Our study revealed that combination therapy with alendronate and calcitriol significantly improved bone mass and femoral neck strength in OVX rats. The results indicated that total BMC values had the strongest correlation with the mechanical strength of the femoral neck.
Several studies [7, 8] have demonstrated the effect of a single treatment of alendronate on the mechanical strength of the femoral neck in OVX rats. In the first study, starting alendronate immediately after OVX at dose of 0.04 mg/kg/day and 1.0 mg/kg/day significantly increased the strength of the femoral neck in OVX rats after 8 weeks of treatment . However, the effects of alendronate on femoral neck strength in these OVX rats were not dose dependent. The second study  showed that the femoral neck strength of rats receiving alendronate at a dose of 3 mg/kg/day was significantly greater than that of OVX control rats, even though alendronate was administered to OVX rats for 4 weeks and started on the second day after OVX. In the present study, the dose of 0.1 mg/kg/day of alendronate increased the maximum fracture loading at the femoral neck in OVX rats, but this was not significant compared with the saline-treated OVX rats. The difference between the two above reports and our results may be due to differences in the period of time from ovariectomy to initiating treatment. It is possible that this period of time may also be partly responsible for differences in results with vitamin D3 treatment between postmenopausal women with osteoporosis [10, 11] and ovariectomized rats [12, 13]. Since osteoporosis associated with estrogen deficiency is a silent disease, when patients start treatment with various drugs, osteoporosis is already well established; therefore, in the present study all treatments were started at 20 weeks after ovariectomy.
Our OVX rats had significantly decreased total BMC, total BMD, cortical BMC, cancellous BMC, cancellous BMD, and total bone area at the femoral neck compared with sham-controls. The combination therapy with alendronate and calcitriol reverted these levels towards those observed in the sham-operated rats. However, these findings, excluding that for total bone area, were also observed with alendronate alone. Therefore, no synergistic effects of alendronate and calcitriol were observed in terms of bone mass in OVX rats.
In terms of the mechanical strength of the femoral neck, the present study showed no difference between alendronate alone or in combination with calcitriol, but only combination treatment of alendronate and calcitriol for 12 weeks significantly improved bone fragility owing to ovariectomy compared with the saline-treated OVX rats. For this reason, we also believe it is of interest that OVX rats treated with the combined treatment showed enhanced total BMC, cortical BMC, and total bone area, which are all correlated with femoral neck strength. Among the three parameters in bone mass, total BMC and cortical BMC were increased by alendronate, and total bone area was improved by calcitriol. Therefore, a combined treatment that reflects differences in functions of the two agents on bone mass, might significantly improve femoral neck strength.
Even though clinical studies have reported correlations between BMD and the incidence of femoral neck fracture in osteoporosis patients , our results found no correlation between BMD and femoral neck strength in rats. In fact, in our study, femoral neck strength was only correlated total BMC, cortical BMC, and total bone area. Our findings are in agreement with several other reports [24, 25]. In healthy rats, femoral neck strength was shown to be correlated only with BMC and not BMD . Meanwhile, in gastrectomized osteopenic rats, BMD was not correlated with femoral neck strength . Furthermore, in another study, in which OVX rats were treat with human insulin-like growth factor-I (IGF-I) alone or in combination with pamidronate, although BMD and BMC were correlated with femoral neck strength, the association was stronger for BMC than for BMD . In addition, cortical bone properties are of great interest in osteoporosis, since it is widely speculated that cortical bone quality does affect fracture risk. In this study, we measured cortical BMC, BMD and cortical thickness as markers of cortical bone quality. Of these, cortical BMC was positively correlated with the femoral neck strength in OVX rats. However, since the femoral neck strength is also affected by other parameters such as external diameter of the femoral neck, hip axis length , cortical porosis, mean degree of mineralization, and osteocytes, it may be difficult to evaluate the determinants of neck strength.
We note several limitations of our study. First, although 12-week-old female rats with growing bones were used in the present study, a baseline control group was not included for comparison. However, as the treatments were started 20 weeks after ovariectomy and lasted 12 weeks, the rats were 8 months old (32 weeks) when treatment started, and 11 months old when bone mass and strength were investigated. Second, the modest number of rats means that the power of the study to demonstrate statistically significant differences was relatively low. With more rats, any synergistic effect of combination alendronate and calcitriol therapy on bone mass and strength might become evident. Finally, we did not examine bone strength of the femoral neck in a configuration simulating a fall to the lateral side. The fall configuration is clinically more relevant because most osteoporotic hip fractures are associated with a fall . In the fall loading configuration, it is important to consider anteversion of the femoral neck. However, this has yet to be clarified in rats. Therefore, we investigated the fracture load at the femoral neck in a direction parallel to the femoral shaft axis; however, the axial loading may influence the fracture types owing to different internal stress distributions. Although approximately half of osteoporotic hip fractures are intertrochanteric in human , all fractures in the present study were found in midcervical region.
Clinical studies have shown that vitamin D3 is effective in increasing BMD and reducing hip fractures in postmenoposal osteoporosis ; however, others have failed to reproduce the same results . On the other hand, in animal studies using OVX rats, vitamin D3 treatment prevented bone loss occurring as a result of estrogen deficiency [12, 13]. Since the bone remodeling period in OVX rats is shorter than that in humans , the reason for the discrepancy between OVX rats and humans is may be related to enhanced remodeling activity in the rat. In addition, the discrepancy may be due to a genetic difference in the sensitivity to vitamin D between rats and humans .
In summary, the present study showed that combination therapy with alendronate and calcitriol significantly restored cortical and cancellous bone loss that was due to estrogen deficiency in OVX rats. Although no synergistic effects of the two agents were found in terms of bone mass at the femoral neck, the combined treatment does reflect the effects of the two agents. On mechanical testing, our results demonstrated that the combined treatment significantly improved bone fragility of the femoral neck in osteopenic conditions.