MON-737 Use of scRNA seq. and Xenium In Situ to determine the Cellular and Molecular Impact of Abaloparatide on the Distinct sites of Fracture healing

Abstract Disclosure: A. Parajuli: None. Fracture (Fx) healing is a multifaceted biological process involving various cell populations from the periosteum, cortex, and bone marrow. Previous research (Wang et al., JBMR 2020) has characterized 3 distinct regions in the Fx callus (Site1: Intramembranous BF along the periosteum, Site 2: Endochondral BF bridging the Fx’ed ends, Site 3: Intramedullary BF between the cortices of broken ends). Recent studies on Abaloparatide (Abl), a PTHrP analog, have demonstrated its distinct effects across these Fx sites. At Site 1, Abl enhances periosteal osteoprogenitor proliferation, leading to increased intramembranous BF. At Site 2, it facilitates chondrocyte-to-osteoblast transition, while at Site 3, it promotes osteocyte transdifferentiation and migration, contributing to intramedullary BF (Parajuli, ASBMR 21-23; Wang et al., JBMR 20). Spatial transcriptomics (Visium) analysis has revealed site-specific molecular changes associated with these processes at 10, 15, and 28 days post-Fx (Parajuli, ASBMR 23). The present study aims to elucidate the cellular dynamics and transcriptomic alterations induced by Abaloparatide at these distinct Fx sites using ScRNA sequencing. A closed, unstabilized tibial Fx model was established in 12-wk-old C57BL/6J mice using a 3-point bending method. Mice received daily subcutaneous injections of either 40 μg/kg Abl or vehicle for 15 days post-Fx. Fx callus regions from 5 mice per group (n=10 total) were microdissected, enzymatically digested, and single cells were isolated and sorted for sequencing using the 10X Genomics 3’ v3 Chromium. Three primary cell clusters were identified within the 3 sites: (1) Stromal cells, characterized by cartilage (Acan, Col2a1, Sox9) and bone markers (Prrx1, Col1a1, Spp1); (2) Hematopoietic cells (Cd14, Itgam); and (3) Endothelial cells (Pecam1). The stromal cluster predominantly consisted of cells from Sites 1 and 2, whereas the hematopoietic cluster was enriched in cells from Sites 1 and 3. Further sub-clustering of the stromal population revealed 8 distinct subpopulations, comprising progenitor cells and early-to-late-stage osteoblasts and chondrocytes. Similarly, the hematopoietic cluster was further resolved into subpopulations associated with immune and inflammatory functions. Abl treatment increased the proportion of osteoblast and chondrocyte subpopulations in Sites 1 and 2, while reducing inflammatory and immune-associated cell populations in Sites 1 and 3. These findings confirm that Abl accelerates BF by promoting osteogenesis at Sites 1, while concurrently mitigating inflammatory responses at Sites 1 & 3, and facilitating cartilage-to-bone transition at Site 2. Spatial transcriptomics (Xenium Insitu), using site-specific gene markers identified in this study, has enabled us to localize the cells in the callus responding to Abl. Presentation: Monday, July 14, 2025