Sutureless fixation of amniotic membrane patch as a therapeutic contact lens by using a polymethyl methacrylate ring and fibrin sealant in a rabbit model.

PURPOSE: To introduce and evaluate a sutureless technique by using a polymethyl methacrylate (PMMA) ring and fibrin sealant to fix an amniotic membrane (AM) patch on the ocular surface as a therapeutic contact lens in a rabbit model. METHODS: PMMA rings were fabricated by duplicating an impression of a rabbit conjunctival fornix. The central cornea of the left eye in 16 rabbits was deepithelialized (diameter = 10 mm). A human AM patch was fixed to the ocular surface by using either a PMMA ring and fibrin sealant or interrupted 10-0 nylon sutures. The fibrin sealant was used to create the PMMA ring-AM complex but not to attach the AM/PMMA ring to the ocular surface. The rabbits were followed up with slit-lamp examination and fluorescein staining for 7 days. Reepithelialization and complications were recorded. RESULTS: The corneal epithelial defect was recovered in each rabbit of both groups after 5 days. In the sutureless group, all membranes remained in place and intact during the follow-up period. One eye was noted to have a partial conjunctival epithelial defect caused by exposure to the PMMA ring. In contrast, >50% of rabbits in the interrupted suture group exhibited complications including conjunctival edema, suture loosening, patch detachment, bleeding, and conjunctival epithelial defects. CONCLUSIONS: The sutureless technique that uses a PMMA ring and fibrin sealant for AM patch placement has a lower incidence of complications than the interrupted suture method. This sutureless technique may promote increased clinical use of AM patch by alleviating patients' pain and shortening surgical time.

[Implantation of modified polyhydroxyethyl methacrylate-polymethyl methacrylate keratoprostheses in rabbit and monkey corneas].

OBJECTIVE: To investigate the biocolonization of polyhydroxyethyl methacrylate (PHEMA) sponge with cornea tissue and evaluate the therapeutic effects of modified porous PHEMA-PMMA (polymethyl methacrylate) Keratoprostheses (KPro) on rabbit and monkey corneas. METHODS: The KPro were made using two-stage polymerization combined with mechanical cutting. The experiment was divided into two groups. In the control group (A group), ten normal rabbit eyes received lamellar implantation of PHEMA sponges. The sponges were obtained 2 weeks, 1, 2, 3 and 4 months after operation. The cell proliferation and neovascularization inside the sponges were observed using light and transmission electron microscopy (TEM) and immunohistochemistry. In the experimental group (B group), the porous PHEMA-PMMA KPros were inserted into the lamellar pockets of ten rabbit corneas and two monkey corneas (stage I operation). The healing process was investigated by slit-lamp microscopy. The anterior lamellar cornea tissues were removed 3 months after surgery, exposing the underneath transparent core (stage II operation). The operated eyes were then followed up for 3 - 6 months. RESULTS: No complication was observed in A group. Under the light microscope, fibroblasts started to grow into the cornea 2 weeks after operation; lots of cells, accompanied with new blood vessels, invaded into the cornea 2 - 3 months after surgery. Invading cells of sponge, as well as keratocyte, were positive for vimentin. Under the electron microscope, the invading cells looked healthy and were surrounded by extracellular matrix and collagen. In B group, eight rabbit eyes which have received KPro implantation, anterior lamellar cornea melting happened in two eyes after the stage I operation. The remaining six corneas retained their central cores during observation after the stage II operation. Two monkey operated eyes were found no complication throughout the whole follow-up. CONCLUSIONS: The PHEMA sponge can obtain a tight fusion with the host cornea. The modified PHEMA-PMMA KPros have obtained a relatively stable therapeutic results after implantation into animal corneas.

[The transfection and expression of IL-1ra gene to the rabbit cornea in situ via cation polymer mediation].

OBJECTIVE: To investigate the efficiency and safety of transfection of PEGFP-IL-1ra plasmid via cation polymer mediation (poly-ethylenimine, PEI) by injection into the corneal stroma. METHODS: Human IL-1ra cDNA fragments were cloned by RT-PCR. Plasmid PEGFP-hIL-1ra recombinants were constructed and transferred into corneal endothelial cells (CEC) via cation polymer mediation. Expression of IL-1ra mRNA and IL-1ra was detected by green fluorescent protein (GFP) and Western-blotting. In the experiment group, 20 microl preparation containing 10 microg plasmid PEGFP-hIL-1ra recombinants and PEI-in-vivo was injected into the corneal stroma of Wistar rats (n = 30). Equivalent PEI-in-vivo solution was injected into another 15 corneas as the controls. Corneas were harvested at different time points (day 1, 3, 6, 14 and 21) after injection. The changes of tissue structure and function after IL-1ra in situ transfection were studied by HE staining, transmission electron microscopy, trypan blue-alizarin red staining and immunohistochemistry. The location and intensity of IL-1ra-GFP fusion protein expression were monitored by fluorescence microscopy. RESULTS: The size of the RT-PCR product of hIL-1ra fragments was approximately 500 bp in agarose gel electrophoresis. Restrictive enzyme digestion analysis of PstI, BamHI and DNA sequence analysis showed that expression of plasmid PEGFP-hIL-1ra recombinants had been constructed successfully. Twelve hours after the transfection of PEGFP-hIL-1ra, GFP fluorescence was detected in 10% - 15% endothelial cells. IL-1ra protein (RMW: 44,000) was detected by Western-blotting. In PEGFP-hIL-1ra treated group, fluorescence was appeared at day 1 in cornea basal epithelial cells, peaked at day 6 in whole cornea, began to weaken at day 14, and only weak fluorescence remained in cornea epithelial cells at day 21. No fluorescence appeared in the control group. No significant pathologic changes could be found in HE stained cornea tissues in both transfected group and the controls. p63 immunocytochemical staining in cornea epithelium was positive in both groups. Trypan blue-alizarin red staining confirmed that there was no damage in cornea endothelial cells. IL-1ra-GFP granules could be found by transmission electron microscope in every layer of cornea in the transfected group, but none in the controls. There was no impairment in the ultrastructure of cells in both groups. CONCLUSIONS: By direct injection of PEGFP-hIL-1ra into corneal stroma and mediated by cation polymer, IL-1ra genes could be transferred and expressed in corneal tissue efficiently and safely, and might provide a novel technique of gene transfection to cornea in situ.