Rapamycin Alleviates Cancellous Bone Loss and Cortical Bone Porosity In Ovariectomized Mice But Not Overall Biomechanical Performance

Background: Recent studies have shown that rapamycin (Rapa) rescues cancellous bone loss of osteoporosis models, but its effects on cortical bone and the biomechanical properties remain uncertain. This study was aimed to determine whether rapamycin improve the quality of long bones in ovariectomy (OVX)-induced osteoporosis.Methods: Thirty female C57BL/6J mice were randomly divided into Sham, OVX and OVX+Rapa group. Mice in the OVX+Rapa group were injected intraperitoneally with 1.5 mg/kg rapamycin daily after ovariectomy. After 12 weeks, the microstructures of femurs and the vascular canal porosity tibiae were analyzed by micro-CT. The compressive stiffness of the distal femurs was calculated with the micro-finite element method, and the bending strength of the tibiae was evaluated with a three-point bending test. Western blot of LC3, P62 was used to assess autophagy activity of bone. The number of osteoclasts was quantified by tartrate-resistant acid phosphatase (TRAP) staining. ELISA detected the concentration of bone-specific alkaline phosphatase (BALP) in serum.Results: OVX led to a decrease in cross-sectional areas of the mid-diaphyses and distal femoral cortical bones, increasing the cortical bone vascular canal porosity from 1.42% to 4.79%. The rapamycin reduced cancellous bone loss and decreased cortical bone vascular canal porosity by 3.9%, but further reduced the thickness of the distal femoral cortical bone by 16.7%, with no significant effect on the cortical bone in the mid-diaphyses. Compared to the Sham group, OVX mice showed a decrease in distal femoral stiffness to 6497 N/mm and a decrease in tibial maximum load to 6.08 N, while rapamycin intervention did not significantly improve the decreased biomechanical properties. Moreover, rapamycin remarkably reduced the TRAP-positive osteoclasts and the concentration of serum BALP by 80.6% and 30.8%, respectively. Autophagy was activated in the OVX group compared with the sham group.Conclusions: This study has demonstrated that rapamycin ameliorates cancellous bone loss and cortical bone porosity in ovariectomized mice but partly reduces the size of cortical bone, while has no effect on improving the biomechanical performance. Additionally, the present study also proved that OVX led to both cancellous and cortical bone loss, with attenuated biomechanical properties of long bones.

Rapamycin Alleviates Cancellous Bone Loss and Cortical Bone Porosity in Ovariectomized Mice but Not Overall Biomechanical Performance

Background: Recent studies have shown that rapamycin (Rapa) rescues cancellous bone loss of osteoporosis models, but its effects on cortical bone and the biomechanical properties remain uncertain. This study was aimed to determine whether rapamycin improve the quality of long bones in ovariectomy (OVX)-induced osteoporosis.Methods: Thirty female C57BL/6J mice were randomly divided into Sham, OVX and OVX+Rapa group. Mice in the OVX+Rapa group were injected intraperitoneally with 1.5 mg/kg rapamycin daily after ovariectomy. After 12 weeks, the microstructures of femurs and the vascular canal porosity tibiae were analyzed by micro-CT. The compressive stiffness of the distal femurs was calculated with the micro-finite element method, and the bending strength of the tibiae was evaluated with a three-point bending test. Western blot of LC3, P62 was used to assess autophagy activity of bone. The number of osteoclasts was quantified by tartrate-resistant acid phosphatase (TRAP) staining. ELISA detected the concentration of bone-specific alkaline phosphatase (BALP) in serum.Results: OVX led to a decrease in cross-sectional areas of the mid-diaphyses and distal femoral cortical bones, increasing the cortical bone vascular canal porosity from 1.42% to 4.79%. The rapamycin reduced cancellous bone loss and decreased cortical bone vascular canal porosity by 3.9%, but further reduced the thickness of the distal femoral cortical bone by 16.7%, with no significant effect on the cortical bone in the mid-diaphyses. Compared to the Sham group, OVX mice showed a decrease in distal femoral stiffness to 6497 N/mm and a decrease in tibial maximum load to 6.08 N, while rapamycin intervention did not significantly improve the decreased biomechanical properties. Moreover, rapamycin remarkably reduced the TRAP-positive osteoclasts and the concentration of serum BALP by 80.6% and 30.8%, respectively. Autophagy was activated in the OVX group compared with the sham group.Conclusions: This study has demonstrated that rapamycin ameliorates cancellous bone loss and cortical bone porosity in ovariectomized mice but partly reduces the size of cortical bone, while has no effect on improving the biomechanical performance. Additionally, the present study also proved that OVX led to both cancellous and cortical bone loss, with attenuated biomechanical properties of long bones.

