The pattern and morphology of cellular infiltration of iris melanocytes implanted into the corneal stroma were studied with a rabbit corneal model. Iris melanocytes are transformed into fibroblast-like cells with a loss of pigment granules, which may reflect the in vivo characteristics of iris melanocytes under pathologic conditions. The metaplastic chararter of iris melanocytes appears to be related to the formation of retrocorneal pigmentation and fibrous membrane.
Animals ; Cell Division ; Cornea/*cytology/pathology/surgery ; Iris/*cytology ; Melanocytes/*cytology/physiology/transplantation ; Metaplasia/pathology ; Rabbits

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

It is important to design a long-period transparent bioactive material for corneal repair in the process of corneal tissue renovation. This article discusses the silk fibroin and formamide blend membranes as a corneal stroma repair material. Silk fibroin solution was mixed with formamide in different proportions to obtain insoluble transparent silk fibroin film by casting method. The blending membranes had excellent mechanical properties, cell compatibility and long-term transparent properties. Rabbit corneal stromal cells were seeded on the sterilized composite films. The rate of cell surface adhesion was over 90% after cells were placed on it for 5 hours. When cells were seeded on blend membranes from one day to seven days, Alma Blue was added to complete medium. Compared with the cell culture plate, there was no significant difference in cell proliferation on formamide/silk films. The results indicated that formamide/silk films might be used as a corneal stroma repair material and worth of further investigatinn
Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; Cell Proliferation ; Cornea ; cytology ; Fibroins ; chemistry ; Rabbits ; Regeneration

A myxoma is a benign tumor found in the heart and in various soft tissues; however, a corneal myxoma is rare. A mucinous mass of unknown etiology was observed on the left cornea of a 32-year-old male patient. We performed deep anterior lamellar keratoplasty using acellular corneal tissue and concurrent amniotic membrane transplantation. Hematoxylin and eosin staining revealed vacuolation of the parenchyma and myxoid change in the corneal tissue that occurred in the anterior half of the corneal parenchyma. We identified a myxoid stroma by Alcian blue staining and observed collagen fibers with denatured stroma by Masson trichrome staining. The patient's visual acuity improved from light perception to 20 / 200, and the intraocular pressure remained within the normal range for one year after surgery. The transplanted cornea survived successfully with well-maintained transparency, and recurrence was not observed one year after surgery.
Adult ; *Cornea ; Corneal Stroma/cytology/*transplantation ; Corneal Transplantation/*methods ; Eye Neoplasms/diagnosis/*surgery ; Humans ; Male ; Myxoma/diagnosis/*surgery

A myxoma is a benign tumor found in the heart and in various soft tissues; however, a corneal myxoma is rare. A mucinous mass of unknown etiology was observed on the left cornea of a 32-year-old male patient. We performed deep anterior lamellar keratoplasty using acellular corneal tissue and concurrent amniotic membrane transplantation. Hematoxylin and eosin staining revealed vacuolation of the parenchyma and myxoid change in the corneal tissue that occurred in the anterior half of the corneal parenchyma. We identified a myxoid stroma by Alcian blue staining and observed collagen fibers with denatured stroma by Masson trichrome staining. The patient's visual acuity improved from light perception to 20 / 200, and the intraocular pressure remained within the normal range for one year after surgery. The transplanted cornea survived successfully with well-maintained transparency, and recurrence was not observed one year after surgery.
Adult ; *Cornea ; Corneal Stroma/cytology/*transplantation ; Corneal Transplantation/*methods ; Eye Neoplasms/diagnosis/*surgery ; Humans ; Male ; Myxoma/diagnosis/*surgery

