Descemet's Membrane Biomimetic Microtopography Differentiates Human Mesenchymal Stem Cells Into Corneal Endothelial-Like Cells.

PURPOSE: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. METHODS: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. RESULTS: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. CONCLUSIONS: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

"Ultrathin" DSAEK tissue prepared with a low-pulse energy, high-frequency femtosecond laser.

PURPOSE: To evaluate the endothelial cell survival and stromal bed quality when creating deep stromal cuts with a low-pulse energy, high-frequency femtosecond laser to produce "ultrathin" tissue for Descemet stripping automated endothelial keratoplasty. METHODS: Seventeen corneas were used for this study. Five corneas were cut with the laser at a depth of 420 to 500 µm to produce a tissue thickness of approximately ≤70 µm. Five corneas served as an uncut comparison group. Vital dye staining and computer digitized planimetry analysis were performed on these corneas. The 7 remaining corneas were cut for scanning electron microscopy evaluation. RESULTS: The mean central posterior stromal thickness of cut corneas was 60.6 µm (range, 43-72 µm). Endothelial cell damage in cut and comparison corneas was 3.92% ± 2.22% (range, 1.71%-6.51%) and 4.15% ± 2.64% (range, 1.21%-7.01%), respectively (P = 0.887). Low-magnification (×12) scanning electron microscopy revealed a somewhat irregular-appearing surface with concentric rings peripherally. Qualitative grading of higher magnification (×50) central images resulted in an average score of 2.56 (between smooth and rough). CONCLUSIONS: Ultrathin tissue for Descemet stripping automated endothelial keratoplasty can be safely prepared with minimal endothelial cell damage using a low-pulse energy, high-frequency femtosecond laser; however, the resulting stromal surface quality may not be optimal with this technique.

Volumetric and ionic regulation during the in vitro development of a corneal endothelial barrier.

Corneal endothelium is responsible for generating an ion flux between the corneal stroma and the anterior chamber of the eye that is necessary for the cornea to remain transparent. However, the ion transport regulatory mechanisms that develop during the formation of the endothelial barrier are not known. In this study, we determined the influence of cell confluence on cell volume and intracellular ionic content on the corneal endothelial cells of rabbits. Our results demonstrate that non-confluent endothelial cells display a hypertrophic volume increase, with higher intracellular contents of potassium and chlorine than those of confluent cells. In contrast, when cells reach confluence and the endothelial barrier forms, cell volume decreases and the intracellular contents of potassium and chlorine decrease. Our genetic analysis showed a higher expression of CFTR and CA2 genes in non-confluent cells, and of the gene KCNC3 in confluent cells. These results suggest that the normal ionic current that keeps the corneal stroma dehydrated and transparent is regulated by cell-cell contacts and endothelial cell confluence, and could explain why the loss of corneal endothelial cells is often associated with corneal edema and even blindness.

Lidocaine effects on corneal endothelial cell ultrastructure.

The purpose of this study was to determine whether intracameral commercial lidocaine 2% induces alterations on the rabbit corneal endothelium. Forty white rabbits received different substances inside the anterior chamber: group (G)1, no substance; G2 and G3 received lidocaine 2% with preservative in aqueous solution; G4 and G5, lidocaine 2% with preservative in gel solution; G6 and G7, the anesthetic preservative (metilparahydroxybenzoate 0.1%); and G8 and G9, lidocaine 2% without preservative in aqueous solution. The animals from G2, 4, 6 and 8 were sacrificed after 1 h, and from G3, 5, 7 and 9 after 24 h after injection of the substance inside the anterior chamber. The corneas were clinically evaluated and assessed by transmission and scanning electron microscopy. G1, 2, 6, 7, 8 and 9 animals had very similar characteristics in clinical, ultrastructural and morphometric evaluations; the G3 and G4 animals showed discrete edema and one animal in G5 had intense corneal edema. We conclude that lidocaine 2% with preservative induces few ultrastructural alterations in the corneal endothelial cells.

Staining ability and biocompatibility of brilliant blue G: preclinical study of brilliant blue G as an adjunct for capsular staining.

