This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na+ concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K+ and Cl- concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage/*toxicity ; Benzophenones/administration & dosage/toxicity ; Benzopyrans/administration & dosage/toxicity ; Bromobenzenes/administration & dosage/toxicity ; Cell Movement/drug effects ; Cells, Cultured ; Diclofenac/administration & dosage/toxicity ; Epithelial Cells/drug effects/metabolism/ultrastructure ; Epithelium, Corneal/cytology/*drug effects/metabolism ; Fluorometholone/administration & dosage/toxicity ; Humans ; L-Lactate Dehydrogenase/metabolism ; Microscopy, Electron, Transmission ; Ophthalmic Solutions ; Propionates/administration & dosage/toxicity

In order to investigate the feasibility of the modified chitosan-gelatin crosslinked membrane (MC-Gel) and chitosan-gelatin crosslinked membrane (CS-Gel) to be a potential biomaterial for corneal regeneration, we evaluated their physicochemical properties and intraocular biocompatibility in this study. White light transmission and permeability of these membranes were detected. Results showed that white light transmission of both membranes was above 90% at 500 nm, which was similar to that of human cornea. The glucose, tryptophan and NaCl permeability of MC-Gel membrane and CS-Gel membrane was better than or similar to those of human cornea. The methylthiazol tetrazolium (MTT) assay was used to assess cell viability and proliferation. Also, interlamellar corneal transplantation was carried out to evaluate ophthalmic biocompatibility of MC-Gel membrane and CS-Gel membrane. Results indicated that MC-Gel membranes could support the proliferation of HCEC and displayed good intraocular biocompatibility when implanted into rabbits. No severe inflammatory reaction occurred after transplantation and the implanted MC-Gel membrane degraded completely 16 weeks post-operation. Due to its good physicochemical properties and biocompatibility, MC-Gel membrane could be a promising candidate material for corneal regeneration.
Animals ; Biocompatible Materials ; chemistry ; Cells, Cultured ; Chitosan ; chemistry ; Cornea ; cytology ; Corneal Injuries ; Cross-Linking Reagents ; Epithelium, Corneal ; cytology ; physiology ; surgery ; Gelatin ; chemistry ; Guided Tissue Regeneration ; methods ; Humans ; Membranes, Artificial ; Rabbits ; Regeneration ; Tissue Engineering ; methods ; Tissue Scaffolds

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.
Animals ; Cell Culture Techniques ; methods ; Cell Line ; cytology ; Epithelium, Corneal ; cytology ; metabolism ; Fluorescent Antibody Technique, Indirect ; Goats ; Limbus Corneae ; cytology ; Stem Cell Transplantation ; Stem Cells ; cytology

OBJECTIVETo investigate the effect of microRNA-205 reduction by antagomirs on adhesion ability of normal human corneal epithelial keratinocytes (NHCEKs).

METHODSAntagomir-205, complementary and inhibitory to microRNA-205, was used to suppress endogenous microRNA-205 in NHCEKs. The adhesion ability of treated NHCEKs was then assessed by cell adhesion assay. Immunoblot and immunohistochemistry were conducted to determine the level of two focal adhesion-related proteins, focal adhesion kinase (FAK) and paxillin (Pax). Phalloidin staining was performed to measure the level of filamentous actin in antagomir-treated NHCEKs.

RESULTSAntagomir-205 markedly reduced the level of microRNA-205 in NHCEKs and significantly enhanced adhesion ability of NHCEKs (P<0.01). Further protein analysis validated that inhibition of microRNA-205 increased the number of phosphorylated FAK and phosphorylated Pax, and decreased filamentous actin.

CONCLUSIONOur findings suggest that microRNA-205 has down-regulating effect on cell motility in NHCEKs.

Base Sequence ; Cell Adhesion ; genetics ; Cells, Cultured ; Epithelium, Corneal ; cytology ; Humans ; Keratinocytes ; cytology ; MicroRNAs ; antagonists & inhibitors ; Oligonucleotide Probes

Limbal epithelial stem cell is extremely significant in the study of ocular surface reconstruction. But it still a problem to identify, purify and culture them. At present, the identification of limbal epithelial stem cell still depends on its associated markers such as P63, ABCG2, integrin, etc. And, there are many kinds of methods to purify the stem cells, e.g. using flow cytometry, attaching to extra-cellular matrix quickly, measuring cell size, etc. But none of them could isolate stem cells effectively. This review summarizes some hot limbal epithelial stem cell markers, purification methods and stem cell niche created methods in vitro, which may meet future research needs for further improvement of the tissue engineering cornea.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Biomarkers ; Cells, Cultured ; Epithelium, Corneal ; cytology ; metabolism ; Humans ; Integrins ; metabolism ; Limbus Corneae ; cytology ; Membrane Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Stem Cell Niche ; cytology ; Stem Cells ; cytology ; metabolism

We report our experience with corneal epithelium, grown in vivo, transplantation in three patients with persistent epithelial defect (PED). The three patients had ocular surface disease unresponsive to standard treatments and were therefore chosen for transplantation. They underwent transplantation of epithelial sheets, grown in vivo, to the most affected eye. In vivo cultivation was carried out in the cornea of a living related donor. After epithelialization was completed, the epithelium grown on an amniotic membrane was harvested gently; it was then transplanted into the patient's eye after debridement of fibrovascular tissue. The cultivated epithelium was completely epithelialized by 2 weeks; it was well-differentiated with well-formed hemidesmosome. On immunohistochemical staining, p63, connexin 43, and Integrin beta4 were expressed in the cells on the epithelial sheet. The PED was covered completely and maintained for 4 weeks in all cases. However, corneal erosion recurred after 5 weeks in two cases. This novel technique demonstrates the corneal epithelial cells can be expanded in vivo successfully on denuded amniotic membrane of a healthy cornea and harvested safely. A corneal epithelial sheet, grown in vivo, can be transplanted to treat eye with a severe ocular surface disease, such as total limbal deficiency.
Adult ; Cell Culture Techniques ; Cells, Cultured ; Corneal Diseases/etiology/pathology/*surgery ; Corneal Transplantation/*methods ; Epithelial Cells/cytology/*transplantation ; Epithelium, Corneal/cytology/*transplantation ; Eye Burns/complications ; Humans ; Limbus Corneae/*pathology ; Male ; Middle Aged ; Stem Cells/*pathology ; Stevens-Johnson Syndrome/complications

BACKGROUNDCornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens. Participation of toll-like receptor (TLR) 2 and TLR4, which are major forms of fungi receptors, may be involved in Aspergillus fumigatus induced immune responses. The objective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-kappaB activation and production of proinflammatory cytokines, and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs). This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.

METHODSAspergillus fumigatus conidia were used to challenge THCE cells. THCE cells were harvested after 0.5, 1, 2 or 4 hours incubation. Real-time quantitative PCR was performed to determine the expression of TLR2, TLR4, TNF-alpha and IL-8. Western blotting was performed to determine the expression of NF-kappaB. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression of TNF-alpha and IL-8. And the release of TNF-alpha and IL-8 in the cell supernatant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.

RESULTSAspergillus fumigatus conidia elicited the expression of TLR2, TLR4, TNF-alpha and IL-8 mRNA in THCEs. Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-kappaB activation, which increased at 30 minutes (increased from 11.35+/-2.74 in the controls to 19.12+/-3.48, P<0.05) and thereafter increased steadily up to 4 hours after challenge (P<0.01). Concomitant with NF-kappaB activation, secretion of TNF-alpha and IL-8 in conidia-challenged cells was increased in a time-dependent manner. Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in inhibition of conidia-induced TNF-alpha and IL-8 secretion (P<0.05), TLR2 antibody and TLR4 antibody together significantly increased inhibition of the conidia-induced secretion of TNF-alpha and IL-8 from THCE cells (P<0.01).

CONCLUSIONAspergillus fumigatus conidia stimulates THCEs inflammatory response through a pathway dependent on TLR2 and TLR4 signaling.

Aspergillus fumigatus ; immunology ; Cells, Cultured ; Epithelium, Corneal ; cytology ; immunology ; Humans ; Interleukin-8 ; biosynthesis ; NF-kappa B ; metabolism ; Toll-Like Receptor 2 ; physiology ; Toll-Like Receptor 4 ; physiology ; Tumor Necrosis Factor-alpha ; biosynthesis

This study was aimed at investigating the cultivation and biological character of corneal epithelial cells (ECs) planted on intact and denued amniotic membrane (AM) as a substrate and trying to find out satisfactory methods for the reconstruction of corneal epithelium using tissue engineering. Rabbit corneal epithelial cells were planted on denuded AM and intact AM respectively. The cultivated corneal epithelial sheet was examined by use of inverted microscope, HE staining pathologyical section and transmission electron microscopy (TEM), and was also detected immunohistochemically. The results revealed that rabbit corneal ECs grew slowly and were difficult to stick and converge on intact AM, whereas they were easy to grow and proliferate on denuded AM. The cultivated corneal ECs showed four to five layers of stratification composed of the basement membrane of AM and multiple layers of corneal ECs showed the presence of CK3. TEM unveiled that the multiple layers of corneal ECs had numerous desmosomal junctions attaching to the basement membrane with hemidesmosomes. Therefore, the above cultivated corneal epithelial sheet can be used as engineering tissue for ocular surface reconstruction.
Amnion ; cytology ; Animals ; Basement Membrane ; cytology ; metabolism ; Cell Adhesion ; physiology ; Cell Culture Techniques ; methods ; Coculture Techniques ; Epithelial Cells ; cytology ; metabolism ; Epithelium, Corneal ; cytology ; metabolism ; Female ; Male ; Rabbits ; Tissue Engineering ; methods

To study the possibility of using hydroxypropyl chitosan-based blend membranes as carriers of corneal cells in tissue engineering, we prepared three kinds of blend membranes labeled hydroxypropyl chitosan/chondroitin sulfate, hydroxypropyl chitosan/gelatin/chondroitin sulfate and hydroxypropyl chitosan/oxidized hyaluronic acid/chondroitin sulfate. The transparency, water content and ability of protein adsorption of the blend membranes were measured. To evaluate the cytocompatibility of the blend membranes with corneal epithelial cells, rabbit corneal epithelial cells were cultured on the surface of the carrier membranes. The morphological characteristics, cell adhesion, cell proliferation and the activity of lactate dehydrogenase (LDH) in the media were investigated. Three kinds of blend membranes had good optical transmittance, suitable water content and ability of protein adsorption. The results showed that the less injury was made to corneal epithelial cells by the hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane than the others. This kind of membrane was favor of the growth and adhesion of corneal epithelial cells. The hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane is a promising carrier of corneal cells and can be used in reconstruction of tissue engineered cornea.
Animals ; Biocompatible Materials ; chemistry ; pharmacology ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chitosan ; chemistry ; Chondroitin Sulfates ; chemistry ; Epithelium, Corneal ; cytology ; Gelatin ; chemistry ; Membranes, Artificial ; Rabbits ; Tissue Engineering ; methods

OBJECTIVE: To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. METHODS: The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. RESULTS: The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. CONCLUSION The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.
Animals ; Cloning, Molecular ; Cricetinae ; Dependovirus ; genetics ; metabolism ; Epithelium, Corneal ; cytology ; metabolism ; Genetic Vectors ; Herpesvirus 1, Human ; enzymology ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection