Gelatin-hydroxyapatite Scaffold Promotes The Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective To evaluate the physicochemical properties of HA-Gel porous scaffolds and its effects on the proliferation and differentiation of rat bone mesenchymal stem cells(r BMSCs)in vitro.The promotion effect of r BMSCs seeded on HA-Gel on bone regeneration was measured in vivo.Methods Rat bone marrow mesenchymal stem cells(rBMSCs)were cultured by whole bone marrow adherent method.Flow cytometry was used to analyze second-generation cell surface markers.After 21 days of mineralization induction,Alizarin red staining was used to identify the osteogenic differentiation ability of the cells.HA-Gel scaffold was fabricated using coprecipitation method with 1,2 and 3circulations(1HA-Gel,2HA-Gel and 3HA-Gel).Scanning electron microscopy(SEM),X-ray diffraction(XRD)and fourier transform infrared spectroscopy(FTIR)were used to evaluate the structural/chemical characterization of the scaffolds.Cellular morphology,viability and proliferation of r BMSC seeded on the scaffolds were examined by SEM,Live/dead staining and CCK8,respectively.The osteogenic differentiation of r BMSC was analyzed by alkaline phosphatase(ALP)staining analysis.Two critical-sized bone defects(diameter 5mm,height 3mm)were created on the skull of rats.12 critixal-sized calvarial defects in 4 rats were generated and randomly filled with r BMSCs-1HA-Gel,r BMSCs-Gel or empty.After 12 weeks,the regeneration bone was evaluated by CBCT,HE and Masson staining.Statistical analysis was performed with SPSS17.0 software,and the test data were all expressed (?)±s.The differences between multiple groups were analyzed by two-way repeated measures analysis of variance.P < 0.05 was considered statistically significant.Results 1.Primary cells were isolated and cultured by whole bone marrow adherent method.Flow cytometry results indicated that the cells positively expressed the surface markers CD90 and CD29,while negatively expressed the hematopoietic markers CD45 and CD11b.Calcium nodules were detected by Alizarin red staining,which indicated the isolated cells have the osteogenic differentiation ability.2.HA-Gel possessed a3-dimensional interconnected porous structure with extensive hydroxyapatite.The main crystalline phase of the composite scaffolds was HA.Besides,composite scaffolds had high porosity(71.11±0.077)% with high degradation rate.r BMSCs spreaded fully and exhibited a spindle-like morphology on 1HA/Gel scaffolds.Compared to 2HA-Gel,3HA-Gel and Gel groups,the results of Live/dead staining and CCK8 showed that the viability and proliferation of r BMSC was enhanced in 1HA-Gel.r BMSC seeded on1HA-Gel significantly induced the ALP expression which was analyzed by ALP staining.3.The results of CBCT,HE and Masson staining demonstrated that the bone volume density and collagen formation was remarkablely greater in r BMSCs-1HA-Gel group than that in the r BMSCs-Gel group and empty group.Conclusion 1HA-Gel scaffold synthesized by co-precipitation method has good biocompatibility,and promotes osteogenic differentiation of r BMSCs in vitro.Moreover,r BMSCs seeded on 1HA-Gel can effectively repair skull defects in vivo.These results reveal a promising strategy for craniofacial bone regeneration in clinical applications.