145. Lymphatic Ablation Accelerates Murine Digit Tip Regeneration Through Enhanced Osteoclast Activity

Neda Vishlaghi, Trisha Ghotra, Monisha Mittal, Ji Hae Choi, Sneha Korlakunta, Mingquan Yan, Janna L. Crossley, Danielle Griswold-Wheeler, Conan Juan, Shiri Gur-Cohen, Babak Mehrara, Robert Tower, Michael Dellinger, Lindsay A. Dawson, Benjamin Levi INTRODUCTION: In the United States, over 2 million individuals suffer from limb loss, resulting in chronic pain and limited form and function. Current treatments are inadequate in supporting tissue regeneration, emphasizing the need to understand post-traumatic endogenous regeneration for devising effective treatment strategies. P3 regeneration parallels a similar response in humans, and is characterized by the phases of inflammation, osteolysis, skin closure, blastema formation, bone differentiation, and matrix deposition. The lymphatic vasculature has been essentially ignored in bone tissue research as prior studies have failed to identify lymphatics in cortical bone during quiescence. In this study, we utilized different transgenic mouse models to elucidate the function of lymphatic vessels and lymphatic signaling during P3 regeneration. MATERIALS AND METHODS: Mice aged between 10-12 weeks underwent a P3 amputation on hind limb digits 2 and 4 of each paw. Samples were harvested on day 5-8 (inflammatory phase and osteolysis), day 10-14 (skin closure and blastema phase), day 16-21 (P3 bone mineralization), and day 28-42 (P3 late-stage bone regeneration). Prox1-eGFP mice were used for the temporal and spatial localization of lymphatic vessels. FLT4CreERT2;iDTR mice were employed for the selective ablation of both capillary and collecting lymphatic vessels. Vegfr3mut/wt mice were utilized to assess the impact of lymphatic hypoplasia on regeneration. Pharmacological inhibition of lymphangiogenesis was achieved using a VEGFR3 inhibitor diet. Surgical removal of the popliteal and inguinal lymph nodes were performed to surgically manipulate lymphatics during the regeneration process. RESULTS: Using a lymphatic-specific reporter line, we discovered that lymphatic vessels are restricted to the ventral side of the digit both during quiescence and following digit amputation throughout the phases of digit tip regeneration. These native and regenerating lymphatics closely surround—but do not invade—the native or regenerated bone. Unexpectedly, genetic, pharmacological, and surgical interventions that reduced lymphangiogenesis accelerated the early osteolysis phase of P3 regeneration. This enhanced regeneration occurred via the transition of myeloid cells to osteoclasts, resulting in faster and more robust digit tip regeneration. The increased myeloid-to-osteoclast transition was due to decreased myeloid cell egress and an elevated transition of myeloid cells to osteoclasts. CONCLUSION: Overall, these findings suggest that therapeutic manipulation of lymphatic vessel dynamics could enhance regenerative mechanisms, offering new insights into the complex interplay between lymphatic vessels, myeloid-to-osteoclast transitions, and tissue regeneration) Lymphangiogenesis patterns during digit tip regeneration) MicroCT images and analysis of VEGFR3 inhibition showing increased bone volume formation) scRNA-seq data confirming lower lymphatic marker expression and higher osteoclast activity. J-L) Histolysis is enhanced after VEGFR3 inhibition during regeneration.