Sequential application of bFGF and BMP-2 facilitates osteogenic differentiation of human periodontal ligament stem cells.

Abstract

Basic fibroblast growth factor (bFGF) promotes cells proliferation and chemotaxis and maintains stemness while inhibits mineralized nodule formation. Bone morphogenetic protein 2 (BMP-2) shows great potential in promoting bone formation. However, sequential application of these two growth factors on periodontal ligament stem cells (PDLSCs) has not been explored. In this study, we aimed to identify the optimal concentration and time of bFGF on PDLSCs proliferation, migration and then investigate the sequential delivery of bFGF and BMP-2 on osteogenic differentiation of PDLSCs in vitro. Periodontal ligament stem cells were isolated by limiting dilution method. Dose-dependent additive effects of bFGF and BMP-2 on PDLSCs were detected. Cell counting assay, cell migration assay, alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to determine different application modalities of bFGF and BMP-2 on proliferation, migration, and osteogenic differentiation of PDLSCs. 50ng/mL bFGF significantly promoted PDLSCs proliferation and chemotaxis while time-dependently inhibited BMP-2 induced ALP activity. Sequential application of 25ng/mL bFGF for first 3days and followed with 50ng/mL BMP-2 for another 9, 18, and 25days significantly promoted PDLSCs osteogenic differentiation. Compared with bFGF and BMP-2 simultaneous group, sequential application of bFGF and BMP-2 group significantly enhanced ALP activity, osteogenesis-related genes and proteins expression and mineral deposition. Sequential application of bFGF and BMP-2 synergistically promoted osteogenic differentiation of PDLSCs, and this sequential application modality of growth factors would provide a new strategy for periodontal regeneration.