Effect of mechano-growth factor on proliferation and differentiation of periodontal ligament stem cells

Abstract

Objective To investigate the effects and molecular mechanisms of mechano-growth factor (MGF) on the proliferation and differentiation of periodontal ligament stem cells (PDLSCs). Methods PDLSCs were obtained using magnetic bead sorting. Flow cytometry was performed to identify biomarkers. The clonogenicity and multidifferentiation potential of PDLSCs were identified by colony-forming unit, osteogenic and adipogenic differentiation assays. A CCK8 assay was used to detect the cell activity under different concentrations of mechano-growth factors (MGF-Ct24E). Western blot was used to detect the protein expression of proliferating cell nuclear antigen(PCNA), Scleraxis, collagen type I alpha 1 (COL1A1）, Osterix, Yes-associated protein (YAP) and phosphorylation Yes-associated protein (P-YAP）in PDLSCs. YAP protein expression was observed by immunofluorescence. Knockdown of YAP expression by a siRNA, detected the expression of PCNA, Scleraxis and COL1A1under MGF-Ct24E in PDLSCs. Results PDLSCs showed high expression of stem cell markers (CD29, CD90 and CD105) and low expression of hematopoietic markers (CD34 and CD45). PDLSCs also have a strong ability to clone. Red calcium junctions were observed by Alizarin red staining, and red lipid droplets were observed by Oil red O staining. After treatment with 50 ng/mL and 100 ng/mL MGF-Ct24E for 24 h, the cell activity of periodontal ligament stem cells was significantly enhanced (P<0.05). The protein expression of PCNA, Scleraxis and COL1A1 was significantly upregulated, and the protein expression of Osterix was significantly downregulated (P<0.05). After treatment with 50 ng/mL mechano-growth factor for 24 h, the phosphorylation of YAP protein was significantly enhanced (P<0.05), and YAP protein was observed to accumulate in the nucleus by immunofluorescence. Following inhibition of YAP expression, PCNA and Scleraxis had significantly downregulated expression caused by MGF-Ct24E (P<0.05). Conclusion MGF promotes proliferation and fibrogenesis via upregulation of YAP activities.

Keywords

• mechano-growth factor
• periodontal membrane
• periodontal ligament stem cells
• periodontal ligament fibroblasts
• yes-associated protein
• phosphorylation-yes-associated protein
• cell proliferation
• fibrogenesis
• osteogenic differentiation
• regeneration and repair