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.
