Decoding Neuroimmune Interactions to Enhance Craniofacial Bone Healing

Cranial bone is richly innervated with sensory nerves that play critical roles in fracture healing by signaling and regulating osteoprogenitor function. This is supported by our prior findings which suggest that small-molecule mediated (i.e. TrkA (tyrosine kinase A) agonists) stimulation of nerve sprouting into calvarial defects enhances bone formation while TrkA inhibition using a chemical-genetic approach abrogates bone repair. In contrast to normal fracture healing, critical-sized, craniofacial bone defects require therapeutic intervention such as the delivery of biomaterials and stem cells. In preliminary studies, we have observed that the delivery of exogenous stem/stromal cells to cranial bone injuries magnifies neural ingrowth associated with bone healing, suggesting that stem cells may regulate bone repair in part by impacting these early-stage neural interactions. To optimize the efficacy of these bioengineering strategies, we will define the neuro-immuno-skeletal signaling axis during bone repair across healing and non-healing cranial bone injuries and elucidate how biomaterial- and stem cell- delivery impacts this signaling axis. We will employ a quantitative light-sheet microscopy (QLSM) platform to visualize and quantitatively describe ?3-tubulin labeled nerve responses to 1-mm and 4-mm full-thickness injuries in the parietal bones of mice. We will combine this information with corresponding single cell RNA sequencing (scRNA-seq) data obtained from cells at the injury sites and the nerve bodies in the trigeminal ganglia which provides sensory innervation to the skull. We propose three Specific Aims: In Aim 1 We will create 1-mm or 4-mm injuries in the parietal bones of 12-week old Baf53b-tdTom mice, in which all peripheral nerves express a tdTomato reporter. We will image macrophages, axons, and osteoprogenitors at multiple time points following injury. Additionally, we will perform timelapse scRNA-seq on cells isolated from both the injury site and trigeminal ganglia and interrogate the reciprocal signaling interactome between early responding macrophages and recruited peripheral nerves in injuries and decipher how these impact osteoprogenitor recruitment and differentiation into osteoblasts. In Aim 2, we will Independently manipulate the magnitude and duration of TrkA+ nerve sprouting using agonists and inhibitors and evaluate the impacts on signaling and cranial bone healing. With either gain or loss of function approaches, changes to the neuro-immuno-skeletal signaling landscape and bone repair outcomes will be assessed using spatial and sequencing methods as described in Aim 1. Lastly, in Aim 3, we will design, fabricate, and test bi-functional scaffolds which provide spatially and temporally controlled release of neurotrophic factors and neural inhibitors to orchestrate the appropriate signaling cascade that maximizes bone healing in critical-sized calvarial injuries in mice. Overall, we will combine information on ligand- receptor signaling into an engineering strategy to maximize the therapeutic outcomes via the neuro-immuno- skeletal signaling that promotes therapeutic cranial bone healing. Project Number: 1R56DE034628-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Warren Grayson (+1 co-PI) | Institution: JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD | Award Amount: $521,362 | Activity Code: R56 | Study Section: Skeletal Biology Development and Disease Study Section[SBDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11336208