El amor y la muerte (2002)

4.1 Introduction

4.2 Materials and methods

4.3 Results

4.1 INTRODUCTION

Chemotherapy has significantly increased the survival rate of patients with malignant

bone tumours (Goorin and Andersen 1991, Winkler 1991). Among these patients, a

number are skeletally immature. Because o f the systemic administration o f these

drugs, bone cells and growth plates are exposed to them. There is evidence in the

literature that these drugs are toxic to osteoblasts and growth plate chondrocytes

(Friedlaender 1984, Wheeler 1995a, Bar-On 1993, Robson 1998). In my study, it has

been shown that chemotherapy drugs reduce cell proliferation and osteogenic

differentiation in MSCs (Chapter 3). Also, it has been shown that chemotherapy has

negative effects on the longitudinal growth in skeletally immature individuals

(Wollner 1980, Glasser 1991, Davies 1994).

In order to minimise the side effects and complications o f chemotherapy on normal

bone tissues, an intensive two-drug chemotherapy regime, in which only cisplatin and

doxorubicin were used, was developed by European Osteosarcoma Intergroup

(Souhami 1997). The results of using cisplatin and doxorubicin in operable

osteosarcoma were shown to be as effective as lengthy, multi-drug TIO regimen

(Meyers 1992). This two-drug regimen is shorter in duration and is better tolerated by

lower. The effects of cisplatin and doxorubicin on the mechanical properties of bones

and growth plates are not clear either. It has been reported that growth arrest is

associated with chemotherapy in patients with malignant bone tumours, and growth

arrest line at the growth plates are visible radiologically in skeletally immature

patients with malignant bone tumours years after chemotherapy treatment (Ogden

1984, Cool 1997). Therefore, the purpose o f this study is to investigate the effects of

systemic chemotherapy on the mechanical properties of adult and skeletally immature

bones. We hypothesize that systemic chemotherapy with cisplatin and

doxorubicin has detrimental effects on growth plates and the mechanical

4.2 MATERIALS AND METHODS

Twenty-four immature Wistar rats at the age of 3-5 weeks and the weight between 90

and 1 lOg, and twelve adult Wistar rats at the age o f 12 months and the weight o f 600g,

were used in this study. They were equally divided into two groups so each group

consisted o f 12 immature and six adult rats. In the experimental group, cisplatin

(Img/Kg body weight) and doxorubicin (1.2mg/Kg body weight), the two-drug

chemotherapy regimen suggested by European Osteosarcoma Intergroup, were given

to the rats intraperitoneally. In patients, these chemotherapy drugs are given via

central venous lines because they can cause vasculitis if they are given via peripheral

veins. The reason to give the chemotherapy drugs to the rats intraperitoneally in my

experiment is that it would have been technically very difficult to give the drugs to the

rats via central venous lines as this is extremely difficult. It is not sensible to

administer the chemotherapy drugs via tail veins either as this can cause vasculitis.

Three injections were given. The interval between each injection was two weeks. In

the control group, normal saline o f the same amount was given intraperitoneally.

Seven days after the third injection, which was five weeks after the first injection, the

rats were killed. Both femora in each animal were harvested and all the soft tissues

surrounding the bones were removed. Mechanical testing was performed using a

six adult and six immature rats in each group, a four-point bending load-to-failure test

was performed on one femur to measure the ultimate bending strength under the

loading rate of 50 Newtons per second (Figure 4-2). Torsion load-to-failure was

performed on the other femur to determine the ultimate torsional strength under the

loading rate o f 5 Newtons per second (Figure 4-3 and 4-4). The specimens were

embedded with bone cement in the testing rig. Sagittal sections o f the proximal tibial

growth plates were prepared by the methods reported in the literature (Brighton 1987).

Briefly, the specimens o f retrieved tibiae were fixed in 10% formal saline, decalcified

in EDTA, dehydrated in an alcohol series, embedded in resin, sectioned using a sledge

microtome, and stained with hematoxylin and eosin. Numbers o f proliferative and

hypertrophic chondrocytes within each column o f the growth plates were determined.

Ten to 15 columns of cells were counted and averaged in each growth plate section. In

the remaining 6 immature rats in each group, the shear load needed to remove the

distal femoral epiphysis from the diaphysis was measured under the loading rate o f 10

Newtons per second (Figure 4-5). The results of histological analysis and mechanical

testing were then analyzed and compared using Student-T tests at p= 0.05. Normality

of the data was justified by Kolmagorov-Smimov tests performed on SPSS-9 software

Figure 4-1. Setup of material testing machine for four-point bending test

Figure 4-3. Testing Rig for ultimate torsion test

Figure 4-4. Bone fractured under torsional load

Figure 4-5 Separation of distal femur physis under shear load

4.3 RESULTS

The body weights o f the rats are shown in Table 4-1. The body weight o f the adult

rats in the control group was 614.7±43.9 grams (mean±S.D) and 598.9±50.6 grams in

the chemotherapy group. The body weight o f the immature rats was 130.5±22.8

grams in the control group and 126.0±17.2 grams in the chemotherapy group. There is

no statistically significant difference in body weights between the control and the

experimental group at the time of sacrificing the rats. The results o f ultimate bending

strength are shown in Table 4-2. The ultimate bending strength of the adult rats in the

control group was 277.2 ± 22.7 newtons and 222.0 ± 51.2 newtons in the

chemotherapy group (p = 0.018). An example of load-deformation curve o f the

four-point bending test is shown in Figure 4-6. The ultimate bending strength o f the

immature rats was 131.7 ± 54.9 newtons in the control group and 91.8 ± 16.1 newtons

in the chemotherapy group (p=0.061). The results o f ultimate torsional strength are

shown in Table 4-3. The ultimate torsional strength o f the adult rats in the control

group was 10.4 ± 2 .5 newtons and was 7.4 ± 2.2 newtons in the chemotherapy group

(p=0.026). The ultimate torsional strength o f the immature rats was 11.0 ± 1.7

newtons in the control group and 9.2 ± 1.9 newtons in the chemotherapy group (p=

0.056). Both ultimate bending and torsional strength reduced significantly in the

results o f ultimate shear strength of the distal femoral physis are shown in Table 4-4.

The ultimate shear strength o f the distal femoral physis was 45.8 ± 9.7 newtons in the

control group and was 35.8 ± 8.5 newtons in the chemotherapy group (p=0.044). The

ultimate shear strength o f the distal femoral physis was reduced significantly in the

chemotherapy group. An example o f load-deformation curve o f the ultimate shear

strength test is shown in Figure 4-7. Histology o f the growth plates showed that in the

control group, there were more proliferative and hypertrophic chondrocytes (Figure

4-8). However, in the chemotherapy group, the numbers of proliferative and

hypertrophic chondrocytes reduced and the cells were smaller (Figure 4-9). It was

also noted that the width o f the growth plate was thinner in the chemotherapy group

than in the control group. The growth plates in the chemotherapy group are less

organized and the cell columns are less well aligned. The septums among the cell

columns in the chemotherapy group are also thinner. The numbers o f proliferative and

hypertrophic chondrocytes are shown in Table 4-5. The average number of

proliferative chondrocytes in the control group was 17.0 ± 3.8 and 11.8 ± 1.8 in the

chemotherapy group (p < 0.001). The average number o f hypertrophic chondrocytes

in the control group was 11.2 ± 2.1 and 6.9 ± 2.0 in the chemotherapy group (p <

Maximum N