Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, China.
Department of Periodontology, School of Stomatology, Shandong University, Jinan, China.
J Periodontal Res. 2019 Aug;54(4):424-434. doi: 10.1111/jre.12644. Epub 2019 Mar 9.
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.
50 ng/mL bFGF significantly promoted PDLSCs proliferation and chemotaxis while time-dependently inhibited BMP-2 induced ALP activity. Sequential application of 25 ng/mL bFGF for first 3 days and followed with 50 ng/mL BMP-2 for another 9, 18, and 25 days 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.
碱性成纤维细胞生长因子（bFGF）可促进细胞增殖和趋化性，维持干细胞特性，同时抑制矿化结节形成。骨形态发生蛋白 2（BMP-2）在促进骨形成方面具有巨大潜力。然而，这两种生长因子在牙周膜干细胞（PDLSCs）上的序贯应用尚未被探索。本研究旨在确定 bFGF 对 PDLSCs 增殖、迁移的最佳浓度和时间，然后研究 bFGF 和 BMP-2 的序贯递呈对 PDLSCs 体外成骨分化的影响。
通过有限稀释法分离牙周膜干细胞。检测 bFGF 和 BMP-2 对 PDLSCs 的剂量依赖性相加效应。细胞计数法、细胞迁移试验、碱性磷酸酶（ALP）活性测定、茜素红染色、实时定量聚合酶链反应（qRT-PCR）和 Western blot 分析用于确定 bFGF 和 BMP-2 对 PDLSCs 增殖、迁移和成骨分化的不同应用方式。
50ng/mL bFGF 显著促进 PDLSCs 增殖和趋化性，同时时间依赖性抑制 BMP-2 诱导的 ALP 活性。前 3 天给予 25ng/mL bFGF，随后再给予 50ng/mL BMP-2 9、18 和 25 天，可显著促进 PDLSCs 成骨分化。与 bFGF 和 BMP-2 同时组相比，bFGF 和 BMP-2 序贯应用组显著增强了 ALP 活性、成骨相关基因和蛋白的表达以及矿化沉积。
bFGF 和 BMP-2 的序贯应用协同促进 PDLSCs 的成骨分化，这种生长因子的序贯应用方式为牙周再生提供了一种新策略。
