A Simple Repair Algorithm for Descemet's Membrane Detachment Performed at the Slit Lamp.

Our study aims to investigate a simple repair algorithm for Descemet's membrane detachment (DMD) following phacoemulsification with Pentacam and anterior segment optical coherence tomography (AS-OCT). Twelve patients with DMD were included in this retrospective study. All cases had persistent corneal edema after phacoemulsification and no improved response to conservative treatment. The repair algorithm consisted of delineating the DMD with the Pentacam and AS-OCT, paracentesis, and intracameral air bubble performed at the slit lamp, followed by immediate supine position. At one month, the final status of Descemet's membrane (DM), best-corrected visual acuity, and incidence of complications were noted. DMD was involved in the visual axis in all cases. The mean interval between phacoemulsification and repair was 5.3 ± 1.2 days. Complete reattachment of DM and corneal clarity occurred in all 12 eyes. Eleven (91.7%) eyes underwent one repair procedure, while one eye (8.3%) underwent a repeat procedure. No adverse events were found. Minor post-intervention complications included temporary increased intraocular pressure due to pupillary block in one eye (8.3%). In conclusion, our modified and simplified repair algorithm for DMD can be performed safely as an outpatient procedure, with accurate delineation using a Pentacam and AS-OCT. It may provide new insight into the timely diagnosis, evaluation, and management of DMD.

Galectin-3 Is a Crucial Immunological Disease Marker in Patients with Fungal Keratitis.

Fungal keratitis, one of the most common infectious eye diseases in China, often results in a poor prognosis due to a delayed diagnosis and the insufficiency of effective therapy. There is an urgent need to identify specific biomarkers for the disease. In this study, we screened out tear proteins in patients with fungal keratitis by microsphere-based immunoassay analysis. Levels of cytokine expression were determined in both human corneal epithelial cell models in vitro and the corneas of patients by western blot, quantitative polymerase chain reaction (qPCR), and immunofluorescence analysis. Neutrophil activation was examined by flow cytometry analysis. The relationship between the cytokine expression and neutrophils was evaluated by immunofluorescence costaining and correlation analysis. These results demonstrated that the galectin-3 expression level was increased in both cell model and patient samples at the early and late stages of fungal keratitis. The neutrophils were significantly activated during the disease course of fungal keratitis. Meanwhile, colocalization and a positive correlation between galectin-3 and neutrophils were observed, suggesting that galectin-3 may play a crucial role in the recruitment of neutrophils and immune regulation of fungal keratitis. In conclusion, galectin-3 could be a key disease marker implying a beneficial immune response in the pathogenesis of fungal keratitis, which might be a target of therapeutic strategy in the future.

A novel clinical dynamic stereopsis assessment based on autostereoscopic display system.

Background: Although it is recognized that dynamic stereopsis is vital in daily life, there is still room for improvement in assessment methods. A novel clinical dynamic stereopsis assessment method was created based on an autostereoscopic display system that did not require additional auxiliary glasses. This study evaluated the optical parameters and clinical performance of the autostereoscopic display system for clinical dynamic stereopsis assessment. Methods: The autostereoscopic dynamic stereopsis assessment device was based on a directional backlight technology. Experiment 1 was performed under the same environmental conditions to compare luminance, crosstalk, and spectrum between the autostereoscopic dynamic stereopsis assessment device and the conventional dynamic random-dot stereopsis measuring instrument. Experiment 2 was an observational, analytic, cross-sectional study involving 135 healthy participants, each of whom was asked to complete measurements on both the autostereoscopic and conventional devices in random order. Stereo acuity, operating time, acceptance, and visual fatigue scores were recorded for clinical evaluation. Results: The autostereoscopic device had brighter luminance (139 and 140 cd/m2 for 2 eyes, respectively), lower crosstalk (4.50% for both eyes), and higher color restoration degree than those of the conventional instrument. Clinically, the novel dynamic stereopsis assessment was as accurate as the traditional method [170" (0.00") and 170" (0.00") respectively; P=0.317], and with more efficiency (166±58.9 and 298±116 s, respectively; P<0.001), higher acceptance (3.36±0.93 and 2.02±0.59 points, respectively; P<0.001), lesser fatigue (0.27±0.46 and 0.73±0.66 points, respectively; P<0.001). The autostereoscopic dynamic stereopsis assessment device with brighter luminance, lower crosstalk, and higher color restoration degree was more effective than the traditional instrument at displaying dynamic clues for clinical dynamic stereopsis assessment. Furthermore, its high-quality image and user-friendly interface provided accurate assessment results in all 3 dynamic stereopsis assessment task conditions, with a higher level of acceptance and lesser visual fatigue, than the traditional assessment method. Conclusions: The autostereoscopic device has excellent functions in both optical parameters and clinical performance, and therefore has the potential to be applied and popularized in future assessments.

