Hydroxyapatite affinity chromatography isolates high-affinity collagen peptides, boosting MC3T3-E1 osteogenesis

Efficiently isolating highly active calcium-binding peptides from collagen remains a challenge, limiting their therapeutic potential for bone health. Conventional methods often yield peptides with suboptimal calcium-binding affinity and thus, reduced osteogenic activity. This bottleneck restricts the development of effective functional foods or supplements for conditions like osteoporosis or osteopenia. A method to selectively enrich peptides with superior calcium-binding capacity and direct osteogenic effects could significantly advance strategies for bone mineral density improvement and skeletal repair.

Researchers employed hydroxyapatite affinity chromatography (HAC) to enrich bovine-bone-derived collagen peptides (CPs). The study focused on isolating peptides with high calcium-binding capacity. The purified fraction, designated F2, was then characterized for its molecular weight distribution and amino acid composition. Structural analyses, including FTIR, SEM, and AFM, were performed to confirm calcium chelation and conformational changes. In vitro osteogenic activity was assessed in MC3T3-E1 osteoblast cells, comparing F2 against unpurified CPs for proliferation, differentiation, and mineralization. Molecular docking was used to predict potential binding of key peptides to osteogenic receptors.

The hydroxyapatite affinity chromatography successfully enriched a specific fraction (F2) of collagen peptides with significantly enhanced calcium-binding capacity. F2 exhibited a 42.44 µg/mg calcium-binding capacity, markedly higher than unpurified CPs. This fraction was predominantly composed of low-molecular-weight (LMW) peptides, ranging from 1-3 kDa, and was notably rich in acidic amino acids like Asp and Glu. Structural analyses confirmed effective calcium chelation by F2, inducing observable conformational changes. In vitro experiments demonstrated F2's superior osteogenic potential: > F2 significantly promoted MC3T3-E1 osteoblast proliferation, differentiation, and mineralization compared to the unpurified CPs, indicating enhanced bioactivity. Molecular docking studies further suggested that key peptides within F2 might interact with osteogenic receptors BMPR1 and TGFBR1, implying the involvement of TGF-β/BMP signaling pathways in its osteogenic effects.