Researches have shown that rapamycin has an advantageous impact on cancellous bone, but its effect on cortical bone or overall biomechanical properties of long bones is rarely reported [9, 15, 16, 18, 23]. The present study verified that rapamycin improved cancellous bone at the distal end of the femur, but we also found a reduction in cortical bone of long bones. We further confirmed that rapamycin did not ameliorate the biomechanical properties reduced by ovariectomy, either in the distal femur or in the tibia. This may be related to the excessive autophagy caused by rapamycin and ovariectomy combined. In addition, this study also confirmed that ovariectomy resulted in cortical bone loss in mice, a phenomenon not observed in some studies of OVX induced osteoporosis, which may be related to the long-term observation of OVX mice.
Studies have shown that rapamycin has a significant effect on reducing cancellous bone loss in osteoporotic animal models, including age-related osteoporosis, alcohol-induced osteoporosis, and postmenopausal osteoporosis [15, 16, 18]. The present study has also confirmed this effect of rapamycin in OVX mice. However, as the treatment strategy of osteoporosis is mainly focused on cancellous bone, there is still a lack of attention on the effect of cortical bone. A study by Wu et al. indicated that in OVX mice with iron accumulation, rapamycin has no effect on cortical bone although it increases cancellous bone mass . Kneissel et al. suggested similar results in the study of OVX mice treated with rapamycin analogue . In the present study, we evaluated the cortical bone not only at the diaphyses, but also at the distal femur as the same level of the cancellous bone. The results have shown that rapamycin has no effect on cortical bone in mid-diaphyses, as had been found in previous studies. However, we did observe further reductions in the area and thickness of the cortical bone in the distal femur. This suggests that contrary to the superior effects on cancellous bone, rapamycin treatment could not alleviate cortical bone loss in OVX mice. In addition, it also implies that the cortical bone from distal femurs responds more rapidly to the intervention of rapamycin than that from mid-diaphyses.
Interestingly, although rapamycin further decreases the bone area and thickness of cortical bone at the end of long bones, it reduced the vascular canal porosity in this section. This suggests that rapamycin reduces the size of the cortical bone but improves its density. The effects of rapamycin on bone tissue are therefore complex.
Since cancellous and cortical bone showed opposite trends under rapamycin treatment, it is necessary to assess the effect of rapamycin on long bone in terms of overall biomechanical function. The results indicate that rapamycin did not improve the biomechanical properties of the long bones of OVX mice, either in terms of stiffness or maximum load. Here, the simulated compressive test was performed on the distal femurs only. For mid-diaphyses of femurs, the biomechanical properties are mainly determined by the cross-sectional bone area, showing no change after the rapamycin treatment.
It is worth mentioning that whether cortical bone loss and attenuated biomechanical properties occur in OVX mice remains controversial. The present study has demonstrated that cross-sectional bone area and cortical bone thickness were decreased in the mid-diaphyses of femurs, which is consistent with the investigation of tibial cortical midshafts from OVX mice . Furthermore, reduced cortical bone cross-sectional area and bone mineral density in the distal femurs were also observed. However, Pereira et al. suggested that for rats 10 weeks after ovariectomy, cancellous bone was damaged but not cortical bone in the distal femur . It is possible that differences in the timing of intervention as well as species differences are responsible for the different results.
Besides, this study supports that OVX results in higher vascular canal porosity, weakening the biomechanical properties of the long bone, which is in good agreement with the study of femoral necks from rats . A study of rats 10 weeks post-OVX also has indicated that estrogen deficiency leads to increased cortical bone vascular canal porosity, which has been proved to closely relate with bone strength [3, 21]. Shah et al. verify this change in cortical vascular canal porosity in a study of rats 4 and 8 weeks after ovariectomy. However, they tend to refute the decreased mechanical properties induced by ovariectomy, because the indentation hardness and elastic modulus are unaffected . The reason for the different results may be that the indentation hardness and elastic modulus and the axial compression stiffness and maximum load measured in this study are evaluated from different aspects of biomechanical properties. Moreover, we believe that the biomechanical properties of long bones could be weakened during prolonged estrogen deficiency states. Different duration after the intervention may be another reason for the different results.
Here, autophagy activity was evaluated to explore the possible mechanisms of changes in bone. Autophagy acts to degrade the impaired proteins or organelles to maintain cell homeostasis, it can be activated by cellular stress such as under situations of energy deficiency, starvation or hypoxia . The increased ratio of LC3-Ⅱ/LC3-Ⅰ and reduced P62 expression serve as indexes of activated autophagic activity [7, 8]. In this study, 12 weeks after the intervention, the OVX group showed markedly higher activity of autophagy in bone tissue when compared with the Sham group, indicating that ovariectomy has an activating function to autophagy. The results are in agreement with previous studies [2, 28]. Estrogen withdrawal, increased oxidative stress and up-regulation of microRNA-119a-3p are the potential factors for autophagic activation in the bone of OVX mice [2, 5, 22]. Rapamycin has been shown to inhibit mTORC1 specifically, leading to increased autophagy activity . Therefore, for bone tissue of OVX mice, autophagy was further activated with rapamycin intervention.
Researches have indicated that inhibition of autophagy results in decreased differentiation and bone resorption [13, 26]. Besides, Gavali et al. reported that estrogen reduces osteoblasts apoptosis and enhances mineralization by promoting autophagy . These findings are supported by Yang et al. . Therefore, it is believed that moderate autophagy is advantageous for the survival or functions of osteoclasts and osteoblasts. However, in this study, after further activating autophagy by rapamycin, TRAP-positive osteoclasts were noticeably reduced in the epiphysis, suggesting the damaged osteoclastogenesis. Also, the expression of BALP was decreased, indicating restricted differentiation of osteoblasts. We suppose that activated autophagy might act as a protective reaction after ovariectomy to resist cellular stress, while excessive autophagy could instead cause damage. This idea is supported by researches for autophagy in the motor neuron. In neurodegenerative disease, autophagy may have a neuroprotective effect by degrading aggregated proteins, but excessive autophagy by rapamycin treatment results in mitochondria damage and apoptosis in motor neuron [12, 29]. Therefore, in this study, it could be speculated that both osteoclasts and osteoblasts are damaged by excessive autophagy simultaneously, but the mechanisms underlying the inconsistent changes in cancellous and cortical bone require further study.
There are still some limitations in this study that need to be recognized. Firstly, in this study, mice received prolonged rapamycin treatment immediately after ovariectomy, other dosages and intervention time of rapamycin treatment should be taken into consideration. Secondly, there is a lack of molecular biology testing to verify the hypothesized mechanisms, such as the differentiation of osteoblasts. Thirdly, the number of tibiae on which high-resolution micro-CT scanning was performed was limited and was only used as a preliminary determination. On the other hand, the advantages of this study were the long duration of the intervention and the detection of different parts of the cortical bone. Also, we performed biomechanical tests on long bones as a functional assessment.
Taken together, rapamycin may not improve the quality of long bone in OVX mice. Excessive autophagy activity might be a possible mechanism. These findings may help to provide a comprehensive view of rapamycin treatment. In the clinical use of rapamycin, especially for postmenopausal women, there is a need to pay close attention to the possible adverse changes in cortical bone. In addition, we have demonstrated that cortical bone loss and attenuated biomechanical properties occur in the long bone after long time OVX, besides of cancellous bone loss.