Secretome of SHED Sheets Facilitates Mandibular Bone Defect Repair by Shifting Macrophages Inflammatory State.

OBJECTIVE: The inflammatory response, especially the inflammatory status of macrophages, is crucial for bone graft integration and regeneration. Cell sheets from various stromal cells exhibit immunomodulatory and pro-osteogenic traits. However, the therapeutic potential of the secretome from human exfoliated deciduous teeth stromal cell (SHED) sheets remains unclear. This study aimed to explore whether SHED sheet-derived secretome (SS-E) aids bone defect repair via macrophage immunomodulation and to clarify the underlying mechanisms. METHODS: SS-E was collected from osteogenically induced SHED sheet supernatants. Its immunomodulatory effects on lipopolysaccharide (LPS)-stimulated inflammatory macrophages were studied using mRNA sequencing and analysis of polarization marker expression. Paracrine osteogenic potential was evaluated by culturing bone marrow mesenchymal stromal cells (BMSC) in the conditioned medium from SS-E-primed macrophages (SS-E-CM). Osteogenic differentiation was quantified through Western blotting, RT-qPCR, alkaline phosphatase (ALP) staining, and Alizarin Red S staining. In vivo, a rat mandibular bone defect model was treated with gelatin methacryloyl (GelMA) hydrogel or GelMA loaded with SS-E (GelMA + SS-E). Bone regeneration was assessed using micro-CT, H&E staining, and immunohistochemistry. RESULTS: SS-E treatment significantly decreased the M1 pro-inflammatory marker iNOS in LPS-stimulated macrophages and increased M2 anti-inflammatory markers (Arg-1, IL-10, CD206). SS-E-CM enhanced BMSC proliferation and upregulated osteogenic factors (RUNX2, ALP, BMP-2), with increased ALP activity and matrix mineralization. In vivo, GelMA + SS-E reduced local inflammation and enhanced new bone formation compared to GelMA alone. CONCLUSION: The SS-E promotes osteogenesis indirectly by redirecting macrophage polarization from a pro-inflammatory to a pro-reparative phenotype. This cell-free, immunomodulatory strategy represents a promising therapeutic approach for complex craniofacial bone reconstruction. TRANSLATIONAL IMPACT STATEMENT: This study presents SS-E, a cell-free secretome derived from stromal cells of human exfoliated deciduous teeth, which safely modulates macrophage-driven inflammation to promote bone regeneration. By combining SS-E with a clinically compatible hydrogel, this immunomodulatory strategy offers a readily translatable approach for enhancing craniofacial bone defect repair without the risks associated with cell-based therapies.