Beyond polarization: single-cell atlas unveils macrophage heterogeneity and neuro-immune crosstalk in bone regeneration.

Bone regeneration is a complex and carefully orchestrated physiological process of bone formation. Immune cells, especially macrophages, play a key role in MSC recruitment, osteoblast differentiation and osteoclast formation. However, the traditional concept of macrophage polarization is inadequate to match its functional diversity and mechanistic complexity. A comprehensive analysis and definition of macrophage heterogeneity in the bone regeneration niche is urgently needed. We analyzed single-cell RNA-seq (scRNA-seq) data of 8148 cells in the bone defect area using multiple methods and focused on macrophages infiltrating the bone regeneration microenvironment. Based on the corresponding scRNA-seq data, we systematically revealed eight heterogeneous subpopulations of macrophages. These subpopulations function as defense responses (DF-macrophages), fibrosis (FB-macrophages), osteogenesis/regeneration (OB-macrophages), bone resorption (OC-macrophages), and programmed death (PD-macrophages). Among them, the OB- and OC-macrophages has the potential to be regulated by neural signals because of their high frequency of G protein-coupled receptor genes (Gprs). Exogenous nerve growth factor (NGF) supplement effectively activated neural reaction and accelerated bone repair combined with inflammation resolution in vivo. Further bulk RNA-seq analysis provided evidence of significant enhancement of anti-inflammatory, regenerative and bone metabolism related genes and functions in macrophages upon exogenous NGF stimulation. In vitro experiments also confirmed the anti-inflammatory of the macrophage treated by the secretion of the neural cells stimulated by NGF. Our study provides evidence for revealing the functional phenotype of macrophages in the bone regeneration environment and provide evidence for the acceptance of neural regulation.