Project Summary/Abstract Bone fractures in the elderly are a serious health issue due to high morbidity and mortality. New therapies are needed to reduce treatment time and decrease the mortality rate. Cellular senescence is closely associated with aging and aging related diseases, including osteoporosis. However, senescent cells (SCs) are heterogenous, and their roles in fracture repair during aging have not been well studied. We recently reported that SCs in callus impaired age-associated fracture repair through TGFβ1 and clearance of total SCs with senolytic drugs (Dasatinib+Quercetin, D+Q) enhanced fracture repair in aged mice only, indicating the difference of SCs in callus between young and aged mice. With cutting-edge technology, single cell RNA sequencing in mesenchymal cells (CD45-CD31-Ter119-) from callus identified 3 clusters of SCs: TGFβ1+CXCR2+, TGFβ1-CXCR2+, TGFβ1+CXCR2. Very interestingly, TGFβ1+/-CXCR2+ clusters strongly inhibited CaMPC growth, indicating they are detrimental SCs (dSCs). CXCR2 signaling promotes cellular senescence. We found that CXCR2 inhibitor specifically removed dSCs and enhanced fracture healing in aged mice, suggesting removal of dSCs, but keep potential beneficial SCs subsets benefit fracture healing in aged mice. To investigate the potential molecular mechanism, we examined the role of TGFβ1 and tissue inhibitor of metalloproteinase 2 (TIMP2), one of the top expressed SASP in dSCs. TGFβ1 neutralizing Ab enhanced fracture healing in aged mice. TGFβ1 and TIMP2 neutralizing Abs synergistically inhibit the effect of SCs on CaMPC growth. The ubiquitin-proteasome system (UPS) plays critical roles in age-associated bone disorders. Ub-proteomics identified PDGFRβ as one of the most ubiquitinated proteins regulating MPC expansion, and its expression is decreased in bone and callus of aged mice. TGFβ1 induces PDGFRβ ubiquitination and degradation, while TIMP2 decreases PDGFRβ receptor phosphorylation and activation. We further found that CXCR2 ligand, CXCL5, which was increased in callus of aged mice specifically induced dSCs in CaMPCs from aged mice. Thus, we hypothesize dSCs accumulate in callus of aged mice where they produce excessive TGFβ1, TIMP2 and CXCLs, resulting in not only a positive loop of dSC generation from CaMPCs via CXCLs, but also increased ubiquitination and decreased PDGFRβ phosphorylation, and a reduced MPC pool, which can be prevented by selectively depleting dSCs via CXCR2 inhibition. In this application, we will 1) fully characterize dSCs phenotypically and examine if dSCs impair fracture healing in aged mice, 2) investigate whether dSCs affect fracture repair of aged mice by regulating PDGFRβ in CaMPCs via TGFβ1 and TIMP2. Our proposal will elucidate the role of a subset of SCs (dSCs) in aging fracture, identify new mechanisms and novel drug targets, which could lead to interventions for geriatric fractures that are of great morbidity, mortality and healthcare costs.