Cutting and Decellularization of Multiple Corneal Stromal Lamellae for the Bioengineering of Endothelial Grafts.

Purpose: Engineered corneal endothelial grafts able to provide numerous functional endothelial cells for the restoration of corneal transparency would be a worthwhile way of replacing donor tissue, which is extremely scarce. The grafts are simply constructed: a biocompatible thin and transparent carrier colonized by a monolayer of cultured endothelial cells (ECs). Here we describe a process able to obtain appropriate carriers by recycling human corneas unsuitable for graft in their original state, but liable to provide multiple thin lamellae when cut with a femtosecond laser as used in refractive surgery. Methods: We selected a robust method of stromal decellularization. To demonstrate that neither this process nor long-term storage hindered cell adherence, lamellae were endothelialized with an EC line. Results: The constructs achieved up to very high EC density (the main quality criterion for regular donor corneas) while remaining transparent and thin. We verified that they could be inserted in the anterior chamber of a human eye, like a conventional endothelial graft. Human decellularized cornea will likely be directly compatible with the recipient cornea and comply with the requirements of health regulatory authorities. Conclusions: This study demonstrates that thin human corneal lamellae could have high potential as carriers in next-generation therapy for endothelial dysfunctions.

Evolution of Corneal Graft Survival Over a 30-Year Period and Comparison of Surgical Techniques: A Cohort Study.

PURPOSE: To compare graft survival between 3 10-year periods and according to surgical techniques performed in the last years. DESIGN: Cohort study. METHODS: setting: Regional center (Besançon University Hospital, France). PATIENTS: All 1132 patients operated on between 1983 and 2014. Graft and patient baseline characteristics, risk factors for failure, surgical procedures, and postoperative corneal status were collected. MAIN OUTCOME MEASURES: Five-year survival rate in the whole cohort; 1-year and 3-year survival rates, respectively, among 88 patients with endothelial dystrophy (ED) or postoperative bullous keratopathy (PBK) operated on using endothelial lamellar keratoplasty (ELK) or penetrating keratoplasty (PK), and among 56 patients with keratoconus operated on using anterior lamellar keratoplasty (ALK) or PK. RESULTS: Between the 1983-1993 and the 2004-2014 periods, overall 5-year graft survival rate increased from 61.4% to 76.5% (P = .0004). The main prognostic factors were preoperative diagnosis, graft endothelial density, and postoperative lens status. After adjusting for these factors, difference in survival rates was no longer significant (hazard ratio 0.90 for the second and 1.17 for the third period, compared to the first, P = .4191). Only 1 graft failure, after PK, occurred among the 56 patients with keratoconus. Among the 88 patients with ED or PBK, the 1-year graft survival was higher with PK (90.6%) than with ELK (60.8%) (P = .0025) but no significance remained after adjustment (hazard ratio 3.22, P = .1304). CONCLUSIONS: Despite numerous changes in graft procedures and surgical techniques, no noticeable improvement in graft survival was found during the last 30 years while taking into account other prognostic factors.

Femtosecond laser cutting of multiple thin corneal stromal lamellae for endothelial bioengineering.

PURPOSE: To assess the feasibility of cutting multiple thin stromal lamellae in human donor corneas using a commercial femtosecond laser (FSL) to provide cell carriers for future endothelial graft bioengineering. METHOD: Eight edematous organ-cultured corneas not suitable for grafting for endothelial reasons were mounted on a Ziemer anterior chamber and cut with a Z6 FSL with 6 successive parallel cuts, from depth to surface. Target thickness of each lamella ranged from 100 to 150 µm depending on initial corneal thickness. Thickness was measured using anterior segment optical coherence tomography before and after cutting on mounted corneas, and on each stromal lamella after detachment. Scanning electron microscopy observation was performed on 4 lamellae and histological cross sections on 1 cornea before detachment. RESULTS: A median of 5 (minimum 3, maximum 7) lamellae was obtained per cornea. All lamellae still attached were the most posterior ones, suggesting that FSL was less efficient because of light scattering by edematous stroma. Cut precision and postdetachment swelling were correlated with anterior-posterior position within the cornea. Median lamella thickness was 127 µm (56-222 µm) before detachment and 196 µm (80-304 µm) after detachment. Surface state was consistent with previously reported FSL lamellar cuts during Descemet stripping automated endothelial keratoplasty. CONCLUSIONS: Up to 7 thin lamellae can be cut in stored corneas with an FSL. This method, once optimized primarily by using deswelled, more transparent corneas, could prove effective for recycling unsuitable donor corneas in corneal bioengineering processes.

Reproducibility of viscoelastic property and intraocular pressure measurements obtained with the Ocular Response Analyzer.

PURPOSE: To analyse the reproducibility of corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) obtained with the ocular response analyzer (ORA). METHODS: This is a prospective study, nonmasked, of eight successive examinations with the ORA device in 60 normal eyes. Using 30 eyes (one eye per subject), the reproducibility was assessed by comparing the first series of four examinations to the second four and by calculating the within-subject coefficient of variation. The correlation and difference with the fellow eye were analysed, respectively. RESULTS: The mean values were 10.7 ± 1.8 mmHg, CRF; 10.6 ± 1.6 mmHg, CH; 15.9 ± 3.9 mmHg, IOPg and 16.2 ± 3.7 mmHg, IOPcc. The reproducibility was significantly different for CRF (5.2 ± 5.9%), CH (7.3 ± 8.6%), IOPg (7.7 ± 6.7%) and IOPcc (10.1 ± 8.0%); p < 0.001. Considering the reproducibility, CRF correlated with CH (rs = 0.55; p < 0.001) and showed to be independent of IOPg and IOPcc. The score spread was best for CRF (2.6 ± 1.5 mmHg; 24.0%) compared to IOPg (4.3 ± 1.5 mmHg; 28.1%) and CH (3.1 ± 1.7 mmHg; 29.9%) and worst for IOPcc (5.5 ± 2.5 mmHg; 34.4%). The lowest difference with the fellow eye was observed for CRF (5.0%; p = 0.09). The correlation with the fellow eye was high, especially for IOPcc and CRF (rs > 0.9; p < 0.001) followed by IOPg and CH (rs > 0.8; p < 0.001). CONCLUSION: The ORA device provides reproducible information on viscoelastic properties of the cornea in normal eyes notably CRF and CH. IOPcc was less reproducible. Four measurements per eye were necessary to reach a 10% precision and six for 5%.