Organ-cultured Corneas Research Articles

Impairment of corneal epithelial wound healing in a TRPV1-deficient mouse.

To examine whether the absence or blockage of an ion channel receptor, transient receptor potential vanilloid subtype 1 (TRPV1), affects the healing of an epithelial injury using an experimental model of an epithelial defect in animal cornea. The expression of TRPV1 in the corneal epithelium was examined using immunohistochemistry in mice and rats. The migration of the corneal epithelium was examined in epithelium-debrided rat cornea in organ culture in the presence or absence of a TRPV1 agonist or its antagonist. Epithelial migration and cell proliferation following the debridement were examined in the cornea of a TRPV1-null mouse. Real-time RT-PCR was performed in samples of healing corneas to analyze the expression pattern of epithelial migration-related components (i.e., IL-6, substance P, and TGF-β1). TRPV1 was detected mainly in the basal layer of mouse or rat corneal epithelium. Adding a TRPV1 receptor agonist to the culture medium enhanced epithelial healing in the rat cornea, and a TRPV1 antagonist retarded it in organ culture. The loss of TRPV1 did not affect the histology of the mouse cornea. In vivo analysis showed the loss of TRPV1-impaired re-epithelialization of the debrided area of the corneal epithelium by the suppression of both cell migration and proliferation. The lack of TRPV1 suppressed the expression of IL-6 and substance P but not of TGF-β1 in response to epithelial debridement in mice. TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing of debrided corneal epithelium in mice.

Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells.

Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

Very Early Endothelial Cell Loss After Simultaneous Corneal Autograft and Allograft

For a better understanding of the very early endothelial cell (EC) loss universally described after all types of keratoplasty, we compared the EC decrease after performing a simultaneous autograft and organ-cultured allograft. A 71-year-old woman presented with a central corneal opacity in her left eye and a profoundly amblyopic right eye with a transparent cornea. Both corneas had a normal EC density (ECD). She underwent a left autograft and a right allograft procedure with an organ-cultured cornea, in which the ECD was determined using a calibrated light microscope with image analysis 48 hours before the surgery, that is, just before the final deswelling step with dextran. The postoperative central ECD was determined using specular microscopy on days (D) 1, 2, 3, 4, 5, 15, 20, 30, 60, 90, 120, and 180. Both grafts were uneventful. For the autograft, the pregraft ECD was 2303 cells per square millimeter, and the cell loss was very low, from 4% (D1) to 3% (D180). For the allograft, the pregraft eye bank ECD was 2787, and this decreased by 32% on D5. On D180, the ECD decrease was almost stabilized at 38%. This difference between the autograft and allograft, both performed in corneas with a normal peripheral endothelial reserve, indicates that the typical very early postoperative decrease in the EC is not caused mainly by surgery-dependent overmortality. It may be mostly artificial, revealing the overestimation of eye bank ECD caused by the technical unfeasibility of strictly considering living ECs and by measuring the ECD several days before grafting. This exceptional case suggests a new paradigm: surgeons graft fewer ECs than they think.

Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.

Comparison of Swollen and Dextran Deswollen Organ-Cultured Corneas for Descemet Membrane Dissection Preparation: Histological and Ultrastructural Findings

To compare the use of swollen tissue versus tissue deswollen by addition of dextran to the medium in the dissection of organ-cultured Descemet membrane (DM) with regard to preparation-related characteristics and ultrastructural findings to optimize transplantation in Descemet membrane endothelial keratoplasty (DMEK). DMs of 20 corneoscleral rims were separated using organ-cultured groups, one immersed in culture medium without dextran (group A) and the other in medium with added dextran for 24 hours (group B). The preparation details were noted. The difficulty of preparation was analyzed using a scoring system (0 = impossible, 10 = easy). By means of a micrometer, ultrathin sections of endothelial layer were obtained. Presence of any residual stroma, thickness of the DM, thickness of the endothelial cell layer, and the smoothness of the lamella were analyzed. In both group A and group B, all 10 usable DMs were available. Mean preparation time was 6.5 ± 1.4 minutes in group A and 6.1 ± 0.9 minutes in group B (P = 0.399). The difficulty score was 7.9 ± 1.9 in group A and 8.0 ± 2.0 in group B (P = 0.726). The total mean thickness of the DM (without the endothelial cell layer) was 13.58 ± 2.81 μm in group A and 12.69 ± 2.06 μm in group B (P = 0.474). The total mean thickness of the endothelial cell layer was 3.99 ± 0.62 in group A and 3.98 ± 0.52 in group B (P = 0.989). Light microscopy and transmission electron microscopy revealed no evidence of any adherent remnants of corneal stroma on any specimen in each group. DM-endothelium grafts for transplantation in DMEK procedures can be surgically prepared from organ-cultured corneal rims in swollen and deswollen conditions. Both separation methods seem to be equivalent in regard to the preparation characteristics of the obtained DM. Nevertheless, clinical studies are still necessary in order to confirm these findings.

