),decreases in total bone tissue area (Figures 4.15
and 4.16), decreases in cortical bone area (Figures 4.19 and 4.20), and decreases in
cortical width (Figures 4.21 and 4.22). Although cancellous bone showed an age-
associated decline, the cortical bone area continued to increase over time, with peak
cortical area acquired at 170 days for the control animals and at 80 days for the
MTX treated animals. Existing studies which monitored the effects of MTX on bone
did not measure the cortical bone response. Therefore, comparisons to previous
work are not possible for cortical morphometry.
The static cortical bone parameters showed similar effects of MTX treatment
for the femur and tibia. However, the dynamic cortical bone parameters, reflecting
osteoblast function, behaved dissimilarly for the different bones. Periosteal
mineralizing surface was significantly reduced for the femur but not for the tibia
(Figures 4.25 and 4.26). The reduction in femoral periosteal mineralizing surface
ranged from 6.3% at 30 days to 33% at 170 days due to MTX treatment. Periosteal
mineral apposition rate was reduced for both the femur and tibia (Figures 4.27 and
4.28). A maximum reduction of 49% and 35% in periosteal mineral apposition rate
was seen at 170 days for the femur and tibia, respectively. Periosteal bone formation
was significantly reduced at all time intervals for the femur but only at 30 days for

128
the tibia (Figures 4.29 and 4.30). The reduction in bone formation on the
periosteum of the femur ranged from 40% at 30 days to 66% at 170 days. Tibial
periosteal bone formation was reduced by 40% at 30 days for the MTX treated rat
compared to controls. MTX treatment affected both the number of osteoblasts
(mineralizing surface) and their activity (mineral apposition rate) for the femur, yet
primarily affected osteoblast activity (mineral apposition) for the tibia. The most
dramatic detrimental effects on the bone dynamics of the tibia occurred at 30 days
with MTX influence subsiding after that time. The MTX effects on the femur were
more gradual and more sustained. Although the mechanisms of bone loss may have
been slightly different and at different rates for the femur and tibia, both bones had
significant cortical bone loss due to MTX treatment. As with the cancellous bone
volume, the decreases in cortical bone area due to MTX treatment can be explained
by the drug’s effects on osteoblast function as documented by reductions in periosteal
mineralizing surface, periosteal mineral apposition rate, and periosteal bone
formation rate. Osteoclast function could not be measured due to the added
thickness of cortical bone sections. However, osteoclastic number and/or activity was
probably increased on the endocortical surface since no fluorescent label was seen
on this surface and there was a significant increase in marrow area in the MTX
treated animals compared to controls.
The biomechanical results do not indicate any consistent significant changes
due to MTX treatment. There was a general trend for the tibia of the MTX treated
animals to have a lower breaking torque (between 13% and 18% reductions) and

129
energy absorbed at failure (between 15% and 34% reductions) than the control
animals (Figure 4.47 and 4.51. respectively). Theoretically, MTX treatment should
cause a decrease in all biomechanical parameters which were measured (breaking
torque, twist angle, energy absorbed, stiffness, and torsional strength).
Histomorphometry confirmed the loss of both cancellous and conical bone mass,
however, the torsional biomechanical tests failed to correlate this loss in bone mass
to compromises in overall bone strength. The failure of the mechanical tests to note
changes in bone strength due to MTX treatment could be attributed to the lack of
sensitivity of the torsional tests or to use of an inappropriate test based on the drug’s
effect on the skeleton.
The following factors may help to explain the lack of sensitivity of the
torsional tests:
(1) There was a large inherent variability between animals when measuring
torsional strength parameters. The variability (standard deviation) of torque,
twist angle, energy absorbed, stiffness, and torsional strength (Tables 4.4 and
4.5) was between 25% and 50% of the parameter’s mean. When the standard
deviation of a sample is half that of the mean, large differences between
group means must exist before statistical significance is recognized.
(2) Errors in preparing the specimen for testing may have added to the variability
of the results. Nicking the bone with the scalpel during the removal of soft
tissue could severely compromise the bone's torsional strength by creating
stress concentrations on the periosteal bone surface. Although extreme care

130
was taken not to perforate to bone surface, this could have occurred
unknowingly and compromised the strength of random bone samples.
(3) The proper embedding procedure for the bone ends was also critical for
accurate torsional assessment. A pure torsional load along the longitudinal
axis of the bone was desired and achieved only if the bone ends were
embedded in the center of the mold. Eccentricity would induce a combined
torsion/bending load and may affect the breaking torque and twist angle.
Care was taken not to induce bending moments through eccentric embedding.
However, slight bending moments were noted during testing of many
specimen. Those specimen with excessive bending moment were either re¬
embedded prior to testing or excluded from the results.
(4) Errors in the measuring device may also contribute to the variability of the
torsional assessment. Electrical noise in the output voltage for both torque
and twist angle were apparent even after low-pass filtering. The noise was
less than 1% of the signal for twist angle yet fluctuated between 1% and 8%
of the maximum and miniumum torque voltage, respectively.
(5) The processing of the raw torque and twist angle data to calculate the
torsional parameters may have induced errors contributing to the variability.
The raw signal was filtered with a digital low pass filter in order to eliminate
low frequency noise yet may have slightly decreased the maximum torque
value recorded. Stiffness was determined to be the slope (change in torque
divided by change in twist angle) of a linear regression run between the

