The Study Of The Biomimetic Construction Of Oriented Lamellar Col/nHAP Composite Scaffold And Mediating Macrophages To Promote Vascularization And Bone Regeneration

The repair and regeneration of bone defects is a a long-term medical science in clinic.Whether bone repair materials can induce angiogenesis and later form a mature and multi-graded vascular network determines the success of bone defect repair.Studies have shown that macrophages,as the core of bone immunity,participating and mediating the angiogenesis of bone repair materials and vascular remodeling process,and play an important role in the vascularization process of bone repair materials.Therefore,it is undoubtedly the most effective strategy at this stage to provide the immunomodulatory properties of bone repair materials so that they can mediate macrophages to promote the endogenous regeneration of blood vessels.In this project,the orientation-induced thermally induced phase separation method is used to biomimize the construction of lamellar oriented type I collagen/nano-hydroxyapatite(Col/n HAP)composite scaffold,froming a molecular composition to nano-microstructure-macrostructure simulation of the multi-level characteristics of bone matrix,to disassed the preparation of nano-micron scale structure of biomimetic scaffold,and the macro-scale external physical stimulation by regulating the interlayer spacing of the lamellar scaffold affects the polarization of macrophages and the secretion of vascular-related factors,and its mechanism of promoting angiogenesis and vascularization to promoting bone defect repair.The research results show that the nano-hydroxyapatite(n HAP)powder prepared by the chemical precipitation method with great crystal phase and uniform size,the Col/n HAP scafold with interlayer spacing of 135 ?m,75 ?m and 35 ?m(Col/n HAP-135,Col/n HAP-75 and Col/n HAP-35)was prepared by the unidirectionally induced thermally induced phase separation method,having a regular oriented lamellar structure and high porosity(>80%).I In vitro degradation results showed that the degradation rate of scaffolds was less than 15% after 12 weeks of degradation,and it was still maintain a relatively complete oriented lamellar structure.The results of co-culture of macrophages with the scaffold show that M?s adhered,grew and proliferated well on the lamellar orientation scaffold,and the Col/n HAP-135 scaffold can better regulate the transition of M?s from M1-to-M2,that down-regulation of pro-inflammatory cytokine TNF-? and up-regulation of anti-inflammatory cytokine TGF-?1,and promote the secretion of pro-angiogenic cytokines(VEGF and PDGF-bb).When the supernatant co-cultured by Col/n HAP-135 scaffold and M?s was co-cultured with human umbilical vein endothelial cells(HUVEC),it showed excellent cell migration and angiogenesis ability.Established a rat skull defect model,the Col/n HAP-135 scaffold showed higher angiogenesis-related genes(VEGF and PDGF-bb)and osteogeni-related genes(Col-I,BMP-2and OCN),H&E staining,VEGF immunohistochemical staining,and Micro-CT results show that the Col/n HAP-135 scaffold is better with vascularization and osteogenesis ability.In summary,the pore size/layer spacing and the orientation structure of the biomimetic scaffold have a significant regulatory effect on M?.Compared with Col/n HAP-random,Col/n HAP-75 and Col/n HAP-35 scaffolds,Col/n HAP-135 oriented scaffolds with larger interlayer spacing can more effectively induce the sequential activation of M1/M2,secrete and express vascular-related cytokines,therefore,it plays an important role in angiogenesis in the early stage of bone defect repair and vascular remodeling in the later stage,and ultimately effectively promote the rapid repair of bone defects.