Inhibition of Galectin-3 Impairs Antifungal Immune Response in Fungal Keratitis.

Galectin-3 is one of the galectin family members which are master regulators of immune homeostasis, especially in infectious diseases. However, its mechanism of immune regulation in fungal keratitis has not been thoroughly studied. Our study is aimed at clarifying the role of galectin-3 in the fungal keratitis mouse model in vivo, thereby providing a new biomarker of antifungal therapy. In our study, aspergillus, the most common pathogenic fungi of fungal keratitis, was identified and isolated by corneal tissue fungus culture. Then, the RNA expression levels of galectin family members in corneas of the mouse model with aspergillus fumigatus keratitis were screened by transcriptome sequencing (RNA-seq). The expression of the galectin-3 was detected by quantitative real-time Polymerase Chain Reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence in the corneal tissue of the fungal keratitis mouse model. Recruitment of neutrophils and the co-immunofluorescence of galectin-3 and related markers in corneal tissue were determined by flow cytometry analysis and immunofluorescence staining. The regulatory role of galectin-3 for proinflammatory cytokines and neutrophils was validated by the knockout mouse model. Galectin-3 knockout deteriorated the condition for the inhibition of galectin-3 was benefecial for fungi to survive and thrive in corneal lesions. These results demonstrated that in the ocular fungal infection, galectin-3 is capable of regulating the pathogenesis of fungal keratitis by modulating neutrophil recruitment. The deterioration of fungal keratitis and increased fungal load in corneal lesions of galectin-3 knockout mice proved the regulatory role of galectin-3 in fungal keratitis. In conclusion, galectin-3 is going to be an essential target to modulate neutrophil recruitment and its related antifungal immune response in fungal keratitis.

Effects of Customized Progressive Addition Lenses vs. Single Vision Lenses on Myopia Progression in Children with Esophoria: A Randomized Clinical Trial.

Purpose: To evaluate the effect of customized progressive addition lenses (CPALs) versus single vision lenses (SVLs) on the progression of juvenile-onset myopia in children with near esophoria. Methods: Ninety-three Chinese children, aged 7-14 years with spherical equivalent refraction (SER) ranging from -0.50 to -4.00 D and near esophoria ≥2Δ, were randomly assigned into a CPALs (n = 46) and an SVLs group (n = 47) for a 2-year, double-masked, randomized trial. The primary outcome measure was the progression of myopia, as determined by cycloplegic autorefraction. A customized near addition, calculated by a regression equation, was prescribed to establish a fixed heterophoria status for each child, which was -3Δ exophoria. Results: Eighty-four (90.3%) of the 93 children completed the 2-year follow-up. The mean initial near addition lenses were 1.65 ± 0.07 D (mean ± SE). The adjusted 2-year myopia progression was 0.23 ± 0.08 D slower in the CPALs group than in the SVLs group (p=0.046). Post hoc analysis found significantly larger treatment effects for CPALs in children without myopic parents (0.47 ± 0.15 D; 95% CI: 0.18-0.76), with lower baseline myopia (0.33 ± 0.09 D; 95% CI: 0.14-0.52; p < 0.05), with higher baseline accommodative lag (0.36 ± 0.11 D; 95% CI: 0.12-0.60; p < 0.05), and with higher baseline near esophoria (0.30 ± 0.10 D; 95% CI: 0.12-0.48; p < 0.05). Conclusion: CPALs exerted a significant but minimal protective effect against myopia progression in Chinese children with esophoric myopia, as compared with SVLs. Regulating near heterophoria and accommodative lag by near addition lenses may not be an appropriate way to prevent myopia progression.

