Boosting TrkA+ Sensory Nerve-to-Bone Interactions Enhances Calvarial Bone Repair and Hedgehog Signaling Activation

The neuroregulatory effects of sensory nerves in bone repair have recently begun to be elucidated, principally through loss of function studies. However, the potential therapeutic efficacy of boosting nerve-to-bone interactions, as well as the elucidation of downstream signaling pathways remains poorly studied. Here, two parallel approaches were utilized to enhance sensory nerve-to-bone interactions using a mouse calvarial bone defect model system. Pharmacologic activation of TrkA with gambogic amide induced bone-associated nerve ingrowth and markedly improved calvarial bone healing. Single-cell RNA sequencing analysis of cells derived from the defect site revealed shifts in cluster proportions, with enrichment of immune cell populations in TrkA agonist-treated mice. Within the skeletal cell lineage, TrkA agonism enhanced osteoblast differentiation while suppressing fibroblastic differentiation. Pathway analysis showed increased activity of Hedgehog, Wnt, BMP, and other osteogenic pathways. Further investigation of intercellular communication identified elevated Hedgehog signaling pathway specifically from DRG neurons. In summary, activation of TrkA-positive sensory nerves stimulates hedgehog pathway activity in local mesenchymal stem and progenitor cells, promoting osteoblast differentiation and bone formation. Our study thus elucidates signaling mechanisms underlying neurogenically-enhanced bone repair. Overall design: 2 samples were analyzed comparing mRNA from mice. One is a control group which received intraperitoneal (IP) injection of vehicle control (10% DMSO). The other is a GA group which received IP injection of gambogic amide. Gambogic amide (GA) was used to activate TrkA. Mice received intraperitoneal (IP) injection of either 100 µL vehicle control (10% DMSO) or GA (1.2 mg/kg in 10% DMSO). Treatment began 24h prior to calvarial injury, and continued daily thereafter for 1 week. Skulls were microdissected 7 days after defect creation. Cranial bone with a defect at the center, with an approximate edge length of 3mm, was minced and subjected to six sequential enzymatic digestions with a mixture containing collagenase type I (1 mg/ml) and collagenase type II (1 mg/ml).