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. 2019 May 11;8(5):446.
doi: 10.3390/cells8050446.

Collagen Peptide Upregulates Osteoblastogenesis from Bone Marrow Mesenchymal Stem Cells through MAPK- Runx2

Jeevithan Elango  1 , Jeyashakila Robinson  2 , Jingyi Zhang  3 , Bin Bao  4 , Nan Ma  5   6 , José Eduardo Maté Sánchez de Val  7 , Wenhui Wu  8   9
Affiliations

Collagen Peptide Upregulates Osteoblastogenesis from Bone Marrow Mesenchymal Stem Cells through MAPK- Runx2

Jeevithan Elango et al. Cells. .

Abstract

Collagen is the most abundant extracellular fibrous protein that has been widely used for biomedical applications due to its excellent biochemical and biocompatibility features. It is believed that the smaller molecular weight collagen, i.e., collagen peptide (CP), has more potent activity than native collagen. However, the preparation of CP from fish bone collagen is a complex and time-consuming process. Additionally, the osteogenic effect of CP depends on its molecular weight and amino acid composition. Considering the above concept, the present work was undertaken to extract the CP directly from Mahi mahi fish (Coryphaena hippurus) bones and test its osteogenic potential using bone marrow mesenchymal stem (BMMS) cells. The hydrolyzed collagen contained triple alpha chains (110 kDa) and a peptide (~1 kDa) and the peptide was successfully separated from hydrolyzed collagen using molecular weight cut-off membrane. CP treatment was up-regulated BMMS cells proliferation and differentiation. Interestingly, CP accrued the mineral deposition in differentiated BMMS cells. Protein and mRNA expression revealed that the osteogenic biomarkers such as collagen, alkaline phosphatase, and osteocalcin levels were significantly increased by CP treatment in differentiated BMMS cells and also further elucidated the hypothesis that CP was upregulated osteogenesis through activating Runx2 via p38MAPK signaling pathway. The above results concluded that the CP from Mahi mahi bones with excellent osteogenic properties could be the suitable biomaterial for bone therapeutic application.

Keywords: Mahi mahi; Runx2; collagen peptide; differentiation; mesenchymal stem cell; mineral deposition; p38MAPK.

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Conflict of interest statement

All authors declared no conflict of interest.

Figures

Figure 1
Figure 1
Tricine SDS-PAGE pattern of Mahi mahi bone collagen. Lane 1: protein standard marker; lane 2: collagen peptide; lane 3: collagen hydrolysate.
Figure 2
Figure 2
Effect of collagen peptide on proliferation (A), differentiation (B), and cellular alkaline phosphatase level (C) of bone marrow mesenchymal stem cells. CP-collagen peptide. * p < 0.05 vs. control.
Figure 3
Figure 3
Haematoxylin and eosin staining of control and collagen peptide (CP)-treated bone marrow mesenchymal stem (BMMS) cells. Scale bars: 100 micrometers.
Figure 4
Figure 4
(A) Histological staining for alkaline phosphatase (i–ii), alizarin red (iii–vi) and von Kossa (v–vi) of control and collagen peptide (CP)-treated bone marrow mesenchymal stem cells (scale bars: 0.1 cm). (B) Quantification of stained area of bone marrow mesenchymal stem cells. The percentage of stained area in bone cells was quantified using ImageJ software (Version 1.52n). CP-collagen peptide, * p < 0.05 vs. control.
Figure 4
Figure 4
(A) Histological staining for alkaline phosphatase (i–ii), alizarin red (iii–vi) and von Kossa (v–vi) of control and collagen peptide (CP)-treated bone marrow mesenchymal stem cells (scale bars: 0.1 cm). (B) Quantification of stained area of bone marrow mesenchymal stem cells. The percentage of stained area in bone cells was quantified using ImageJ software (Version 1.52n). CP-collagen peptide, * p < 0.05 vs. control.
Figure 5
Figure 5
Immunocytochemistry of control and collagen peptide (CP)-treated bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cells treated with primary antibody (anti-Col1α2) overnight and DyLight 594-conjugated secondary antibody (scale bars: 75 micrometers). I–ii, iii–iv, and v–vi: 7, 14 and 21 days treated BMMS cells, respectively.
Figure 6
Figure 6
Osteogenic mRNA expression of collagen peptide-treated bone marrow mesenchymal stem cells. ALP: alkaline phosphatase; CP: collagen peptide. * p < 0.05 vs. control.
Figure 7
Figure 7
Western blot analysis of collagen peptide (CP)-treated BMMS cells. * p < 0.05 vs. control.
Scheme 1
Scheme 1
Schematic illustration of extraction steps involved in isolation of collagen peptide from Mahi mahi bone. MWCM: molecular weight cut-off membrane.
See this image and copyright information in PMC

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