Fabrication and Characterization of Melt-Electrowritten Hierarchical PCL Scaffolds for Conjunctival Tissue Engineering.

Conjunctival tissue loss resulting from trauma, inflammatory diseases, or surgical interventions remains a major clinical challenge and highlights the need for reliable tissue substitutes. Additionally, advanced three-dimensional in vitro systems are required for studying conjunctival biology, evaluating therapeutics, and supporting the principles of the 3Rs. This study aimed to develop a hierarchically structured melt-electro-written (MEW) poly(ε-caprolactone) (PCL) scaffold as a platform for generating bilayered conjunctival constructs. The scaffold comprised a dense apical network to support epithelial attachment and a porous basal region to facilitate fibroblast infiltration and extracellular matrix formation. To enhance cell-material interactions, scaffolds were functionalized with a polydopamine coating. Morphology, surface modification, and mechanical properties were characterized by scanning electron microscopy, Raman spectroscopy, and tensile testing. Human conjunctival fibroblasts and epithelial cells were sequentially seeded to establish a bilayered model, followed by immunocytochemical evaluation. MEW enabled reproducible fabrication of thin scaffolds with defined apical and basal pore architectures. Compared with a conventional nonhierarchical box-pattern scaffold, the hierarchical design increased ultimate tensile stress from 0.32 to 0.83 MPa, reaching the mean value measured for fresh porcine conjunctiva, while producing little change in Young's modulus. PDA functionalization enhanced cellular attachment, spreading, and scaffold colonization. Sequential seeding generated a bilayer-like construct within seven days, characterized by preferential fibroblast integration within the porous basal region and formation of a CK13-positive epithelial layer predominantly localized to the dense apical surface. In conclusion, the developed scaffold supports the generation of bi-layered conjunctival constructs and represents a promising platform for conjunctival tissue engineering and standardized ocular surface models for advanced in vitro research.