Osteoporosis (OP) predominantly affects elderly individuals. Stem cells show potential for treating OP. However, animal models with normal immune function can eliminate implanted human cells. This study utilized naturally aging NOD/SCID mice, which exhibit immunodeficiency, to create a human osteoporosis model. This approach helps to minimize the premature immune clearance of transplanted allogeneic or xenogeneic cells in preclinical studies, allowing for a more accurate replication of the clinical pharmacological and pharmacokinetic processes involved in stem cell interventions for osteoporosis. NOD/SCID mice were fed until 12, 32, and 43 weeks of age, respectively, and then euthanized. We harvested lumbar vertebra for Micro‐Computed Tomography (Micro-CT) scanning and pathological examination. Additionally, we performed biomechanical testing of lumbar vertebra to assess the severity of osteoporosis. We utilized real-time RT-PCR to assess gene expression changes associated with bone metabolism, aging, inflammation, oxidative stress, and the Tgf-β1/Smad3 signaling pathway. In addition, the protein expression levels of P16, Tgf-β1 and Smad3 were detected using Western Blotting (WB). In comparison to 12-week-old mice, the 32-week-old and 43-week-old mice displayed significantly sparser and fractured trabeculae in their lumbar vertebra, lower bone mineral density (BMD), and changes in bone microstructural parameters (**P<0.01, ***P<0.001). Additionally, compared to 12-week-old mice, the 32-week-old and 43-week-old mice exhibited decreased expression of osteogenic genes (Alp, Opg, Sp7, Col1a1), increased expression of osteoclastic gene (Rankl), the number of TRAP-positive osteoclasts significantly increased in 32-week-old and 43-week-old mice compared to 12-week-old mice. The expression of genes related to aging and inflammatory (P16, Il-1β, Tnf-α) increases with advancing age (*P<0.05, **P<0.01, ***P<0.001). The expression of oxidative stress-related genes (Sod1, Sod2, Foxo3, Nrf2), as well as Tgf-β1 and Smad3 decreased with age (*P<0.05, **P<0.01, ***P<0.001). As age increases, the levels of P16 protein increase, Tgf-β1 and Smad3 proteins decrease. Our study successfully replicated osteoporosis models in NOD/SCID mice at both 32 and 43 weeks, with the latter exhibiting more severe osteoporosis. This condition seems to be driven by factors such as aging, inflammation, oxidative stress, and the Tgf-β1/Smad3 signaling pathway.
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