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 200 μg/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.

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 200 μg/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.