Crosslinked modified decellularized rabbit conjunctival stroma for reconstruction of tissue-engineered conjunctiva in vitro

Conjunctival reconstruction is an essential part of ocular surface restoration, especially in severe conjunctival disorders. Decellularized conjunctival tissues have been used in tissue engineering. In this study, we investigated the feasibility of constructing tissue-engineered conjunctiva using stem cell (human amniotic epithelial cells, hAECs), and cross-linked modified decellularized rabbit conjunctival stroma (DRCS-Asp-hEGF), and decellularized rabbit conjunctiva stroma (DRCS). With phospholipase A2 and sodium dodecyl, DRCS were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, as confirmed by DNA quantification, histology, and immunofluorescence. The results of Fourier transform infrared, Alcian blue staining and human epidermal growth factor (hEGF) release assays showed that DRCS-Asp-hEGF was successfully prepared via crosslinking with aspartic acid (Asp) and modified by hEGF at pH 7.7. The hAECs were positive for octamer-binding transcription factor-4 and ABCG2 cell markers. The hAECs were directly placed on the DRCS and DRCS-Asp-hEGF for five days respectively. Tissue-engineered conjunctiva was constructed in vitro for five days, and the fluorescence staining results showed that hAECs grew in monolayers on DRCS-Asp-hEGF and DRCS. Flow cytometry results showed that compared with DRCS, the number of apoptotic cells stained in DRCS-Asp-hEGF was small, 86.70 +/- 0.79% of the cells survived, and 87.59 +/- 1.43% of the cells were in the G1 phase of DNA synthesis. Electron microscopy results showed that desmosome junction structures, which were similar to the native conjunctival tissue, were formed between cells and the matrix in the DRCS-Asp-hEGF.