Naoshi Shinozaki

Surgical Reconstruction of the Ocular Surface in Advanced Ocular Cicatricial Pemphigoid and Stevens-Johnson Syndrome

PURPOSE Ocular cicatricial pemphigoid and Stevens-Johnson syndrome often cause ocular damage and blindness not amenable to surgical correction. We present a new surgical technique for reconstructing affected eyes. METHODS Fourteen eyes of 11 patients with cicatricial keratoconjunctivitis (seven patients with cicatricial pemphigoid and four with Stevens-Johnson syndrome; average age +/- S.D., 55.5 +/- 25.4 years) were treated with a combination of allograft limbal transplantation, amniotic membrane transplantation, and tarsorrhaphy, followed every 15 minutes by artificial tears derived from the patients blood serum. Eight eyes required concomitant penetrating or lamellar keratoplasty because of corneal opacity. RESULTS With a mean follow-up of 143 days (range, 10 to 608 days), we achieved successful ocular surface reconstruction in 12 eyes, with minimal recurrence of symblepharon. Failure occurred in two eyes (one each in 9- and 10-year-old boys) that developed corneal infiltration and vascularization. CONCLUSIONS A combination of allograft limbal transplantation, amniotic membrane transplantation, and tarsorrhaphy, followed by the use of serum-derived tears, can reconstruct the ocular surface in most cases. Although in this study the follow-up period was short and relatively few patients were studied, this approach appears to offer an alternative to keratoprosthesis for treating severe cicatricial keratoconjunctivitis with dry eye.

Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation.

BACKGROUND Conditions that destroy the limbal area of the peripheral cornea, such as the Stevens-Johnson syndrome, ocular pemphigoid, and chemical and thermal injuries, can deplete stem cells of the corneal epithelium. The result is scarring and opacification of the normally clear cornea. Standard corneal transplantation cannot treat this form of functional blindness. METHODS We performed and evaluated 70 transplantations of corneal epithelial stem cells from cadaveric eyes into 43 eyes of 39 patients with severe ocular-surface disorders and limbal dysfunction. Medical treatment had failed in all patients. The patients had a mean preoperative visual acuity of 0.004 (only being able to count the number of fingers presented by the examiner) in the affected eyes, which satisfies the criteria for legal blindness in most countries. In 28 eyes, we also performed standard corneal transplantation. Stem-cell transplantations were performed as many as four times on 1 eye if the initial results were not satisfactory; 19 eyes had multiple transplantations. Patients were followed for at least one year after transplantation. RESULTS A mean of 1163 days after stem-cell transplantation, 22 of the 43 eyes (51 percent) had corneal epithelialization; of the 22 eyes, 7 eyes had corneal stromal edema and 15 eyes had clear corneas. Mean visual acuity improved from 0.004 to 0.02 (vision sufficient to distinguish the largest symbol on the visual-acuity chart from a distance of 1 m) (P<0.001). The 15 eyes in which the cornea remained clear had a final mean visual acuity of 0.11 (the ability to distinguish the largest symbol from a distance of 5 m). Complications of the first transplantation included persistent defects in the corneal epithelium in 26 eyes, ocular hypertension in 16 eyes, and rejection of the corneal graft in 13 of 28 eyes. The epithelial defects eventually healed in all but two of the eyes. CONCLUSIONS Transplantation of corneal epithelial stem cells can restore useful vision in some patients with severe ocular-surface disorders.

Transplantation of amniotic membrane and limbal autograft for patients with recurrent pterygium associated with symblepharon

AIM Treatment of recurrent pterygium associated with symblepharon requires both suppression of fibrosis and reconstruction of limbal barrier. To achieve this, human amniotic membrane was transplanted and limbal autografts performed. METHODS Four patients with severe symblepharon resulting from multiple surgeries for pterygium were treated. Human amniotic membrane was obtained at caesarean section and preserved until surgery. After excision of the fibrous tissues, the amniotic membrane was placed on the sclera, and a limbal autograft transplantation was performed using limbal tissues taken from the affected eye. RESULTS Recurrence of symblepharon was not observed in any of the patients and significant suppression of the subconjunctival fibrosis was achieved. Ocular movement improved in all cases. Complete remission of pterygium regrowth occurred in three cases, and a slight (about 1 mm) recurrence occurred in one case. The limbal donor site showed the presence of mild depressions without the formation of pseudopterygium. CONCLUSION Transplantation of human amniotic membrane with a limbal autograft appears to be a promising surgical treatment for reconstructing the ocular surface in patients with recurrent pterygium associated with symblepharon.

Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes

A biointeractive collagen-phospholipid corneal substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed an overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and regeneration (despite MPCs known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea. In addition, the glucose and albumin permeability were comparable to those of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal source materials.