131
minimum and maximum torque. However, stiffness is an instantaneous
parameter; the stiffness changes as torque and twist angle change. The bone
stiffness is usually greatest at the beginning of the test and decreases just prior
to failure. The change in instantaneous stiffness is illustrated in Figure 3.2
where the slope of the torque/angle curve changes with twist angle. Using
the linear regression simplifies the stiffness to an average throughout the
range of torques and twist angles during testing.
(6) Errors in torsional strength could have been produced by a non-representative
polar moment of inertia value. The calculation of the polar moment of
inertia was made adjacent to the spiral torsional fracture, however, in some
cases the failure produced a comminuted fracture and the geometry had to be
assessed more distant to the fracture site.
Torsional tests may not be the most appropriate biomechanical assessment for
the effect of MTX on bone. Torsional strength is based, almost exclusively, on mid-
diaphyseal cortical bone and primarily periosteal cortical bone. Although the
morphometric measurements of tibial and femoral cortical area were significantly
reduced for the MTX treated animals, the geometric property of polar moment of
inertia was not significantly different, except at 170 days (Figure 4.56 and 4.57).
Polar moment of inertia is a measure of the distribution of bone about the centroid
of the cross-section. Bone on the periosteal surface, being farther from the centroid,
is more important in the calculation of polar moment of inertia and contributes more

132
to the torsional strength and load at failure of the bone. Cortical bone loss due to
MTX occurred primarily on the endocortical surface. This pattern of bone loss
would minimally affect the torsional properties of the bone. Although there was
significant cortical thinning due to MTX treatment, greater bone loss would need to
occur before torsional strength would be significantly compromised. Perhaps a more
sensitive measure of changes in mechanical strength due to MTX treatment would
involve crush tests of cancellous bone, either in the proximal tibia or vertebrae.
The dual-energy x-ray absorptiometry results did not reveal consistent changes
in bone mineral density due to MTX treatment. However, the femur of the MTX
treated animals had a small (2.5%) but statistically significant lower bone mineral
density at 80 days when compared to the control animals (Figure 4.58). Dual-energy
x-ray absorptiometry has been used for both animal and human studies to document
changes in bone density. Raab et al. (58) states that even a 5.0% difference in bone
mineral density (BMD) would be hard to detect using DEXA due to the inherent 1 %
to 2% coefficient of variation of the machine and the large variation in bone mineral
density between subjects or animals within the same treatment group. The BMD
coefficient of variation for the machine and bone samples used for this study was
between 1.5% and 2.8% (Appendix E). Although, the femoral BMD was found to
be significantly different at 80 days between MTX-treated and age-matched control
animals, given the coefficient of variation of the measuring device, this statistical
difference would not reflect a valid difference between the groups.
Histomorphometry confirmed significant changes in bone mass, yet DEXA was not

133
able to pick up these differences in these small bone samples due to the lack of
precision of the instrument.
Clinical studies (52,59,73), with the exception of Gnudi et al. (31), have
documented MTX-induced osteopenia by citing fracture incidence and bone pain.
These indices, although important clinically, do not have the precision to quantify
bone changes resulting from drug treatment. Bone fracture is the inevitable endpoint
following months or years of drug-induced bone dissipation. Additionally, fracture
healing is not dependent on bone density. Gnudi et al. (31), in an attempt to
quantify bone changes, used single photon absorptiometry (SPA) to evaluate bone
mineral content at two positions in the forearm following MTX treatment in
osteosarcoma patients. SPA was used to document losses of bone mass which occur
during MTX treatment which may not be severe enough to cause immediate fracture
but would increase the patient’s risk of fracture in the future. Gnudi did find
significant differences in bone mineral content of humans on high doses of MTX.
A similar noninvasive radiographic method, dual energy x-ray absorptiometry, was
used in the present study to document changes in bone mass in a rat model.
Although histomorphometric analysis was able to identify changes in both cancellous
and cortical bone mass, DEXA failed to perceive these changes in the rat skeleton.
Clinical studies have suggested that, although methotrexate has been proven
to induce osteopenia, the detrimental bone effects are reversible after completion of
therapy without longitudinal clinical data to back up these hypotheses (59,73). This
prediction is based on radiographic evidence of normal fracture healing after MTX

134
withdrawal. As mentioned before, fracture healing is not a valid means to assess
bone quantity or quality since fractures heal even in the presence of profound
osteoporosis in adults. If the patients from these clinical studies (59,73) experienced
fractures due to MTX-induced osteopenia, the healing of the fracture does not
necessarily indicate the bone mass has improved. The results from the present
study, which used precise experimental methods to assess bone quality and quantity,
do not predict recovery from the MTX insult. Cancellous and cortical bone mass
and bone formation were depressed in the MTX treated animals even 170 days after
treatment. The osteoblastic activity did not show signs of recovery and bone mass
remained depressed. Insight into the observed lack of recovery from MTX treatment
may be found in the pharmacology of MTX.
The major fraction of the MTX dose is removed from the plasma within the
first hour in both rats (69) and humans (71,77). However, MTX is transported into
the tissues and is tightly bound to proteins within the tissue. Even at low MTX
doses, the membrane transport mechanism, shuttling MTX into the cells, becomes
saturated (69). The amount of MTX bound to the proteins within the tissue is
approximately the same for low and high doses of MTX. The amount of protein-
bound MTX within the cell is the determining factor for MTX cytotoxicity (66).
Scheufler et al. (70) determined the half-life of MTX in the liver, kidney, and bone
marrow to be very long (up to 23 days) in rats after a single high-dose of MTX (31
mg/kg). These researchers attribute this slow clearance of MTX in these tissues to
the strong binding of MTX to dihydrofolate reductase and the slow replacement of

