The impact of biomechanics on corneal endothelium tissue engineering.

The integrity of innermost layer of the cornea, the corneal endothelium, is key to sustaining corneal transparency. Therefore, disease or injury causing loss or damage to the corneal endothelial cell population may threaten vision. Transplantation of corneal tissue is the standard treatment used to replace malfunctioning corneal endothelial cells. However, this surgery is dependent upon donor tissue, which is limited in supply. Hence, tissue engineers have attempted to construct alternative transplantable tissues or cell therapies to alleviate this problem. Nevertheless, the intrinsic non-dividing nature of corneal endothelial cells continues to foil scientists in their attempts to yield large numbers of cells in the laboratory for use in such novel therapies. Interestingly, the contribution of the biomechanical properties of the underlying extracellular matrix (ECM) on cell division, tissue development and maintenance has been extensively investigated in other many cell types. However, the impact of biomechanics on corneal endothelial cell behaviour is relatively unexplored. Here, we describe contemporary tissue engineering solutions aimed at circumventing donor tissue scarcity. We review the ECM structure and biomechanical features of corneal endothelial cells. We discuss the alterations of ECM in endothelial disease development and progression and point out the role of ECM in developing a tissue-engineered corneal endothelium. We highlight the main biomechanical cues, including topographical and mechanical features, that impact cellular behaviors. Finally, we discuss the influence of biomechanical cues on cell and tissue development, and how corneal endothelial cells response to individual biomechanical stimuli in tissue engineering, which have implications for designing an engineered endothelium and maintaining cell function.

Evaluation of reconstructed human corneal endothelium sheets made with porcine Descemet's membrane in vitro and in vivo.

PURPOSE: To identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo. METHODS: Denuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery. RESULTS: No significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na+/K+-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 ± 52.30 cells/mm2. After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 ± 56.23 µm at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 ± 78.24 cells/mm2 in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM. CONCLUSIONS: Seeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty.

Integrin: Basement membrane adhesion by corneal epithelial and endothelial cells.

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet's membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFß1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization medium.

Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane-endothelium (DM-EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM-EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM-EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM-EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM-EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM-EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.

[Density of the endotheliocytic layer of the cornea of the eyeball as a function of age].

This article presents the results of measuring the number of corneal endotheliocytes in a unit area of descemet membrane surface in 546 volunteers of different ages. The average values of the density of the corneal posterior epithelium for the age intervals 40-49, 50-59, 60-69, 70-79, 80 years and older are shown, a constant decrease in the number of endotheliocyte cells as the number of years lived increases.

[Construction of corneal endothelium deficiency model by cryo-injury and its application].

OBJECTIVE: To set up an animal model of corneal endothelium deficiency and test the bio-safety and pump function of B4G12 cells with this model. METHODS: Thirty SD rats were divided into cryo-injury group and cryo-injury with B4G12 cell transplantation group. Models of corneal endothelium deficiency were created by cryo-injury with liquid nitrogen, and then B4G12 cells were transplanted into the eyes by anterior chamber injection. Corneas were checked under slit lamp and confocal microscope observations at some specific time points, pathological staining was also performed. RESULTS: The corneal endothelial cells were removed completely from the Descemet's membrane after cryo-injury. Cells transplanted firmly stuck to the Descemet's membrane. The corneas in the cryo-injury group were swelling with haze, while in the transplantation groups, the corneas restored transparence and normal thickness 2 weeks after the surgery. Confocal microscope and HE staining confirmed that in the cryo-injury group, the Descemet's membrane was denuded and the cornea stroma layer was in edema, on the contrary, in the other group, the transplanted cells completely covered the Descemet's membrane. CONCLUSION: Cryo-injury can be used for building the rat model of corneal endothelium deficiency, no neoplasm and inflammation reaction were found during the observation after B4G12 cells transplanted and B4G12 cells had the pump function in vivo.

Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans.

The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat-mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 µm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 µm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches.

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank.

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.

Identification of the preoperative and perioperative factors that predict postoperative endothelial cell density after Descemet membrane endothelial keratoplasty: A retrospective cohort study.