Changes in Anterior and Posterior Corneal Elevation in Patients With Allergic Conjunctivitis.

Purpose: To evaluate corneal elevation changes in patients with allergic conjunctivitis (AC) and to analyze their correlations with ocular allergy signs and corneal biomechanical alterations. Methods: Thirty patients (30 eyes) with AC and twenty normal subjects (20 eyes) were included in this prospective study. All participants underwent a complete ocular examination, including corneal tomography by Pentacam and corneal biomechanics evaluation by Corvis ST. AC patients were evaluated for their eye rubbing frequency and ocular allergic signs. Results: The elevation at the thinnest location (TE) and the central location (CE), the elevation difference at the thinnest location (TED) and the central location (CED), and the mean value of elevation difference in the central 4 mm zoom (MED) of both the anterior and posterior corneal surface were significantly higher in the AC group than in the normal group (p < 0.05 for all). In AC patients, only anterior corneal elevation parameters were positively correlated with eye rubbing frequency and ocular allergy sign severity (p < 0.05 for all), while the tomography and biomechanical index (TBI) was positively correlated with the elevation parameters of both the anterior and posterior corneal surfaces (p < 0.05 for all). Conclusion: AC patients carry an increased risk of corneal ectasia. Posterior corneal elevation parameters are sensitive and reliable predictors of keratoconus (KC) risk in AC patients. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04299399, identifier [NCT04299399].

Effect of Dual Endothelin Receptor Antagonist on a Retinal Degeneration Animal Model by Regulating Choroidal Microvascular Morphology.

OBJECTIVE: Clinical studies have found that increasing levels of plasma endothelin-1 (ET-1) might inhibit choroidal blood flow (BF) and promote choroidal vasoconstriction. This study was designed to investigate ET-1 levels and its effect on choroidal microvascular morphology in a retinitis pigmentosa (RP) animal model. METHODS: Mice with retinal degeneration (rd10) were intragastrically administered bosentan, a dual endothelin receptor antagonist. We detected plasma ET-1 levels using an enzyme-linked immunosorbent assay (ELISA) kit at P14, P21, and P28 and evaluated ET-1 expression in RPE/choroid/sclera complexes using western blot and whole mount immunofluorescence staining at P28. Retinal thickness was measured using hematoxylin and eosin (H&E) staining at P28. At the same time, we also estimated choroidal microvascular densities using vascular luminal casting with a scanning electron microscope (SEM). RESULTS: Plasma ET-1 levels were increased significantly in rd10 mice at P21 (65.48 ± 24.83 pg/ml) and P28 (85.89 ± 20.23 pg/ml) compared with C57BL/6J mice at P21 (33.52 ± 16.33 pg/ml) and P28 (42.38 ± 17.53 pg/ml); the expression of ET-1 was also upregulated in RPE/choroid/sclera complexes at P28. Bosentan inhibited ET-1 expression in plasma (P < 0.05) and RPE/choroid/sclera complexes at P28 in rd10 mice. Choroidal microvascular densities were decreased in rd10 mice, and bosentan could weaken these changes. CONCLUSION: Plasma and local ET-1 was elevated in an animal model of RP, suggesting that it likely participates in the pathological progression of retinal degeneration and may thus provide a new intervention target. ET-1 blockade might exert its protective effect by elevating choroidal microvascular density via inhibition of ET-1.

Corneal Opacity Leading to Multiple Myeloma Diagnosis: A Case Report and Literature Review.