135
poisoned cells by non-poisoned cells. This research would indicate that 93% of the
drug should be cleared from the bone marrow approximately 112 days after cessation
of treatment. Given that the last MTX treatment for the rats in the present study
occurred 19 days after the initiation of treatment, the drug should be cleared from
the bone marrow after 130 days. Therefore, the last time point (170 days) should
theoretically reflect the tissue’s response after 93% of MTX has been cleared from
the tissue. However, chronic treatment with MTX would tend to increase protein-
bound MTX and prolong tissue clearance. Although the daily dosage of MTX used
in the present study was relatively low, the duration of exposure to MTX was fairly
long (10 days). Chronic exposure to MTX would increase intracellular MTX more
than fewer exposures of higher MTX dose. The effects of MTX on cellular function
(DNA replication) is cumulative — the longer the exposure, the longer the recovery
from the MTX insult. It appears that in the present study, MTX had not cleared
from the bone marrow. The osteoprogenitor stem cells had not recovered by 170
days and, therefore, the histomorphometric indices of bone quantity and activity
remained depressed. Perhaps a longer interval between the cessation of MTX
treatment and skeletal assessment would provide adequate time for MTX clearance
and the return of normally functioning osteoprogenitor stem cells.
High doses of methotrexate are typically followed by leucovorin rescue in the
clinical setting in hopes of saving the normal functioning cells from destruction.
Leucovorin and MTX competitively bind to receptors on the cell membrane (77),
which theoretically could decrease the amount of MTX entering the cell. The use

136
of leucovorin rescue was not mentioned in any of the clinically-based articles
reviewed except Gnudi et al. (31), who incorporated leucovorin rescue into their
chemotherapy protocol. Leucovorin rescue has not been used in methotrexate
animal studies (8,23,24,35,74)since leucovorin would potentially moderate the effects
being investigated. Although not specifically identified, leucovorin rescue may be a
factor involved in bone recovery following MTX treatment noted in the clinical
studies (59,73). Perhaps the bone’s recovery is delayed without the use of leucovorin.
However, it has been postulated by Koizumi et al. (44) that by-products of protein-
bound MTX breakdown (MTX-polyglutamates) interfere with leucovorin rescue of
normal, non-neoplastic tissue. These recent findings would indicate leucovorin rescue
may not be as effective as previously believed.
With advances in chemotherapy drugs, an increasing number of patients are
overcoming their cancer and extending their life expectancy. As suggested by the
results of the present study, some chemotherapy drugs may produce secondary
diseases such as osteoporosis. Osteoporosis can have serious life-long consequences.
Since Methotrexate inhibits osteoblast function, it might be advantageous when
treating patients with MTX to also administer osteoporosis drugs known to enhance
bone formation, such as sodium fluoride or pulsatile parathyroid hormone.
Summary
The results of this study indicate that MTX had negative effects on both
cortical and cancellous bone of Sprague-Dawley rats, even 170 days after treatment.

137
The drug decreased cancellous bone volume, decreased conical bone area, decreased
cancellous and cortical bone formation, and increased cancellous bone resorption.
The observed effects of MTX on these bone properties were expected immediately
following treatment. However, it was hypothesized that MTX would be cleared from
the tissues by 170 days, allowing osteoblast and osteoclast function to return to
normal. Osteoblast and osteoclast recovery was not observed at the conclusion of the
study and, as a result, cancellous and cortical bone mass remained depressed in the
MTX treated animals. Radiological (DEXA) and biomechanical assessments failed
to disclose consistent differences in the bones of the MTX-treated and control
animals.
MTX inhibits DNA synthesis through its binding to dihydrofolate reductase.
Therefore, MTX would decrease replication of osteoblasts and osteoclasts.
Decreases in osteoblast number and activity in this study confirm MTX’s effects on
the osteoblast line. However, osteoclast numbers increased. This would indicate that
MTX depressed only bone formation and not bone resorption through selective
interference in osteoblast replication.
Chemotherapy agents have been designed to have toxic effects on rapidly
growing neoplastic cells. While the short-term toxicity during therapy to the
cancerous target cells is of primary importance, the long-term consequences to
normal tissue may be an equally important consideration. This study confirms that
MTX-induced osteopenia can persist after completion of chemotherapy.

138
Recommendations for Future Work
Based on the findings of this study, the following areas of additional research
are suggested to enhance the understanding of the effects of MTX on the skeleton:
(1) Repeat the study outlined in this document, yet extend the time for recovery
prior to skeletal assessments. This study would assess the time required,
following MTX treatment, for normal cell function to return to the skeleton.
(2) Repeat the study outlined in this document yet adjust the recovery time
(suggestion # 1), and have two groups of MTX treated animals. One group
would receive four courses of MTX (20 days) and the other group would
receive only two courses (10 days). This would enable evaluation of the
relationship between duration of MTX treatment and the recovery of
skeleton.
(3) Investigate the skeletal effects of using high doses of MTX in conjunction with
leucovorin rescue, high doses of MTX without leucovorin rescue, and lower
doses of MTX with and without leucovorin rescue using a rat model.
Duration of MTX treatment could also be varied in this study.
(4) Investigate the effects of prophylactic osteoporosis treatment (ie. pulsatile
PTH or sodium fluoride) used in conjunction with chemotherapy for the
prevention of MTX-induced osteopenia.
(5) Analyze the bone mineral density of human patients undergoing
chemotherapy treatment using DEXA. This longitudinal study should include

139
pre-treatment bone scans, scans during chemotherapy treatment, and scans at
intervals following treatment.

