# 3D bioprinting of a bilayered, tissue engineered cornea > **NIH NIH R01** · STANFORD UNIVERSITY · 2024 · $496,338 ## Abstract Corneal blindness is a leading cause of visual impairment, affecting an estimated 12.5 million people worldwide. Corneal transplantation, or keratoplasty, is the only curative treatment for corneal blindness, yet the cadaveric donor tissue required for this sight-restoring procedure is available to less than 2% of patients worldwide. While several different tissue-engineered corneal transplants have been proposed, designing therapies with the long- term transparency and regenerative capacity of a human donor cornea remains a formidable challenge. The native cornea is a multi-layered tissue, with each layer playing a distinct role in the overall corneal function; however, most tissue-engineered corneas to date have been monolithic structures. Here we propose leveraging recent advances in 3D bioprinting to fabricate a bilayered, tissue-engineered cornea, with each layer fabricated from a customized biomaterial formulation with optimized print parameters to achieve the desired biofunctionality. This technology builds on our recently reported family of UNION bioinks that form cohesive, strong interfaces between distinct biomaterials when 3D-printed into a tissue-engineered construct. In Aim 1, we design a 3D-printed stromal layer with encapsulated corneal stromal stem cells (CSSCs) that resists contraction, remains transparent, and secretes pro-regenerative paracrine signals. While CSSCs have the potential to reduce scarring, their contractile phenotype leads to deformation of engineered tissue, altering the shape, size, and transparency over time. We hypothesize that covalent crosslinking of a chemically modified collagen type I bioink will enable printing of a mechanically robust hydrogel with ordered fibrils that guide cell alignment and phenotype. Our preliminary data demonstrate that bio-orthogonal azide-alkyne click chemistry crosslinking resists cell-induced deformation and promotes cell alignment. In parallel, in Aim 2, we design an acellular 3D-printed basement membrane layer that promotes corneal epithelial cell (CEC) migration and forms a cohesive interface with the 3D-printed stromal layer. CECs are known to alter their migration in response to properties of their underlying basement membrane, which is distinct from the stromal layer. Clinical translation requires a cohesive interface between these two layers to maintain a stable graft. We hypothesize that a thin layer of printed protein can be formulated to promote migration of endogenous CECs while maintaining a cohesive interface with the underlying stromal layer through bio-orthogonal covalent crosslinks. The bilayer construct will be evaluated in an ex vivo rabbit eye model to quantify the migration of endogenous CECs in response to secreted signals from the encapsulated CSSCs. In Aim 3, we evaluate the translational potential of the 3D-printed, bilayered corneal tissue in a preclinical rabbit model of keratoplasty. The rabbits undergo a 5.0- mm keratectomy to excise a central cornea... ## Key facts - **NIH application ID:** 10780824 - **Project number:** 1R01EY035697-01 - **Recipient organization:** STANFORD UNIVERSITY - **Principal Investigator:** Sarah C Heilshorn - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $496,338 - **Award type:** 1 - **Project period:** 2024-01-01 → 2027-12-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10780824 ## Citation > US National Institutes of Health, RePORTER application 10780824, 3D bioprinting of a bilayered, tissue engineered cornea (1R01EY035697-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10780824. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*