SAT-737 Contribution of sost-Positive Osteocytes to Bone Formation during Fracture Repair

Abstract Disclosure: Y. Wang: None. S. Won-Kim: None. D.D. Bikle: None. Fracture (Fx) repair occurs at 3 different sites of the callus: intramembranous bone formation (IBF) developing from the outer layer of the periosteum of the cortex; endochondral bone formation (EndoBF) developing within the bridge region of the Fx; intramedullary bone formation (IMBF), developing within the marrow of the ends of broken bones. IMBF is a transient process without the involvement of chondrocytes or prx1 cre labeled stem cells from the periosteum but rather originating from cortical osteocytes. The mechanisms of IMBF remain unknown. To address this issue, we investigated the origin of the progenitors and mechanism of progenitor differentiation involved in IMBF by lineage tracing, immunohistochemistry (IHC) and histology. A unilateral closed mid tibial Fx was made in 12 wks old mice. We first determined the progenitors contributing to Fx repair by tracing the cell fate of sost expressing cells in the sostTdT mice [tamoxifen regulated sost cre mice X Rosa TdTomato (TdT) mice]. Tamoxifen (Tam) was given 1 day prior to Fx. At D2 post Fx, no bone had yet formed in the IMBF site. 23% of the sost- positive osteocytes appeared in the cortical bone (CB) of the fractured bone ends close to the IMBF. A subset of these cells co-expressed the stem cell marker Sox2 (15%), and leptin receptor (LepR, 55%) as identified by IHC. Similarly, at D2 post Fx no sost-positive cells were observed in the newly formed woven bone in the IBF site or in the cartilage in the EndoBF site. By D14 post Fx, newly formed bone was observed in the IMBF site, and sost positive cells appeared in the bone surface in both the IMBF and IBF sites. These cells no longer expressed Sox2, but co-expressed osteocalcin. Interestingly, LepR was expressed in sost positive cells in the IMBF sites, but not in the IBF sites. By D21 post Fx, sost positive cells were embedded inside the newly formed bones at both the IMBF and IBF sites, and continuously co-expressed LepR in the IMBF sites. No sost positive cells were observed in the cartilage in the EndoBF site at D14 and D21 post Fx. We then determined the Fx repair in osteocyte depleted mice (dmp1DTR mice) [floxed diphtheria toxin (DT) receptor (floxed DTR) mice X tam regulated DMP-1cre mice]. Tam and DT (50 ng/mouse) were given for 5 days starting 1 day prior to Fx. At D21 post Fx, H&E staining revealed 56 % of osteocytes were depleted at the CB of fractured bone ends which reduced bone formation in the IMBF site (BV/TV: 2.1 % in dmp1DTR mice vs. 4.5 % in the controls, p< 0.05) and IBF site (BV/TV: 3.3 % in dmp1DTR mice vs. 4.0 % in the controls, p = 0.09) when compared with the controls. Our data indicate that during Fx repair, sost expressing osteocytes from the broken bone ends appear to reprogram, migrate to the intramedullary (LepR+ and LepR- cells) and intramembranous sites (LepR- cells only), differentiate into osteoblasts, and contribute to IMBF and IBF. Presentation: Saturday, July 12, 2025