APPENDIX A
CANCELLOUS BONE FIXATION, DEHYDRATION AND METHYL
METHACRYLATE EMBEDDING
I. Fixation
Both cancellous and cortical bone were placed in fixative immediately following
harvest. Phosphate buffered formalin is best for preservation of bone cells, however,
alcohol formalin was the fixative chosen for this study since it is able to better
preserve fluorescent markers in the bone matrix.
Alcohol Formalin
80% ETOH 90 ml
37% formaldehyde solution 10 ml
Bones were placed in fixative for at least 24 hours and stored in the refrigerator.
After fixation the bone were transferred to small 20 ml glass vial with 70% ETOH
and refrigerated until embedding procedures begin.
140

141
II. Dehydration
Cancellous bone to be embedded in methyl methacrylate was thoroughly dehydrated
according to the following schedule. All bones were refrigerated in alcohol solutions
in separate vials.
70% ETOH
1-2 days
95% ETOH
2 days
100% ETOH
1 day
100% ETOH
1 day
XYLENE
1 day
All times indicated above are minimum times in solution. Bones can remain in
solutions up to 3 extra days except xylene (1 day maximum).
III. Embedding
Preparation and use of methyl methacrylate solutions for infiltration and embedding
should be carried out under a fume hood. The following list the steps in preparing
the solutions:
1. Approximately 2 g of calcium chloride pellets were placed in a large Nalgene
container. The calcium chloride removes the excess water from the methyl
methacrylate. Methyl methacrylate was poured into the container and shaken
well (see recipes for solutions I-IV for proper amount). The methyl
methacrylate was allowed to sit for approximately 30 seconds and then poured
through a filter and the desired amount was measured.

142
Dibutyl phthalate and benzoyl peroxide were measured and added to the
methyl methacrylate in an Erlenmeyer flask. The flask was covered with
parafilm and stirred for 4-6 hours. The recipes for solutions I-IV follow:
Solution I
methyl methacrylate
85 ml
dibutyl phthalate
15 ml
Refrigerated bone in solution for at least 3 days.
Solution II
methyl methacrylate
85 ml
dibutyl phthalate
15 ml
benzoyl peroxide
1 g
Refrigerated bone in solution for at least 3 days
Solution III
methyl methacrylate
85 ml
dibutyl phthalate
15 ml
benzoyl peroxide
2.5 g
Refrigerated bone in solution for at least 3 days
Solution IV
methyl methacrylate
170 ml
dibutyl phthalate
30 ml
benzoyl peroxide
5 g
Did not refrigerate, left bone in solution at room temperature overnight.

143
3. After changing to solution IV the bones were placed in a vacuum/dessicator
for 6-8 hours with the vials uncapped. The vacuum was released and
reapplied every hour. The bones were then oriented in the vial with the
anterior aspect, which was ground flat, flush with the bottom of the vial and
placed in a water bath at approximately 43°C overnight. Care was taken not
to apply too much heat initially to the specimen, excess heat causes the methyl
methacrylate solution to bubble which would compromise the bone block for
sectioning. The following morning the temperature was turned up slightly to
finalize the polymerization. Proper hardness of the plastic is indicated when
a probe could not penetrate the plastic more than 2-3 mm.
4.
After cooling, the vials were broken to release the plastic block for sectioning.

APPENDIX B
MODIFIED VON KOSSA STAIN
The 4 ¿¿m sections of cancellous bone were placed on gelatinized slides and
then stained with a modified Von Kossa stain using a tetrachrome counterstain. The
following section details the procedure for preparation of gelatinized slides and the
Von Kossa stain.
Gelatinized Slides
1. Slides were first cleaned with the following series of solutions:
70% ETOH
2 minutes
95% ETOH
2 minutes
95% HC1/ETOH
2 minutes (50 ml HC1 per 450 ml 95% ETOH)
95% ETOH
2 minutes
95% ETOH
2 minutes
2. Gelatin solution (Houpt’s Adhesive) was prepared by adding (enough for 50
slides):
dH:0
300 ml
300 bloom gelatin (Sigma) 5.25 g
Solution was heated to 60 °C then the glygerin was added
glycerin 10 ml
Solution was stirred for 1-2 hours.
144

145
3. Cleaned slides were arranged in coplin jars and the gelatin solution was
poured over the slides until the jar was approximately 2/3 full (to cover
usable portion of slide with gelatin). Slides were soaked in gelatin for 1 hour,
then removed and placed in slide trays to dry overnight.
Von Kossa Staining Technique
1. Slides, with the bone sections adhered, were soaked in 2-methoxyethylacetate
in coplin jars for at least 3 days to dissolve the plastic prior to staining. Each
day the 2-methoxyethylacetate was changed to a fresh solution.
2. The recipes for the four staining solutions follow:
I. Silver Nitrate Solution
3 g silver nitrate
60 ml dH,0
This solution degrades with exposure to light so it was shielded with
aluminum foil encased couplin jar prior and during use. Solution was
filtered before use.
II. Sodium Carbonate-formaldehyde solution
5 g sodium carbonate (anhydrous)
25 ml formaldehyde
75 ml dH20

