Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure.Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood.Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 μJ from 0.80 μJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy.Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Utilization rate and usage patterns of phakic and pseudophakic donor corneas recovered by the Singapore Eye Bank

Purpose To compare the utilization rate and usage patterns of pseudophakic and phakic donor corneas recovered by the Singapore Eye Bank. Methods Records of local donor corneas recovered by the Singapore Eye Bank from 2012 to 2017 were examined. Corneas that were deemed suitable for clinical use were stratified into phakic and pseudophakic groups. We examined the basic demographic pattern of both groups and the initial type of surgery/ies that the corneas were suitable for based on tissue parameters such as time from harvesting, stromal clarity, the clear central corneal area, the presence of Descemet’s membrane tears or defects, and endothelial cell density and quality. We also identified the types of corneal grafts that the corneas were eventually used for; Penetrating Keratoplasty (PK), Anterior Lamellar Keratoplasty (ALK), Endothelial Keratoplasty (EK). Finally, the overall utilization rates for each group were determined. Results A total of 986 corneas deemed suitable for transplant were analyzed, 908 (92%) were phakic and 78 were pseudophakic (8%). The average age of pseudophakic donor corneas was (65 ± 8 yrs. old) and there was a slight male preponderance for both groups (55%). Age adjusted analysis of pseudophakic corneas showed the endothelial cell density (ECD) (mean: 2327 ± 47.1 cells/mm2) and clear area (mean: 7.0 ± 0.7 mm) were lesser than phakic corneas. The percentage of pseudophakic corneas that were of EK standard (ECD >2500 cells/mm2) were lower compared to phakic corneas (37% and 77% respectively, p < 0.001). There was significant correlation between previous cataract surgery and the endothelial cell count of the donor corneas (p < 0.001), and regression analysis also showed a strong association of ECD with cataract surgery in reference to non-cataract surgery (-478.8 (95% CI-576.9 to -380.7). The overall utilization rate for pseudophakic corneas was 58% compared to that of phakic corneas at 83%. The most common reason for pseudophakic corneas not to be utilized was due to the presence of Descemet’s membrane (DM) tears or defects under the main or side port incision created during phacoemulsification (30%). Phakic corneas were used primarily for optical grafts 84% (mainly EK) while pseudophakic corneas were used mostly for therapeutic/tectonic grafts 47% (mainly ALK or patch grafts). Conclusion Compared to phakic donor corneas, pseudophakic corneas generally have lower overall tissue quality leading to lower uptake by surgeons and lower utilization rates. Eye banks must continuously refine their donor acceptance criteria and engage surgeons to optimize utilization of each recovered tissue.

Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation

ObjectiveTo study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with postkeratoplasty infections.Methods and AnalysisThe antimicrobial activity of Kerasave was determined after 0, 3 and 14 days of incubation at 2°C–8°C, inoculating Kerasave and the control medium with 105–106 colony forming units (CFU) of Candida albicans (CA), Fusarium solani (FS), Aspergillus brasiliensis (AB), Staphylococcus aureus (SA), Enterococcus faecalis (EF), Bacillus subtilis spizizenii (BS), Pseudomonas aeruginosa (PA), Enterobacter cloacae (EC) and Klebsiella pneumoniae (KP). Log10 reductions at different time intervals were determined by assessing the number of viable CFU using the serial dilution plating technique.ResultsAfter 3 days, Kerasave induced the highest log10 decrease in the concentrations of KP, PA, CA and EC (5.37, 4.15, 2.97 and 2.67, respectively; all p<0.001). The log10 decreases of SA and EF were 2.27 and 2.11, respectively (all p<0.001). The lowest log10 decrease was observed in BS, AB and FS concentrations (0.25, 0.30 and 0.67, respectively; p<0.001 for BS and AB and p=0.004 for FS). After 14 days, the microbial count of CA, FS, SA, EF, PA and EC further decreased (p=0.006 for FS; p<0.001 for the others).ConclusionKerasave effectively reduced or kept unchanged the microbial concentration of almost all tested strains after 3 days. Thus, this novel medium represents a valuable tool to control the microbial contamination of human donor corneas during hypothermic storage for up to 14 days before transplantation.

Reliability and efficiency of corneal thickness measurements using sterile donor tomography in the eye bank

To evaluate the reliability and efficiency of sterile pachymetric measurements of donor corneas based on tomographic data using two different methods: a “manual” and a “(semi-)automated” method. Twenty-five (25) donor corneas (50%) stored in MI and 25 (50%) in MII were imaged 5 times consecutively using an anterior segment OCT (AS-OCT). The central corneal thickness (CCT) was measured both with the manual measurement tool of the AS-OCT (= CCTm) and with a MATLAB self-programmed software allowing (semi-)automated analysis (= CCTa). We analyzed the reliability of CCTm and CCTa using Cronbach´s alpha (α) and Wilcoxon signed-Rank Test. Concerning CCTm, 68 measurements (54.4%) in MI and 46 (36.8%) in MII presented distortions in the imaged 3D-volumes and were discarded. Concerning CCTa, 5 (4%) in MI and 1 (0.8%) in MII were not analyzable. The mean (± SD) CCTm was 1129 ± 6.8 in MI and 820 ± 5.1 µm in MII. The mean CCTa was 1149 ± 2.7 and 811 ± 2.4 µm, respectively. Both methods showed a high reliability with a Cronbach´s α for CCTm of 1.0 (MI/MII) and for CCTa of 0.99 (MI) and 1.0 (MII). Nevertheless, the mean SD of the 5 measurements was significantly higher for CCTm compared to CCTa in MI (p = 0.03), but not in MII (p = 0.92). Sterile donor tomography proves to be highly reliable for assessment of CCT with both methods. However, due to frequent distortions regarding the manual method, the (semi-)automated method is more efficient and should be preferred.

Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

How to Predict the Suitability for Corneal Donorship?

Background: In Germany, more than one-third of donor corneas harvested are not suitable for transplantation. We evaluated the factors associated with the usability of donor corneas. Method: Data from 2032 consecutive donor corneas harvested at the Rhineland-Palatinate Eye Bank in Mainz, Germany, were retrospectively analyzed. Factors of interest were age, sex, lens status, cause of death, cardiopulmonary resuscitation (CPR), death-to-explantation-interval (DEI), and the influence of these factors on the proportion of discarded donor corneas. Factors associated with endothelial cell density (ECD) were analyzed in a linear regression mixed model. Results: Higher donor age, male gender, pseudophakic lens status, and longer DEI were associated with significantly reduced ECD. With respect to DEI, the estimated cell loss was 7 ± 2 cells/mm2/hour (p < 0.001). Age was associated with a lower ECD of 6 ± 2 cells/mm2 per year (p = 0.001). Female ECD was 189 ± 44 cells/mm2 higher than male ECD (p < 0.001). Pseudophakic eyes had 378 ± 42 cells/mm2 less compared with phakic eyes (p < 0.001). Cause of death did not affect the ECD. Of note, 55% and 38% of corneas harvested on the second and third postmortem day, respectively, and 45% of corneas from donors older than 80 years were still suitable for transplantation. Conclusions: In the context of a growing need for donor corneas, we do not recommend limiting donor age and collection time to 24 h or excluding oncology donors, as is the practice in many countries. Therefore, we propose a mathematical model for better donor preselection.

Human corneal endothelial cells grafts to replace cadaveric donor corneas

Human corneal endothelial cells (HCEC) grafts to replace cadaveric donor corneas have made tremendous progress in recent years, and this article highlights the most recent discoveries and important achievements in this area. Cell injection therapy with cultured HCEC has just finished its first clinical research, which showed promising results. This follows on the heels of the success of autologous limbal stem cell transplantation in bioengineered ocular tissue transplantation. Transplantation techniques like bioengineered endothelium sheet transplantation and full-thickness corneal transplantation are likely to become more prevalent in the future decade. This goal will be achieved by a combination of current advancements in HCEC propagation, high-quality culture media, and 3D or 4D tissue culture, which will be the trend in the future.

Semiquantitative Criteria in the Eye Bank That Correlate with Cornea Guttata in Donor Corneas

Background Cornea guttata may not be recognized in the eye bank and recent studies have displayed that guttae are transplanted in about 15% of cases in varying severities. The purpose of this study was to establish semiquantitative criteria for the detection of cornea guttata in donor corneas in the eye bank. Methods In this retrospective cohort study, preoperative endothelial pictures of donor corneas were collected and classified according to the post-penetrating keratoplasty cornea guttata grade into three distinct groups: group 1 consists of healthy corneas with no guttae (guttata grade 0); group 2 constitutes corneas with mild asymptomatic cornea guttata (guttata grade +); and group 3 comprises corneas with advanced widespread cornea guttata (guttata grade ++/+++/++++). The preoperative pictures of each group were then individually analyzed using the following five semiquantitative criteria: The number and the area of the cell-depleted surfaces, the presence of less than 50% of the cells having a hexagonal or a circular shape, the presence of cell membrane defects and interruptions, the presence of blebs in the cell membrane, and the presence of groups of cells with a distinct whitish color. Results In total, 262 patients were included in this study, with a total number of 1582 preoperative donor corneal endothelial pictures. Out of those pictures, groups 1, 2, and 3 encompassed 995 (62.9%), 411 (26.0%), and 176 (11.1%) pictures, respectively. Three out of the five eye bank criteria were found to correlate with postoperative cornea guttata with a highly significant p value of < 0.001. These three criteria are the presence of less than 50% of the cells having a hexagonal or a circular shape, the presence of cell membrane defects and interruptions and, the presence of blebs. The presence of groups of cells with a distinct whitish color was only a weak predictive factor for cornea guttata (p = 0.069). There was no statistically significant correlation between the number and the area of cell-depleted surfaces and postoperative cornea guttata with a p = 0.181. Conclusion Three semiquantitative criteria that can be detected in the eye bank using inverted light microscopy seem to correlate with postoperative cornea guttata: The presence of blebs, the presence of cell membrane defects and interruptions, as well as endothelial pictures with less than 50% of the cells having a hexagonal of circular shape. The presence of groups of cells with a distinct whitish color appears to be a weak predictor of cornea guttata.