Digital light processing programs shape-morphing hydrogels into undulating 3D scaffolds supporting corneal limbal epithelial organization

This study developed a rapid single-step Digital Light Processing strategy for fabricating shape-morphing hydrogel scaffolds that reproduce key topographical features of the human corneal limbal epithelial stem-cell niche. A photocrosslinkable bioink containing methacrylated collagen I, hyaluronic acid, and silk fibroin was patterned using alternating high and low UV doses to create spatial differences in crosslinking density. Incubation at 37 °C induced preferential shrinkage of the lower-UV regions, producing stable undulating grooves and ridges through a temperature-dependent silk-fibroin conformational transition. Mechanical testing showed that UV dose strongly controlled hydrogel stiffness and that silk fibroin further altered the temperature dependence of the material. At 37 °C, low-UV CHS hydrogels had a Young’s modulus of approximately 19 kPa compared with 11 kPa for CH hydrogels, while high-UV CHS and CH gels reached approximately 340 and 249 kPa, respectively. At room temperature, low-UV CHS gels were substantially softer at approximately 6 kPa, supporting the conclusion that silk-fibroin β-sheet formation at 37 °C contributes to stiffening and shape morphing. Primary human limbal-derived corneal epithelial cells adhered and proliferated effectively on silk-fibroin-containing scaffolds and remained viable for at least three weeks. Cells preferentially stratified within the softer, low-UV grooves of patterned scaffolds, reaching up to approximately five layers, while flat gels showed limited stratification. Mechanotransduction was also spatially regulated: confluent epithelial cells on softer regions displayed greater nuclear YAP localization than those on stiffer regions. The engineered epithelium developed physiologically relevant apicobasal organization, with P63-positive progenitor cells enriched basally, PAX6-positive differentiated cells located more apically, increasing CK3 expression, tight-junction organization, and progressive laminin-5 basement membrane deposition. Overall, the study demonstrates that programmable hydrogel stiffness and temperature-driven shape morphing can be combined to generate a scalable biomimetic corneal limbal niche model with controlled mechanical and topographical cues.