Reconstruction of Tissue-Engineered Conjunctiva Using Human Amniotic Epithelial Cells and Decellularized Porcine Conjunctiva Matrix With RADA16-I Peptide Hydrogel in a Perfusion Culture System.

Extensive conjunctival defects present significant clinical challenges due to limitations of current treatments including donor scarcity, immune rejection, and suboptimal graft outcomes. In this study, we developed a tissue-engineered conjunctiva by integrating decellularized porcine conjunctival matrix (DPCM), human amniotic epithelial cells (hAECs), RADA16-I peptide hydrogel, and a perfusion culture system. The DPCM was prepared using a phospholipase A2 decellularization method and exhibited effective decellularization (93.7% DNA removal) while preserving native extracellular matrix composition. Tissue-engineered constructs were fabricated by seeding hAECs suspended in 0.5% RADA16-I peptide hydrogel onto DPCM scaffolds and cultured under perfusion conditions. Under perfusion culture, hAECs formed stratified cell layers with high viability (> 90%). Compared to static culture, perfusion significantly enhanced hAEC density, distribution uniformity, and proliferation (Ki67+), while maintaining stemness marker ABCG2 expression. After 3 days, the constructs showed significant upregulation of conjunctival epithelial markers (CK4, CK13, Muc5AC) at both protein and transcriptional levels, indicating successful transdifferentiation of hAECs toward a conjunctival epithelial phenotype. This integrated approach generates a functional tissue-engineered conjunctiva that promotes hAEC proliferation, stemness maintenance, and differentiation into conjunctival epithelial cells, offering a promising alternative for ocular surface reconstruction.