Non-invasive measurement of transparency, arcus senilis, and scleral rim diameter of corneas during eye banking

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0-33.6), 38.3 (24.3-75.4) and 57.9 (33.9-90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3-10.6), 10.5 (8.2-11.5), and 11.1 (9.9-12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.

Setting up organ‐cultured corneas pre‐cutting by a French blood center‐eye bank

AbstractPurpose In France, the cutting of endothelial graft for DSAEK is almost exclusively performed by surgeons directly in the operating room whereas in the US more than 2/3 are now precut by eye banks (EB) technicians using 4°C storage. Among the French Blood Center EB, the EB of St‐Etienne received the first agreement from the French National Agency for Medicines and Health products for precutting and distribution of corneas. Aim: to report our first experience with DSAEK precut by technicians using organ‐cultured (OC) corneasMethods OC corneas with an initial endothelial cell density (ECD) &gt;2200 cells/mm2 (by image analysis) were precut by two trained technicians using a Moria microkeratome. Corneas were stored in CMax (Eurobio) and deswelled in CJet (5% Dextran T500) 20h prior to pre‐cutting. Central corneal thickness (CCT) was measure by ultrasound before and after cut with a single pass of the 350μm head. Microbiological testing was done with blood culture bottles 24h after precuttingResults Twelve corneas with initial ECD of 2724±269 cells/mm2 (mean±SD), 19±4 days of OC and CCT of 548±50 μm were cut and sent to 4 centers (Feb to May 2013). Final CCT was 194±33μm (166 to 277). All surgeries were uneventful but rebubbling was necessary in 6 cases and 1 graft with eccentric trephination by the surgeon did never adhereConclusion Precutting of OC corneas by EB allows quality and microbiology controls. Three improvements are ongoing: OCT thickness map before, during and after cut, use of the microkeratome double‐pass technique, and ink marking of the centre to guide final trephination by surgeons. Grants: ONDAINE, AOL CHU 2012

Femtosecond laser cutting of multiple ultrathin corneal stromal lamellae for endothelial graft bioengineering

AbstractPurpose The bioengineering of endothelial grafts (EG) is developing in several countries as an answer to the shortage of donor corneas worldwide. They are composed of a thin transparent biocompatible carrier evenly covered with tightly packed fully functional endothelial cells. Multiple options exist for the carriers. In our laboratory, we are focused on the optimization of human donor tissue because the well‐organised cornea donation in France is not the most limiting point. Aim: to present the preparation of ultrathin stromal lamellae from a single human corneaMethods Human organ‐cultured corneas unsuitable for graft were used either with our without deswelling in Dextran containing medium. They were mounted on a artificial anterior chamber and cut with a femtosecond laser (femto LDV Z6, Ziemer) with aplanation. Six horizontal parallel cuts were realized on a 9 mm diameter area without vertical section. Cornea was then trephine at 8.00mm on a punch trephine. Thickness map was obtained before and after the cut with an AS‐OCT equipped with a custom adapter for ex vivo corneas. Lamellae were separated under an operating video‐microscope and again observed with OCT. Quality of surface were analysed with scanning electron microscopy and multiphotonic confocal microscopy with SHG for collagen fiber imagingResults Seven regular lamellae of approximately 100μm were obtained on a single cornea and easily detached. Characterisation of surface quality is ongoingConclusion Femtosecond laser cutting of multiple lamellae on a single human cornea could allow obtaining a sufficient number of thin transparent and fully biocompatible carriers for EG bioengineering

