Phosvitin phosphopeptides and peptides-calcium chelate promote calcium deposition in a three-cell co-culture system by modulating the OPG/RANKL/RANK signaling pathway

Phosvitin (PV) is a highly phosphorylated protein found in egg yolk. PV hydrolysate (PPP) has excellent calcium-binding capacity, although its ability to promote calcium deposition and the underlying mechanism are unclear. This study aimed to explore the calcium deposition capacity and mechanisms of PPP and PPP-Ca. The fraction 3 (PPP3) was isolated by anion-exchange chromatography and exhibited the best calcium-binding activity (98.11 ± 0.91%). To mimic the microenvironment of bone formation, a three-cell co-culture model of intestinal epithelial cells-osteoblasts-osteoclasts (Caco-2/MC3T3-E1/RAW264.7) was established. The alkaline phosphatase results indicated that PPP3 and PPP3-Ca significantly promoted osteoblasts differentiation compared to CaCl₂. Correspondingly, PPP3, PPP3-Ca, and CaCl₂ reduced tartrate resistant acid phosphatase content by 43.84%, 46.92% and 17.21%, respectively, suggesting that PPP3 and PPP3-Ca effectively inhibited osteoclasts differentiation. The molecular mechanism by which PPP3 and PPP3-Ca inhibited osteoclasts bone resorption involved regulating mRNA expression of the OPG/RANKL/RANK pathway. Notably, PPP3-Ca demonstrated higher pro-calcium deposition in co-cultured cells, about 1.70 times higher than CaCl₂. PPP3-Ca dependent high phosphorylation exerted a pro-mineralization effect, as analyzed by scanning electron microscopy and energy dispersive spectroscopy. Overall, this study suggested that PPP3-Ca as organic calcium was more effective than CaCl₂ in promoting calcium deposition.