Repairing Effect and Mechanism of the 4-Dimensionally Printed Limbal Stem Cell Strategy on Corneal Alkali Burns in Large Animals

Alkali burn of corneas can induce corneal stromal fibrosis and limbal stem cell deficiency, which destroys corneal epithelial homeostasis, leading to scarring and impaired vision. Although stem cell therapy has shown potential therapeutic contributions to corneal injuries, it still faces the challenges of difficult retention and low survival rates due to the limitations of corneal curvature and an abnormal microenvironment. In this work, a 4D-printed chitosan-based hydrogel (4D-CTH) was prepared to load limbal stem cells (LSCs) for the regulation of epithelial microenvironment homeostasis and the repair of alkali-burned corneas. 4D-CTH, which has good biocompatibility and a regular spatial structure, was proven to be a candidate for use as a tissue engineering carrier that supplies highly active LSCs to a cornea injured by alkali. Both in vitro and in vivo studies confirmed that treatment with 4D-CTH + LSCs can provide more efficient corneal repair for alkali burn injuries compared to epidermal growth factor, which is the traditional treatment method for treating burned corneas. Based on single-cell sequencing analysis, 4D-CTH can markedly increase the proportion of LSCs in corneal tissue by promoting the residence and growth of LSCs. Additionally, 4D-CTH loaded with LSCs can inhibit and reverse corneal fibrosis by interfering with fibroblast differentiation, which is closely related to the down-regulation of cytochrome c oxidase subunit VIc expression by LSCs, thereby inhibiting oxidative phosphorylation in fibroblasts. In conclusion, this work not only confirmed the feasibility of 4D-CTH + LSCs for the treatment of corneas burned by alkali but also clarified the regulation mechanism of corneal epithelial homeostasis by 4D-CTH + LSCs, providing theoretical support and an application paradigm for corneal tissue engineering therapy.