Preconditioned Donor Corneal Thickness for Microthin Endothelial Keratoplasty

To describe a novel surgical technique to produce thin endothelial grafts for Descemet stripping automated endothelial keratoplasty (DSAEK). Thirteen human cadaveric corneas in organ culture were randomized into conventional (n = 7) and microthin (n = 6) DSAEK groups. Grafts in the conventional DSAEK group were prepared using the conventional DSAEK technique of a single microkeratome pass with a 350-μm cutting head. Corneas in the microthin group were preconditioned to achieve a target central thickness of 530 μm before graft dissection with a 350-μm microkeratome head. Preconditioning involved stromal dehydration under pachymetric control using sterile airflow for 15-second increments. Donor and graft thicknesses were assessed with optical coherence tomography, and endothelial viability with trypan blue and alizarin red staining. Mean endothelial graft thickness obtained using the microthin DSAEK technique was 106 μm (SD, 32 μm) compared with 177 μm (SD, 33 μm) obtained using conventional DSAEK technique (P = 0.0024). Donor preconditioning yielded a predicted reduction of 100 μm in graft stromal thickness at a rate of 1.5 μm/s and mean duration of 72 seconds. The average anterior lamella thickness (cut depth) obtained in microthin and conventional DSAEK groups were 424 and 431 μm, respectively (P = 0.84). There was no difference in endothelial viability between the 2 groups. There were no corneal perforations during graft preparation in this study. Donor preconditioning by pachymetry-controlled stromal dehydration achieved significantly thinner endothelial grafts compared with the conventional DSAEK technique without compromise to endothelial viability or graft wastage.

Multikinase-Inhibitoren als therapeutischer Ansatz bei neovaskulärer AMD: In-vitro-Evaluation der Sicherheit von Axitinib, Pazopanib und Sorafenib zur intraokularen Anwendung

Multikinase inhibitors (MKI) interfere effectively at different levels of the neovascularisation cascade. Early clinical and experimental data suggest that MKIs represent a promising novel approach for the treatment of neovascular age-related macular degeneration (AMD). However, so far little is known about the biocompatibility of MKIs regarding human ocular cells. This in vitro study investigates and compares the biocompatibility of three MKIs, axitinib, pazopanib, and sorafenib regarding ocular cells of the anterior and posterior segments, as well as organ-cultured donor corneas. Primary human optic nerve head astrocytes (ONHA), trabecular meshwork cells (TMC), and retinal pigment epithelium (RPE), human corneal endothelial and lens epithelial cells (CEC and LEC) were treated with different concentrations of axitinib, pazopanib, or sorafenib (0.1 to 100 µg/mL). To simulate oxidative stress, the cells were additionally co-incubated with 400 µM hydrogen peroxide. Induction of cell death and cellular viability were examined by live-dead assay and tetrazolium dye reduction assay (MTT). In addition, the influence of the three substances on human corneal endothelium was evaluated in seropositive donor corneas in organ culture by phase contrast microscopy. Up to a concentration of 7.5 mg/mL of the substances tested in any cell type examined, no toxic effects were found. Even after 10 days of incubation of organ-cultured donor corneas with 7.5 µg/mL, axitinib, pazopanib, or sorafenib, no evidence for endothelial toxicity was found. All three MKIs tested, axitinib, pazopanib, and sorafenib showed a good biocompatibility on the investigated ocular cells. Even under conditions of oxidative stress, there were no toxic effects up to a concentration of 7.5 µg/mL. Only at higher concentrations, there was a dose-dependent decrease in cellular viability and pronounced induction of cell death. These effects on cellular viability and induction of cell death appeared to be stronger with pazopanib, followed by sorafenib, than with axitinib.

Risk Factors for Donor Cornea Contamination

Microbiological contamination is a common cause for elimination of organ-cultured donor corneas. The aims of the present study were to analyze contamination rates and identify risk factors for contamination. Retrospectively, the contamination rates of 4546 organ-cultured corneas and the causative species were studied. The impact of sex, age, death-to-explantation interval, explantation technique, cause of death, and mean monthly temperature on contamination rate was analyzed. The median annual contamination rate was 5.3% (range: 3%-19%). Most contaminations were of fungal origin (61.9%), with Candida species (45%) being predominant. Bacterial contaminations (34.4%) were dominated by Staphylococcus species (12.8%). Sex, donor age, and mean monthly temperature had no statistically significant influence on the contamination rate. The median death-to-explantation interval of contaminated corneas (44 hours) was longer than that of sterile corneas (39 hours; P < 0.001; n = 4437). Cardiopulmonary failure was associated with the highest contamination rate (13.6%) of all death causes. The switch from whole globe to in situ excision was followed by a temporary increase in contamination rate (12.5%-19.4%). Although the genesis of donor cornea contamination seems to be multifactorial, resident species from physiological skin flora are the main contaminants indicating that the donor corpses could be the main source of microbiological contamination. A change in the explantation technique was followed by an increase in the contamination rate.

