Sympathetic nerve inhibition enhances calvarial bone repair via senescent macrophage-induced osteogenesis and angiogenesis

Abstract Bone regeneration is a tightly coordinated process involving multiple cellular and molecular components, with emerging evidence highlighting the pivotal role of the nervous system, especially the sympathetic nervous system, in modulating skeletal repair. However, the mechanistic details of neuro-skeletal interactions during bone healing remain elusive. Here, we inhibited peripheral sympathetic nerves using 6-hydroxydopamine (6-OHDA) in a murine calvarial defect model and performed single-cell RNA sequencing on the injury sites at 7 and 14 days post-injury to delineate the cellular landscape underlying regeneration. Our analyses revealed activation of neurogenesis-associated pathways and dynamic crosstalk between neural and skeletal cells following injury. Sympathetic nerve inhibition significantly enhanced calvarial bone repair, characterized by downregulation of Capn6 in suture mesenchymal cells, increased formation of H-type blood vessels, and the emergence of a distinct macrophage subset exhibiting senescence-associated phenotypes. Importantly, pharmacological clearance of senescent cells by senolytic agents abrogated the regenerative benefits conferred by sympathetic blockade. Mechanistically, sympathetic inhibition promoted angiogenesis and osteogenesis by facilitating interactions between suture mesenchymal cells and endothelial cells, while the senescent-like macrophages contributed to bone repair via secretion of osteogenic cytokines. Collectively, these findings uncover a critical role of sympathetic nerves in regulating the bone healing niche and identify potential therapeutic targets to enhance skeletal regeneration. These insights may pave the way for the development of neuromodulatory or senescence-targeted therapies to promote bone repair in challenging clinical scenarios such as cranial bone defects, non-union fractures, or aging-associated impaired healing.