负载骨形态发生蛋白2水凝胶诱导牙髓干细胞的成骨分化

Bone morphogenetic protein 2-loaded hydrogel induces osteogenic differentiation of dental pulp stem cells

背景:前期研究证明,甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶支架可促进人牙髓干细胞的增殖及分化。水凝胶负载骨形态发生蛋白2被认为是骨修复中有前景的材料。目的:观察负载不同质量浓度骨形态发生蛋白2的甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶对人牙髓干细胞成骨向分化的诱导作用。方法:制备含0,50,100,200μg/mL骨形态发生蛋白2的甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶,分别记为GelMA/TDM、BMP-2(50 ng/mL)GelMA/TDM、BMP-2(100 ng/mL)GelMA/TDM、BMP-2(200 ng/mL)GelMA/TDM,检测复合水凝胶对骨形态发生蛋白2的体外缓释性能。采用改良组织块酶消化法提取人牙髓干细胞,分别接种于4种水凝胶表面,采用CCK-8法检测细胞增殖,DAPI染色检测细胞黏附;对各组水凝胶表面的人牙髓干细胞进行成骨诱导,进行碱性磷酸酶染色、碱性磷酸酶活性检测与茜素红染色,采用RT-PCR法检测相关成骨基因(Runx2、骨形态发生蛋白2、骨桥蛋白、骨钙素、Ⅰ型胶原)表达。结果与结论:①甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶可持续释放骨形态发生蛋白2长达21 d,在第3-6天释放较快,第6天之后释放趋于平稳;②4种水凝胶均可促进人牙髓干细胞的增殖,其中以BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶的作用最明显;相较于GelMA/TDM复合水凝胶,BMP-2-GelMA/TDM复合水凝胶可促进人牙髓干细胞的黏附,其中以BMP-2(200 ng/mL)-GelMA/TDM复合水凝胶的作用最明显;③相较于GelMA/TDM复合水凝胶,BMP-2-GelMA/TDM复合水凝胶可提升碱性磷酸酶活性、钙结节含量与相关成骨基因表达,综合分析显示BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶的作用更明显;④结果表明,BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶促进牙髓干细胞成骨向分化的能力更明显。

BACKGROUND:Previous studies have shown that gelatin modified by methacrylic anhydride(GelMA)/treated dentin matrix(TDM)composite hydrogel scaffolds can promote the proliferation and differentiation of human dental pulp stem cells.Hydrogels loaded with bone morphogenetic protein 2 are considered to be promising materials for bone repair.OBJECTIVE:To observe the effect of GelMA/TDM composite hydrogels loaded with different concentrations of bone morphogenetic protein-2 on the induction of osteogenic differentiation of human dental pulp stem cells.METHODS:GelMA/TDM composite hydrogels containing 0,50,100,and 200μg/mL bone morphogenetic protein 2 were prepared,denoted as GelMA/TDM,bone morphogenetic protein-2(50 ng/mL)GelMA/TDM,bone morphogenetic protein-2(100 ng/mL)GelMA/TDM,and bone morphogenetic protein-2(200 ng/mL)GelMA/TDM,respectively,and the in vitro sustained release of bone morphogenetic protein 2 from the composite hydrogels was detected.Human dental pulp stem cells were extracted by the modified tissue block enzymatic digestion method and seeded on the surfaces of the four kinds of hydrogels.Cell proliferation was detected by CCK-8 assay and cell adhesion was detected by DAPI staining.Human dental pulp stem cells on the surfaces of each group of hydrogels were induced to osteoblastically,and alkaline phosphatase staining,alkaline phosphatase activity detection,and alizarin red staining were performed.RT-PCR was used to detect the expression of related osteogenic genes(Runx2,bone morphogenetic protein 2,osteopontin,osteocalcin,and type I collagen).RESULTS AND CONCLUSION:(1)The GelMA/TDM composite hydrogel could continuously release bone morphogenetic protein 2 for up to 21 days,with a faster release from day 3 to day 6 and a steady release after 6 days.(2)All four kinds of hydrogels could promote the proliferation of human dental pulp stem cells,among which the bone morphogenetic protein-2(100 ng/mL)-GelMA/TDM composite hydrogel had the most obvious effect.Compared with the GelMA/TDM composite hydrogel,the bone morphogenetic protein-2-GelMA/TDM composite hydrogel could promote the adhesion of human dental pulp stem cells,among which the bone morphogenetic protein-2(200 ng/mL)-GelMA/TDM composite hydrogel had the most obvious effect.(3)Compared with the GelMA/TDM composite hydrogel,the bone morphogenetic protein-2-GelMA/TDM composite hydrogel could increase the activity of alkaline phosphatase,the content of calcium nodules,and the expression of related osteogenic genes.Comprehensive analysis showed that the effect of the bone morphogenetic protein-2(100 ng/mL)-GelMA/TDM composite hydrogel was more obvious.(4)The results show that the bone morphogenetic protein-2(100 ng/mL)-GelMA/TDM composite hydrogel has a more obvious ability to promote the osteogenic differentiation of dental pulp stem cells.