Bovine collagen peptides compounds promote the proliferation and differentiation of MC3T3-E1 pre-osteoblasts.

Junli Liu,Yihu Wang,Shujun Song,Shaoyan Si,Bingxin Xu,Kai Feng,Ming Ma,Bing Zhang,Yanchuan Guo,Jigong Wu,Effie C Tsilibary

doi:10.1371/journal.pone.0099920

Abstract

ObjectiveCollagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells.MethodsMouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0.ResultsCell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days.ConclusionsBovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis.

Highlights

Collagen peptides (CPs) are the hydrolysate components of collagen and are known to have efficacy against various pathologic conditions [1,2]
No significant differences were observed between the optical density (OD) values of the compounds and untreated (CN) and CP compound-treated cells at 3 days
The OD values of the MC3T3-E1 cells after treatment with 0.6–6 mg/mL bovine CP compounds for 7 days were much higher than those of the CN cells (p,0.01, Figure 1A), and the best concentration of bovine CP compounds for proliferation at 7 days was 3 mg/mL (p,0.01, Figure 1B)

Summary

Introduction

Collagen peptides (CPs) are the hydrolysate components of collagen and are known to have efficacy against various pathologic conditions [1,2]. CP compounds from different species can produce a variety of active peptides with different bioactivities, such as high angiotensin I-converting enzyme inhibitory and antioxidant activities found in active peptides derived from fish or squid skin gelatins [4,5]. C2 (with cell adhesive properties) and E1 (with cell adhesive and antioxidant properties), have been isolated from bovine tendon collagen. The peptides supported faster wound closure than collagen under normal as well as stressed conditions [6]. Oral intake of specific bioactive CPs reduced skin wrinkles and had positive effects on dermal matrix synthesis [7]. Hydrolyzed collagen intake increased bone mass in growing rats trained with running exercise [8]

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Conclusion