Targeted Isolation of Novel Calcium-Binding Peptides via Hydroxyapatite Affinity and Their Promotion Effect on Osteogenesis in MC3T3-E1 Cells.

Collagen peptides (CPs) have emerged as a promising food-derived strategy to enhance calcium utilization and osteogenesis, notably through specific calcium binding via chelation. However, conventional separation methods often fail to efficiently isolate peptides with high calcium-binding affinity, resulting in products with limited osteogenic activity. In this study, hydroxyapatite affinity chromatography (HAC) was employed, leveraging its dual ion-exchange and affinity properties, to enrich bovine-bone-derived CPs with high calcium-binding capacity. The purified fraction (F2) exhibited a significantly higher calcium-binding capacity (42.44 µg/mg) than unpurified CPs and was enriched in low-molecular-weight (LMW) peptides (1-3 kDa) rich in acidic amino acids (Asp and Glu). After calcium chelation, structural analysis (e.g., FTIR, SEM, and AFM) confirmed its effective calcium-binding capacity and conformational changes. In addition, in vitro assays demonstrated that F2 markedly promoted MC3T3-E1 osteoblast proliferation, differentiation, and mineralization compared with CPs, presenting enhanced osteogenic bioactivity. Molecular docking further indicated that key peptides may bind to the osteogenic receptors BMPR1 and TGFBR1, raising the possibility that the TGF-β/BMP signaling pathways could be involved. Collectively, our findings indicate that HAC is an effective strategy for enriching osteogenic CPs with high calcium-binding and osteogenic activity, offering a scalable strategy for producing potent peptides applicable to bone health management and therapeutics. PRACTICAL APPLICATIONS: This study provides a scalable method for extracting highly active calcium-binding peptides from collagen peptides using a hydroxyapatite column. These purified peptides can be developed into functional food ingredients or dietary supplements to support bone health. By improving calcium delivery efficiency and direct osteogenic activity, this approach offers a science-based strategy for next-generation bone health products.