Applied mechanical loading to mouse hindlimb acutely increases skeletal perfusion and chronically enhanced vascular porosity

Blood supply is essential for osteogenesis, yet its relationship to load-related increases in bone mass is poorly defined. Herein, we aim to investigate the link between load-induced osteogenesis and the blood supply (bone perfusion and vascular porosity) using an established osteogenic noninvasive model of axial loading. Accordingly, 12 N mechanical loads were applied to the right tibiae of six male C57BL6 mice at 10–12 wk of age, 3 times/wk for 2 wk. Skeletal perfusion was measured acutely (postloading) and chronically in loaded and contralateral, nonloaded hindlimbs by laser-Doppler imaging. Vascular and lacunar porosity of the cortical bone and tibia load-related changes in trabecular and cortical bone was measured by nanoCT and micro-CT, respectively. We found that the mean skeletal perfusion (loaded: nonloaded limb ratio) increased by 56% immediately following the first loading episode (vs. baseline, P < 0.01), and a similar increase was observed after all loading episodes, demonstrating that these acute responses were conserved for 2 wk of loading. Loading failed, however, to engender any significant chronic changes in mean perfusion between the beginning and the end of the experiment. In contrast, 2 wk of loading engendered an increased vascular canal number in the tibial cortical compartment (midshaft) and, as expected, also increased trabecular and cortical bone volumes and modified tibial architecture in the loaded limb. Our results indicate that each episode of loading both generates acute enhancement in skeletal blood perfusion and also stimulates chronic vascular architectural changes in the bone cortices, which coincide with load-induced increases in bone mass. NEW & NOTEWORTHY This study investigated modifications to the blood supply (bone perfusion and intracortical vascular canals) in mechanoadaptive responses in C57BL6 mice. Each episode of mechanical loading acutely increases skeletal perfusion. Two weeks of mechanical loading increased bone mass and cortical vascular canal number, while there was no chronic increase in hindlimb perfusion. Our findings suggest that the blood supply may participate in the processes that govern load-induced bone formation.

Hyperglycemia compromises Rat Cortical Bone by Increasing Osteocyte Lacunar Density and Decreasing Vascular Canal Volume

Uncontrolled diabetes is associated with increased risk of bony fractures. However, the mechanisms have yet to be understood. Using high-resolution synchrotron micro-CT, we calculated the changes in the microstructure of femoral cortices of streptozotocin-induced hyperglycemic (STZ) Wistar Albino rats and tested the mechanical properties of the mineralized matrix by nanoindentation. Total lacunar volume of femoral cortices increased in STZ group due to a 9% increase in lacunar density. However, total vascular canal volume decreased in STZ group due to a remarkable decrease in vascular canal diameter (7 ± 0.3 vs. 8.5 ± 0.4 µm). Osteocytic territorial matrix volume was less in the STZ group (14,908 ± 689 µm3) compared with healthy controls (16,367 ± 391 µm3). In conclusion, hyperglycemia increased cellularity and lacunar density, decreased osteocyte territorial matrix, and reduced vascular girth, in addition to decreasing matrix mechanical properties in the STZ group when compared with euglycemic controls.

The effects of growth rate and biomechanical loading on bone laminarity within the emu skeleton

The orientation of vascular canals in primary bone may reflect differences in growth rate and/or adaptation to biomechanical loads. Previous studies link specific canal orientations to bone growth rates, but results between different taxa are contradictory. Circumferential vascular canals (forming laminar bone) have been hypothesized to reflect either (or both) rapid growth rate or locomotion-induced torsional loading. Previous work on the hindlimb biomechanics in the emu shows that the femur and tibiotarsus experience large shear strains, likely resulting from torsional loads that increase through ontogeny. Here, we test how growth rate and biomechanical loading affect bone laminarity in wing and hindlimb elements from growing emu (2–60 wks). If laminar bone is an adaptation to torsion-induced shear strains, it should increase from juveniles to adults. Alternatively, if bone laminarity reflects rapid growth, as has been shown previously in emu, it should be abundant in fast-growing juveniles and decrease with age. Transverse mid-shaft histological sections from the limb bones (femur, tibiotarsus, humerus, ulna, and radius) were prepared and imaged. Growth rates were measured using fluorescent bone labels. Vascular canal orientation was quantified using laminarity index (proportion of circumferential canals). Principal components analysis was performed to convert highly correlated variables (i.e., mass, age, growth rate, and shear strain) into principal components. Random-intercept beta regression modeling determined which principal components best explained laminarity. The fastest growth rates were found in young individuals for all five skeletal elements. Maximum growth rate did not coincide with peak laminarity. Instead, in the femur and tibiotarsus, elevated laminarity is strongly correlated with adult features such as large size, old age, and modest growth rate. This result is contrary to predictions made based on a previous study of emu but is consistent with results observed in some other avian species (penguin, chicken). Shear strain in the caudal octant of the femur and tibiotarsus is positively correlated with laminarity but has a weaker effect on laminarity relative to mass, age, and growth rate. Laminarity in the wing elements is variable and does not correlate with ontogenetic factors (including mass, age, and growth rate). Its presence may relate to relaxed developmental canalization or a retained ancestral feature. In conclusion, ontogeny (including growth rate) is the dominant influence on vascular canal orientation at least in the hindlimb of the emu.