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. 2014 Jun 13;9(6):e99920.
doi: 10.1371/journal.pone.0099920. eCollection 2014.

Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

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Free PMC article

Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

JunLi Liu et al. PLoS One. .
Free PMC article

Abstract

Objective: Collagen 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.

Methods: Mouse 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.

Results: Cell 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.

Conclusions: Bovine 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of bovine CP compounds on the proliferation of MC3T3-E1 cells.
The proliferation of MC3T3-E1 cells was measured by MTT assays after treatment with CP at 0.1–6 mg/mL (A) or 0.3 and 3 mg/mL (B) for 3–7 days. Data are expressed as means ± standard deviation (n = 6). *p<0.05, **p<0.01, Significant difference from CN. CP: bovine collagen peptides compounds; CN: control.
Figure 2
Figure 2. Alteration of the cell cycle distribution of MC3T3-E1 cells by bovine CP compound treatment.
Cell cycle analyses were performed by FCM scanning. Representative histograms of MC3T3-E1 cells in the CP-treated and CN groups at 4–5 days are shown. Data are expressed as means ± standard deviation (n = 4). *p<0.05, **p<0.01, Significant difference from CN. CP: bovine collagen peptides compounds; CN: control.
Figure 3
Figure 3. Real-time-PCR analysis of the expressions levels of different osteoblast markers after bovine CP compound treatment.
Real-time PCR analyses of the Runx2 (A), ALP (B), Col1α1 (C), and OC (D) mRNA levels in MC3T3-E1 cells induced with osteogenic medium after treatment with 3 mg/mL CP or no treatment (CN) for 7 or 14 days are shown. The results were normalized by the mRNA levels of β-actin as a housekeeping gene. Data are expressed as means ± standard deviation (n = 4). *p<0.05, **p<0.01, Significant difference from CN. CP: bovine collagen peptides compounds; CN: control.
Figure 4
Figure 4. Osteogenic activities of bovine CP compounds in MC3T3-E1 cells.
The ALP activity (A), OC amount (B), and Runx2 expression (C, D) were determined by colorimetric analysis, ELISA, and western blotting analysis, respectively. MC3T3-E1 cells induced with osteogenic medium after treatment with 3 mg/mL CP or no treatment (CN) for 7 or 14 days are shown. The expression of Runx2 was normalized by that of β-actin as a housekeeping gene (C, D). Data are expressed as means ± standard deviation (n = 3). *p<0.05, **p<0.01, Significant difference from CN. CP: bovine collagen peptides compounds; CN: control.
Figure 5
Figure 5. Influence of bovine CP compounds on mineralization in MC3T3-E1 cells.
The calcium deposits in the mineralized matrix were analyzed by alizarin red staining. MC3T3-E1 cells induced with osteogenic medium after treatment with 0.3–3 mg/mL CP or no treatment (CN) for 14 or 21 days are shown. Data are expressed as means ± standard deviation (n = 4). *p<0.05, Significant difference from CN. CP: bovine collagen peptides compounds; CN: control.

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References

    1. Kawaguchi T, Nanbu PN, Kurokawa M (2012) Distribution of prolylhydroxyproline and its metabolites after oral administration in rats. Biol Pharm Bull 35: 422–427. - PubMed
    1. Schadow S, Siebert HC, Lochnit G, Kordelle J, Rickert M, et al. (2013) Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates. PLoS One 8: e53955. - PMC - PubMed
    1. Moskowitz RW (2000) Role of collagen hydrolysate in bone and joint disease. Semin Arthritis Rheum 30: 87–99. - PubMed
    1. Alemán A, Giménez B, Montero P, Gómez-Guillén MC (2011) Antioxidant activity of several marine skin gelatins. LWT Food Sci Technol 44: 407–413.
    1. Alemán A, Pérez-Santín E, Bordenave-Juchereau S, Arnaudin I (2011) Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Res Int 44: 1044–1051.

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Grant support

This work was supported by The cooperation project from Technical institute of Physics and Chemistry,CAS (Grant No. LHS- DBSW2013-01).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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