Corneal Endothelial Blebs Induced in Scleral Lens Wearers.

SIGNIFICANCE: In the bleb phenomenon, some endothelial cells transiently lose their specular reflection. This has been reported during contact lens wear and goggle-induced hypoxia or hypercapnia. PURPOSE: The purposes of this study were to determine whether blebs appear after scleral lens wear and if their appearance is influenced by lens clearance and to compare bleb and cell sizes. METHODS: Twenty-one subjects were fitted with two similar scleral lenses with different targeted clearances of 200 and 400 µm (the SL200 and SL400, respectively). Each lens was worn unilaterally for 25 minutes, whereas the other eye served as a control. Before and after lens wear, the endothelium was photographed using specular microscopy. The number of blebs and measurements of the areas of cells and blebs were analyzed. Paired t tests compared differences in the areas of cells and blebs. Differences in median bleb number were evaluated using the Wilcoxon test. RESULTS: After wearing the SL200 and SL400 lenses, respectively, 9 and 14 subjects had at least one bleb. The median bleb number after wearing lenses was significantly different (SL200, 0.00; SL400, 1.00; P = .02). Bleb and cell areas were significantly different (blebs, 293 ± 28; cells, 370 ± 32 µm; P < .0001). CONCLUSIONS: After 25 minutes of wearing scleral lenses with each of the two targeted clearances, SL400 induced significantly more blebs than did SL200, suggesting evidence of reduced oxygen and/or increased carbon dioxide levels under scleral lenses fitted with excessive clearance. Blebs may occur more in smaller cells.

The presence of a feeder layer improves human corneal endothelial cell proliferation by altering the expression of the transcription factors Sp1 and NFI.

Based on the use of tissue-cultured human corneal endothelial cells (HCECs), cell therapy is a very promising avenue in the treatment of corneal endothelial pathologies such as Fuchs' dystrophy, and post-surgical corneal edema. However, once in culture, HCECs rapidly lose their phenotypic and physiological characteristics, and are therefore unsuitable for the reconstruction of a functional endothelial monolayer. Expression of NFI, a transcription factor that can either function as an activator or a repressor of gene transcription, has never been examined in endothelial cells. The present study therefore aimed to determine the impact of a non-proliferating, lethally irradiated i3T3 feeder layer on the maintenance of HCEC's morphological characteristics, and both the expression and stability of Sp1 (a strong transcriptional activator) and NFI in such cells. The typical morphology of endothelial cells was best maintained when 8 × 103/cm2 HCECs were co-cultured in the presence of 2 × 104 cells/cm2 i3T3. HCECs were found to express both Sp1 and NFI in vitro. Also, the presence of i3T3 led to higher levels of Sp1 and NFI in HCECs, with a concomitant increase in their DNA binding levels (assessed by electrophoretic mobility shift assays (EMSA)). Specifically, i3T3 increased the expression of the NFIA, NFIB and NFIC isoforms, without a noticeable increase in their mRNAs (as revealed by gene profiling on microarray). Gene profiling analysis also identified a few feeder layer-dependent, differentially regulated genes whose protein products may contribute to improving the properties of HCECs in culture. Therefore, co-culturing HCECs with an i3T3 feeder layer clearly improves their morphological characteristics by maintaining stable levels of Sp1 and NFI in cell culture.

Oxygen Tension Beneath Scleral Lenses of Different Clearances.