Alkylphosphocholines for intraocular lens coating

To assess the effect of alkylphosphocholine (APC)-coated intraocular lenses (IOLs) on human lens epithelial cell (LEC) proliferation in vitro and the corneal biocompatibility of APC in an organ culture model of human donor eyes. Research Laboratory for Experimental Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Experimental study. Six foldable IOLs differing in optic material were incubated with APC, washed in phosphate-buffered saline, and dried overnight. Intraocular lenses of the same lot served as uncoated controls. Each rehydrated IOL was placed in 1 well of a 24-well plate containing proliferating human LECs. Cell survival was tested by the tetrazolium-dye reduction assay 5 days later. Biocompatibility of the human corneal endothelium was assessed by a 24-hour incubation of human donor corneas with different concentrations of APC before the live/dead assay was performed. Human LEC proliferation was attenuated by APC-coated IOLs (P=.001). Coated hydrophilic acrylic IOLs were more effective inhibitors of human LEC proliferation than coated hydrophobic acrylic or silicone IOLs (P=.001). Alkylphosphocholines were well tolerated by the corneal endothelium in an organ culture model of human donor corneas up to a concentration of 1 mM (n = 12). Results show that APCs are suitable agents for IOL coating without linker molecules. Coated IOLs can inhibit human LEC proliferation and were well tolerated by human donor corneas in organ culture.

Evaluation of the HB&amp;L System for the Microbiological Screening of Storage Medium for Organ-Cultured Corneas

Aims. To compare HB&L and BACTEC systems for detecting the microorganisms contaminating the corneal storage liquid preserved at 31°C. Methods. Human donor corneas were stored at 4°C followed by preservation at 31°C. Samples of the storage medium were inoculated in BACTEC Peds Plus/F (aerobic microorganisms), BACTEC Plus Anaerobic/F (anaerobic microorganisms), and HB&L bottles. The tests were performed (a) after six days of storage, (b) end of storage, and (c) after 24 hours of preservation in deturgescent liquid sequentially. 10,655 storage and deturgescent media samples were subjected to microbiological control using BACTEC (6-day incubation) and HB&L (24-hour incubation) systems simultaneously. BACTEC positive/negative refers to both/either aerobic and anaerobic positives/negatives, whereas HB&L can only detect the aerobic microbes, and therefore the positives/negatives depend on the presence/absence of aerobic microorganisms. Results. 147 (1.38%) samples were identified positive with at least one of the two methods. 127 samples (134 identified microorganisms) were positive with both HB&L and BACTEC. 14 HB&L+/BACTEC− and 6 BACTEC+/HB&L− were identified. Sensitivity (95.5%), specificity (99.8%), and positive (90.1%) and negative predictive values (99.9%) were high with HB&L considering a 3.5% annual contamination rate. Conclusion. HB&L is a rapid system for detecting microorganisms in corneal storage medium in addition to the existing methods.

Expression of an anti-CD4 single-chain antibody fragment from the donor cornea can prolong corneal allograft survival in inbred rats

AimTo investigate whether expression of an anti-CD4 antibody fragment (scFv) by a lentivector-transduced donor cornea can prolong rat corneal allograft survival.MethodsInbred Fischer 344 rats received penetrating corneal allografts from Wistar-Furth...