OBJECTIVE: To evaluate the effectiveness and biocompatibility of brilliant blue G (BBG) for capsular visualization for continuous curvilinear capsulorrhexis. METHODS: The capsular staining ability of BBG was evaluated at graded concentrations of 10.0, 1.0, 0.5, 0.25, 0.1, and 0.01 mg/mL in enucleated pig's eyes. The biocompatibility of BBG was assessed in rat's eyes for 2 months. The eyes were analyzed using light, fluorescence, transmission electron, and scanning electron microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) was used to detect apoptotic cells, and endothelial cell counts were analyzed using scanning electron microscopy. The results were compared using indocyanine green and trypan blue. RESULTS: The BBG improved capsular visualization, and a complete capsulorrhexis could be performed. In the rat model, no apparent toxic effect was observed using biomicroscopy during 2 months. Histologically, BBG showed satisfactory biocompatibility. Apoptotic cell death of the endothelial cells was detected in only the trypan blue group. In contrast to BBG, indocyanine green and trypan blue showed degeneration of corneal endothelial cells using transmission and scanning electron microscopy. CONCLUSION: The BBG contributed to better capsular visualization and caused no apparent complications to the corneal endothelium.Clinical Relevance The BBG is effective and safe capsular staining for continuous curvilinear capsulorrhexis.

Long-term outcome of iron-endocytosing cultured corneal endothelial cell transplantation with magnetic attraction.

The long-term efficacy and safety of transplanting iron-endocytosing cultured corneal endothelial cells (CECs) with magnetic attraction were evaluated. Rabbit corneas were subjected to cryo-injury to detach CECs. Cultured rabbit CECs (RCEC) were exposed to spherical iron powder and then injected into the anterior chamber, after which a neogium magnet was fixed on the eyelid for 24 hr to attract the cells to Descemet's membrane (RCEC-iron group, n=4). An RCEC group (cryo-injury and injection of normal cultured RCEC, n=4) and a Cryo group (cryo-injury without injection of RCEC, n=4) served as controls. Intraocular pressure was measured for 12 months after surgery. Corneal findings on slit lamp biomicroscopy, RCEC density, the electro-retinogram (ERG), and residual iron in the ocular tissues were evaluated at final assessment. Intraocular pressure did not increase in any group throughout 12 months of observation. At the final assessment, the average corneal edema score of the RCEC-iron group was significantly lower than that of the RCEC or Cryo groups (p=0.021). The average CEC density of the RCEC-iron group was 2581+/-230 cells mm(-2) (mean+/-SD), whereas no CECs were observed on the inner surface of the central cornea in the RCEC and Cryo groups. No significant differences of the ERG (a- and b-wave amplitudes, and b-wave/a-wave ratio) were detected among the groups. Iron powder was not detected by Berlin blue staining in the ocular tissues of the RCEC-iron group. Apoptotic cells were not observed in the endothelium by terminal transferase-mediated nick-end labeling. Transplanted iron-endocytosing RCEC remained viable for 12 months after surgery. There were no detectable ocular complications after the transplantation of iron-endocytosing cultured RCEC. Magnetic attachment of iron-endocytosing CECs can be an effective and safe method for corneal endothelial repair.

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.

PURPOSE: To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. METHODS: Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. RESULTS: When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. CONCLUSIONS: FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.

Acute effects of H-7 on ciliary epithelium and corneal endothelium in monkey eyes.

PURPOSE: Topical or intracameral administration of H-7 doubles outflow facility and reduces intraocular pressure in cynomolgus monkeys, by relaxing and expanding the trabecular meshwork (TM) and Schlemm's canal (SC). Since H-7 may have anti-glaucoma potential, we determined its effects on the corneal endothelium and ciliary epithelium for safety considerations. METHODS: Following topical H-7, aqueous humor flow (AHF), corneal endothelial transfer coefficient (k(a)) and anterior chamber (AC) entry of i.v. fluorescein were measured by fluorophotometry; AC aqueous protein concentration ([Protein](AC)) was determined by Lowry assay; and corneal thickness and endothelial cell density and morphology were measured by ultrasonic pachymetry and specular microscopy respectively. Following intracameral H-7, specular and/or light and electron microscopy of the corneal endothelium or ciliary epithelium were performed. RESULTS: Following unilateral topical H-7: (1) AHF and k(a) were essentially unchanged at 0.5--3.0, 3.5--6.0, and 0.5--6.0 hr, with an insignificant increase from 0.5--1.5 hr; (2) [Protein]( AC) was insignificantly increased at 1-1.5 hr but had returned to baseline by 2.5 hr; (3) entry of i.v. fluorescein into aqueous or cornea was modestly and transiently increased; (4) the central cornea thickened significantly at 1--2.5 hr, gradually returning to baseline 2.5 hr after H-7, while peripheral corneal thickness was less affected; (5) corneal endothelial cell borders became indistinct by 1 hr, but cell morphology was recovering by 3--5 hr and had completely returned to normal by 24 hr; (6) corneal endothelial cell density was unchanged at 5--24 hr. Following intracameral H-7, no significant changes were observed in corneal endothelial cell density or morphology by specular microscopy, nor in corneal endothelial or ciliary epithelial morphology by light and electron microscopy. CONCLUSIONS: A facility-effective intracameral dose of H-7 had no discernible structural effect on the corneal endothelium or ciliary epithelium. It is not yet clear whether carefully chosen topical doses of H-7 or analogues can enhance outflow facility without meaningfully affecting the cornea and ciliary processes.