146
III. Farmer’s Diminisher
(A) 10 g sodium thiosulfate
100 ml dH20
(B) 1 g potassium ferricyanide
10 ml dH,0
50 ml of solution A was mixed with 2.5 ml of solution B immediately
before use. This solution is only stable for 20 minutes.
IV. Tetrachrome solution
1.8 g tetrachrome
60 ml dH20
Solution was stirred for at least 2 hours and was filtered before use.
3.Slides were taken through the following steps of alcohol to distilled water:
100% ETOH
2 minutes
95% ETOH
2 minutes
70% ETOH
2 minutes
40% ETOH
2 minutes
dH-,0
2 minutes
4. Slides were transferred to coplin jar encased in aluminum foil with solution
I for 15 minutes or until bone stains dark.
5. Slides were transferred to coplin jar and rinsed with dH:0 three times,
allowing to soak for 1 minute with each change.

147
6.
7.
8.
9.
10.
11.
12.
13.
Slides were transferred to coplin jar with solution II for 2 minutes. Time is
very critical for this solution.
Again slides were rinsed with dH-,0 two times, allowing to soak for 1 minute
with each change.
Slides were transferred to solution III for 30 seconds. Again, time is critical
in this solution.
Slides were rinsed in running tap water with a gentle flow for 20 minutes.
Slides were then rinsed in dH;0 two times, allowing to soak for 1 minute with
each change.
Slides were transferred to solution IV for 40 minutes.
Slides were then rinsed with dH20 three times, allowing to soak for 1 minute
with each change.
Slides were taken through the following solutions:
70% ETOH 1 minute (critical)
100% ETOH 1 minute (critical)
xv lene
3 minutes
14.
xylene 3 minutes
Slides were then coverslipped with permount and analyzed.

APPENDIX C
CORTICAL BONE FIXATION, DEHYDRATION,
AND EMBEDDING IN BIOPLASTIC
Fixation
Cortical bone specimens were fixed in alcohol formalin as discussed in Appendix A.
Dehydration
Cortical bone specimens were dehydrated with the following sequence of solutions:
70% ETOH
2 days minimum
95% ETOH
2 days minimum
100% ETOH
10 changes, one change every 2 hours
Acetone
10 changes, one change every 2 hours
Embedding
Embedding of cortical bone requires two steps. The first step involves creating a thin
layer of hardened bioplastic on the bottom of the vial without the bone specimen.
The second step involves placing the bone specimen on the first layer and adding
another layer of bioplastic to cover the specimen. Mixing and hardening the
bioplastic are covered in the following paragraphs:
148

149
1. 100 ml of TAP clear lite resin (#00191) was poured into a disposable plastic
beaker. 3 drops of catalyzer 207 (#08478) was added to the resin. Solution
should be stirred for 1-2 hours.
2. Bioplastic solution was poured into vials and placed in vacuum/dessicator
uncapped for 6-8 hours (only for part 2 when bone specimens is included).
Vacuum was released every hour and then reset.
3. Vials were then placed in water bath set to approximately 43 °C overnight to
harden.
4. Bioplastic embedded cortical bone specimen were then allowed to cool,
released from the vial, and prepared for sectioning.

APPENDIX D
COMPUTER CODE FOR IMAGE ANALYSIS
I. Program for determining cancellous bone volume
ft Donna Wheeler
ft Orthopaedics 392-4251
ft Static Bone Parameters for Trabecular Bone
ft File: TBVS.MCR
ft VIDAS system
ft 12/3/92
ft
ft This program will measure the following static trabecular bone parameters
ft BV = total bone volume (mm2)
ft TV = total volume of the sampling area (mm2)
ft TBV = percent trabecular bone = BV/TV (%)
#
ft Set the image size and variables needed by the system
ft
InitField FIELDPERIM, REF ARE A, TOT ALARE A, ARE AP
global ID,BV,TV,TBV
x=y = 1
x_st = y_st = x_sz=y_sz=0
appen=0
ID = "12345"
BV=TV = TBV=0.0
ft
ft Database management
ft
fname = "tbv"
for i = 1, i < 20, i = i -E1: write
read "Enter data file name (8 char max): ",fname
fv[]=ID,BV,TV,TBV
ans = "y"
write "Do you want to append ",fname
read ans
if ans= = "n"
150

151
DBerase fname
DBcreate fname,"fv"
DBclose fname
endif
ft
ft Pick scale for measuring
ft
scalgeom l,"2.5/1.6",_OFF,_ON
ft
ft
ft Calibration of system - Initial Offset and Gain
ft
ft
tvon
for i = 1, i < 20, i = i -h 1: write
write "adjust light to prevent overload"
write "invoke 1st three neutral density filters"
write "set objective to 2.5xand isobar to 1.6x"
clall
loadlut "grey"
tvon
write "Place First slide on the scope and focus"
write "set OFFSET and GAIN to produce appropriate contrast"
pause
loadlut "tvonline"
write "Use cursor to adjust brightness"
adjustint 255,127,_ON
ft
ft Prompt first for 1st slide
ft
loadlut "grey"
clearallio
tvon
read "Enter 1st slide id: ",ID
ft
ft Main Loop
ft
while(ID! = "0")
ft
ft Reset variables
ft
BV = TV = TBV=0.0
ft
ft Start processing