PURPOSE: To evaluate the relative partial pressure in oxygen (pO2) at the corneal surface under Boston XO2 scleral lenses (SL) fitted with targeted clearances of 200 and 400 µm (SL200 and SL400). METHODS: During this prospective study, the right eyes of eight normal subjects were fitted with SL200 and SL400. Clearance, validated after 5 minutes of wear with an optical coherence tomograph, was used with lens thicknesses to calculate transmissibility and estimate pO2. Corneal pO2s were measured with an oxygen electrode after 5 minutes of (1) corneal exposure to calibrating gases with various pO2 or of (2) SL wear. Decays in pO2 were modeled to an exponential. Linear regression between exponent k of these decays and calibrating gas pO2s allowed for the calculation of corneal pO2 under SL. Differences between pO2s beneath SL200 and SL400 were tested with a mixed ANOVA. RESULTS: The estimated transmissibility based on thicknesses and clearances (239.7 ± 34.7; 434.5 ± 33.2 µm) predicted a corneal pO2 of 8.52 ± 0.51 and 6.37 ± 0.28% for SL200 and SL400. These values were close to measured pO2: 9.07 ± 0.86 and 6.19 ± 0.87% (mean ± SEM) (P < .05) for SL200 and SL400, respectively. Both pO2 measurements fall short of the theoretical values needed to prevent hypoxia during lens wear (9.9% and above). CONCLUSIONS: As shown in vivo for the first time, an 18-mm scleral lens fitted with a 400-µm clearance reduces the oxygen tension available to the cornea by 30% compared to a similar lens fitted with a 200-µm clearance after 5 minutes of wear.

Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

PURPOSE: To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. METHODS: Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or ß-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. RESULTS: Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and ß) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. CONCLUSIONS: This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents.

Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.

PURPOSE: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. METHODS: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. RESULTS: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. CONCLUSIONS: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.

Intra-ocular pressure variation associated with the wear of scleral lenses of different diameters.

PURPOSE: To evaluate the variation of intra-ocular pressure during scleral lens wear, and the influence of the lens diameter on the results. METHODS: This is a prospective, randomized study performed on Caucasian subjects (16 F; 5 M), aged 24.7 + 4.1 y.o. A diurnal variation pattern (IOPg) was established, then, transpalpebral IOP (IOPt) was taken before and during SL wear. One eye, randomly fitted with a 15.8 diameter SL (L1), was compared to the fellow eye, fitted with an 18 mm SL of the same design, thickness and material (L2). Anterior segment tomography was taken pre-and after lens removal. RESULTS: Baseline IOPg (L1:15.2 + 3.1 mm HG; L2: 15.1 +/- 2.8 mm) did not reveal significant diurnal variations. Wearing L1, IOPt rose from 10.1 + 1.9 mm HG to 14.4 + 5.5 mm HG after 4.5 + 0.3 hrs, while with L2, it rose from 9.2 + 2.1 mm HG to 14.4 + 4.8 mm Hg. This difference is statistically significant based on time but not on lenses. Anterior segment parameters did not vary except for the anterior chamber volume (L1: -1.53 + 7.61 mm3; L2: -3.47 + 6.4 mm3), and for the corneal thickness (+2.1% with L1 and L2). CONCLUSION: These results suggest that, as evaluated with a non-standard transpalpebral methodology, IOP during scleral lens wear may be increased in average by 5 mm Hg, regardless of the lens diameter. More work is needed to confirm if practitioners should be warned when using SL on populations at risk for glaucoma.

Characterization of wound reepithelialization using a new human tissue-engineered corneal wound healing model.

PURPOSE: The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. METHODS: Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. RESULTS: Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that alpha v beta6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. CONCLUSIONS: The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.

Optimization of culture conditions for porcine corneal endothelial cells.

PURPOSE: To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology. METHODS: PCEC cultures were seeded at an initial cell density of 400 cells/cm(2) in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm(2) in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm(2) in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 microg/ml), ascorbic acid (10, 20, 40 microg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology. RESULTS: Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium. CONCLUSIONS: Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.

Morphometry of corneal endothelium in patients with corneal guttata.

