Project Summary The demand for human donor tissue for treatment of corneal blindness far outpaces the supply, necessitating an innovative bioengineered approach for corneal regeneration. However, current laboratory-made constructs are insufficient due to lack of long-term transparency and underwhelming regenerative capacity. Recent studies suggest that cell therapies using transplanted corneal mesenchymal stromal cells (MSCs) help prevent corneal scar formation and restore corneal transparency. To propel this strategy toward clinical translation, both an effective cell delivery system and a precise understanding of their regenerative effects will be required. 3D bioprinting—in which cells and matrix components are precisely patterned—is a promising technique for creating customizable corneal constructs for implantation. We have previously demonstrated a versatile, cell-friendly 3D bioprinting platform for corneal MSCs. In my proposed research, I will apply 3D bioprinting for bioorthogonally- crosslinked collagen hydrogels with encapsulated human corneal MSCs to fabricate highly regenerative corneal stroma-like constructs. This system will allow for control over the corneal MSC microenvironment to optimize and leverage their regenerative potential. I specifically aim to overcome two common challenges in bioengineered corneal tissue that both result in loss of transparency: (1) tissue contraction over time due to cell- imposed forces, and (2) lack of cell and matrix organization that mimics the hierarchical structure of the native corneal stroma. I will test the hypotheses that (Aim 1) crosslinking collagen hydrogels with a covalent bioorthogonal chemistry increases stability against contraction from corneal MSCs without hindering their ability to secrete pro-regenerative factors and (Aim 2) aligning the collagen fibril microstructure through 3D bioprinting guides the organized deposition of nascent matrix to facilitate sustained transparency. These constructs will be evaluated in vivo (Aim 3) in a rabbit keratectomy model to assess restoration of corneal thickness, stromal integrity, and optical transparency; integration of the hydrogel into the host; re-epithelialization; surface inflammation and scarring; and phenotype of the transplanted corneal MSCs. Together, these results will be critical for understanding the effect of the surrounding 3D matrix on the regenerative capacity of corneal MSCs. My training will be supported by Sarah Heilshorn, Ph.D. (Materials Science & Engineering), an expert on biomaterials for regenerative medicine, and David Myung, M.D., Ph.D. (Ophthalmology), an attending physician who specializes in corneal surgery and diseases of the eye. In addition to expanding my scientific technical skills, my training plan includes development of mentorship, scientific writing, and presentation skills; training in research ethics; and enhancement of collaboration skills through a series of on-campus courses, workshops and seminars as...