Artificial human corneas: Scaffolds for transplantation and host regeneration

Purpose To review the development of artificial corneas (pros-theses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

Tissue-Engineered Recombinant Human Collagen-Based Corneal Substitutes for Implantation: Performance of Type I versus Type III Collagen

PURPOSE To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

Measurement of IL-4 in tears of patients with seasonal allergic conjunctivitis and vernal keratoconjunctivitis

To elucidate the mechanism of ocular surface allergic disease, we focused on IL‐4, which is one of the key factors in regulating IgE production, and thus determined the concentration of IL‐4 in tears, IL‐4 concentration was determined in the tears of 15 patients with seasonal allergic conjunctivitis, 15 vernal keratoconjunctivitis (VKC), 10 giant papillary conjunctivitis (GPC), 10 patients with non‐allergic conjunctivitis and post‐cataract surgical conjunctivitis as intermediate conjunctivitis, and 10 normal subjects using a highly sensitive sandwich ELISA. The mean level of IL‐4 in normal controls was low, and seasonal allergic conjunctivitis, VKC and GPC showed a significant elevation (P < 0.05), respectively. IL‐4 of VKC and GPC were also significantly higher than allergic conjunctivitis, and non‐allergic conjunctivitis and post‐cataract surgical conjunctivitis were not higher than normal. These results raise the possibility that the increased level of IL‐4 in tears could play a role in allergic disease and its severity in patients.

Hydroxypropyl Methylcellulose for the Treatment of Severe Dry Eye Associated with Sjögren's Syndrome

Our purpose was to evaluate the efficacy of a new formulation of methylcellulose, preservative-free 0.5% hydroxypropyl methylcellulose (HPMC), for the treatment of dry eye. In the clinical part of our study, two groups of dry-eye patients, those with Sjogrens syndrome (SS) and those without (non-SS), were treated topically with 0.5% HPMC and evaluated for symptoms, ocular surface vital staining, tear breakup time (BUT), and tear evaporation rate from the ocular surface at 40% ambient humidity (TEROS40). In the in vitro part of the study, rose bengal uptake was measured in human conjunctival epithelial cells, which were cultured and incubated with or without 0.5% HPMC. Although symptoms improved in both groups, rose bengal and fluorescein staining and BUT improved significantly only in the SS group. TEROS40 increased for 30 min after instillation of 0.5% HPMC, but not after the use of 0.1% sodium hyaluronate or salinebased artificial tears. Rose bengal uptake by cultured conjunctival epithelial cells was blocked by 0.5% HPMC. These findings suggest that 0.5% HPMC provides long coverage of and protection for the ocular surface. Patients with severe dry eye, such as in SS, are good candidates for this treatment.

Innervated human corneal equivalents as in vitro models for nerve-target cell interactions

A sensory nerve supply is crucial for optimal tissue function. However, the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood. Engineered tissue substitutes can provide controllable environments in which to study tissue innervation. We have therefore engineered human corneal substitutes that promote nerve in‐growth in a pattern similar to in vivo re‐innervation. We demonstrate that these nerves (a) are morphologically equivalent to natural corneal nerves; (b) make appropriate contact with target cells; (c) can generate action potentials; (d) respond to chemical and physical stimuli; and (e) play an important role in the overall functioning of the bioengineered tissue. This model can be used for studying the more general topics of nerve ingrowth or regeneration and the interaction between nerves and their target cells and, more specifically, the role of nerves in corneal function. This model could also be used as an in vitro alternative to animals for safety and efficacy testing of chemicals and drugs.

Collagen-poly(N-isopropylacrylamide)-based membranes for corneal stroma scaffolds.

Purpose To investigate the feasibility of using the biocompatibility of collagen-based blended biomaterials as cell-delivery systems in ocular surface reconstruction in vivo. Methods Collagen-based composites that were blended with synthetic acrylamide-based polymers [poly(N-isopropylacrylamide), pNIPAAm] were transplanted into corneal pockets of white rabbits, with a 3-mm epithelial window. Epithelial cells were allowed to migrate onto the polymer. Transplanted eyes were examined daily for up to 30 days, after which animals were killed for histologic examination. Immunohistochemistry was performed for vimentin, &agr;–smooth muscle actin (&agr;-SMA), CD4, and CD8. Gold-chloride staining was performed to observe neuronal regrowth. Human amniotic membranes (AMs) and sham-operated corneas served as controls. All animals received topical antibiotics (levofloxacin) without the use of steroids or other immunosuppressive agents. Results The pNIPAAm polymer allowed smooth epithelialization of the cornea, which was similar to the epithelialization observed in sham controls and AM-transplanted eyes. Histology revealed that epithelium overlying the polymer was bundled into several layers, without the orientation observed with AM and sham controls. The polymer gradually thinned and was gradually replaced by host tissue. Vimentin- and &agr;-SMA–positive cells were found in stromal pockets up to 1 month following polymer transplantation. These cells were responsible for slight subepithelial haze near the wound edge. CD4- and CD8-positive lymphocytes were also observed in the vicinity of the polymer. Gold-chloride staining showed nerve regrowth in the wound edge after 1 month and subepithelial branches after 3 months. Conclusion Collagen–pNIPAAm blended polymers may be effective as biomaterials to be used in the early stages of lamellar stromal replacement.