We don’t graft as many endothelial cells as we think (part 2): comparison of cell loss after autograft and organ cultured allograft in the same patient

AbstractPurpose Reasons for endothelial cell density (ECD) decrease after corneal grafts remain unclear. We recently demonstrated that eye bank ECD (ebECD) overestimates the pool of viable EC really grafted to patients (Pipparelli.IOVS2011;52:6018) because standard ebECD cannot take account of acellular areas or area covered by dying EC. Autograft is a very rare situation where ECD can be easily determined by specular microscopy just before graft and almost all grafted EC are living. Aim: to compare EC decrease after simultaneous autograft and organ cultured (OC) allograft in the same patientMethods A 71 year‐old woman presented with pseudophakic corneal edema in left eye and amblyopic right eye with a normal cornea. She underwent two penetrating keratoplasties (PK): autograft of the left eye and allograft of the right eye with an OC cornea. Follow‐up at day (D)1, 5, 15, 20 and 30 comprised slit lamp examination and non‐contact central specular microscopy (Topcon SP2000P)Results Both grafts were uneventful. For the allograft, ebECD 48h before graft was 2787 cells/mm² and postop ECD decreased of 39%, 40%, 40% and 43% at D5, 15, 20 and 30. For the autograft, pregraft specular ECD was 2017 cells/mm² and postop ECD were 1941, 1934, 1924, 1909 and 1900 cells/mm² respectively at D1, 5, 15, 20 and 30Conclusion This exceptional case confirms a dramatic discrepancy between ebECD and very early postop ECD, likely to be explained mainly by an overestimation of the true EC pool of viable cells by standard eye banks count, rather than by overmortality due to peri‐operative events, since almost no early cell loss is observed during autograft

In Vitro Quantification of the Stiffening Effect of Corneal Cross-linking in the Human Cornea Using Radial Shearing Speckle Pattern Interferometry

To quantify the magnitude of the stiffening effect of corneal cross-linking (CXL) by studying intact human corneas exposed to physiological pressure transients. Nine organ-cultured human corneas mounted in artificial anterior chambers were studied. A radial shearing speckle pattern interferometer was used to measure changes in corneal strain following an increase in artificial anterior chamber pressure from 15.0 to 15.5 mmHg before and after treatment. Measurements were taken from all corneas with their epithelium intact before all underwent epithelial debridement. Three specimens were used as controls and did not receive any further treatment; three underwent riboflavin/ultraviolet A (UVA) CXL using 30 minutes of 370-nm irradiation at 3 mW/cm2 following epithelial removal and saturation with 0.1% riboflavin; and three were fixed with the cross-linking agent 2.5% glutaraldehyde. Strain measurements were repeated after these treatments. Young's moduli of individual corneas were calculated by mathematical analysis. Mean donor age was 81.7 years. Before treatment, the mean Young's moduli of the control, riboflavin/UVA CXL, and glutaraldehyde-fixed corneas did not differ significantly: 0.46±0.03, 0.48±0.03, and 0.47±0.03 MPa, respectively. Following treatment these values changed to 0.46±0.2, 2.06±0.22, and 3.48±0.41 MPa, respectively. In proportional terms, this was equivalent to an increase in corneal Young's modulus by a factor of 4.3 (P<.05) following riboflavin/UVA CXL and 7.3 (P<.05) after glutaraldehyde fixation. Riboflavin-UVA CXL increases the stiffness of organ-cultured corneas by a factor of more than four. This finding quantifies the efficacy of CXL in a physiologic configuration.

Effects of Variation in Depth and Side Cut Angulations in LASIK and Thin-flap LASIK Using a Femtosecond Laser: A Biomechanical Study

To study the corneal biomechanical effects of varying LASIK flap depth and side cut angulations and evaluate the relative contribution of the lamellar and side cuts using a femtosecond laser and radial shearing speckle pattern interferometry (RSSPI). Forty-two organ-cultured human corneas were divided into a control group and three investigative groups, each undergoing different incision types at both 90- and 160-μm depth using a femtosecond laser. In the first group, typical LASIK flaps were created; in the second group, only the bed was cut (delamination); and in the third group, side cuts alone were affected. Corneal strain was measured using RSSPI before and after treatment following an increase in hydrostatic pressure from 15.0 to 15.5 mmHg and again after 1 week of incubation in culture medium. The flap group demonstrated a weakening of strength related to the depth of cut, with strain increasing by 9% and 32% at 90 and 160 μm, respectively. Similar changes, 9% and 33%, were observed following execution of side cuts to the same depths. By contrast, strain increase following delamination showed no relationship with depth, increasing by 5% in both instances. When the side cut angle was made more acute, strain decreased with a 2% strain increase being measured after a 90-μm, 150° side cut was created. No significant changes occurred during the period of organ culture. Vertical side cuts through corneal lamellae rather than horizontal delamination incisions contribute to the loss of structural integrity during LASIK flap creation. Angulating side cuts such that the stromal diameter of the flap exceeds its epithelial diameter can decrease this effect.