Low postoperative endothelial-cell density (ECD) plays a key role in graft failure after Descemet-membrane endothelial keratoplasty (DMEK). Identifying pre/perioperative factors that predict postoperative ECD could help improve DMEK outcomes. This retrospective study was conducted with consecutive adult patients with Fuchs-endothelial corneal dystrophy who underwent DMEK in 2015-2019 and were followed for 12 months. Patients underwent concomitant cataract surgery (triple-DMEK) or had previously undergone cataract surgery (pseudophakic-DMEK). Multivariate analyses assessed whether: patient age/sex; graft-donor age; preoperative ECD, mean keratometry, or visual acuity; triple DMEK; surgery duration; surgical difficulties; and need for rebubbling predicted 6- or 12-month ECD in the whole cohort or in subgroups with high/low ECD at 6 or 12 months. The subgroups were generated with the clinically relevant threshold of 1000 cells/mm2. Surgeries were defined as difficult if any part was not standard. In total, 103 eyes (95 patients; average age, 71 years; 62% women) were included. Eighteen eyes involved difficult surgery (14 difficult graft preparation or unfolding cases and four others). Regardless of how the study group was defined, the only pre/perioperative variable that associated significantly with 6- and 12-month ECD was difficult surgery (p = 0.01, 0.02, 0.05, and 0.0009). Difficult surgery also associated with longer surgery duration (p = 0.002). Difficult-surgery subgroup analysis showed that difficult graft dissection associated with lower postoperative ECD (p = 0.03). This association may reflect endothelial cell loss due to excessive graft handling and/or an intrinsic unhealthiness of the endothelial cells in the graft that conferred unwanted physical properties onto the graft that complicated its preparation/unfolding.

Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors.

PURPOSE: Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH. METHODS: Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca(2+)](i)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively. RESULTS: Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR. CONCLUSIONS: Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

Preloading Trifolded Grafts for Descemet Membrane Endothelial Keratoplasty Affects Scroll Formation.

PURPOSE: The trifolded, endothelium-in approach to Descemet membrane endothelial keratoplasty (DMEK) facilitates tissue insertion into the anterior chamber. We hypothesized that preloading the trifolded donor grafts in a cartridge for 48 hours before insertion would induce biomechanical changes that decrease their scrolling tendency compared with those loaded immediately before insertion. METHODS: Ten Descemet membrane donor grafts, peeled and cut to 8.0 mm, were prepared by a single eye bank technician. Each graft was trifolded and pulled into a DMEK cartridge and stored for 48 hours. They were then pulled with microforceps into a petri dish filled with balanced salt solution. A video was recorded of the graft becoming a scroll over a 2-minute period. Each graft, serving as its own control, was then trifolded, pulled into the cartridge, and the process repeated. Images from 1, 5, 10, 60, and 120 seconds were extracted from video recording of the procedures. Scroll width was analyzed by graders masked to group assignment. A paired t test was used to determine differences in scroll width at each time point between the 48-hour and instant trifolding conditions. RESULTS: All grafts scrolled after removal from the cartridge into balanced salt solution. We measured a significant difference at all time points 1 through 120 seconds (4.02 preloaded vs. 2.91-mm instant trifold, P = 0.035). CONCLUSIONS: Preloading DMEK grafts in a trifolded configuration for 48 hours reduces the scrolling tendency of Descemet membrane for at least 2 minutes.

Molecular evidence of senescence in corneal endothelial cells of senescence-accelerated mice.