BACKGROUND A multiple myeloma patient presenting with corneal opacity and blurred vision as chief complaint is rare. CASE REPORT A 54-year-old woman with a 9-month history of blurred vision without other systemic symptoms consulted an ophthalmologist. The patient had bilateral diffuse corneal opacity at the corneal epithelium and anterior stroma under slit-lamp examination. Decreased corneal endothelial cells density was found by microscopy. During consultation, the patient was noted to have an anemic face. Laboratory analysis and bone marrow were investigated. Serum protein electrophoresis revealed a raised serum kappa paraprotein band (12.4 g/L). The erythrocyte sedimentation rate (ESR) was accelerated to 49 mm/h (normal <20mm/h). There was mild kidney impairment. The blood urea increased to 8.1 mmol/L (normal <7.1 mmol/L) and creatinine increased to 158 µmol/L (normal <133 µmol/L). Then, a bone marrow biopsy was performed, showing 26% pleomorphic plasma cells (normal <15%). The patient was eventually diagnosed as having MM and was treated with systemic chemotherapy. CONCLUSIONS Blurred vision due to corneal opacity can be an initial presentation of MM, of which ophthalmologists should be aware.

Valsalva Retinopathy in Twin-Pregnancy: A Case Report and Literature Review.

BACKGROUND Valsalva retinopathy is a rare but sight-threatening condition with sudden visual loss. Twin-pregnancy is known as a confirmed risk factor for Valsalva retinopathy. CASE REPORT A 21-year-old twin-pregnant Chinese woman with 31 weeks of gestation complained of a sudden decrease of visual acuity in her right eye for five hours. Based on the patient's medical history, clinical findings, laboratory testing and optical coherence tomography (OCT) imaging studies, Valsalva retinopathy was confirmed. Initially, we managed the patient with Nd: YAG (neodymium-doped yttrium aluminum garnet) laser to puncture the posterior portion of the vitreous. As the laser failed to puncture the posterior hyaloid face due to dense premacular hemorrhages, we switched to conservative treatment. We reviewed the case with the patient 12 weeks after an uneventful vaginal delivery. Her right visual acuity had spontaneously increased to 6/6 with no active medical intervention. CONCLUSIONS Valsalva retinopathy can be caused by twin-pregnancy. Patients with Valsalva retinopathy can be managed conservatively with careful prognosis following failed laser treatment. Ophthalmologists and obstetrician should coordinate properly and pay more attention to Valsalva retinopathic patients with twin-pregnancy.

Reconstruction of auto-tissue-engineered lamellar cornea by dynamic culture for transplantation: a rabbit model.

To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin ß4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3-, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63-, ABCG2-). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function.

Determination of anterior segment changes with Pentacam after phacoemulsification in eyes with primary angle-closure glaucoma.

BACKGROUND: To investigate changes of anterior segment morphology in primary angle-closure glaucoma after phacoemulsification using the Pentacam system. DESIGN: Prospective, interventional study, Zhongshan Hospital of Sun Yat-sen University. PARTICIPANTS: Eighty-five eyes from 60 patients with primary angle-closure glaucoma undergoing phacoemulsification. METHODS: Intraocular pressure was measured by Goldmann applanation tonometry. Anterior segment morphology was assessed using the Pentacam camera. MAIN OUTCOME MEASURES: The intraocular pressure, central anterior chamber depth, peripheral anterior chamber depth, anterior chamber volume, pupil diameter and anterior chamber angle preoperative and 3 months postoperative. RESULTS: A total 78 eyes of 55 patients with primary angle-closure glaucoma were included in the analysis. Thirty-two eyes (41.0%) had acute primary angle-closure glaucoma, and 46 eyes (59.0%) had chronic primary angle-closure glaucoma. In both groups, statistically significant decreases in intraocular pressure and increases in anterior chamber volume, central anterior chamber depth, peripheral anterior chamber depth and anterior chamber angle inferiorly, nasally, temporally and superiorly were observed at 3 months after phacoemulsification (all P < 0.001). CONCLUSIONS: Phacoemulsification induces significant changes in anterior segment morphology in primary angle-closure glaucoma accompanied by a significant fall in intraocular pressure in the short term.

Long-term cultivation of human corneal endothelial cells by telomerase expression.

