Subcutaneously Engineered Decalcified Bone Matrix Xenografts Promote Bone Repair by Regulating the Immune Microenvironment, Prevascularization, and Stem Cell Homing

Immunoregulatory and vascularized microenvironments play an important role in bone regeneration; however, the precise regulation for vascularization and inflammatory reactions remains elusive during bone repair. In this study, by means of subcutaneous preimplantation, we successfully constructed demineralized bone matrix (DBM) grafts with immunoregulatory and vascularized microenvironments. According to the current results, at the early time points (days 1 and 3), subcutaneously implanted DBM grafts recruited a large number of pro-inflammatory M1 macrophages with positive expression of CD68 and iNOS, while at the later time points (days 7 and 14), these inflammatory cells gradually subsided, accompanying increased presence of anti-inflammatory M2 macrophages with positive expression of CD206 and Arg-1, indicating a gradually enhanced anti-inflammatory microenvironment. At the same time, the gradually increased angiogenesis was observed in the DBM grafts with implantation time. In addition, the positive cells of CD105, CD73, and CD90 were observed in the inner region of the DBM grafts, implying the homing of mesenchymal stem cells. The repair results of cranial bone defects in a rat model further confirmed that the subcutaneous DBM xenografts at 7 days significantly improved bone regeneration. In summary, we developed a simple and novel strategy for bone regeneration mediated by anti-inflammatory microenvironment, prevascularization, and endogenous stem cell homing.