PROJECT SUMMARY Successful regenerative medicine approaches require harnessing the appropriate cell signals at the right time to direct host tissue functions. These signals are often informed by the natural regenerative processes controlled during development and homeostasis by mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (EVs), which allow a cell-based yet cell-free approach for downstream regenerative technologies. This multidisciplinary, MPI proposal brings together a team of two senior investigators leading regenerative medicine-focused group with complementary strengths, co-investigators with critical roles, and industrial partner RoosterBio, Inc. Together, we will create and test an innovative enabling technology to stably incorporate EVs to a biomaterial intended for tissue engineering and regenerative medicine applications. Specifically, we will use a novel azide-based click chemistry technique to controllably immobilize EVs to silk fibroin as a demonstrative application, but immobilization can also be done on other biomaterials, substrates, or surfaces, or even tissues. We chose silk as our biomaterial in this application given its FDA-approved status and wide use. We hypothesize that “azide-clickable" MSC-derived EVs (which we will refer to simply as “Az-EVs”) will have more stable immobilization to silk fibroin biomaterials than unmodified EVs, and this will result in higher regenerative potency. To test this hypothesis and provide proof-of-concept applications, we will pursue four specific aims: Aim 1 - Demonstrate and validate Az-EV immobilization to silk fibroin-based materials; Aim 2 - Demonstrate the MSC-mimicking effects of Az-EVs immobilized to silk in vitro; Aim 3 - Demonstrate the regenerative effects of Az-EVs in a mouse chronic wound healing model; Aim 4 - Demonstrate the regenerative effects of Az-EVs in a rat tissue engineered vascular graft model. Partner RoosterBio, Inc. will “industrialize” (scale-up) the production of MSC-derived Az-EVs for commercialization to make available to other researchers and clinicians. This research will provide insight to the efficacy of this novel selective EV immobilization technology to efficiently direct EV delivery within a biological system of interest. Our proof-of-concept studies will demonstrate how utilization of this regenerative technology can aid in treating chronic wounds and enabling TEVGs with improved patency rates.

PROJECT NARRATIVE The next generation of technologies for regenerative medicine application will be fueled by cell- derived, self-packaged therapeutics known as extracellular vesicles (EVs). We will develop a process that makes these nano-sized therapeutics attachable to a biomaterial, like Velcro®, for tissue repair and regeneration. Additionally, our industry partner will modify their workflow to make the attachable EVs widely available and easy to use for downstream consumers, including those that are advancing regenerative medicine.