PURPOSE: To investigate senescent evidence in corneal endothelial cells (CECs) of the senescence-accelerated mouse (SAM), which is considered a suitable animal model for the further study of the senescent mechanism in CECs. METHODS: Thirty-six male mice from a senescence resistant mouse strain (SAM R1) and a senescence-prone strain (SAM P8) at various ages (1, 6, and 12 months) were analyzed in this study. The endothelial cell density (ECD) and cell viability were detected using trypan blue and alizarin red dyes while the senescent cells were observed by senescence-associated beta-galactosidase (SA-beta-Gal; pH 6.0) staining. In addition, ultrastructure was observed using an electron microscope. The senescence-related genes (p16(INK4a), p19(ARF), p21(WAF1/CIP1), and p53) in the CECs were visualized via immunohistochemistry and were quantitatively detected using real-time polymerase chain reaction (PCR). Signal proteins of phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2) were detected by western blot analysis. RESULTS: Our results indicated that the ECD values decreased with increasing age in both the SAM-R1 and SAM P8 series where the values in the older SAM p8 series decreased even lower than in the older SAM R1 series. The mean decreased rate was 2.276% per month in the SAM R1 and 2.755% per month in the SAM P8 series. In addition, changes in the senescence-like ultrastructure were observed in the CECs of both strains, and the increase in the positive staining of SA-beta-Gal was observed in both strains as well. It is worth noting that such changes were more significant in the SAM P8 strain. Immunohistochemical detection assays indicated the expression of p-ERK 1/2, p16(INK4a), p19(ARF), p21(WAF1/CIP1), and p53 (nuclear localization for each) in each age group analyzed. Furthermore, the results of real-time PCR studies showed an increase in the expression of p16(INK4a) mRNA as a function of age in the SAM R1 strain and in the early senescence stage of the SAM P8 strain in addition to an increase in the expression of p21(WAF1/CIP1) and p53 mRNA as a function of age in the SAM P8 strain (no significant increase was observed in the SAM R1 strain). Additional results from western blot analysis demonstrated an age-related increase in the quantity of the p-ERK 1/2 proteins in both strains. CONCLUSIONS: The SAM R1 and SAM P8 strains represent suitable models for the study of CEC senescence in vivo. In addition, the progression of cellular senescence in CECs occurs more quickly in the SAM P8 strain as opposed to the SAM R1 strain. Our results also indicate that the p16(INK4a) signaling pathway may play a key role in the early stages of senescence in CECs while the p53/p21(WAF1/CIP1) signaling pathway may exert its principle effect in the late stages of senescence in CECs. Further study is still required about the role of the mitogen-activated protein kinase (MAPK) signaling cascade in the process of senescence in CECs.

Characterization of the collagen in the hexagonal lattice of Descemet's membrane: its relation to type VIII collagen.

To investigate the nature of the hexagonal lattice structure in Descemet's membrane, monoclonal antibodies were raised against a homogenate of bovine Descemet's membranes. They were screened by immunofluorescence microscopy to obtain antibodies that label Descement's membrane. Some monoclonal antibodies labeled both Descemet's membrane and fine filaments within the stroma. In electron microscopy, with immunogold labeling on a critical point dried specimen, the antibodies labeled the hexagonal lattices and long-spacing structures produced by the bovine corneal endothelial cells in culture; 6A2 antibodies labeled the nodes of the lattice and 9H3 antibodies labeled the sides of the lattice. These antibodies also labeled the hexagonal lattice of Descemet's membrane in situ in ultrathin frozen sectioning. In immunofluorescence, these antibodies stained the sclera, choroid, and optic nerve sheath and its septum. They also labeled the dura mater of the spinal cord, and the perichondrium of the tracheal cartilage. In immunoblotting, the antibodies recognized 64-kD collagenous peptides both in tissue culture and in Descemet's membrane in vivo. They also recognized 50-kD pepsin-resistant fragments from Descemet's membranes that are related to type VIII collagen. However, they did not react either in immunoblotting or in immunoprecipitation with medium of subconfluent cultures from which type VIII collagen had been obtained. The results are discussed with reference to the nature of type VIII collagen, which is currently under dispute. This lattice collagen may be a member of a novel class of long-spacing fibrils.

The spatial organization of Descemet's membrane-associated type IV collagen in the avian cornea.

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.

