Periosteal Matrix-derived Hydrogel Promotes Bone Repair Through Macrophage M2 Polarization Regulation Coupled With Enhanced Angio-and Osteogenesis

Objective:Preparation of an injectable periosteal extracellular matrix hydrogel and evaluation for its role in events of immune inflammation,angiogenesis and osteogenic differentiation during bone healing.Methods : Decellularized periosteum extracellular matrix(PEM)hydrogel was prepared by enzyme digestion method.Histological staining was used to observe the decellularized tissue structure and component retention,and then quantitatively determine the DNA and collagen content.Using type I collagen hydrogel as a universal substrate control,the microstructures of the hydrogels were observed by SEM.The potential biological functions of PEM hydrogels were inferred by mass spectrometry analysis.After co-culture with Raw264.7 cells for 3 days,the expression regulation of macrophage markers in vitro by PEM hydrogels was evaluated by q PCR and immunofluorescence.After co-cultivation with HUVECs for 6 h,the angiogenic activity of PEM hydrogels in vitro was evaluated by endothelial cell tube-like formation assay.After co-cultivation with osteoblasts for 14 days,the effects of PEM hydrogels on expression of osteoblast differentiation-related genes were evaluated by q PCR and immunofluorescence.After the PEM hydrogel was injected into a rat calvarial critical-sized defect model in vivo,conventional and special histological staining were applied to detect angiogenesis,new bone formation and maturation.Immunofluorescence was used to detect macrophage polarization in vivo.And Micro-CT was used to evaluate mineralized bone formation.Results:Decellularized periosteum extracellular matrix was effectively removes from nuclear components but the collagen and GAG components were retained well.Its gelation product PEM hydrogel has a similar microstructure compared to type I collagen hydrogel.Mass spectrometric analysis suggested that PEM components have the potential effects of promoting cell adhesion,migration,enhancing angiogenesis,and inducing M2 polarization of macrophages.In vitro cell experiments showed that compared with type I collagen hydrogel,PEM hydrogel exhibited better M2 polarization induction of macrophages,up-regulated the expression levels of CD206 and Arg1 but down-regulated the expression levels of IL-1?and i NOS,and promoted a greater number of endothelial cell ring formation and caused higher expression levels of osteogenesis-related marker expression(Runx2,ALP,Col1?1,OCN,and OPN).In vivo experiments in critical-sized bone defects also showed that PEM hydrogel promoted the M1-M2 phenotypic transition of macrophages at the early stage of(3 days),and increased the number and diameter of blood vessels at the middle stage(14 days),and made earlier bone formation and maturation at the later stage(4 weeks and 8 weeks).Conclusion:Compared with type I collagen hydrogel of one single component,PEM hydrogel has a unique biochemical composition,showing superior regulatory effects in terms of early immune regulation,latter angiogenesis,osteogenic differentiation and other repair events.These biological effects are combined with the natural process of bone regeneration to ultimately accelerate bone repair.Therefore,PEM hydrogel has a broad application prospect in bone tissue engineering.