=tfc =tfc =tfc =*fc ^
152
#
tvon
loadlut "grey"
if(_STATUS = =21) \ break
tvinp 1
tvoff
Have user mark region of interest
for i = l,i<20,i = i + l:write
write "Mark REGION OF INTEREST"
write "upper left comer then lower right comer"
drawframe 1,1 ,x_st,y_st,x_sz,y_sz,_ON
clear 2,0
clovl 2
copywind 1,2,x_st,y_st,x_sz,y_sz,x_st,y_st, 1 ,_OFF
Sharpen edges of image and separate into two phases
delin 2,3,50,7, OFF
for i = l,i<20,i = i-Fl:write
write "identify grey level of trabeculae with cursor"
dis21ev 3,4,0,100,_ON, ON,3
display 4
Scrap small areas and close small holes
thingrey 5,6,1, OFF,_OFF
scrap" 4,5, OFF,0,15, ON, ON
close 5,6,7,255,1
scrap 6,7, OFF,0,20, ON, ON
Identify bone areas and measure
loadlut "grey"
identframe 7,8, ON, 5, x_sz, y_sz, x_st, y_st ,_OFF ,_OFF, ON
Measf 8
clear 9,255
greygr 8,9.9,0,0,0
contour 9,2,10,7,255, ON, OFF
loadlut "grey"
display 10
#

153
tt Calculate area and perimeter values
tt
BV=TOT ALARE A
TV = REFAREA
TBV = BV/TV
tt
tt
tt Add data to database
tt
tt
DBopen fname,"nn"
DBappend fname
DBclose fname
tt
tt
ft Prompt for next slide
tt
loadlut "grey"
clearallio
tvon
for i = 1, i < 20, i = i -P1: write
write "place next slide on scope and focus"
write "adjust GAIN and OFFSET if needed"
read "Enter next slide ID or <0>to quit:", ID
tt
tt
endwhile
tt
tt Clean up system and list data
tt
tvoff
loadlut "grey"
outlist fname, OFF

154
II. Program for determining cortical bone parameters
tt Donna Wheeler Orthopaedics 2-4251
tt Purpose: Analysis of static X-sectional properties of bones
tt file: xsects.mcr
tt Prepared for VIDAS imaging system
tt updated 2/10/93
tt
tt
tt This program will provide calculations for area properties
tt for a bone cross-section (marrow area, cortical area, and J)
tt
tt Initialize parameters and variables
tt
resetpar
InitField REF AREA, TOT ALARE A
InitObj DM AX, CGRAVX, CGRA V Y, ELLIPSE A, ELLIPS EB, OB JLABEL
InitD ios ELLIPS EA, ELLIPSEB, LENGTH, DISTANCE2P
external UNITXY,SCALEX,SCALEY,CGRAVX,CGRAVY
global ID,len,t,tmean,a,b,J,xarea,barea,marea,angle,xc,yc,txx,tyy,tsd
x=y=0
xc=yc=0
appen=len=0
t=tmean = xarea=barea=marea = a = b = J = txx = ty y=tsd=0.0
DMAX = angle=0.0
ID = "12345"
UNITXY = "mm"
tt
tt variable definition:
tt tmean - mean of all thicknesses sampled (mm)
tt a = maximum radius from ELLIPSEA (mm)
tt b = minimum radius from ELLIPSEB (mm)
tt J = polar moment of inertia for hollow ellipse using a & b (mm4)
tt xarea = cortical bone area of x-section (mm2)
tt barea = total area of bone and marrow space (mm2)
tt marea = area of marrow (mm2)
tt
tt
tt Set up database
tt
fname = "xsect"
for i = 1, i < 20, i = i -t-1: write
read "Enter data file name (8 char max): ",fname
fv [] = ID, barea, marea, xarea, tmean. a, b, J

155
ans = "y"
write "Do you want to append ",fname
read ans
if ans = = "n"
DBerase fname
DBcreate fname," fv"
DBclose fname
endif
ft
ft set up database to calculate the mean cortical thickness
ft
bone[] = DMAX,angle,t,txx,tyy
DBerase "bonedb"
DBcreate "bonedb","bone"
ft
ft Set contrast and calibrate system
ft
scalgeom Image = 1,scale = "2.5/1.25",inter = ON, List=_ON
clall
loadlut File = "grey"
tvon
for i = l, i<20,i = i + l:write
write "place first slide on scope and focus"
write " adjust initial gain and offset on controller"
pause
loadlut File = "tvonline"
for i = l, i < 20, i = i + l:write
write "Use cursor to adjust brightness"
adjustint 255,127,_ON
ft
ft Prompt first for 1st slide
ft
loadlut "grey"
clearallio
tvon
for i=l, i<20,i = i-+-1:write
read "Enter 1st slide id: ",ID
ft
ft Main Loop
ft
while(ID! = "0")
ft
ft
ft Stan processing

156
#
tvon
loadlut "grey"
if(_STATUS ==21) : break
tvinp 1
tvoff
display 1
#
# Digitize around outside and inside perimeter of bone section
# Measure areas
#
for i = l,i<15,i=i + l:write
ovcolour (8)
write "digitize around OUTSIDE perimeter"
eraseoutside 1,2,3,255
dis21ev 2,4,0,254, ON, OFF,3
identify 4,4, ON, OFF
Measf 4
barea=TOTALAREA
for i=l, i< 15,i = i + l:write
loadlut "grey"
display 2
write "digitize around INSIDE perimeter"
eraseinside 2,5,3,255
display 5
dis21ev 5,6,0,254, ON, OFF,3
display 6
identify 6,7, ON, OFF
Measf 7
xarea=TOT ALAREA
marea=barea-xarea
#
# Determine mean cortical thickness - new method
#
display 6
identify 6,7, ON, OFF
Measo 7
clovl 7
step = 30
for i = l, i< 15,i = i + l:write
write " The mean wall thickness of the specimen will be measured"
xc = int(CGRA VX/SCALEX)
yc = int(CGRAVY/SCALEY)
Gcircle 7,xc,yc,5,-l,0

