ABSTRACT: Bone autograft healing is a highly orchestrated process that integrates immune activation, vascular ingrowth, and osteogenic remodeling. To define the molecular and cellular programs driving early autograft integration, bulk and single-cell RNA sequencing was used to analyze graft-associated tissues over 14 days in a murine periosteal-mediated autograft model. Global transcriptomic analysis revealed rapid and dynamic remodeling, with maximal gene expression changes occurring within the first week. The first 48 h were dominated by pro-inflammatory signaling, including TNF, IL-1, TLR, and MAPK pathways, accompanied by transcriptional signatures of phagocytosis and cellular clearance. These early inflammatory programs gave way to pro-regenerative signals, including activation of HIF-1, PI3K-AKT, Wnt, and BMP pathways, coincident with angiogenesis, osteogenesis, and matrix deposition. By day 14, extracellular matrix production and remodeling predominated, marked by metalloproteinase activity and structural matrix gene enrichment. Single-cell RNA sequencing revealed that donor-derived (eGFP+) graft cells were rare and transient, whereas host-derived immune cells were progressively replaced by myofibroblasts, endothelial cells, and neurogenic cell types, including GABAergic neurons and IGSF21+ dendritic cells, suggesting active neurovascular crosstalk during healing. Together, these data define a temporal immune-to-regenerative cascade in bone autograft repair and highlight candidate cellular and molecular targets to enhance graft performance.
