# Free amino acid–rich egg yolk protein hydrolysate promotes osteogenesis of MC3T3-E1 cells association with β-catenin nuclear translocation

## TL;DR

A protein hydrolysate from egg yolk promotes bone cell development and could help treat bone diseases.

## Contribution

The study identifies a new functional food ingredient with potential for preventing or improving bone metabolic diseases.

## Key findings

- Y-PEP enhanced osteoblast differentiation and mineralization in a dose-dependent manner.
- Y-PEP increased β-catenin levels and promoted its nuclear translocation, upregulating osteogenesis-related genes.
- The amino acid composition of Y-PEP synergistically supports osteogenesis through metabolism and signaling.

## Abstract

Promoting osteogenesis is a key approach to preventing and improving bone metabolic diseases.

This study investigated the impact of free amino acid-rich egg yolk protein hydrolysate (Y-PEP) on MC3T3-E1 osteoblast differentiation and mineralization.

Free amino acids in Y-PEP were quantified using a high-speed amino acid analyzer. MC3T3-E1 cells were osteogenically induced with Y-PEP (5–100 μg/mL), and cell viability (72 h), alkaline phosphatase activity and collagen synthesis (day 9), as well as calcium deposition and osteocalcin production (day 18) were assessed. Moreover, runt-related transcription factor 2/osterix mRNA (qRT-PCR) and total/cytosolic/nuclear β-catenin and β-catenin phosphorylation (western blot) were measured.

Twenty-one amino acids, including leucine, lysine, arginine, glutamic acid, and valine, were identified and quantified in Y-PEP by comparing retention times and peak areas with amino acid mixture standard solutions. Y-PEP concentrations below 100 μg/mL had no impact on MC3T3-E1 osteoblast viability. Y-PEP enhanced osteoblast differentiation markers in a dose-dependent manner at concentrations from 5 to 100 μg/mL and promoted mineralization markers in mature osteoblasts dose-dependently at 25–50 μg/mL. The pro-osteogenic effect of Y-PEP may involve increasing total cellular β-catenin levels and promoting β-catenin nuclear translocation rate to upregulate transcription of osteogenesis-associated genes. The osteogenic activity of Y-PEP may result from the synergistic effects among signal transduction, metabolism, and mineral handling driven by its complex amino acid composition.

Y-PEP can promote osteogenesis in MC3T3-E1 cells and has the potential to serve as a functional food ingredient for preventing or improving metabolic bone diseases.