OBJECTIVE: To study the morphology of the corneal endothelium in patients diagnosed with corneal guttata using an image processing algorithm based on a contour detection method. DESIGN: Retrospective observational case series. PARTICIPANTS: Twenty-four subjects with known corneal guttata. METHODS: Two hundred eight images of corneal endothelium, captured with a noncontact specular microscope were analyzed using the Contour method, which demonstrates endothelial cell density (ECD), coefficient of variation of cell area, percentage of 4- to 8-sided cells as well as the number, area, and coefficient of variation of corneal guttata. MAIN OUTCOME MEASURES: The number, surface area, and coefficient of variation of corneal guttata. RESULTS: Corneal position had no significant effect on ECD or on the percentage of endothelial cells with 4, 5, 7, or 8 sides. However, the coefficient of variation of images taken from the central cornea was significantly larger than those taken at the 2- and 6-o'clock positions. In addition, the percentage of hexagonal cells was significantly lower in pictures of the central position compared to those located in the upper paracentral position. The numbers and surface areas of guttata were significantly larger in pictures of the central compared to some paracentral positions. Subjects who had previously undergone cataract surgery with intraocular lens (IOL) implantation did not show different areas of corneal guttata, but exhibited a significantly lower cell density (1825+/-582) compared with unoperated patients (2400+/-457/mm2). Analogously, the only significant change observed in paired comparisons between the operated eye of patients with unilateral cataract extraction with IOL implantation and their unoperated fellow eye was a lower cell density obtained in operated eyes. Compared with normal subjects, subjects with corneal guttata were shown to have a significantly lower ECD, a lower proportion of hexagonal cells, and a higher coefficient of variation of cell area in the central cornea. CONCLUSION: This study supports the finding that corneal guttata mainly affect the central corneal area. A future prospective study using the described Contour detection method would be helpful to evaluate more accurately the risks associated with the evolution of corneal guttata into Fuchs' dystrophy.

Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction.

PURPOSE: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. METHODS: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. RESULTS: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. CONCLUSIONS: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency.

[Amnion and ocular surface problems]. / Amnios et problèmes de surface oculaire.

The amniotic membrane, the most internal placental membrane, has various properties useful in ophthalmology. Collected on delivery by elective Caesarean section, the amnion is prepared under sterile conditions, and, usually, cryopreserved until its use as a biological bandage or as a substrate for epithelial growth in the management of various ocular surface conditions. Specifically, the amnion is used to : (1) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or to (2) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. In this last condition, either hereditary or acquired after a thermal or a chemical burn, corneal stem cells, located at a transitional zone between the cornea and conjunctiva, are lost. These cells are essential for renewal of corneal epithelium in normal and in diseased states. The loss of these cells leaves the corneal surface free for invasion by conjunctival epithelium. Not only, does conjunctival epithelium support the development of vascularisation on the normally avascular cornea, but some conjunctival cells differentiate into mucus secreting goblet cells. Such a change in phenotype leads to loss of corneal transparency and visual disability. The removal of this fibro-vascular outgrowth in combination with transplantation of both amniotic membrane and corneal stem cells are used to treat this condition. The amnion stimulates the proliferation of less differentiated cells which have the potential to reconstruct the cornea. This potential is at the origin of the hypothesis that the amnion may provide an alternative niche for limbal stem cells of the corneal epithelium. It abounds in cytokines and has antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties, in addition to allowing, like fetal skin does, wound healing with minimal scar formation. These desirable properties are responsible for the increasing use of amniotic membrane in ophthalmology. The complete understanding of the mechanisms of action of amniotic membrane for ocular surface diseases has yet to be understood. Once revealed by research, they may provide new pharmacological avenues to treat ocular surface diseases.

Predicting estimates of oxygen transmissibility for scleral lenses.

BACKGROUND/PURPOSE: Although scleral contact lenses are prescribed with increasing frequency, little is known about their long-term effects on ocular physiology. The main goal of this paper is to predict values of oxygen transmissibility of scleral lens systems by applying the concept of resistors in series to parameters characteristic of current scleral lenses. A second aim is to find the maximal lens and post-lens tear layer thickness combinations above which hypoxia-induced corneal swelling would be found. METHODS: Theoretical calculations were used to predict the oxygen transmissibility of scleral lens systems, considering several material permeabilities (Dks 100-170), varying lens thicknesses (250-500 µm), the known tear permeability (Dk of 80) and expected post-lens tear layer thicknesses (100-400 µm). The Holden-Mertz Dk/t criteria of 24 Fatt units for the central cornea and the Harvitt-Bonanno criteria of 35 Fatt units for the limbal area were used as reference points. RESULTS: Our calculations of oxygen transmissibility, with varying tear layer and lens thicknesses, ranged from 10 to 36.7 at the scleral lens centers and from 17.4 to 62.6 at the peripheries. Our calculations of maximum central lens thicknesses show a practical range of 250-495 µm, in conjunction with a post-lens tear layer thickness of 100-250 µm. CONCLUSION: Our computations show that most modern scleral lenses, with recommended fitting techniques, should lead to some level of hypoxia-induced corneal swelling. Recommendations are made to minimize hypoxia-induced corneal swelling: highest Dk available (>150) lens with a maximal central thickness of 250 µm and fitted with a clearance that does not exceed 200 µm.

