Effect of Bone Morphogenetic Protein (BMP)-2, -4, and -7 on Proliferation and Osteogenic Differentiation in Human Periodontal Ligament Stem Cells (PDLSCs)

Abstract

Aim: To assess the effects of bone morphogenetic protein (BMP)-2, BMP-4, and BMP-7 on human periodontal ligament stem cells (PDLSC) proliferation and osteogenic differentiation. Methods: PDLSCs were isolated by an immunomagnetic method. Cell surface antigen (CD146, CD44, and CD34) expression and pluripotency (osteogenic and adipogenic) were determined. In doseresponse and time-course experiments, cultured PDLSCs were treated with individual BMPs, a combination of BMPs in 1:1 ratios, and a mixture of all three BMPs (1:1:1 ratio). In the dose-response experiments, PDLSCs were incubated for 12 d with medium containing BMPs at a dose of 0, 10, 25, 50, or 100 ng/ml. In the time-course experiments, PDLSCs were administered 50 ng/ml BMPs for different times (0, 3, 6, 12, or 24 d). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and an enzyme kit were used to quantify cell growth and alkaline phosphatase (ALP) activity. Immunohistochemistry and western blotting were used to assess the expression of osteogenic differentiation-related proteins, i.e., osteocalcin, bone sialoprotein, collagen type I, and collagen type III. Results: PDLSCs expressed CD146 (93%) and CD44 (91.2%) but not CD34 (1.8%). All cells exhibited osteogenic and adipogenic potential. PDLSC proliferation and ALP activity in PDLSCs treated with individual BMPs and BMPs combinations increased in a dose- and time-dependent manner; proliferation and ALP activity were greater upon treatment with BMP combinations than upon treatment with individual BMPs. Compared with those in the control group, osteogenic differentiation-related proteins levels in PDLSCs treated with 50 ng/ml BMPs for 12 d were markedly increased, whereas no obvious differences were detected between the groups subjected to different BMP treatments. Conclusion: BMP-2, BMP-4, and BMP-7 alone and in combination promoted the development and osteogenic differentiation of PDLSCs, and the promotion of these cellular processes was more pronounced upon treatment with BMP combinations.