Project Summary Injuries of the growth plate account for nearly 20% of all fractures in children and often result in impaired bone growth. Once damaged, cartilage tissue of the growth plate is often replaced by unwanted bone tissue. This “bony bar” can cause angular deformities of the bone or stunt longitudinal bone growth. Angiogenesis is known to precede bony bar formation after growth plate injury, yet the mechanistic underpinnings of this (and by extension the means to prevent it) are poorly understood. Recent studies from our laboratory have demonstrated an essential role for skeletal sensory nerves in the regulation of skeletal cells and tissues. Most skeletal neurons are NGF (Nerve growth factor) responsive TrkA (Tropomyosin receptor kinase A) fibers, which our group has shown to interact with skeletal tissues to influence skeletal development and repair. Most recently, we reported that TrkA+ neurons regulate vascular proliferation and angiogenesis. This work has led us to the entirely new hypothesis that skeletal sensory neurons may regulate response to growth plate injury, potentially via regulation of angiogenesis, and that this could be targeted therapeutically. To assess this hypothesis, our studies are divided into two Specific Aims: Specific Aim 1. Identify molecular determinants of sensory neural regulation of growth plate injury response. We will utilize multi-tissue single cell RNA sequencing (scRNA-Seq) to map the neuroskeletal interactions after growth plate injury. Analysis of both the injured physis and neural soma in the corresponding lumbar dorsal root ganglia (DRG) neurons will recreate the neuro-skeletal interactome before and after injury. Next, experiments will be performed within TrkAF592A transgenic animals, in which injury site innervation and bony bar formation are attenuated. Our Aim 1 hypotheses are that: (1) chemical-genetic neuronal inhibition may disrupt pathologic bony bar formation, and growth plate angiogenesis, and (2) single cell sequencing will map the neuro-skeletal interactome and predict neural regulatory mechanisms. Specific Aim 2. Target sensory neurons via a small molecule inhibitor to prevent growth plate innervation and bony bar formation. In Aim 2, we will utilize a small molecule inhibitor of TrkA (AR786) in order to pharmacologically target TrkA signaling so as to inhibit growth plate injury site innervation, vascularity and bony bar formation. Studies will be performed in Thy1-YFP reporter animals which permit visualization of nerve ingrowth during growth plate repair. Our Aim 2 hypothesis is that small molecule TrkA inhibition during growth plate injury will prevent injury site innervation and pathologic bony bar formation.