The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo.

Research on the stability of a rabbit dry eye model induced by topical application of the preservative benzalkonium chloride.

BACKGROUND: Dry eye is a common disease worldwide, and animal models are critical for the study of it. At present, there is no research about the stability of the extant animal models, which may have negative implications for previous dry eye studies. In this study, we observed the stability of a rabbit dry eye model induced by the topical benzalkonium chloride (BAC) and determined the valid time of this model. METHODS AND FINDINGS: Eighty white rabbits were randomly divided into four groups. One eye from each rabbit was randomly chosen to receive topical 0.1% BAC twice daily for 2 weeks (Group BAC-W2), 3 weeks (Group BAC-W3), 4 weeks (Group BAC-W4), or 5 weeks (Group BAC-W5). Fluorescein staining, Schirmer's tests, and conjunctival impression cytology were performed before BAC treatment (normal) and on days 0, 7, 14 and 21 after BAC removal. The eyeballs were collected at these time points for immunofluorescence staining, hematoxylin and eosin (HE) staining, and electron microscopy. After removing BAC, the signs of dry eye in Group BAC-W2 lasted one week. Compared with normal, there were still significant differences in the results of Schirmer's tests and fluorescein staining in Groups BAC-W3 and BAC-W4 on day 7 (P<0.05) and in Group BAC-W5 on day 14 (P<0.05). Decreases in goblet cell density remained stable in the three experimental groups at all time points (P<0.001). Decreased levels of mucin-5 subtype AC (MUC5AC), along with histopathological and ultrastructural disorders of the cornea and conjunctiva could be observed in Group BAC-W4 and particularly in Group BAC-W5 until day 21. CONCLUSIONS: A stable rabbit dry eye model was induced by topical 0.1% BAC for 5 weeks, and after BAC removal, the signs of dry eye were sustained for 2 weeks (for the mixed type of dry eye) or for at least 3 weeks (for mucin-deficient dry eye).

A modified symblepharon ring for sutureless amniotic membrane patch to treat acute ocular surface burns.

The objective of this study is to evaluate a sutureless technique by using a modified symblepharon ring to fix an amniotic membrane (AM) patch on the ocular surface to treat acute ocular burns. Seventy-five patients with acute ocular burns of total 75 eyes graded III to VI were enrolled in this study. They were randomly divided into two groups. Thirty-nine eyes received the sutureless AM patch with a modified symblepharon ring, and the other 36 eyes underwent the conventional sutured AM patch as control. The time and the rate of epithelialization, corneal neovascularization, and complications were recorded. Both the operation time and the time to epithelial closure in the sutureless group were much shorter than that in the suture group (P < .01). The rate of reepithelialization in the sutureless group was higher than in the suture group (P < .05). The rate of the vascularization and symblepharon were lower in the sutureless group than in the suture group (P < .05). The conjunctival sac contraction occurred only in the eyes with grade V and VI in the sutureless group and was later than in the suture group (P < .05). This modified method is simple, minimally invasive, free from trauma, and more effective compared with controls.

A novel procedure for treating canalicular obstruction by re-canaliculisation and bicanalicular intubation.

AIM: The aim of this study was to evaluate a new procedure for treating canalicular obstruction by re-canaliculisation and bicanalicular intubation (RC-BCI). METHODS: Thirty adult patients (32 eyes) with canalicular obstruction were treated with RC-BCI from September 2005 to December 2007 at Zhongshan Ophthalmic Centre (Guangzhou, China). Silicone tubes were left in place for 2-3 months and were removed when patients had relief by tearing. Patients were evaluated postoperatively by symptoms, lacrimal irrigation and satisfaction rate. RESULTS: Mean follow-up time after tube removal was 21.5 (range 6-26) months. Twenty-six eyes (81.25%) had complete epiphora relief, two eyes (6.25%) had partial relief and four eyes (12.5%) had no improvement after the removal of the tubes. One eye (3.13%) had lower punctum splitting 2 months after the surgery. The overall satisfaction rate was 93.3% in 30 patients. No other complications occurred. CONCLUSION: Our findings demonstrated that the RC-BCI was an effective procedure for treating canalicular obstruction with few complications.

