Effect Of BFGF-loaded Thermosensitive Hydrogel On Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective:In this study,bFGF-loaded chitosan/β-sodium glycerophosphate/gelatin thermosensitive hydrogel was synthesized to study its effect on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells,so as to provide experimental basis for the clinical application of bFGF in repairing alveolar bone defects.Methods:Chitosan/β-sodium glycerophosphate/gelatin thermosensitive hydrogel was prepared,and its characterization was observed under electron microscope after freeze-drying.Hydrogel was soaked in simulated body fluid to detect hydrogel degradation ability in vitro.The rat bone marrow mesenchymal stem cells were isolated and identified,and co-cultured with hydrogel,live/dead cell staining was used to detect the toxicity of thermosensitive hydrogel.The relatively optimum concentration of bFGF-loaded thermosensitive hydrogel for promoting proliferation of rat bone marrow mesenchymal stem cells was screened by CCK-8 method and used for subsequent studies.The experiment included blank control group,thermosensitive hydrogel group and bFGF-loaded thermosensitive hydrogel group,the effects of bFGF-loaded thermosensitive hydrogel with on osteogenic differentiation of rat bone marrow mesenchymal stem cells were observed by alkaline phosphatase staining,alizarine red staining and semi-quantitative analysis of calcium nodules.The effect of bFGF-loaded thermosensitive hydrogel on osteogenic gene m RNA expression of rat bone marrow mesenchymal stem cells was detected by real-time fluorescence quantitative PCR.Results:1.Chitosan /β-sodium glycerophosphate/gelatin thermosensitive hydrogel had good temperature sensitivity,biodegradability and biocompatibility,the freeze-dried hydrogel was loose and porous,and the pore size of the freeze-dried hydrogel was20-80μm by scanning electron microscopy.2.Rat bone marrow mesenchymal stem cells were successfully isolated.The bFGF-loaded thermosensitive hydrogel could significantly promote the proliferation of rat bone marrow mesenchymal stem cells in a dose-dependent manner,and the relatively optimal concentration was 100ng/ml.3.Alkaline phosphatase staining results showed that the expression of alkaline phosphatase increased in the bFGF-loaded thermosensitive hydrogel group,and increased with the increase of osteogenic induction days.The staining degree of alkaline phosphatase in the bFGF-loaded thermosensitive hydrogel group was higher than that in blank control group and thermosensitive hydrogel group.Alizarine red staining and semi-quantitative analysis of calcium nodules showed that the formation of calcium nodules in the bFGF-loaded thermosensitive hydrogel group was more than that in the blank control group and the thermosensitive hydrogel group.4.qRT-PCR results showed that the m RNA expression levels of ALP,BMP-2 and Runx2 in the bFGF-loaded thermosensitive hydrogel group were higher than those in the thermosensitive hydrogel group and the blank control group,and the expression levels of corresponding osteogenic genes in the thermosensitive hydrogel group were higher than those in the blank control group.Conclusion:1.Successfully preparing the chitosan/β-sodium glycerol phosphate/gelatin thermosensitive hydrogel with good temperature sensitivity,biodegradability and biocompatibility.2.The bFGF-loaded thermosensitive hydrogel can significantly promote the proliferation of rat bone marrow mesenchymal stem cells,and the proliferation promoting ability is positively correlated with the concentration of bFGF in a certain range.The relatively optimal concentration of bFGF-loaded thermosensitive hydrogel is 100ng/ ml.3.The bFGF-loaded thermosensitive hydrogel can promote osteogenic differentiation of rat bone marrow mesenchymal stem cells and increase the expression of osteogenic genes ALP,BMP-2 and Runx2 m RNA.