2.5. Experimental osteoporosis

The Food and Drug Administration of the United States has established guidelines for using animals in preclinical testing of agents intended for the prevention or treatment of human postmenopausal osteoporosis (Guidelines for preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis 1994). It recommends the use of ovariectomized rats and larger animals, because of the relationship of human osteoporosis to estrogen depletion. Studies must include histologic evaluation of the bones. Furthermore, in addition to determinations of bone density and biochemical markers of bone turnover, the use of biomechanical testing is suggested to evaluate the propensity to develop fragility fractures.

The ovariectomized rat is considered an appropriate model for studying human menopausal osteoporosis because of many similarities in their pathophysiological mechanisms (Kalu 1991, Wronski & Yen 1991, Frost & Jee 1992). In both species, bone loss is most rapid after the onset of estrogen deficiency, and it is characterized by an increased bone turnover, resorption exceeding formation (Gallagher et al. 1972, Delmas et al. 1983, Wronski et al. 1988a, for review see Kalu 1991). Furthermore, in both species, bone loss is greater in the trabecular than in the cortical bone (Yamazaki & Yamaguchi 1989), intestinal absorption of calcium is decreased (Arjmandi et al. 1994), and the skeletal responses to current prevention and treatment modalities are similar (Wronski et al. 1989b). These regimens include exercise (Donahue et al. 1988, Tuukkanen et al. 1991, Peng et al. 1994a, for review see Tuukkanen 1993), estrogen replacement therapy (Aitken et al. 1972, Wronski et al. 1988b, Kalu et al. 1991a), as well as treatment with bisphosphanates (Seedor et al. 1991, Wronski et al. 1991a) and calcitonin (Hayashi et al. 1989, Mazzuoli et al. 1990).

Two models of experiments are widely used with the ovariectomized rats. The aged rat model uses animals 6-24 months of age, and the mature rat model uses animals about 3 months of age (for review see Kalu 1991). Although the aged rat model has many of the characteristics to look for in an animal model of postmenopausal osteoporosis, mature rats are used more often, because they, in comparison to the aged rats, are cheaper, easily available, and the effects of ovariectomy on their skeleton are manifested within a reasonable time. Furthermore, the characteristics of bone loss are mostly similar to those of the aged rat model (Kalu 1991).

In ovariectomized rats, the proportion of cancellous bone decreases significantly, the trabecular bone volume in the secondary spongiosa of the long bones being about 10-15 %, as compared with 20-30% of the sham-operated controls (Wronski et al. 1993, Tsurukami et al. 1994, Yamaura et al. 1994). However, this decrease is greatly inhibited by therapeutic interventions with estrogen (Kalu et al. 1991b, Evans et al. 1994), calcitonin (Wronski et al. 1991b) and bisphosphonates (Wronski et al. 1993, Lepola et al. 1995). Partial prevention is also achieved by moderate exercise (Tuukkanen 1993).

Bone resorption has been shown to increase about 40% within a week following ovariectomy, as monitored by measuring the urinary excretion of [³H]; of previously [³H];-tetracycline prelabeled rats (Cecchini et al. 1997). The increase of bone resorption is also significantly inhibited by treatment with estrogen (Turner et al. 1993, Goulding et al. 1996, Cecchini et al. 1997), calcitonin (Mühlbauer & Fleisch 1995), and bisphosphonates (Mühlbauer & Fleisch 1995, Frolik et al. 1996).

After ovariectomy, decreased bone biomechanical properties have been detected in rats. Peng et al. (1994b), using the mature rat model, found a decrease of maximum load in the bending of tibial shaft (8,7%) and in the loading of femoral neck (15,8%) in ovariectomized rats. The values for stiffness did not differ among the groups. Accordingly, using the same model, Sœgaard et al. (1994) revealed a significant decrease in the maximum load of femoral neck in the ovariectomized rats. No differences in the values of deformation at maximum load or of energy absorption capacity were found. In the study of Lepola et al. (1995) ovariectomy reduced the maximum load values in compression of the femoral neck by 12%. No significant effects by ovariectomy were observed concerning deformation or rigidity. Bagi et al. (1997) found reduced values for strength and stiffness of femoral necks obtained from ovariectomized rats as compared with those from the sham-operated controls. Lauritzen et al. (1993), using 6-month-old rats, showed a significant reduction of femoral midshaft ultimate load and stiffness, as well as of femoral neck stiffness in ovariectomized rats relative to non-ovariectomized controls. Furthermore, ovariectomy induced a 16,6% loss of the maximum torque capacity of tibia in the study of Peng et al. (1994a).

On the other hand, there are also findings indicating no significant ovariectomy-induced changes in the biomechanical properties of long bones (Toolan et al. 1992, Bagi et al. 1993). Sato et al. (1997) found no differences in the strength of the femoral neck between baseline, sham-operated and ovariectomized rats when 9-month-old rats were used, and suggested limited utility of this measurement in aged rats. In fact, even findings showing improved biomechanical properties after ovariectomy have been published. Aerssens et al. (1993), using the mature rat model, found a significant increase in torsional strength, in deformation energy to fracture, and in angular deformation of the torsional tested femurs. In the study of Lepola et al. (1995) the maximum load in three-point bending of femur was elevated by 7% after ovariectomy compared with sham-operated rats. Also the values for rigidity and deformation were increased. Accordingly, using 4-month-old rats, Bagi et al. (1995) found increased ultimate torque values in the ovariectomized rats relative to the shams.

Altered bone cross-sectional geometry, usually seen as widening of the medullary cavity of long bones (Turner et al. 1989), affects the cross-sectional moment of inertia, and may thus affect the bone biomechanical properties (Hayes & Gerhart 1985). The contradictory findings regarding bone biomechanical properties may, in addition to variations in the age and strain of the rats, and in the time after operation, be a consequence of these geometrical properties. For example, in all above studies showing improved biomechanical properties, the moment of inertia was increased, obviously due to geometrical adaptation of the bone to altered conditions (Aerssens et al. 1993, Bagi et al. 1995, Lepola et al. 1995). The ovariectomy-induced decline in bone mechanical strength seems to be more evident in the femoral neck than in the long bones of the rats. This may be explained by the fact that mechanical strength of the femoral neck is considerably determined by trabecular bone (Martens et al. 1983), which is more susceptible to estrogen deficiency than cortical bone (Turner et al. 1987). However, the cancellous bone of the femoral neck in rats may be less important for strength properties than in humans, because the femoral neck of rats contains much more cortical bone than that of humans (Bagi et al. 1997).

Ovariectomy-induced reduction of bone biomechanical strength has been shown to be suppressed by treatment with estrogen (Shen et al. 1995) and bisphosphonates (Katsumata et al. 1995, Kaastad et al. 1997).