Corneal endothelial toxicity of different lidocaine concentrations.

PURPOSE: To examine the potential damaging effect on the corneal endothelium of unpreserved lidocaine in concentrations of 1%, 5%, and 10%. SETTINGS: Department of Ophthalmology, Charité Medical Faculty, Humboldt University, Berlin, Germany. METHODS: Experimental porcine corneas (n = 18) were exposed to 100 microL of unpreserved lidocaine hydrochloride at concentrations of 1%, 5%, and 10% for 60 minutes. Additional corneas (n = 6) were treated with lidocaine hydrochloride 1% for 30 minutes to simulate clinical conditions. Balanced salt solution (BSS((R))) served as a control to evaluate corneal endothelial cell damage using Janus Green photometry. Morphology, damage pattern, and changes in the ultrastructural appearance of corneal endothelial cells were examined by light and scanning electron microscopy. RESULTS: Lidocaine 1% used for 30 or 60 minutes did not cause significantly more corneal endothelial damage (mean 3.00% +/- 0.76% [SD] and 3.26% +/- 1.00%, respectively) than in the control group (mean 3.32% +/- 0. 86%) (P >.01). Significant corneal endothelial cell loss was observed with lidocaine 5% (mean 10.7% +/- 6.4%) (P <.001) and lidocaine 10% (42.3% +/- 17.0%) (P <.001). CONCLUSION: Experimental exposure of corneal endothelial cells to higher concentrations of lidocaine resulted in significant cell loss, indicating that the 1% concentration only should be used clinically.

Comparison of preservative-free bupivacaine vs. lidocaine for intracameral anesthesia: a randomized clinical trial and in vitro analysis.

PURPOSE: To determine whether intracameral bupivacaine hydrochloride 0.5% is as effective as lidocaine hydrochloride 1.0% in controlling discomfort of patients during phacoemulsification and posterior chamber intraocular lens implantation. In rabbits, corneal endothelial cell function, ultrastructure, and viability were evaluated after in vitro perfusion of bupivacaine 0.5%. METHODS: In a double-masked, controlled trial, 48 eyes of 48 patients with uncomplicated age-related cataract were randomly assigned to receive bupivacaine 0.5% or lidocaine 1.0% intracamerally before phacoemulsification with a posterior chamber intraocular lens. Outcome measures such as pain, visual acuity, amount of sedation, length of surgery, pupil size, intraocular pressure, corneal clarity, and anterior chamber reaction were compared. In laboratory studies, paired rabbit corneas were evaluated by endothelial cell perfusion with either bupivacaine 0.5%, bupivacaine 0.5% and glutathione bicarbonate Ringer solution in a 1:1 ratio or bupivacaine 0.5% buffered to a pH of 7.0. The paired control corneas were perfused with glutathione bicarbonate Ringer solution and rates of corneal swelling were determined. Cell ultrastructure and viability were also evaluated. RESULTS: In the randomized trial, there was no significant difference in the pain patients had during surgery or in the early or late postoperative period. No statistically significant difference was seen between the two groups in terms of pupil size, intraocular pressure, corneal edema, anterior chamber reaction, or visual acuity immediately after the operation or on postoperative day 1. Paired rabbit corneas perfused with bupivacaine 0.5% and bupivacaine 0.5% buffered to a pH of 7.0 swelled significantly (P<.001, P = .009, respectively), and had corneal endothelial cell damage. Dilution of the bupivacaine 1:1 with glutathione bicarbonate Ringer solution prevented corneal edema and damage to the corneal endothelium. Endothelial cell viability was also decreased after perfusion of bupivacaine 0.5% (P<.001). CONCLUSIONS: Clinically, bupivacaine 0.5% is as effective as lidocaine 1.0% for anesthesia during phacoemulsification and posterior chamber intraocular lens implantation. However, in vitro perfusion of bupivacaine 0.5% damaged the corneal endothelium of rabbits except when the drug was diluted 1:1 with glutathione bicarbonate Ringer solution. Surgeons who use 0.2 to 0.5 ml of intracameral bupivacaine 0.5% should be aware of its potential to cause endothelial cell damage because of its lipid solubility. The bupivacaine 0.5% should be diluted at least 1:1 with balanced salt solution before intracameral injection, followed immediately by phacoemulsification. The surgeon should ensure that the bupivacaine 0.5% is nonpreserved and packaged in single-use vials or flip-top containers.

