Time-Dependent Effects of Chronic Hypoxic Exposure on Microarchitecture of Different Skeletal Sites in Mice

Chen, Yanli, Suying Zhu, Doudou Hao, Zhiyou Shi, Yang Zhong, Suyuan Wang, and Yunhong Wu. Time-Dependent Effects of Chronic Hypoxic Exposure on Microarchitecture of Different Skeletal Sites in Mice. High Alt Med Biol. 00:00–00, 2026.—High-altitude hypoxia accelerates bone loss by disrupting bone metabolic homeostasis. Currently, hypoxic effects on bone and their link to exposure time remain undefined. This study aimed to systematically evaluate the dynamic effects of chronic hypoxic exposure on bone homeostasis and bone microstructure in different skeletal sites through in vivo and in vitro experiments. Male C57BL/6J mice were exposed to 5,500 m simulated hypoxia for 16 weeks. Micro-CT analyzed cervical (C3–C4), lumbar (L4–L6) vertebrae, and femurs. Histopathological analysis assessed bone tissue and multiorgan pathology. In vitro experiments, a 5% hypoxic culture system was established to detect the proliferation and differentiation capabilities of MC3T3-E1 osteoblasts. After 12 weeks of hypoxic exposure, compared with the control group, the trabecular separation (Tb.Sp) of the mouse femur was significantly increased, and the bone mineral content was significantly decreased. After 16 weeks of exposure, the trabecular bone volume/tissue volume (Tb.BV/TV) and bone mineral density of the cervical vertebrae, lumbar vertebrae, and femurs were significantly reduced. H&E staining showed osteoblasts in multiple bones of hypoxic mice shifted to quiescent states, and long-term hypoxia induced multi-organ damage (pulmonary, renal, colonic, skeletal muscle). 5% hypoxia significantly downregulated collagen type I and alkaline phosphatase (ALP) mRNA levels in MC3T3-E1 cells, inhibited ALP activity and mineralized nodule formation, and reduced osteoblast differentiation capacity. Chronic hypoxic exposure induces bone loss in multiple skeletal sites of mice in a time-dependent manner, with femur showing significantly higher sensitivity to hypoxic exposure than the cervical and lumbar vertebrae. The underlying mechanism may be related to hypoxia inhibiting osteoblast function and disrupting the balance of bone metabolism. Long-term hypoxia also causes pathological damage to multiple organs. This study provides evidence for clarification of the pathological mechanism of hypoxic bone injury and lays a foundation for the subsequent development of targeted intervention strategies for hypoxia-related bone diseases.