157
clear 8,0
loadlut "ident"
for i=0,i<360/step,i = i + l
Gvector 8,xc,yc,xc + int(300*cos(step*i/_DEG)),yc-int(300*sin(step*i/_DEG)),i+2
endfor
clear 9,0
multiply 8,6,9,255
display 9
DBopen "bonedb","bone"
while 1
Measo 9
if STATUS: break
angle = (OBJLABEL-2)*step
txx = DMAX*cos(angle/(_PI*2))*SCALEX
tyy = DMAX*sin(angle/(2*_PI))*SCALEY
t=(txxA2+tyy^2)XX5
DBappend "bonedb"
endwhile
stat[32]: =0.0
measdbst "bonedb","t",15,_OFF,_ON,0.00,100.0,100.0,"stat","*"
tmean=stat[6]
tsd = stat[7]
for i = 1, i< 15,i = i + l:write
write "Mean wall thickness: ",stat[6],"+/-",stat[7]," ",UNITXY
pause
DBerase "bonedb"
DBcreate "bonedb","bone"
loadlut "grey"
ft
ft Determine major and minor axes for elliptical model
# [using ELLIPSEA and ELLIPSEB parameters]
#
#
display 6
identify 6,10, ON, OFF
Measo 10
a = ELLIPSEA/2
b = ELLIPSEB/2
J=_PI*(a*b^3 +b*a^3-(a-tmean)*(b-tmean)*((b-tmean)^2+(a-tmean)A2))/4
#
#
# Add data to database
#
#

158
DBopen fname,"nn"
DBappend fname
DBclose fname
#
#
# Prompt for next slide
#
loadlut "grey"
clearallio
tvon
for i = 1, i < 20, i=i -P1: write
write "place next slide on scope and focus"
write "adjust gain and offset if needed"
read "Enter next slide ID or <0>to quit:", ID
#
#
endwhile
#
# Clean up system and list data
#
tvoff
loadlut "grey"
outlist fname, OFF

APPENDIX E
DEXA REPEATABILITY STUDY
The reliability and reproducibility of the DEXA as a means to assess bone
mineral density was evaluating by a three phase experiment. For each phase of the
experiment, the femur and the tibia of two specimen were scanned 10 times.
I. To determine the inherent variability of the scanner, repeated scans of the
same bones were made without repositioning the bone between scans.
II. To determine the variability associated with repositioning the specimen,
repeated scans were made removing and replacing the bones between scans.
III. To determine if calibrating the scanner (Quality Assurance) affected the
measurements, repeated scans were made (without repositioning) before and
after calibration.
The coefficient of variation (%CV) was calculated for bone mineral content
(BMC), AREA, and bone mineral density (BMD) for parts I and II and a Students
t-test was used to determine if there were differences in BMC, AREA, and BMD
before and after calibration for part III. All statistical test were run on SAS software
(PC-SAS version 6.03,Cary, NC) with a significance level set a p=0.05.
159

160
The results for part I and II and presented in Table E.l.
Table E. 1 Results of DEXA Reliability Study
Analysis
Bone
DEXA
parameter
Mean
Standard
Deviation
%cv
PART I.
NO Reposition
Femur
BMC (g)
0.486
0.024
4.47
AREA (cm2)
2.05
0.087
4.25
BMD (g/cm2)
0.224
0.0035
1.56
Tibia
BMC (g)
0.335
0.018
5.37
AREA (cm2)
1.6846
0.054
3.18
BMD (g/cm2)
0.199
0.0055
2.78
PART II.
Reposition
Femur
BMC (g)
0.474
0.021
4.54
AREA (cm2)
2.06
0.101
4.93
BMD (g/cm2)
0.23
0.003
1.42
Tibia
BMC (g)
0.346
0.017
4.89
AREA (cm2)
1.71
0.055
3.24
BMD (g/cm2)
0.201
0.005
2.82
There was not a significant difference in the BMC, AREA, or BMD values before
or after calibration (p>0.05).
These results indicate that repositioning the bone between trials does not
increase the coefficient of variation. Also the calibration procedure does not
significantly change the scan information. For the parameter of interest, BMD, the
coefficient of variation was below 2% for the femur and below 3% for the tibia. This
is within the standards accepted for DEXA scan for human and is comparable to the
coefficient of variation acheived by others (2,3,32,48,67).