BACKGROUND: The shortage of donor corneas is a severe global issue, and hence the development of corneal alternatives is imperative and urgent. Although attempts to produce artificial cornea substitutes by tissue engineering have made some positive progress, many problems remain that hamper their clinical application worldwide. For example, the curvature of tissue-engineered cornea substitutes cannot be designed to fit the bulbus oculi of patients. OBJECTIVE: To overcome these limitations, in this paper, we present a novel integrated three-dimensional (3D) bioprinting-based cornea substitute fabrication strategy to realize design, customized fabrication, and evaluation of multi-layer hollow structures with complicated surfaces. METHODS: The key rationale for this method is to combine digital light processing (DLP) and extrusion bioprinting into an integrated 3D cornea bioprinting system. A designable and personalized corneal substitute was designed based on mathematical modelling and a computer tomography scan of a natural cornea. The printed corneal substitute was evaluated based on biomechanical analysis, weight, structural integrity, and fit. RESULTS: The results revealed that the fabrication of high water content and highly transparent curved films with geometric features designed according to the natural human cornea can be achieved using a rapid, simple, and low-cost manufacturing process with a high repetition rate and quality. CONCLUSIONS This study demonstrated the feasibility of customized design, analysis, and fabrication of a corneal substitute. The programmability of this method opens up the possibility of producing substitutes for other cornea-like shell structures with different scale and geometry features, such as the glomerulus, atrium, and oophoron.
Artificial Organs ; Bioprinting ; Cornea/cytology* ; Humans ; Models, Theoretical ; Printing, Three-Dimensional ; Tensile Strength ; Tissue Engineering/methods* ; Tissue Scaffolds

The efficiency and safe range of Lipofectamine2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 microg/ml) or bcl-xl (10 microg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-xl into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-xl could be detectable, and the positive rate reached the peak-on the posttransfection day 3 (48.3%), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.
Cations/administration & dosage ; Cornea/cytology ; Gene Therapy ; Keratinocytes/cytology ; Keratinocytes/*metabolism ; Liposomes ; Transfection ; bcl-X Protein/biosynthesis ; bcl-X Protein/*genetics

INTRODUCTIONTo determine the differentiation of human limbal epithelial cells in tissue culture.

MATERIALS AND METHODSEpithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR).

RESULTSThe colony-forming efficiency of primary cultured (P0) cells was about 19.35 +/- 6.46% (mean +/- SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P <0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 +/- 1.27)% (n = 7, P0) and (3.96 +/- 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse.

CONCLUSIONSHuman limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

Cell Differentiation ; Coculture Techniques ; Connexin 43 ; metabolism ; Cornea ; cytology ; Epithelial Cells ; Humans ; Immunohistochemistry ; Keratins ; metabolism ; Limbus Corneae ; cytology ; Protein Precursors ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; Tissue Culture Techniques

The purpose of this study was to elucidate the origin and cellular composition of retrocorneal membranes (RCMs) associated with chemical burns using immunohistochemical staining for primitive cell markers. Six cases of RCMs were collected during penetrating keratoplasty. We examined RCMs with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) staining and immunohistochemical analysis using monoclonal antibodies against hematopoietic stem cells (CD34, CD133, c-kit), mesenchymal stem cells (beta-1-integrin, TGF-beta, vimentin, hSTRO-1), fibroblasts (FGF-beta, alpha-smooth muscle actin), and corneal endothelial cells (type IV collagen, CD133, VEGF, VEGFR1). Histologic analysis of RCMs revealed an organized assembly of spindle-shaped cells, pigment-laden cells, and thin collagenous matrix structures. RCMs were positive for markers of mesenchymal stem cells including beta-1-integrin, TGF-beta, vimentin, and hSTRO-1. Fibroblast markers were also positive, including FGF-beta and alpha-smooth muscle actin (SMA). In contrast, immunohistochemical staining was negative for hematopoietic stem cell markers including CD34, CD133 and c-kit as well as corneal endothelial cell markers such as type IV collagen, CD133 except VEGF and VEGFR1. Pigment-laden cells did not stain with any antibodies. The results of this study suggest that RCMs consist of a thin collagen matrix and fibroblast-like cells and may be a possible neogenetic structure produced from a lineage of bone marrow-derived mesenchymal stem cells.
Adult ; Aged ; Antigens, CD/metabolism ; Cornea/*cytology/pathology ; Cytokines/metabolism ; Endothelial Cells/cytology/metabolism ; Female ; Fibroblasts/cytology/metabolism ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins/metabolism ; Male ; Mesenchymal Stromal Cells/cytology/metabolism ; Middle Aged ; Stem Cells/cytology/*metabolism

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.
Alkalies/*toxicity ; Amnion/*cytology ; Animals ; Cornea/*injuries ; Corneal Diseases/chemically induced/therapy/*veterinary ; Culture Media, Conditioned/*pharmacology ; Epithelial Cells/*physiology ; Humans ; Male ; *Rabbits