基于软骨内成骨过程的血管化组织工程移植物构建OA

Objective To construct a tissue-engineered graft based on the process of endochondral ossification,induce vascular ingrowth,and establish blood supply in the graft center in order to promote the repair of the tissue defect.Methods Preparation of human bone marrow mesenchymal stem cells(hBMSCs)aggregates:after the gelatin microcar-riers were combined with hBMSCs,the dispersed microcarriers would automatically gather together after the chondrogenic induction medium was cultured for a period of time,which was called hBMSC aggregates.Construction of Indian hedgehog(IHH)protein-mediated hBMSC aggregates chondrogenic differentiation culture system:set different concentrations of IHH protein[0 ng/mL(control group),25 ng/mL,100 ng/mL,300 ng/mL IHH],the survival and proliferation of hBMSCs at different IHH concentrations were detected by live-dead staining and 5-ethynyl-2'-deoxyuridine(EdU)pro-liferation assay;SRY-box transcription factor 9(SOX9)immunofluorescence staining,collagen type X(COLX)immuno-histochemistry,Goldner staining and scanning electron microscopy were used to detect the chondrogenic differentiation and hypertrophy of hBMSC aggregates in each group after 21 days of chondrogenic induction.Vascular ingrowth in each group was evaluated by hematoxylin and eosin stain(H&E stain),vascular endothelial growth factor(VEGF)and platelet en-dothelial cell adhesion molecule(CD31/PECAM-1)immunofluorescence staining at 12 weeks after the graft was implanted into the femoral muscle bag model of nude mice.Results After 72 hours of chondrogenic differentiation,the cell survival rate of each group was more than 95%,only a small number of dead cells were observed,and the cells still had proliferation ability.The results in vitro showed that SOX9 were expressed in all four groups.Among them,the number of SOX9 positive cells in the 100 ng/mL IHH treatment group was significantly higher than that in the other three groups,and the chondrogenic induction efficiency was the highest.The results of COLX showed that the distributions of COLX in the 100 ng/mL and 300 ng/mL IHH treatment groups were higher than that in the other two groups.The results of Goldner staining and scanning electron microscopy showed that osteoid deposition and mineralization occurred in the 100 ng/mL and 300 ng/mL IHH treatment groups,and the 300 ng/mL IHH group was more obvious.In the ectopic implantation ex-periment,compared with the control group,the numbers of red blood cells and blood vessels in the 100 ng/mL and 300 ng/mL IHH pretreatment groups were significantly higher,and the expressions of VEGF and CD31 were also signifi-cantly higher.Conclusion Exogenous IHH protein can significantly promote chondrogenic differentiation,hypertrophy,maturation and mineral deposition of hBMSC aggregates,and can induce vascularization in the center of the aggregates af-ter ectopic implantation.