Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency.

PURPOSE: This study was performed to evaluate the potential of a collagen-based membrane, collagen vitrigel (CV), for reconstructing corneal epithelium in the stromal wound and limbal stem cell deficiency (LSCD) models. METHODS: Three groups of rabbits were used in the stromal wound model: CV affixed using fibrin glue (CV + FG group, n = 9), fibrin glue only (FG group, n = 3) and an untreated control group (n = 3). In the LSCD model, one group received CV containing human limbal epithelial cells (CV + hLEC group, n = 2) and the other was an untreated control (n = 1). Gross observation, including fluorescent staining, pathological examination, immunohistochemistry and electron microscopy, was used to evaluate the effect of CV on the corneal epithelium. RESULTS: In the stromal wound model, fluorescent staining showed that epithelial reconstruction occurred as rapidly in the CV + FG group as it did in the control group. The pathological examination proved that the CV supported a healthy corneal epithelium in the CV + FG group, whereas FG led to hypertrophy and inappropriate differentiation of corneal epithelium in the FG group. In the LSCD model, the corneas in the CV + hLEC group showed sustained tissue transparency with good epithelialization, low inflammatory response and reduced neovascularization. However, the control cornea was translucent and showed high amounts of inflammation and neovascularization. CONCLUSION: We have demonstrated that CV supports corneal epithelial differentiation and prevents epithelial hypertrophy, in addition to serving as a scaffold for hLEC transplantation, without complications.

Synthesis and characterization of a chondroitin sulfate-polyethylene glycol corneal adhesive.

PURPOSE: To describe the synthesis of a chondroitin sulfate-polyethylene glycol (CS-PEG) adhesive and characterize its physical and biological properties in vitro and in vivo. SETTING: Johns Hopkins University and a research facility, Baltimore, Maryland, USA. METHODS: Metabolic activity (WST-1 reagent) was used to evaluate the cytocompatibility of the adhesive with rabbit primary epithelial, stromal, and endothelial cells. Full-thickness corneal incisions (3.0 mm) in ex vivo porcine eyes were sealed with the adhesive, and burst pressure was evaluated to determine the effectiveness of the material in maintaining intraocular pressure (IOP). Finally, a partial-thickness incision was made in a swine cornea and then sealed using the adhesive. Two weeks postoperatively, both eyes were enucleated and examined grossly and histologically. RESULTS: In vitro results showed cytocompatibility of the tissue adhesive with corneal cells and an ability to seal full-thickness corneal incisions exposed to IOPs of 200 mm Hg and higher. Histological evidence from in vivo data confirmed that the CS-PEG material is biodegradable, induces minimal inflammatory response, resists epithelial cell ingrowth, and does not induce scar formation. CONCLUSIONS: The new adhesive was effective in restoring IOP and withstanding pressures greater than 200 mm Hg after being applied to a full-thickness corneal incision. The adhesive material was biocompatible with the 3 types of cells found in corneal tissue. When the adhesive was implanted in a live swine model, no adverse side effects were observed.

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels.

The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase-polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment.