体外组织培养模式的构建及转化生长因子促进牙髓细胞向成牙本质细胞诱导分化的研究

目的通过构建体外组织培养模式,观察牙髓细胞向成牙本质细胞诱导分化的潜力及转化生长因子-β1(TGF-β1)的促进作用。方法获取人第三磨牙牙髓组织,剪碎后与牙本质陶瓷粉(CDP)及2μg/mL TGF-β1混合,将混合物以骨基质明胶(BMG)为载体构建牙髓组织培养模型,在培养3、7、14和21 d后,通过HE和组织特异性染色鉴定有无骨-牙本质基质成分形成,免疫组化检测细胞有无牙本质特异性蛋白——牙本质涎蛋白(DSP)的表达。结果牙髓组织经TGF-β1诱导培养后可进一步矿化,甲苯胺蓝和Mallory染色表明在牙髓组织的中心出现骨-牙本质样基质的沉积,免疫组化检测牙髓细胞开始出现DSP的表达。结论成功构建体外牙髓组织培养模型,该模型能维持牙髓细胞的生长和存活,可用于体外对牙髓细胞的诱导分化观察;该模型模式下TGF-β1可明显促进牙髓细胞向成牙本质细胞的分化诱导潜能。

Construction of Tissue Culture Model in vitro and Investigation of Transforming Growth Factor Promoting Pulp Fibroblast to Differentiate into the Odontoblast

OBJECTIVE: To construct the tissue culture model in vitro, and investigate the potential of dental pulp fibroblast differentiating into the odontoblast and the promoting role of transforming growth factor beta1 (TGF-beta1) on it. METHODS: Human pulps were cultured for 3, 7, 14 and 21 days on bone matrix gelatin (BMG) in DMEM cultural medium supplemented with TGF-beta1. The characteristics of matrix were studied through toluidine blue and Mallory stain. Meanwhile, the expression of dentin salivary protein (DSP) on the pulp cells was investigated with immunohistochemical staining. RESULTS: This experiment found that the pulp tissue in vitro were able to develop into more progressive stage, and some pulp fibroblast cells to differentiate into odontoblast-like cells. Toluidine blue and Mallory staining analysis revealed the localized deposition of mineralized bone-dentin matrix that was detected at the site of dental pulp cells. Immunohistochemical analysis proved that the DSP synthesized in these cells with the presence of TGF-beta1. CONCLUSION: The results demonstrate that this culture condition can maintain the phenotype of human pulp tissue. The model of organ culture is suitable to study the development of pulp tissue in vitro. TGF-beta1 can promote the potential of pulp cell into odontoblast, which provides an academic basis for tooth repair and regeneration.