Effect of intracameral anesthesia on the corneal endothelium.

PURPOSE: To evaluate the effects of intracameral anesthesia on the corneal endothelium. SETTING: Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Japan. METHODS: This study comprised 24 eyes of 12 white rabbits. One eye of 3 rabbits each was injected with preservative-free lidocaine at concentrations of 0.02, 0.2, or 2% and the fellow eye injected with balanced salt solution (BSS) as a control. The anesthetic agent was injected into the anterior chamber using a bimanual technique. Immediately after enucleation, the cornea was examined by scanning electron microscopy. RESULTS: Scanning electron microscopy revealed no abnormal findings in the eyes injected with lidocaine 0.02 or 0.2% when compared with eyes in the control group. Scanning electron microscopy of the eyes injected with lidocaine 2% showed irregular hexagonal endothelial cells and a significant loss of microvilli. CONCLUSION: Intracameral anesthesia with high concentrations of lidocaine risks corneal endothelial damage but at the low concentration usually used in cataract surgery did not appear to have an adverse effect.

Efficacy and safety of gentamicin and streptomycin in Optisol-GS, a preservation medium for donor corneas.

Increasing reports of gentamicin-resistant bacteria contaminating donor corneas and causing endophthalmitis have indicated that preservation of corneal storage media with 100 micrograms/ml of gentamicin alone needs reevaluation. We investigated the stability and possible cytotoxicity of streptomycin as a supplement to gentamicin in Optisol corneal storage medium. The combination of gentamicin and streptomycin in Optisol solution was stable at room temperature for at least 4 weeks and inhibited the growth of Staphylococcus aureus, S. epidermidis, alpha hemolytic streptococci, Streptococcus Group D, Propionibacterium acnes, Escherichia coli, and diphtheroids, but not Pseudomonas aeruginosa. The addition of vancomycin did not significantly improve the antibacterial effectiveness of the gentamicin and streptomycin combination when stored at 4 degrees C. The growth of 15 of 20 clinical ocular isolates of Ps. aeruginosa was suppressed by the gentamicin-streptomycin combination. Streptomycin in concentrations of up to 1,000 micrograms/ml did not decrease the mitotic activity of corneal endothelial cells as evaluated by the in vitro incorporation of tritiated thymidine or cause cytotoxicity. The addition of 200 micrograms/ml of streptomycin to Optisol corneal storage medium containing 100 micrograms/ml of gentamicin may significantly improve activity against gentamicin-sensitive and gentamicin-resistant contaminants.

Advances in corneal preservation.

The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance and have been primary goals in the development of corneal storage media. In our investigational studies we have specifically addressed the improvement of the quality of donor tissue after 4 degrees C storage, the extension of corneal preservation time, the enhancement of corneal wound healing, and the reduction of the normal progressive loss of endothelial cells postkeratoplasty. Specifically we have developed in vitro HCE cell and epithelial cell culture models that can accurately reflect the response of human corneal tissue in vivo. These models have been utilized to study the effects of growth factors and medium components in relation to their biocompatibility and efficacy in the development of improved corneal preservation solutions. Our laboratory investigated in vitro conditions that allowed human corneal endothelium to shift from a nonproliferative state, in which they remain viable and metabolically active, to a proliferative, mitotically active state. Isolation techniques developed in our laboratory have enabled the establishment of primary and subsequent subcultures of human corneal endothelium that retain the attributes of native endothelium. These in vitro conditions maintain HCE cells in a proliferative state, actively undergoing mitosis. A quantitative bioassay has been developed to determine the effects of various test medium in the stimulation or inhibition of DNA synthesis. In attempting to learn more about the events that occur during in vitro endothelial cell isolation, cell reattachment, extracellular matrix interaction and migrating during subculture, SEM was done on isolated HCE cells incubated in CSM. These studies suggest that the components of the extracellular matrix modulate the growth response of HCE cells, and play a role in regulating proliferation and migration. These observations are important in view of the fact that anterior chamber environment limits cell regeneration of the endothelium, and supports wound healing via cell migration. In vivo, it is the complex interaction of the HCE cell and the extracellular matrix that signal the cell to respond to cell loss in this manner. As our knowledge of human corneal endothelium has increased so has our anticipation of developing the "optimum" medium. Thus additional components have been added to this basic medium to address specific complications encountered with 4 degrees C corneal preservation. Antioxidants, additional energy sources, and other nutritive substrates have been used to supplement and further define a chondroitin sulfate-based medium. These changes have been a part of our new awareness that, even at 4 degrees C, the cornea is metabolically active.(ABSTRACT TRUNCATED AT 400 WORDS)