Project Summary/Abstract Volumetric Muscle Loss (VML) is the most devastating and chronic musculoskeletal injury in which a substantial amount of muscle is lost due to trauma or surgery and can be life-threatening. Sadly, the current standard of care, which includes surgical transplantation of allografts or synthetic acellular scaffolds followed by physical therapy, is limited in efficacy. Tissue-engineered cell-laden synthetic scaffolds are promising; however, cell-based therapies exhibit immune rejections and inflammation chances. On the contrary, extracellular vesicles (EVs) have been proven to demonstrate a strong potential as a ‘cell-free’ regenerative medicine to promote muscle regeneration. However, the therapeutic efficacy of EVs depends on their parent cells’ state and can be engineered to promote myogenesis, leading to functional muscle regeneration. Hence, the long-term objective of this R03 project is to establish the foundation for developing ‘customized’ therapeutic nEVs as a promising regenerative medicine for VML treatment. Specifically, the primary research goal of this pilot R03 is to develop customized therapeutic exosomes (a sub-type of EVs, nEVs) using controlled piezoelectric stimulation of parent adipose-derived mesenchymal stem cells (ADSCs) to enhance myogenesis in recipient muscle cells. There are two primary hypotheses. First, nEV formation and content can be customized, i.e., modulated by controlled pre-conditioning parent cells, i.e., by influencing the cellular microenvironment. Second, the customized nEV cargo can influence biological functions in recipient cells, affecting myogenesis. To test the hypotheses, we have designed two specific aims. In Specific Aim 1, we will determine the impact of piezoelectric stimulation on nEV formation and content from ADSCs. Briefly, we will utilize controlled piezoelectric stimulation to analyze its influence on ADSCs-derived nEV formation and content. We will use Small RNA sequencing to identify modulated micro-RNA (miRNAs) in the piezoelectric stimulation pre-conditioned nEVs (piezo-nEVs). In Specific Aim 2, we will determine the myogenic efficacy of piezo-nEVs. Specifically, we aim to understand how piezo- nEVs interact with recipient C2C12s muscle cells and influence myogenic differentiation. We will determine the definite pathways by which piezo-nEVs interact with C2C12s. We will utilize standard immunohistochemistry and western blotting to study the mechanistic interactions of the piezo-nEVs with recipient C2C12s. Successful completion of this project will yield the following outcomes: 1) the development of customizable nEVs using piezoelectric stimulation pre-conditioning of parent ADSCs, 2) an in-depth fundamental analysis of how piezoelectric stimulation can influence the development of therapeutic nEVs with modulated miRNA, and 3) determine the therapeutic efficacy of piezo-nEVs in terms of influencing myogenesis. The results from this pilot R03 project will serve as a proof-of-concept for follow-up R01 studies and set the foundation for developing customized therapeutic nEVs as a promising medicine for functional muscle regeneration and treating VML.

Project Narrative This R03 project aims to establish the foundation for developing an extracellular vesicle (EV)-based therapy for treating critical Volumetric Loss Defects (VML) injuries caused by traumatic incidents such as road accidents, major falls, or fire breakouts. Specifically, in this project, we will develop a new class of customizable therapeutic EVs by controlled pre-conditioning of parent cells and understand the role of the therapeutic EVs in influencing myogenesis towards healthy muscle formation. The results of this pilot project will set the basis for future elaborate studies that will introduce a full-proof EV-based regenerative medicine therapy for treating patients with critical VML injuries or other injuries that require muscle regeneration.