INTRODUCTIONIn this study, we have developed and optimised a novel gelatin-chitosan (GC) substrate for use as a cellular carrier for tissue-engineered conjunctival epithelium.

MATERIALS AND METHODSThe substrate was fabricated by casting and the mechanical properties of the substrate, including tensile strength and elongation, were measured. Using the MTT, cell proliferation assay with rabbit conjunctival fibroblasts, we optimised the G:C ratio to enhance cytocompatibility. Rabbit conjunctival epithelial cells were immunostained using monoclonal antibodies for keratin 4 and pancytokeratin to investigate the biological effects of the GC substrate on the proliferation and differentiation of epithelial cells.

RESULTSWe found that increasing the amount of gelatin resulted in an increase in elasticity (from 1:9 to 1:1 ratio), reaching a maximum (101.89% +/- 7.13%) at a ratio of 1:1. The MTT assay showed that the proliferation of conjunctival fibroblasts significantly increased from 0.068 +/- 0.017 to 0.177 +/- 0.011 (P = 0.014) as the gelatin was increased from 20% (1:4) to 50% (1:1). Additional studies using tissue-cultured conjunctiva explants showed that these explants grew well on the substrate, forming a multilayered epithelium. Cell morphology on this substrate was similar to that of cells grown on culture dishes alone. Positive staining of keratin 4 and pancytokeratin indicated that the substrate supported normal differentiation of conjunctival epithelial cells.

CONCLUSIONBy enhancing the proportion of gelatin, both the mechanical and biological properties of the chitosan substrate were improved. The results also suggest that this GC biomembrane may be a useful candidate for reconstructive tissue engineering of the conjunctiva.

Animals ; Biocompatible Materials ; Cell Proliferation ; Cells, Cultured ; Chitosan ; Conjunctiva ; cytology ; metabolism ; physiology ; Elasticity ; Epithelium ; physiology ; Gelatin ; Keratins ; metabolism ; Rabbits ; Tensile Strength ; Tissue Engineering

PURPOSE: To investigate the short-term efficacy of topical immunosuppressive agents on the survival of cultivated allo-conjunctival equivalents. METHODS: Twenty-five eyes of New Zealand white rabbits were included. Temporal conjunctivae were trephined to a diameter of 7.5 mm, and then cultured allo-conjunctival epithelial cells on amniotic membrane were transplanted onto them. Various immunosuppressants including steroid, cyclosporine, and rapamycin were applied topically four times a day for a week. Epithelial defects and graft edema were graded daily. Numbers of inflammatory cells were measured in H&E. PKH26 and cytokeratin 4 and 7 were immunostained. RESULTS: Earlier epithelialization was observed in 1% steroid-treated eyes and defects persisted significantly in 0.5% CsA applied eyes. In histology, PKH26 positive cells considered as donor cells were only found in 1% steroid or 0.01% rapamycin applied eyes. 1% steroid- or 0.01% rapamycin-applied eyes both showed positive staining for keratin-4 and -7. Inflammatory cells were less found in 1% steroid or 0.01% rapamycin treated eyes. CONCLUSIONS: Topical steroid or rapamycin can help to suppress acute inflammation and enhance the acute survival of transplanted conjunctival cells.
Administration, Topical ; Animals ; Cell Count ; *Cell Transplantation ; Cells, Cultured ; Conjunctiva/*cytology ; Cyclosporine/pharmacology ; Epithelial Cells/metabolism/*transplantation ; Female ; Fluorescent Antibody Technique, Indirect ; Graft Survival/*drug effects ; Immunosuppressive Agents/*pharmacology ; Keratin-4/metabolism ; Keratin-7/metabolism ; Male ; Organic Chemicals/metabolism ; Prednisone/pharmacology ; Rabbits ; Sirolimus/pharmacology ; Transplantation, Homologous