Cloning and Characterization of a Novel Endo-Type Metal-Independent Alginate Lyase from the Marine Bacteria Vibrio sp. Ni1.

The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K+ slightly enhances the activity, while Fe2+ and Cu2+ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.

Survey report on keratoplasty in China: A 5-year review from 2014 to 2018.

To provide the general information on corneal transplantation (CT) in China, China Cornea Society designed a questionnaire on CT from 2014 to 2018 and entrusted it to 31 committee members for implementation of the survey nationwide. This article presents the results of the survey and compares the indicators used in the survey and those in the annual statistical report released by the Eye Bank Association of America (EBAA). The number of corneal transplantations completed by the 64 hospitals from 2014 to 2018 was respectively 5377, 6394, 7595, 8270 and 8980, totally 36,616 (22,959 male and 13,657 female). The five largest hospitals by the number of corneal transplantations completed 15,994 surgeries in total, accounting for 43.68% of all the surgeries performed in the 64 hospitals. The most common indication for corneal transplantations was corneal leukoma (7683, 20.98%), followed by bacterial keratitis (4209, 11.49%), corneal dystrophies (4189, 11.44%), keratoconus (3578, 9.77%) and corneal perforation (2839, 7.75%). The main surgical techniques were penetrating keratoplasty (PK) (19,896, 54.34%), anterior lamellar keratoplasty (ALK) (13,869, 37.88%). The proportion of PK decreased from 57.97% in 2014 to 52.88% in 2018 while the proportion of ALK increased from 36.04% in 2014 to 37.92% in 2018. The geographical distribution of keratoplasties performed in China is unbalanced. PK and ALK were the main techniques of CT and corneal leukoma, bacterial keratitis and corneal dystrophies were the main indications for CT in China.

Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

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.

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.

Rapidly constructed scaffold-free cornea epithelial sheets for ocular surface reconstruction.

PURPOSE: To develop a centrifugal cell seeding method for rapid and efficient reconstruction of ocular surface with limbal stem cell deficiency (LSCD) in rabbits. METHODS: The orthogonal design method was used to optimize centrifugation parameters for cell seeding. Methylthiazol tetrazolium proliferation assay, colony-forming efficiency, and flow cytometry were used to study cell viability. Histology, electron microscopy, and immunocytochemistry were evaluated for centrifugation-constructed cornea epithelial sheets (CCCESs). The rabbit eyes with LSCD were treated with or without CCCES for in vivo evaluation. RESULTS: The 80.04% attached cells with 98.04% viability were achieved using optimal cell seeding density at 9 × 10(5) cm(-2) with centrifugation at 1800 rpm for 4 min. The 0.4% glycerin was added in the medium to increase the surface tension and osmotic pressure to optimal condition for obtaining higher cell density. The three-layer epithelial sheets were rapid constructed, which displayed the characteristics of normal corneal epithelium. In vivo transplantation, labeled cells of CCCES were detected at 30 days. CCCES reconstructed the LSCD corneal epithelia without conjunctivalization and neovascularation, evidenced by positive K3 and negative K4, Muc5AC. CONCLUSION: The scaffold-free corneal epithelial sheets were rapidly constructed using optimal centrifugation procedure, which was demonstrated to reconstruct ocular surface with LSCD.

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.