Preparation, characterization, and osteogenic activity mechanism of casein phosphopeptide-calcium chelate

Abstract

Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 105 mg/kg. The test results of Ultraviolet–Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 μg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.

A Comprehensive Review of Self-Assembled Food Protein-Derived Multicomponent Peptides: From Forming Mechanism and Structural Diversity to Applications

“…The DHTEK−zinc complex formed more stable spherical particles with a particle size of 100−500 nm, which could dissociate and rechelate during gastrointestinal digestion, thus achieving the protection and transport of zinc ions. 102 Casein phosphopeptide, derived from milk proteins, could form stable NPs, which have also been reported to serve as delivery carriers for calcium, 103 zinc, 104 and iron. 105 To summarize, the potential usage of FPDMPs as effective delivery vehicles for minerals could not only guard the stability of metal ions by interacting with them but also modulate the self-assembly behavior of peptides and bestow new functions and properties to them, which hold immense potential for applications across diverse fields, including food science, biomedicine, and nanomaterials.…”

Structural characterisation of deer sinew peptides as calcium carriers, their promotion of MC3T3-E1 cell proliferation and their effect on bone deposition in mice

“…When the organic ligand combines with the metal ion calcium, it causes the original absorption peak to shift or disappear or a new absorption peak to appear. 39 Therefore, the changes in the absorption wavelength and absorption intensity of the UV absorption spectrum are used to determine whether a new substance has been produced. Generally, the absorption peaks of peptide bonds in amino acid molecules are located between 200 and 300 nm.…”

Biocompatibility and osteoinductive ability of casein phosphopeptide modified polyetheretherketone

“…The findings were consistent with the previous studies. Wen Huang et al (2022) prepared CPP-calcium chelate and found it had positive effects on the proliferation and differentiation of MC3T3-E1 cells in vitro . Higher cell adhesion and proliferation and better cell adhesion morphology on PEEK-CPP samples might be relevant to the deeper valleys and grooves and higher hydrophilicity on PEEK-CPP surface.…”

“…Furthermore, some studies showed that CPP could facilitate the transcellular transport of Ca 2+ Li et al, 2021 found that CPP feeding substantially increased the femur index, serum Ca 2+ and serum osteocalcin levels, and femoral calcium content in rats. Moreover, in a study on the osteogenic ability of CPP-calcium chelate, it was proven that there were 321 differently expressed genes found in MC3T3-E1 cells and CPP might stimulate the osteogenic differentiation through AMPK signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and Wnt signaling pathway, and the expression of NOTUM, WIF1, LRP4, APOD, OGN, and IGF1 (Huang et al, 2022).…”

“…As we know, Ca 2+ are the main component of teeth and bones. Thus, CPP, which typically functions with amorphous calcium phosphate (ACP), has been applied as a carrier for Ca 2+ to improve remineralization and inhibit demineralization of enamel surfaces and has also been proven to have osteoinductive ability ( Cross et al, 2007 ; Dashper et al, 2016 ; Dawood et al, 2018 ; Alawadhi et al, 2020 ; Sionov et al, 2021 ; Huang et al, 2022 ). Therefore, CPP has great potential for research on implant surface modification and bone tissue engineering.…”