ABSTRACT In elderly individuals, bone aging can lead to dysregulation of the bone immune microenvironment, resulting in the formation of osteoporotic microenvironment. Consequently, this can cause delayed bone fracture healing and early implant failure. Regarding osteoporotic microenvironment, the interaction between bone mesenchymal stem cells (BMSCs) and macrophages has gained increasing attention. Aged BMSCs exhibit a senescence-associated secretory phenotype (SASP). And SASP, together with macrophage inflammaging, contributes to the inflammatory microenvironment leading to osteoporosis and delayed bone healing. USP7, as a key deubiquitinase regulating cellular senescence and inflammation, has been found to promote senolysis when inhibited. This study investigated the effects of USP7 inhibition on aged BMSCs and bone marrow derived macrophages (BMDMs), as well as their interactive patterns. Through mRNA-Seq analysis, it was found that USP7 inhibition induced senolysis and osteogenic differentiation in aged BMSCs. For senescent BMDMs, USP7 inhibition suppressed NLRP3 inflammasome pathway and enhanced efferocytosis capacity through EPSIN1/LRP1 pathway, enabling them to efficiently clear apoptotic aged BMSCs caused by USP7 inhibition. Furthermore, by administering P5091 systemically to aged mice, it was discovered that USP7 inhibition could rescue bone loss and promote osseointegration of aged C57BL/6J mice via BMDMs efferocytosis and BMSCs senolysis. This study highlights the potential of USP7 inhibition in promoting senolysis in aged BMSCs and enhancing the efferocytosis capacity of senescent BMDMs, which exerts a synergistic therapeutic effect by inducing senescent cell apoptosis and immune clearance. The "kill and bury" model proposed in this study for aged BMSCs-BMDMs crosstalk holds promise for future mechanistic research and drug development. Lay summary The inflammatory osteoimmunology state is an important mechanism underlying age-related osteoporosis and delayed osseointegration. The interaction between BMSCs and macrophages may represent crucial therapeutic targets. On one hand, this study reveals that USP7 depletion could induce apoptosis of aged BMSCs through senolysis, while on the other hand, USP7 depletion could promote macrophage efferocytosis via the EPSIN1/LRP1 pathway, thereby clearing apoptotic aged BMSCs. This process simultaneously contributed to the clearance of senescent cells and the promotion of M2 polarization in macrophages. Moreover, in vivo experiments also demonstrate that USP7 inhibition improved age-related osteoporosis and osteoporotic osseointegration by promoting senolysis and efferocytosis in aged mice, highlighting USP7 as a potential therapeutic target for bone aging-related diseases.

USP7 Inhibition Promotes Mesenchymal Stem cell-Macrophage Crosstalk in Age-related Osteoporosis via Efferocytosis and Senolysis