Graduate Student University of California, Santa Barbara, United States
Abstract: Musculoskeletal tissues possess limited potential for self-repair and large-scale injuries severely disrupt regeneration. Thus, great efforts have been made for the development of bioinstructive, extracellular matrix (ECM)-mimetic materials that guide mesenchymal stem cell (MSC) activity toward structural and functional tissue restoration. The design of these biomaterials relies on a comprehensive understanding of ECM components that confer its biomodulatory properties and direct wound healing; yet, it was only recently discovered that extracellular vesicles (EV) are natively embedded within ECM deposited by MSC in vitro (ECM-EV). This discovery has revealed a critically understudied mechanism of cell-environment interactions with significant implications for engineering cellular signals for regeneration. The bulk of current MSC EV research addresses those secreted into conditioned culture medium (liquid-EV), shown to be a primary mechanism of MSC paracrine signaling and exert regenerative functions within biomaterials in various tissues. Here, we characterize key differences in MSC liquid-EV and ECM-EV identity and compare their regenerative properties in collagen I biomaterials toward novel musculoskeletal tissue engineering strategies. We describe methods to isolate these two populations of EV from the same MSC culture in vitro and confirm EV enrichment by surface marker expression (Western blot), morphology (TEM), and size distribution (nanoparticle tracking analysis). We show that these EV differentially express tetraspanins (CD9, CD63, CD81) and possess unique proteomic profiles, evidenced by dSTORM microscopy and SDS-PAGE, respectively. Additionally, we incorporate these EV in collagen biomaterials to interrogate their ability to drive MSC proliferation, differentiation, and ECM deposition. Our results will contribute to a growing body of literature aiming to understand characteristic differences between liquid-EV and ECM-EV and elucidate the role of tissue-resident EV in cell sensing of the microenvironment.
