缓释BMP-2透明质酸-肝素支架的制备及其对牙髓干细胞分化的研究

Preparation of hyaluronic-heparin scaffold capable of sustainably releasing BMP-2 and the study of its influence on the differentiation of dental pulp stem cells

目的:为了使透明质酸(hyaluronic acid,HA)具备负载和释放生长因子的能力,设计制备透明质酸-肝素(hyaluronic acid-heparin,HA-Hep)支架作为骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)缓释载体,并探讨其对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质分化的影响。方法:化学方法制备不同投料比HA-Hep材料。通过傅立叶变换红外光谱与元素分析技术表征肝素与HA的接枝位点、效率等特点。扫描电镜观察该材料微观形态和结构特点。借助静电吸附法将BMP-2加载至HA-Hep,并测绘其释放曲线。通过测定DPSCs增殖情况评价该材料的生物相容性。qPCR检测成牙分化相关基因的表达水平,评估HA-Hep负载BMP-2对DPSCs成牙分化的影响。结果:HA-Hep微观呈疏松多孔网状结构,能够缓释BMP-2达28 d,且释放速率随材料肝素含量比例的增加呈减缓趋势。HA-Hep对DPSCs增殖有促进作用,且与材料浓度呈正相关;qPCR结果表明,与对照组相比,载BMP-2的HA-Hep材料组牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、Ⅰ型胶原蛋白(typeⅠcollagen,Col-1)和牙本质基质蛋白-1(dentin matrix protein-1,DMP-1)表达水平在7、14 d均显著升高(均P<0.001)。结论:肝素修饰HA能延缓BMP-2释放,HA-Hep负载BMP-2可上调DPSCs成牙基因(DSPP、Col-1、DMP-1)的表达,有助于生长因子在细胞分化中发挥长期效应。

Objective:In order to enable hyaluronic acid(HA)to load and release growth factors,a hyaluronic acid-heparin(HA-Hep)scaffold was designed as a sustained-release carrier for bone morphogenetic protein 2(BMP-2),and the effect of the scaffold on odontogenic differentiation of dental pulp stem cells(DPSCs)was studied.Methods:HA-Hep materials with different ratios were prepared using chemical methods.The connection position and efficiency of heparin and hyaluronic acid were detected by Fourier transform infrared spectroscopy and elemental analysis.The microstructure and structural characteristics of the materials were observed by scanning electron microscope.BMP-2 was loaded into HA-Hep by electrostatic adsorption method,and its release curve was measured and drawn.To evaluate the biocompatibility of HA-Hep by detecting the proliferation of DPSCs.The expression levels of odontogenic differentiation-related genes were detected by qPCR to evaluate the effect of HA-Hep loading BMP-2 on odontogenic differentiation of dental pulp stem cells.Results:Microscopically,HA-Hep showed a loose porous network structure.The release of BMP-2 was sustained for 28 days,and the release rate slowed down with the increase of heparin content.HA-Hep promoted the proliferation of DPSCs was positively correlated with the materials concentration.qPCR results showed that the expression levels of dentin sialophosphoprotein(DSPP),typeⅠcollagen(Col-1),and dentin matrix protein-1(DMP-1)in BMP-2-loaded HA-Hep material group were significantly higher than those in the control group at 7 and 14 days(all P<0.001).Conclusion:Heparin-modified HA can delay the release of BMP-2.HA-Hep loaded with BMP-2 can up-regulate the expression of odontoblast genes(DSPP,Col-1,and DMP-1)in DPSCs,which contributes to the long-term effect of growth factors in cell differentiation.