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.

Abstract

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.