SUMMARY Perianal fistulas (PAF) occur in 30-40% of Crohn’s disease patients and their complications lead to a significant impairment in quality of life. Current treatments are effective in less than 50% of cases. The overall objective of this proposed study is to develop a biostimulatory nanofiber-hydrogel composite (NHC) plug to promote tissue healing and to test its efficacy to repair PAF in clinically relevant animal models. Previous work from the Mao and Selaru labs laid the foundation for this study. Specifically, we have established novel rodent and swine models of PAF that faithfully recapitulate the biology of PAF in patients. In parallel, we have developed a first-generation injectable biodegradable nanofiber-hydrogel composite (NHC) from poly(e-caprolactone) (PCL) nanofibers covalently bonded to hyaluronic acid (HA) hydrogel; and demonstrated its ability to deliver adipose stem cells (ADSCs) and repair PAF by conditioning inflammatory responses, permitting host cell infiltration, and facilitating angiogenesis and progressive remodeling. Building on these preliminary results, we plan to engineer a second generation NHC plug with enhanced mechanical strength and integrity as an off-the-shelf device and optimize its biostimulatory activities to induce more favorable cellular responses governing fistula healing. We hypothesize that this NHC plug with structurally, mechanically, and biofunctionally optimized features will effectively promote angiogenesis and soft tissue restoration; and serve as a carrier for ADSCs and ADSC- derived exosomes to further improve the PAF tissue healing. To test this hypothesis and demonstrate its translational potential, we will pursue three specific aims (1) develop a collagen nanofiber-based NHC plug with optimal mechanical properties and porous structure for ease of implantation to support perianal fistula healing, (2) utilize a rat model to evaluate the efficacy for PAF healing when used alone or in conjunction with ADSCs or ADSC-derived exosomes, and elucidate the pro-regenerative mechanism by assessing cell infiltration, angiogenesis, extracellular matrix deposition, and tissue remodeling, and (3) utilize a swine model to demonstrate the synergistic effect in tissue remodeling by combining the optimized NHC plug with ADSC-derived exosomes. This study will deliver a translatable off-the-shelf biomimetic NHC microporous plugs for the delivery of ADSC-derived exosomes capable of promoting soft tissue remodeling and healing for PAF repair.