APPENDIX F
SAS PROGRAMS FOR STATISTICAL ANALYSIS
I. Program for statistical analysis of cancellous bone parameters
data donna.pt;
infile ’c:\data\pt2.txt’ lrecl = 300;
input id $ no trt $ time grp $
tbv dl mr perim lbg;
proc sort data = donna.pt;
by grp;
proc means data=donna.pt n mean std min max;
by grp;
var tbv dl mr lbg;
proc glm data=donna.pt;
class trt time;
model tbv dl mr lbg = trt time trt*time;
means trt / duncan;
means time /duncan;
proc sort data=donna.pt; by time;
proc glm data=donna.pt;
class trt ;
model tbv dl mr lbg = trt;
by time;
means trt / duncan;
proc sort data=donna.pt; by trt;
proc glm data=donna.pt;
class time;
model tbv dl mr lbg = time;
by trt;
means time/ duncan;
run;
161

162
II. Program for statistical analysis of biomechanical parameters
data donna.biometho;
infile 'c:\data\biometho.txt’ lrecl = 300;
input id $ limb $ time trt $ grp $ no
slope torque angle E a J S100;
proc sort data = donna.biometho;
by limb grp;
proc means data=donna.biometho n mean std min max;
by limb grp;
var slope torque angle E J SI00 ;
proc glm data=donna.biometho;
class trt time;
model slope torque angle E S100 = trt time trt*time;
by limb ;
means trt / duncan;
means time /duncan;
proc sort data=donna.biometho; by limb time;
proc glm data=donna.biometho;
class trt ;
model slope torque angle E S100 = trt;
by limb time;
means trt / duncan;
proc sort data=donna.biometho; by limb trt;
proc glm data=donna.biometho;
class time;
model slope torque angle E S100 = time;
by limb trt;
means time/ duncan;
run;

163
III. Program for statistical analysis of DEXA parameters
data donna.scan;
infile 'c:\data\scan.txt’ lrecl=300;
input id $ trt $ time grp $ no cat $ limb $
bmc area bmd;
proc sort data=donna.scan;
by limb cat grp;
proc means data=donna.scan n mean std min max;
by limb cat grp;
var bmd ;
proc glm data=donna.scan;
class trt time;
model bmd = trt time trt*time;
by limb cat;
means trt / duncan;
means time /duncan;
proc sort data=donna.scan; by limb cat time;
proc glm data=donna.scan;
class trt ;
model bmd = trt;
by limb cat time;
means trt / duncan;
proc sort data=donna.scan; by limb cat trt;
proc glm data=donna.scan;
class time;
model bmd = time;
by limb cat trt;
means time/ duncan;
run;

APPENDIX G
QUICK REFERENCE FOR ABBREVIATIONS
Table G.l Standard Abbreviations
Abbreviation
Description
Cn.BV/TV
Cancellous Bone Volume
Cn.Oc.S
Cancellous Osteoclast Surface
Cn.LBG
Cancellous Longitudinal Bone Growth
Cn.MS
Cancellous Mineralizing Surface
Cn.MAR
Cancellous Mineral Apposition Rate
Cn.BFR/BS
Cancellous Bone Formation Rate
Ct.T.Ar
Total Cortical Tissue Area
Ct.Ma.Ar
Cortial Marrow Area
Ct.Ar
Cortical Area
Ct.Wi
Cortical Width
J
Polar Moment of Inertia
Ps.Ms
Periosteal Mineralizing Surface
Ps.MAR
Periosteal Mineral Apposition Rate
Ps.BFR
Periosteal Bone Formation Rate
Tu
Torque at Failure
9u
Twist Angle at Failure
Eu
Energy Absorbed at Failure
S
Stiffness
Tu
Torsional Strength
BMD
Bone Mineral Density
164

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BIOGRAPHICAL SKETCH
Donna L. Wheeler was bom on February 20, 1962, in Jacksonville. Florida.
After attending elementary school in Atlanta, Georgia, and Ft. Lauderdale, Florida,
she entered Duncan U. Fletcher Jr. and Sr. High School. She graduated as
salutatorian of her class in June of 1980. After graduation she attended Vanderbilt
University in Nashville, Tennessee, where she pursued a degree in biomedical
engineering as well as played varsity basketball. In August of 1982 she transferred
to the University of Florida to continue her education. She was awarded a Bachelor
of Science degree in mechanical engineering with high honors in May of 1985. She
continued her studies at the University of Florida as a graduate student in
mechanical engineering where she used novel experimental stress analysis techniques
for orthopaedic applications. This work culminated in the thesis "The Use of
Holographic Interferometry to Measure Surface Deformations of the Femur due to
a Cementless Prosthesis" and the award of a Master of Science degree in mechanical
engineering in December of 1987.
She was hired as a research scientist in the Department of Orthopaedics at
the University of Florida upon completion of her master's degree. During this time,
she became interested in metabolic bone diseases and shifted her emphasis from
mechanics to physiology and histology. In August of 1989 she became a doctoral
173

174
student in the Department of Exercise and Sport Sciences at the University of
Florida. Through this education and her appreciation of orthopaedic problems
associated with osteosarcoma and osteoporosis, she became interested in
chemotherapy-induced osteoporosis. This interest culminated in the dissertation
entitled, " The Short- and Long-term Effects of Methotrexate on the Rat Skeleton"
as partial fulfillment of the degree requirements of a Doctor of Philosophy in
exercise and sport sciences conferred in December of 1993.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
James É. Graves, Chau-
Associate Scientist of Exercise and Sport Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Scott K. Powers
Professor of Exercise and Sport Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
)avid T. Lowenthal
^Fofessor of Exercise and Sport Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the jié^ree of/Pocton^of Philosophy.
rofessor of Mechanical Engineering

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is,fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
\s.y.c. Ldat
ik
4^
I ronski
Associate Professor of Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Robert A. Vander Griend
Associate Professor of Medicine
This dissertation was submitted to the Graduate Faculty of College of Health
and Human Performance and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree^ of Doctor ot/Philosophy.
December 1993
Dean, College of Health and Human Performance
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08557 1023