Tissue engineering of feline corneal endothelium using a devitalized human cornea as carrier.

The difficulties in obtaining good quality tissue for the replacement of corneas of patients suffering from endothelial dysfunctions have prompted us to evaluate the feasibility of producing a tissue-engineered (TE) corneal endothelium using devitalized human stromal carriers. Thus, corneal substitutes were produced by seeding cultured feline corneal endothelial cells on top of previously frozen human corneal stromas. After two weeks of culture to allow attachment and spreading of the seeded cells, the TE corneal endothelium was stained with alizarin red for endothelial cell count and fixed for histology, immunofluorescence labeling, scanning and transmission electron microscopy. Histology and Hoechst staining showed that there were no remaining cells in the devitalized stroma. After seeding, histology and transmission electron microscopy showed that the TE corneal endothelium formed a monolayer of tightly packed cells that were well adhered to Descemet's membrane. Scanning electron microscopy corroborated that the cells covered the entire posterior corneal surface and had an endothelial morphology. Alizarin staining showed that mean cell counts were 2272 +/- 344 cells/mm(2), indicating that the cell density was appropriate for grafting. The TE feline corneal endothelium also expressed the function-related proteins Na(+)/HCO(3)(-), ZO-1, and Na(+)/K(+)-ATPase alpha1, and could easily be marked with a fluorescent tracker. This study demonstrates the feasibility of reconstructing a highly cellular and healthy corneal endothelium on devitalized human corneal stromas.

Impact of cell source on human cornea reconstructed by tissue engineering.

PURPOSE: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. METHODS: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. RESULTS: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. CONCLUSIONS: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.

Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function.

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.

Does endothelial cell density correlate with corneal diameter in a group of young adults?

PURPOSE: In children, but not in the elderly, an association exists between corneal diameter and endothelial cell density (ECD). We tested whether such an association also held true in young adults. METHODS: The eyes of 35 healthy subjects (mean age, 23.1 +/- 3.1 years) were photographed by using a video camera and a noncontact endothelial microscope. Both sets of images were analyzed with image software and the contour method to measure corneal diameter, ECD, and endothelial coefficients. Axial lengths, refractive errors, and corneal curvatures were measured by using an A-scan ultrasonic biometer and kerato-refractometer. Measurements, averaged for the right and left eyes, were analyzed depending on (1) use of contact lenses, (2) ametropia, and on whether (3) axial length or (4) corneal diameter was above or below group means. Differences were tested for statistical significance with independent t tests and association with the Pearson correlation coefficient. RESULTS: ECD, corneal diameter, and spherical equivalent refraction were 3022 +/- 262 cells/mm2, 12.0 +/- 0.5 mm, and -3.1 +/- 2.5 D, respectively. The only significant differences between wearers and nonwearers of contact lenses were the spherical refractive equivalent and axial length. There was no correlation between ECD and corneal diameter or axial length. CONCLUSIONS: As opposed to previously reported results in children, but as found in the elderly, there is no correlation between ECD and corneal diameter in young adults. Therefore, corneal size cannot be considered a determinant of ECD in young adults.

Biocompatibility and light transmission of liposomal lenses.

PURPOSE: To validate the biocompatibility and transmittance properties of contact lenses bearing intact liposomes. These liposomal lenses loaded with therapeutics can be used as ophthalmic drug delivery systems. METHODS: The biocompatibility of soft contact lenses, coated with liposomes was evaluated through in vitro direct and indirect cytocompatibility assays on human corneal epithelial cells, on reconstructed human corneas and on ex vivo rabbit corneas. The direct and indirect transmission spectra of liposome-covered lenses were also evaluated to test if they transmit all wavelengths of the ultraviolet-visible spectrum, to thereby fulfill their optical function, without gross alteration of the colors perception and with a minimum of light dispersion. RESULTS: Contact lenses bearing layers of stable liposomes did not induce any significant changes in cell viability and in cell growth, compared with lenses bearing no liposome. Elution assays revealed that no cytotoxic compound leaks from the lenses whether bearing liposomes or not. Histological analyses of reconstructed human corneas and ex vivo rabbit corneas directly exposed to liposomal lenses revealed neither alteration to the cell nor to the tissue structures. Contact lenses bearing layers of liposomes did not significantly affect light transmission compared with control lenses without liposome at the wavelength of maximal photopic sensitivity, i.e., 550 nm. In addition, the contact lenses afford more eye protection in the ultraviolet spectrum, compared with the control lenses. CONCLUSIONS: Liposomal contact lenses are biocompatible and their transmittance properties are not affected in the visible light range.

Morphometry of cells and guttae in subjects with normal or guttate endothelium with a contour detection algorithm.

PURPOSE: To develop a semiautomatic method to analyze morphology of cells and guttae in corneal endothelium. METHODS: Specular endothelial pictures from 42 and 21 subjects with healthy and guttate corneas, respectively, were analyzed independently by two observers with cell contour-extracting routines. One observer also analyzed healthy endothelia with the Corner method (Bambi). Differences between observers and between methods in mean cell area (MCA), coefficient of variation (CV), and percentage of cells with five, six, or seven sides were tested for significance with paired t tests. The Contour analysis of pictures with guttae included their mean area. RESULTS: There were no significant differences in MCA, CV, or the percentage of cells with five, six, or seven sides between the measurements obtained on repeated analysis by the same observer or on a second analysis performed by a different observer with the Contour method. However, the differences between the Contour and Bambi methods were statistically significant for MCA (337.5 +/- 37.7 vs. 327.7 +/- 36.5), CV (0.32 +/- 0.05 vs. 0.30 +/- 0.05), and percentage of cells with six and seven sides, but not for the percentage of five-sided cells. In subjects with guttata, the MCA was 561 +/- 170 microm, and the mean area of guttae was 1,538 +/- 849 microm. CONCLUSIONS: This detection algorithm is repeatable and reproducible, and it generates a cell border overlay useful in analyzing the morphology of cells and guttae. The analysis of corneal guttae could become a useful follow-up procedure to discriminate between patients with corneal guttata and Fuchs dystrophy.

Overwear of contact lenses: increased severity of clinical signs as a function of protein adsorption.

This study compared the clinical behavior of disposable and frequent replacement Acuvue and 1-Day contact lenses. Each type of lens was worn on one eye according to the schedule recommended by the manufacturer, and on the other eye for a longer period of time, up to 30 days in length. Both type of lenses were prescribed on a daily-wear basis. The amount of protein collected from the lenses was measured using two spectrophotometric protein assays. Visual acuity and comfort, along with several other clinical signs, were classified according to Cornea and Contact Lens Research Unit (CCLRU) scales, and possible associations between each of these signs and the amount of protein extracted from the lenses was tested. A comparison between the lens worn on the compliant eye with the lens worn on the noncompliant eye allowed us to measure the impact of overwear on ocular health and subjective clinical findings. After four months of study, the overwear of Acuvue and 1-Day lenses significantly increased the amount of protein bound on the contact lenses, as well as the severity of upper conjunctival papillae, upper lid conjunctival hyperemia, and limbal congestion. Even if reduced values for visual acuity and noninvasive break-up time (NIBUT) were identified, these variations were not found to be statistically significant. The clinical implications of this study would allow a practitioner to identify, according to the variations in several clinical signs, a patient who overwears contact lenses, so that action may be taken to reduce possible deleterious effects on ocular health.