Minimizing Endothelial Cell Loss Caused by Orientation Stamps on Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To quantify endothelial cell loss (ECL) caused by orientation stamps on prestripped and preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts, and to examine a method for reducing ECL using a smaller stamp. METHODS: Ten prestripped and 10 preloaded DMEK grafts were prepared with S-stamps. Ten additional preloaded DMEK grafts were prepared with both an S-stamp and a smaller F-stamp in different paracentral areas of the graft. The footprint of each stamp was measured using ink on cardstock. DMEK grafts were stored in viewing chambers filled with 20 mL of Optisol-GS for 3 days at 4°C. ECL was quantified using Calcein-AM staining and FIJI Weka Segmentation. RESULTS: S-stamps on prestripped DMEK grafts contributed an average ECL of 1.1% ± 0.5% (range: 0.6%-2.2%) toward total graft damage, whereas S-stamps on preloaded DMEK grafts contributed approximately twice that amount (average ECL: 2.0% ± 0.7%, range: 1.3%-3.1%, P = 0.004). Overall ECL for prestripped grafts (average: 7.1% ± 3.3%, range: 3.3%-13.7%) and preloaded grafts (average: 11.3% ± 4.2%, range: 6.9%-19.4%) was similar to previous reports. The footprint of the S-stamp was approximately 45% larger than that of the F-stamp. In 10 preloaded grafts marked with both stamps, the S-stamp caused an average ECL of 1.9% ± 0.6% (range: 1.2%-3.2%), whereas the smaller F-stamp caused an average ECL of 1.0% ± 0.2% (range: 0.8%-1.4%, P = 0.0002). CONCLUSIONS: Loss of endothelial cells associated with graft-stamping was greater in preloaded tissue than in prestripped tissue and was less with a smaller F-stamp than with a larger S-stamp. Using a smaller stamp could help minimize ECL in prestripped and preloaded DMEK grafts.

Microkeratome-assisted ultrathin Descemet's stripping automated endothelial keratoplasty: A randomized trial comparing single-pass versus double-pass technique.

Purpose: To compare the outcomes of two techniques, for preparation of microkeratome-assisted ultrathin grafts for Descemet's stripping automated endothelial keratoplasty (DSAEK). Methods: The study involved 20 eyes of 20 patients with pseudophakic bullous keratopathy, randomized into two groups. Group 1 eyes underwent microkeratome-assisted DSAEK using the single-pass technique for lenticule preparation, whereas group 2 eyes underwent microkeratome-assisted DSAEK using the double-pass technique. Patients were followed up till 6 months, postoperatively. Best-corrected visual acuity (BCVA) at final follow-up was considered as the primary outcome measure, whereas graft thickness (GT) contrast sensitivity and endothelial cell loss were considered as the secondary outcome measures. A P value of <0.05 was considered as statistically significant. Results: Baseline characteristics of two groups were comparable. The mean central GT was comparable in both groups at 6 months follow-up [group 1: 98 ± 24.46 µm, group 2: 129 ± 31.46 µm (P = 0.18)]. Both groups fared equally in terms of BCVA (P = 0.33). Contrast sensitivity was significantly better in group 1 eyes (P = 0.045). A statistically significant negative correlation was found between postoperative BCVA and postoperative GT (R = -0.728, P = 0.016). The percentage endothelial cell loss was slightly higher in group 2 eyes, although not statistically significant. Two eyes in group 2 experienced complications during lenticule preparation. None of the eye experienced any complication in the postoperative period. Conclusion: Both techniques provided grafts with comparable thickness and endothelial cell loss and were associated with comparable BCVA, at final follow-up visit. The contrast sensitivity was, however, better in eyes receiving grafts prepared with the single-pass technique.

Comparison of preservation and transportation protocols for preloaded Descemet membrane endothelial keratoplasty.

BACKGROUND/AIMS: Descemet membrane endothelial keratoplasty (DMEK) preparation is technically demanding and is a limiting factor for uptake of this kind of surgery. Supply methods that simplify the procedure for surgeons are key to increasing uptake. This study compares two different shipping protocols for DMEK. METHODS: An 8.5 mm DMEK graft was punched, marked and loaded for transportation in two different conditions: (A) endothelium trifolded inwards in organ culture conditions (n=7) and (B) endothelium rolled outwards in hypothermic conditions (n=7). Tissues were shipped from Italy to the UK, then analysed for orientation, endothelial cell density, denuded areas, cell mortality, triple viability staining (Hoechst/ethidium homodimer/calcein AM (HEC)), immunolocalisation of ZO-1 and Na/K-ATPase proteins, visualisation of actin filaments using phalloidin and histological analysis using H&E on paraffin-embedded sections. RESULTS: All tissues clearly showed the mark used for graft orientation. After shipping in condition A, there was an increase in cell mortality of 8.1% and in denuded areas of 22.4%, whereas for condition B there was an increase in cell mortality of 14.2% and in denuded areas of 34.3% after shipping. HEC staining revealed areas of viable cells and apoptotic cells, with large denuded areas found in the periphery for condition B and within folds for condition A. CONCLUSIONS: Prestripped preloaded DMEK grafts retained sufficient viable cells for transplantation, with condition A (endothelium-in) offering the advantage of greater flexibility of use due to a longer shelf-life. HEC analysis provides further detailed information as to the status of DMEK grafts and should be used in future similar studies.

