Triple Gene-Knockout Xenogeneic Bone Repair Material Achieves Synergistic Optimization of Bone Repair Immunomicroenvironment and Bone Regeneration Via Antigen-Immunity-inflammation Axis Modulation

Bone defect repair confronts a dual challenge of complex immune rejection microenvironments and osteogenic impairment, severely restricting the clinical application of xenogeneic bone graft materials. The dynamic interplay between xenogeneic antigens (α-Gal, Neu5Gc, and SDa) and the host immune system establishes an "antigen-immunity-inflammation" alliance that activates complement cascades, recruits immune cell infiltration, and drives pro-inflammatory macrophage polarization, thereby inducing chronic inflammation and fibrosis. Crucially, this pro-inflammatory microenvironment constitutes the critical determinant of osteogenic failure. Consequently, reshaping the bone repair immunomicroenvironment may fundamentally overturn this paradigm. In this study, we constructed a triple gene-edited and Urist-processed xenogeneic decalcified bone matrix system (3KODBM). Through a synergistic strategy combining genetic editing to knock out major xenogeneic antigen genes (GGTA1, CMAH, and B4GALNT2) with the Urist method to eliminate residual antigenic epitopes, we effectively attenuated immune rejection. Furthermore, we integrated genetic editing with physicochemical modification to synergistically remodel the immunomicroenvironment, promote M1-to-M2 macrophage polarization, and release osteogenic factors such as TGF-β, thereby facilitating bone repair. Our findings demonstrate that 3KODBM significantly downregulates the IL-17A/RORγt inflammatory pathway, reduces fibrosis, and markedly promotes osteogenic markers including Runx2 and Osterix. Mechanistically, by establishing an immune rejection "brake" through antigen gene knockout and an osteogenic "booster" via TGF-β/calcium signaling activation, we modulate the microenvironment to achieve substantial bone defect restoration. This study illuminates the therapeutic prospects of synergistically attenuating xenotransplantation immune rejection, offering an innovative gene-editing bone repair strategy to overcome immune rejection challenges in xenogeneic bone grafts.