Calcium and vitamin D are essential for combating osteoporosis. However, enhancing the anti-osteoporosis effects of the supplements remains a challenge. This study introduced an optimized multiple emulsion micro-reaction method for preparing vitamin D3/calcium phosphate microparticles. The microstructure and characteristics of vitamin D3/hydroxyapatite microparticles (VD3@HAP) and vitamin D3/calcium hydrophosphate microparticles (VD3@CHP) were analyzed using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and stress testing. The calcium absorption and anti-osteoporosis effects of VD3@HAP and VD3@CHP were evaluated in situ and in vivo, respectively. FTIR and XRD analyses confirmed that the microparticles retained the spectral characteristics of calcium phosphates, while VD3@HAP exhibited randomly distributed pores on its surface, enhancing both the loading capacity and photostability of vitamin D3 compared to VD3@CHP. Additionally, the vitamin D3/calcium phosphate microparticles prepared by this method significantly increased bone mineral density and decreased serum alkaline phosphatase levels. Notably, VD3@HAP demonstrated superior calcium absorption, reduced marrow cavity area, and improved osteoporosis symptoms in the rat model compared to VD3@CHP, as evidenced by absorption assay, X-ray analysis, and HE staining. Thus, the multiple emulsion micro-reaction method effectively prepared vitamin D3/calcium phosphate microparticles that enhanced the anti-osteoporosis effects of supplementation, particularly VD3@HAP. We anticipate that VD3@HAP could serve as a safe and effective calcium supplement with significant potential for preventing and treating osteoporosis.
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