Evaluation of interface quality in organ-cultured lamellar corneal transplants.

BackgroundWith increasing numbers of lamellar keratoplasties, eye banks are challenged to deliver precut lamellar donor tissue. In Europe, the most common technique of corneal storage is organ culture which requires a deswelling process before surgical processing. The aim of this study was to investigate the influence of different deswelling times on the cutting plane quality after microkeratome-assisted lamellar dissection.MethodsEight paired donor corneas (16 specimens) not suitable for transplantation were organ cultured under standard conditions at the Eye Bank of the Ludwig-Maximilians Universität, Munich, Germany. Pairs of corneal buttons were analyzed during the deswelling process in dextrane-containing medium. While one cornea was cut at an early time point during the deswelling process and put back into deswelling medium thereafter, the partner cornea was completely deswollen and dissected after 72 hours. Specimens were then further processed for scanning electron microscopy. Surface quality was assessed both digitally using Scanning Probe Imaging Processing software, and manually by three blinded graders.ResultsThe corneal buttons processed at the beginning of the deswelling process had a smoother surface when compared to the partner cornea that was cut at the end of the deswelling process. In our setting, no relevant difference was detectable between manual and automated microkeratome dissection.ConclusionFor lamellar keratoplasty, organ-cultured corneas should be processed at an early stage during the deswelling process. We interpret the smoother dissection plane during early deswelling as a result of mechanical properties in a highly hydrated cornea.

Comparison of in situ Corneoscleral Disc Excision versus Whole Globe Enucleation in Cornea Donors Regarding Microbial Contamination in Organ Culture Medium – a Prospective Monocentric Study over 9 Years

Background: Corneas needed for keratoplasty can be harvested using two techniques: whole globe enucleation and in situ excision of the corneoscleral disc. This study evaluates the rate of microbial contamination of the donor cornea organ culture medium according to the method of retrieval. Methods: All donor corneas of our cornea bank received between January 1, 2001 and December 31, 2009 put into organ culture and microbiologically tested were prospectively analyzed for microbial contamination of the organ culture medium. Results: 2,805 donor corneas could be included in this study in total. 975 of them were retrieved by whole globe enucleation (group 1) and 1,830 by in situ corneoscleral disc excision (group 2). 15 corneas of group 1 (1.5%) and 46 corneas of group 2 (2.5%) showed a contamination of the organ culture medium. The difference was shown not to be statistically significant (p = 0.082). Conclusion: The rate of microbial contamination in organ-cultured donor corneas does not seem to be dependent on the method of their retrieval.

Poloxamines for Deswelling of Organ-Cultured Corneas

Background: Poloxamines are amphiphilic tetrofunctional block copolymers composed of four polyoxyethylene-polyoxypropylene arms joined to a central ethylene diamine bridge. Their safe profile allows diverse pharmaceutical and biomedical applications. Aim: To assess their use for corneal deswelling using a porcine model of organ culture (OC). Methods: Five poloxamines (T90R4, T904, T908, T1107 and T1307) were dissolved in a standard commercial OC medium (control) to reach 350 mosm kg^–1. In vitro cytotoxicity was tested using MTT assay on human corneal epithelial and endothelial cell (EC) lines and on primary human corneal fibroblasts. Paired porcine corneas stored in OC for 3 days were assigned for 48 h to a poloxamine medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, mortality, stromal thickness and transparency were evaluated before and after deswelling. Post-deswelling, EC viability/mortality was determined using a fluorescent live/dead assay. Results: Besides similar corneal thickness reduction and transparency improvement, T908, T1107 and T1307 decreased EC loss (5.4 ± 1.7% vs. 9.9 ± 2.6% in controls (p < 0.001)) and mortality, improved EC morphometry and reduced endothelial lesions compared to dextran. Conclusion: On this porcine model, poloxamines T908, T1107 and T1307 appear as good candidates to replace dextran for the deswelling. Experiments on human corneas are now necessary to confirm their efficiency and safety profile in OC.