Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells.

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH-G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH-G 20:80 membranes than other proportions. Consequently, CH-G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH-G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH-G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

Entrapment of conjunctival goblet cells by desiccation-induced cornification.

PURPOSE: To evaluate the effects of desiccating stress on conjunctival goblet cell density and morphology and the expression of cornified envelope precursors by the ocular surface epithelia. METHODS: Experimental dry eye (EDE) was created in C57BL/6 mice. Real-time PCR evaluated the expression of cornified envelope (CE) precursor proteins (involucrin and small proline-rich [Sprr] -1a, -1b, -2a, -2b, -2f, and -2g proteins), the cross-linking transglutaminase 1 enzyme (Tg-1) and Muc5AC mRNA transcripts by the ocular surface epithelia. Laser scanning confocal microscopy evaluated the expression of the CE precursor proteins Tg-1 and Muc5AC in cryosections. Tg-1 activity was measured by a fluorescein cadaverine assay. Muc5AC concentration was measured by ELISA. RESULTS: Levels of involucrin; Sprr-1a, -1b, -2a, -2b, -2f, and -2g; and Tg1-1 mRNA transcripts in ocular surface tissues increased in response to desiccating stress. Expression and activity of Tg in the conjunctiva markedly increased after EDE. Desiccating stress caused progressive loss of mucin-filled goblet cells. The apical portion of the remaining conjunctival goblet cells became entrapped by adjacent stratified apical epithelia expressing increased levels of cornified envelope precursors. CONCLUSIONS: Exposure to desiccating stress stimulates ocular surface epithelia to produce cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them. This effect is accompanied by loss of mucin-filled goblet cells and entrapment of mucin contents in the remaining ones by cornifying cells that block the egress of mucin contents to the ocular surface. This mechanism may contribute to the conjunctival mucin deficiency that develops in dry eye.