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

The cornea serves as a transparent protective barrier for the eye, with epithelial homeostasis and renewal critically dependent on limbal epithelial stem cells residing in a specialized niche characterized by stromal invaginations that form limbal crypts. Elegant advanced in vitro models of the limbal niche have been described, but most are technologically demanding, requiring multi-step fabrication that limits scalability. We developed a simple and rapid strategy to fabricate 3D scaffolds with undulating topography using a shape-morphing hydrogel concept. A photocrosslinkable bioink composed of methacrylated collagen, hyaluronic acid, and silk fibroin was patterned using grayscale UV projection. Digital Light Processing technology enabled spatially controlled heterogeneous crosslinking densities in a single-step process. The undulating topography was then formed through differential shrinking dynamics of the hydrogel at 37 °C; specifically, the volume of areas exposed to lower UV doses decreased while areas exposed to higher UV doses remained stable. The addition of silk fibroin proved essential for both temperature-dependent shape morphing and sustained corneal epithelial cell adhesion and growth. The undulating scaffolds supported epithelial stratification for at least three weeks in culture. Physiologically relevant corneal mechanotransduction and apicobasal organization was achieved, with nuclear YAP localization in soft areas, P63-positive progenitor cells enriched in basal layers and PAX6-positive differentiated cells in apical layers. Additionally, progressive epithelial maturation was demonstrated by increased CK3 expression, establishment of tight junctions, and the deposition of a basement membrane. STATEMENT OF SIGNIFICANCE: Paschalidis et al. This work offers dual significance. First, we introduce an innovative, simple and single-step bioprinting approach to create 3D scaffolds with complex topography. Using Digital Light Processing technology, a silk fibroin-containing photocurable bioink is exposed to spatially heterogeneous UV doses. This enables differential crosslinking densities in the printed scaffold, which undergoes programmable shape-morphing. Second, we applied this methodology to engineer advanced corneal models that recapitulate the structural features of the epithelial stem cell niche, an undulating topography. This addresses critical needs in human disease modeling and preclinical testing while reducing animal use. The resulting model sustains three-week cultures and faithfully mimics physiological epithelial organization, with progenitor markers expressed in basal layers and differentiation markers indicating barrier function in apical layers.