Evaluation and Quality Assessment of Prestripped, Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To determine graft quality and feasibility of Descemet membrane endothelial keratoplasty (DMEK) grafts that are prestripped and preloaded into injectors by eye bank technicians before shipping to surgeons. METHODS: DMEK grafts (n = 31) were prepared from donor corneas and preloaded into Straiko Modified Jones tubes and set inside viewing chambers filled with 20 mL of Optisol-GS. Preloaded grafts were evaluated using specular microscopy and slit-lamp biomicroscopy. Endothelial cell loss (ECL) was captured by vital dye staining and quantified using FIJI. A subset of preloaded tissues was subjected to a shipping validation and 5-day storage assay. Fourteen additional DMEK grafts (not preloaded) were examined to quantify damage resulting from prestripping alone. RESULTS: Specular microscopy was able to be performed for all preloaded tissues. Average ECL for preloaded tissues quantified by vital dye staining and FIJI after overnight storage was 16.8% ± 5.9%, and differed from slit-lamp ECL estimation by an average of 5.3% ± 3.6%. The average damage caused by prestripping alone was 9.3% ± 5.9%, and it was significantly less than that of preloaded tissues (P < 0.01). Average ECL for preloaded tissues subjected to round-trip shipping events was 18.5% ± 12.4%, and ECL for tissues stored at 4°C for 5 days after preloading was 13.1% ± 9.5%. CONCLUSIONS: It is possible to prepare, evaluate, and ship DMEK grafts loaded inside a glass carrier and viewing chamber. The ability to evaluate tissues after processing allows for adherence to the Eye Bank Association of America Medical Standards, and for surgeons to receive the most accurate tissue information.

In Vivo Imaging and Morphometry of the Human Pre-Descemet's Layer and Endothelium With Ultrahigh-Resolution Optical Coherence Tomography.

PURPOSE: To visualize in vivo and quantify the thickness of the posterior corneal layers: the acellular pre-Descemet's layer (PDL), Descemet's membrane (DM), and endothelium (END) in healthy subjects, using ultrahigh-resolution optical coherence tomography (UHR-OCT). METHODS: A research-grade, 800-nm UHR-OCT system with 0.95-µm axial resolution in corneal tissue was used to image in vivo the posterior cornea in healthy subjects. The system offers approximately 98 dB sensitivity for 680 µW optical power incident on the cornea and 34,000 A-scans/s image acquisition rate. This study comprised 20 healthy subjects, aged 20 to 60 years. The thickness of the PDL, DM, and END layers was measured both with a custom, automatic segmentation algorithm and manually. RESULTS: The boundaries and structure of the posterior corneal layers were clearly visible in the UHR-OCT images. The average thickness was measured to be 6.6 ± 1.4 µm (PDL), 10.4 ± 2.9 µm (DM), and 4.8 ± 0.4 µm (END), which agrees well with published data from ex vivo studies. Both the END and DM thickness showed minor spatial variations, whereas the PDL showed up to 2× thickness change for different locations on the same cross-sectional corneal image or over the entire imaged region of the cornea. CONCLUSIONS: Our data indicate that all three layers of the posterior cornea can be clearly visualized in vivo and their thicknesses measured precisely with UHR-OCT. Although the PDL thickness showed large spatial variations, the thickness of the DM and END layers was consistent over the entire imaged region of the cornea.