Microstructures and Anti-Osteoporosis Effects of Vitamin D₃/Calcium Phosphate Microparticles Prepared by Multiple Emulsion Micro-Reaction Method
Rui Yang a , Guohui Yan c , Chunling Li a,b , Chen Sun b , Bo Liu b , Fengzhi Zhou b , Qianqian Zheng b , Yuqi Sun b
- a The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, 121000, PR China
- b The School of Pharmacy, Jinzhou Medical University, Jinzhou, 121000, PR China
- c The School of Clinical Medicine, Fujian Medical University, Fujian, 350000, PR China
Abstract
This study introduces an innovative method for creating vitamin D3-loaded calcium phosphate microparticles using a multiple emulsion micro-reaction technique. The resulting formulations—VD3@HAP and VD3@CHP—were evaluated for structural integrity, calcium absorption, and anti-osteoporosis effects. VD3@HAP showed superior results, making it a promising alternative to traditional calcium supplements.
Introduction
Osteoporosis, a global health concern, affects over 200 million people worldwide. Despite the known benefits of calcium and vitamin D in bone health, current supplementation methods face challenges such as poor absorption, degradation of vitamin D3, and low bioavailability. The study proposes a dual-functional supplement system that not only delivers calcium and vitamin D3 more effectively but also improves their stability and therapeutic impact.
Materials & Methods
Researchers used Naâ‚‚HPOâ‚„, CaClâ‚‚, and vitamin D3 to create a W/O/W (water/oil/water) multiple emulsion, which served as the framework for forming microparticles. Two types were created:
VD3@HAP: Vitamin D3-loaded hydroxyapatite microparticles
VD3@CHP: Vitamin D3-loaded calcium hydrophosphate microparticles
The microparticles were characterized through FTIR, XRD, SEM, and stress testing. In vivo testing involved rat and mouse models for calcium absorption, bone mineral density (BMD), and biochemical markers such as alkaline phosphatase (ALP).
Figure 1: Characterization of vitamin D3/calcium phosphate microparticles. The FTIR (green zone) showed the characteristic absorption peaks of HAP and CHP, respectively. The XRD (yellow zone) displayed the typical diffraction peaks corresponding to the crystal structures of different calcium phosphates.
Figure 2: SEM analysis showed that HAP microparticles had a porous structure, enabling vitamin D3 to be absorbed into the surface, while CHP microparticles lacked pores, causing vitamin D3 to only adhere externally. This structural difference gave VD3@HAP a higher loading capacity. In light exposure tests, VD3@HAP also demonstrated significantly greater photostability than VD3@CHP, maintaining more of its vitamin D3 content under strong light conditions (4500 lx), while calcium levels remained stable in both.
Results
VD3@HAP exhibited:
Higher vitamin D3 loading capacity due to porous structure
Superior photostability under stress testing
More efficient calcium absorption in the intestine
 Figure 3: Vitamin D3/hydroxyapatite microparticles (VD3@HAP) showed superior calcium absorption and permeability compared to VD3@CHP in in situ intestinal perfusion tests. In vivo, rats treated with VD3@HAP demonstrated a significant increase in bone mineral density (BMD) and a notable reduction in alkaline phosphatase (ALP) levels, both indicators of improved bone health. These effects were stronger than those observed in the VD3@CHP group and comparable to the commercial supplement Caltrate.
In osteoporosis-induced mice, VD3@HAP significantly increased bone mineral density and decreased serum ALP levels, outperforming VD3@CHP and matching the performance of Caltrate (a commercial calcium supplement).
 Figure 4: Vitamin D3/calcium phosphate microparticles significantly improved osteoporosis symptoms in treated mice. X-ray analysis showed denser bone structure in the VD3@HAP group, while HE staining confirmed better trabecular formation, higher osteocyte density, and reduced marrow cavity size compared to the untreated osteoporosis group. These improvements were comparable to those seen in mice treated with Caltrate.
Conclusion
The study demonstrates that vitamin D3/calcium phosphate microparticles, especially VD3@HAP, offer an effective and biocompatible solution for osteoporosis treatment. This emulsion-based synthesis method provides improved vitamin D3 stability, enhanced calcium absorption, and measurable therapeutic benefits. VD3@HAP shows strong potential for clinical application in bone health supplementation.
References
Koo S, Lee EJ, Xiong H, Yun DH, McDonald MM, Park SI, Kim JS. Real-Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases. Angew Chem Int Ed Engl. 2024 Feb 5;63(6):e202318459. doi: 10.1002/anie.202318459. Epub 2023 Dec 29. PMID: 3810541
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