Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.
Cell Count ; Cell Division/drug effects ; Cells, Cultured ; Corneal Stroma/cytology/*drug effects ; Fibroblasts/cytology/drug effects ; Fluoresceins/*pharmacology/toxicity ; Fluorescent Dyes/*pharmacology/toxicity ; Humans ; Microscopy, Fluorescence

OBJECTIVETo explore the possibility that using the bovine corneal stroma to provide a suitable carrier on which the cells can grow for tissue engineering cornea.

METHODSNine fresh bovine corneas were selected. Each cornea was cut into 2 pieces, and exposed to 0.25% trypsinase for various lengths of time (20 minutes, 40 minutes, and 60 minutes) to get the stroma part with least cells and maintaining the collagen fibers arrangement. Samples obtained from each group were examined with scanning electron microscopy and HE staining. The left ones were freeze-dried and sterilized. The various concentrations of extraction were used to cultivate human fibroblasts, and a 3-(4,5-dimethylthiazol-2-yl)-2, (MTT)-based colorimetric assay was taken to evaluate the exhistance of 5-diphenyltetrazolium bromide cytotoxinic effects. Then the proper corneal stroma was used as a carrier to cultivated the rabbit corneal limbal cells which were planted on it in a concentration of 2 x 10(5)/cm2 in vitro. The cell-carrier samples were sent for scanning electron microscopy and HE staining.

RESULTSThe corneal stroma had the least cells in the group acted with typsin for 60 minutes, while the collagen fibers arrangement was not so orderly as before. The extractions showed no significant difference in cell culture, and no obviously harmful effect on the cell growth. The rabbit corneal limbal cells presented a stratified growth on the bovine corneal stroma.

CONCLUSIONThe bovine corneal stroma without cells prepared using the typsin and lyophilization can be a suitable carrier for cell culture in vitro.

Animals ; Biocompatible Materials ; toxicity ; Cattle ; Cell Separation ; methods ; Cells, Cultured ; Corneal Stroma ; cytology ; Fibroblasts ; cytology ; drug effects ; Humans ; Limbus Corneae ; cytology ; drug effects ; Materials Testing ; Rabbits

BACKGROUNDProstaglandin E2 (PGE(2)) is a key modulator of dendritic cells (DCs) function, and cornea-derived transforming growth factor beta 2 (TGF-β(2)) promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE(2) is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells (CSCs) and whether PGE(2) and TGF-β(2) have additive effects in this immunosuppressive mechanism.

METHODSBone marrow-derived DCs (BM-DCs), splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE(2) in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β(2) antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class II (MHCII), were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake.

RESULTSHigher concentration of PGE(2) was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC II, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC II, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β(2) antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC II and T cell stimulatory capacity.

CONCLUSIONSPGE(2) contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE(2) and TGF-β(2) have additive effects on the immunosuppression of BM-DCs.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Corneal Stroma ; cytology ; metabolism ; Culture Media, Conditioned ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; Dimethyl Sulfoxide ; pharmacology ; Dinoprostone ; antagonists & inhibitors ; metabolism ; Flow Cytometry ; Lipopolysaccharides ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transforming Growth Factor beta2 ; metabolism ; Xanthones ; pharmacology

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Cell Division/drug effects ; Cell Line ; Cells, Cultured ; Corneal Stroma/*cytology/drug effects ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2/*pharmacology ; Humans ; Immunotoxins/pharmacology ; *N-Glycosyl Hydrolases ; Plant Proteins/*pharmacology ; Rabbits ; Ribosome Inactivating Proteins, Type 1