Harnessing the Inflammatory Response to Promote Regeneration in Mammals

ABSTRACT Bone and soft tissue regeneration after amputation remains an enigmatic phenomenon in human biology, contrasting starkly with the regenerative abilities displayed by other bone injuries (e.g. fracture). Macrophages have emerged as key players in creating regeneration-permissive environments, promoting bone regrowth when properly guided. However, the mechanisms underlying macrophage-mediated regeneration remain poorly understood, leaving unanswered questions regarding their role in human regeneration and potential therapeutic applications. To address these challenges, we leverage a comparative model of bone regeneration and scar formation within a single organism, the mouse. Focusing on distal fingertip amputations (P3) resulting in regeneration and proximal amputations (P2) leading to scar formation, we investigate macrophage subtypes and their impact on neighboring cells to promote bone and soft tissue regeneration. Our preliminary data identify a unique population of macrophages that associate with growing bone and express growth factors (i.e. BMPs) known to be essential for bone regeneration. These cells are found in regenerative injuries and not found in scar- forming injuries suggesting a potential role specific to regeneration. Moreover, this population has a unique metabolic profile compared to macrophages found in scar-forming injuries, suggesting a connection between cellular metabolism and regenerative potential. This project will test the role of macrophages in guiding digit regeneration and their potential as therapeutic agents. In Aim 1, we will test if the unique metabolic and signaling profile of P3-macrophages is essential for regeneration. Using mouse transgenic macrophage-depletion models and advanced analysis techniques combining single cell RNA sequencing and spatial transcriptomics, we aim to identify specific molecular signals macrophages use to instruct neighboring cells. We will additionally test the requirement for specific metabolic and signaling profiles by knocking out key proteins in macrophages and determining their influence on regeneration. In Aim 2, we will determine whether P3-macrophages can promote a regenerative environment during fibrotic scarring. Through cell isolation, macrophage depletion, and metabolic manipulation, we aim to explore the regenerative impact of P3-macrophages in a non-regenerative context. In conclusion, our project aims to unravel the intricate role of macrophages in regeneration using a mouse model. By identifying the specific functions and metabolic profiles of regenerative macrophages, we hope to unlock the potential of macrophages as therapeutic targets for engineering regeneration-permissive environments. This research provides valuable insights into how the body naturally harnesses macrophages to guide bone and soft tissue regeneration and offers potential avenues for regenerative medicine.