In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
Cell Proliferation/*drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Corneal/*cytology ; Epithelium, Corneal/*cytology ; Nerve Growth Factor/*pharmacology

A novel cornea tissue scaffold material was prepared with N-vinly pyrrolidome (NVP) and a biodegradable crosslinking agent by radical polymerization, using azoisobutyronitrile (AIBN) as initiator. Water absorption test and contact angle measure were conducted, and the degradation process of material was investigated. The biocompatibility evaluation was carried out by implantation of material in the rabbits, and by cell culture. The water absorption was over 104%, the contact angle was lower than 41degrees, and the degradation speed in vitro kept steady. The results of implantation in the rabbits showed that the material was almost degraded 3 months later and lots of collagen and cornea stroma cells appeared in it,but there was no inflammation around it. The result of epithelial cells culture showed that the cells conglutinated on the material, but no remarkable cytotoxicity was noted.
Animals ; Biocompatible Materials ; chemistry ; Cornea ; cytology ; Epithelium, Corneal ; cytology ; Materials Testing ; Nitriles ; chemistry ; Prostheses and Implants ; Pyrrolidinones ; chemistry ; Rabbits ; Tissue Engineering

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

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

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

The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.
Stem Cells/cytology ; Stem Cell Transplantation/*methods ; Middle Aged ; Microscopy, Electron ; Male ; Keratin-3/biosynthesis ; Immunohistochemistry ; Humans ; Epithelium, Corneal/cytology/*metabolism/*pathology/*transplantation ; Corneal Diseases/*therapy ; Burns/*surgery/therapy ; Biological Dressings ; Amnion/*ultrastructure ; Adult

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

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

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers' group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.
Variation (Genetics)/*genetics ; RNA, Ribosomal, 18S/genetics ; Phylogeny ; Oceans and Seas ; Humans ; Geologic Sediments/*parasitology ; Epithelium, Corneal/cytology ; Epithelial Cells/parasitology ; DNA, Mitochondrial/genetics ; Animals ; Acanthamoeba/*genetics/*isolation & purification

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.
Trypsin/pharmacology ; Stem Cells/*cytology/drug effects ; Rabbits ; Limbus Corneae/*cytology/drug effects ; Humans ; Epithelium, Corneal/*cytology/drug effects ; Edetic Acid/pharmacology ; Cells, Cultured ; Cell Culture Techniques ; Cell Count ; Animals