Tectonic lamellar keratoplasty with acellular corneal stroma in high-risk corneal transplantation.

PURPOSE: Tectonic lamellar keratoplasty (TLKP) is a primary surgical procedure to improve the condition of the recipient bed in high-risk corneal transplantation. It is usually performed for a secondary optical penetrating keratoplasty (PKP). The present study was undertaken to explore a new strategy for TLKP using acellular corneal stroma (ACS) prepared by decellularization. METHODS: ACS for TLKP was prepared from cat cornea by decellularization. The efficiency of the decellularization was examined by hematoxylin and eosin (H&E) staining and through DNA content analysis. Twenty-eight New Zealand white rabbits, as recipients, were assigned to one of two groups that had different material for their TLKP. The TLKP was combined with a central optical PKP as a single-stage procedure. Either ACS or fresh cat corneal lamella, 11.25 mm in diameter, was used for the TLKP in these two groups. After TLKP, a 6.5-mm full-thickness cat cornea was placed in the central cornea of each recipient rabbit for PKP. Clinical outcomes and the histology of the transplants were compared post-operatively. RESULTS: ACS for TLKP prolonged the survival of the transplants. The mean survival time of the transplants in the ACS group (36.4±4.3 days) was longer than for those in the control group (14.0±2.2 days, p<0.05). The ACS group showed a significantly smaller neovascularization area compared to the control group. The areas of corneal neovascularization were 5.3±1.1 mm² and 45.2±4.9 mm² (p<0.05), respectively, after two weeks, and 25.1±4.7 mm² and 105.3±12.4 mm² (p<0.05), respectively, after four weeks. Histology revealed that fewer inflammatory cells were infiltrating the transplants in the ACS group than those in the control group. CONCLUSIONS: The use of ACS for TLKP prolonged the survival of corneal transplants, reduced corneal neovascularization, and prevented from infiltration of inflammatory cells. It is a feasible and effective strategy to prolong the survival of transplants in high-risk corneal transplantation.

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction.

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.

Development and characterization of acellular porcine corneal matrix using sodium dodecylsulfate.

PURPOSE: The objective of this study was to produce a porcine corneal acellular matrix (ACM) and assess its possibility for biomedical applications. METHODS: Porcine corneas were treated with various concentrations of sodium dodecylsulfate for different lengths of time. Optimal conditions for processing the ACM were noted regarding removal of all cellular components and retention of the spatial arrangement of the corneal stroma. The physical characteristics (including water absorption and light transmittance), biomechanics, and cytotoxicity of the ACM were also found to be conserved. Subsequently, ACM was transplanted into the interlaminar stroma of rabbit corneas. The transparency and structures of the collagen fibers were determined. RESULTS: By immersing corneal tissues in isotonic buffer containing 0.1% sodium dodecylsulfate for 7 hours, we were able to produce an ACM whose cells were completely removed, without disrupting collagen layer structure. Although water absorption and light transmittance of the ACM decreased when compared with natural corneal stroma, ACM showed similar biomechanical properties and biocompatibility as natural ones. After xenotransplantation into rabbit corneal stromal layers for 4 weeks, both ACM and rabbit corneas showed complete transparency. Almost 1 year postoperatively, the corneas remained transparent with regular stromal structures and ACM appeared stable in situ without deliquescence or immunological rejection. CONCLUSIONS: A simple and valid method to produce decellularized corneal matrix has been successfully developed. These acellular matrices similar to natural corneas in structure, strength, and transparency have tremendous potential for corneal transplantation as ideal implants for donors and for tissue engineering applications as suitable scaffolds.

The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold.

This study was to develop a method using phospholipase A(2) (PLA(2)) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200 U/ml PLA(2) and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA(2) and SD were 0.35+/-0.04 U/mg dry weight and 4.3+/-0.8 ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8+/-2 days, and their transparency was restored in 84+/-11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.