Thiolated Chitosan/Calcium Carbonate Composite Scaffolds Loaded With BMP-2-derived Peptide P24 Induce Bone Regeneration

Objective:To prepare thiolated chitosan/calcium carbonate composite scaffolds loaded with P24 peptide(TCS-P24/CA).Then to test the characterization and factor release characteristics of the scaffold and to evaluate the effect of the TCS-P24/CA scaffold on osteogenesis and bone regeneration in vitro and in vivo.In order to find suitable bioactive factor carriers and promote the development of bone tissue engineering composites and provide theoretical reference for clinical application.Research methods:Rat BMSCs were isolated by adherent growth method and cultured in vitro to identify their surface markers.The TCS-P24/CA scaffolds were prepared by in situ co-precipitation,physical adsorption and freeze drying methods.The three-dimensional structure and the morphology of cells on the surface of the scaffolds were observed by scanning electron microscope(SEM),and the factor release characteristics of the scaffolds were determined.The in vitro cytocompatibility and osteogenic induction of the scaffolds were detected.SD rats were selected as the experimental model and 5mm cranium defects were prepared.24 SD rats were randomly divided into 4 groups:group A without any material,group B with TCS/CA scaffold,group C with TCS-5%P24/CA scaffold and group D with TCS-10%P24/CA scaffold.The animals were sacrificed 4 and 8 weeks after operation,and the repair of bone defects were evaluated by Micro-CT and histological examination.SPSS 20.0 statistical software was used to analyze the data.All quantitative data are presented as mean±SD.The student's t-test was performed to assess statistical significance of results between groups.Values of P<0.05 were accepted as statistically significant.Significance level was presented as either*P<0.05 or**<0.01.Research results:BMSCs presented long fusiform morphology and had the capacity of amplification in vitro.Flow cytometry analysis showed that the surface markers expression of BMSCs were in accordance with MSCs.The prepared TCS-P24/CA scaffold showed the majority of the pores have a diameter from 90-120?m.SEM showed that the TCS-P24/CA scaffold had a three-dimensional porous network structure with high porosity.At the same time,most of the BMSCs showed spindle shape on TCS/CA,TCS-5%P24/CA and TCS-10%P24/CA scaffolds.In vitro,the continuous release of P24 lasted for 29 days.BMSCs co-cultured with scaffolds in vitro showed no evident toxicity and positive expression of alp,as well as the expression of osteogenesis-related genes and the formation of calcium nodules were increased.Micro-CT and histological examination demonstrated that the effect of repairing critical bone defect in TCS-10%P24/CA group was significantly better than that in TCS-5%P24/CA,TCS/CA group and blank group,and the quantitative measurement of BMD in TCS-10%P24/CA group was higher than that in TCS-5%P24/CA,TCS/CA group and blank group.Conclusions:These results demonstrated that P24 peptide can have beneficial effect on the bone generating properties of TCS-P24/CA scaffolds.TCS-P24/CA scaffolds could sustained release of P24 peptides for 29 days.In conclusion,this study demonstrated that TCS-P24/CA scaffolds can enhance osteogenesis in vitro and promote bone regeneration in vivo,suggesting that TCS-P24/CA scaffolds are potential bone graft substitutes in bone tissue engineering.