Experimental Study Of Gelatin Microsphere Aggregates(C-GMSs) Scaffolds Promoting Vascularized Bone Regeneration

Bone defects caused by tumors and trauma are common clinical diseases.With the intensification of the aging population in our country,the incidence of bone defects is increasing.At present,the main clinical treatment is bone transplantation,but there are problems such as immune rejection,infection,nonunion and so on.Bone tissue engineering is considered to be the most promising bone defect repair strategy;compared with traditional autograft and allograft,it does not cause additional trauma to the recipient,and reduces the risk of infection and recipient rejection.However,insufficient vascularization has become a serious problem in bone repair.In the process of damage and repair of large bone tissue,angiogenesis at the injury site determines the degree of damage and repair.In recent years,the research on the vascularization of tissue-engineered bone has attracted much attention,and some progress has also been made.In the process of tissue engineering,scaffold materials play a crucial role in cell behavior,tissue formation,and repair of tissue defects.As a natural polymer material,gelatin has the advantages of high biocompatibility,biodegradability,and promotion of cell adhesion and proliferation,and has been widely used in tissue engineering scaffold research.Objective EDC/NHS cross-linked gelatin microsphere aggregates(C-GMSs)scaffolds were constructed.First,the promoting effect of C-GMSs scaffold on MC3T3-E1 osteogenic cell proliferation and osteogenic differentiation,as well as the promoting effect on the proliferation and angiogenic differentiation of human aortic endothelial cells(HAEC),was investigated at the cellular level.Secondly,the promoting effect of C-GMSs on the formation of vascularized tissue engineered bone was investigated in vitro and subcutaneously in nude mice.Finally,the repair effect of C-GMSs scaffold complex on large bone defects was investigated.Method Gelatin microspheres were prepared by water-in-oil single emulsion method and C-GMSs were prepared by cross-linking with EDC/NHS.The structure and particle size of the microspheres were characterized by optical microscopy and scanning electron microscopy.Methacrylated gelatin(Gel-MA)was synthesized,and Gel-MA macroporous hydrogel(G-MCG)scaffold was prepared by using a temperature-sensitive gelatin microsphere porogen.HAEC and MC3T3-E1 cells were co-cultured on C-GMSs scaffolds to form macroscopic cell-scaffold complexes,and the co-growth of HAEC and MC3T3-E1 cells on microspheres was verified by mitochondrial fluorescent labeling technology;The effects of the two scaffolds on cell viability were detected by calcein/PI staining;the morphology of complex cells was detected by F-actin staining;cell proliferation was detected by MTSblue.After 21 days of in vitro culture,the complexes were stained with H&E to evaluate the formation of endothelialized tissue-engineered bone.Osteogenic differentiation of MC3T3-E1 cells was assessed by alizarin red staining and immunohistochemical staining for alkaline phosphatase(ALP),RUNT-related transcription factor 2(RUNX2),collagen type I(Col-1),osteocalcin(OCN)and osteopontin(OPN).Endothelialization levels in the complexes were assessed by immunohistochemical staining for platelet endothelial cell adhesion molecule(CD31),vascular endothelial growth factor(VEGF),von Willebrand factor(v WF),and hypoxiainducible factor 1α(HIF-1α).Subsequently,the vascularization and ossification levels of the cell-C-GMSs scaffold complex were detected by subcutaneous implantation experiments in nude mice.Finally,rat femoral critical size bone defect model was established,and the effect of the unloaded C-GMSs scaffold in inducing angiogenesis and promoting bone regeneration in vivo was evaluated by various methods such as imaging and histology.Result C-GMSs microspheres with particle sizes ranging from 100 to 500 μm were successfully prepared and sorted.Mitochondrial fluorescence localization data showed that HAEC and MC3T3-E1 cells could be co-cultured on C-GMS scaffolds.Calcein/PI staining results showed that cells cultured in C-GMSs scaffolds had higher viability compared with G-MCG scaffolds.F-actin staining results showed that HAECs and MC3T3-E1 cells could stretch in the microenvironment of the C-GMSs scaffold on the surface,showing normal spindle or polygonal shape.The complex immunohistochemical results confirmed that the C-GMSs scaffold could promote the formation and vascularization of tissue-engineered bone.In the in situ repair experiment of rat femoral defect,it was found that C-GMSs can better recruit autologous cells,combined with its own rapid degradation,and achieved vascularized bone regeneration and its integration with host bone within 50 days.The above results show that the C-GMSs scaffold with high specific surface area can provide a suitable living environment for osteoblasts and hemangioblasts,and promote their proliferation and differentiation.Moreover,C-GMSs scaffolds can recruit autologous therapeutic cells in situ to achieve rapid repair of bone defects.Conclusion In conclusion,the EDC/NHS cross-linked C-GMSs scaffold was constructed in this study,which can not only promote the construction of vascularized tissue engineered bone in vitro,but also effectively induce the rapid repair of critical femoral defects in rats.The above results indicate that the C-GMSs scaffolds reported in this study have potential clinical application prospects in the fields of bone tissue engineering vascularization and bone defect repair.