Abstract Volumetric Muscle Loss (VML) is a pervasive clinical challenge with poor functional outcomes and long-term pain even after gold standard autograft treatment. Currently there is a poor understanding of mechanisms and root cause of pain after VML beyond the observation that patients are commonly afflicted with long-term pain. The overall goal of the parent grant is to define the critical roles of immune and FAP cells in VML injury and repair and to develop newly engineered immunomodulatory and regenerative treatment strategies that will provide a foundation to translate into clinical treatments for VML. The initial grant proposed to focus on developing novel immunomodulatory and regenerative therapies and to measure muscle and limb function after regeneration. The overall research goal of this supplement proposal is to expand on the current Aims and to evaluate changes in pain, sensory neurons and immune mechanisms of pain after VML. We will further assess whether novel immunomodulatory and stem cell therapies are able to reduce long term pain after VML. To expand the evaluations of limb function and pain we propose to incorporate assessments of tactile allodynia in the limbs of the mice using Von Frey filaments, and dynamic weight bearing to measure load distribution on each limb. Sensory neurons innervation of the muscle will be evaluated by staining sections for CGRP, NF200, and TRPV1. We will also assess the neurological changes that often lead to chronic pain by marking afferent neurons in the dorsal root ganglia (DRG). Immune cells in the DRG will be assessed by spectral flow cytometry for neutrophils, monocytes, macrophages, and T-cells. Lastly we will expand on immune mechanisms of pain by including new assessments of lipid mediator biosynthesis via LC-MS-based lipidomics from the muscle and DRG. The overall research outcomes of the proposed supplementary work will: 1) generate preliminary data to motivate the use of PEG-MAL resolving delivery for chronic musculoskeletal pain applications, and 2) expand research capacity and measurements of the current work to include new measures of pain and sensory neuron function and inflammation. The proposed work will have a significant impact on the understanding of pain after VML injury and the development of new therapeutic approaches to resolve chronic pain in VML patients. This award supplement will additionally provide research and career development training to promote diversity in health-related research. The proposed neuroscience graduate student, Dominique Granville, will receive training in: peripheral nerve sensory mechanisms, musculoskeletal trauma models, pre-clinical pain sensitization and behavioral metrics, DRG isolation, histology and flow cytometry, biomaterial fabrication, and lipid mediator lipidomics analyses; as well as training through an individualized development plan, research and career diversity seminars and workshops, attending and presenting at conferences, and c...