Claude J. Giasson

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.

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.

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.

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.

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 Descemets 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.

Dorzolamide and corneal recovery from edema in patients with glaucoma or ocular hypertension.

PURPOSE To investigate whether dorzolamide alters corneal hydration control in patients with glaucoma or ocular hypertension. METHODS Pachymetry, tonometry, and endothelial cell density were measured by a masked observer in 19 subjects with bilateral glaucoma or ocular hypertension. They were treated with 2% dorzolamide in one eye, and with saline in the other, before wearing contact lenses under patched eyes. Corneal thickness, measured each 30 minutes up to 4.5 hours after contact lens removal, enabled estimation of percentage recovery per hour and time for 95% of corneal thickness recovery for both eyes. Seven patients repeated this test after 1 year of dorzolamide use, and their results were compared with those of the preceding year. RESULTS After induction of hypoxic corneal edema, there was no significant difference between paired corneas in swelling levels (60.0+/-11.8 and 59.8+/-12.9 microm) (P = .94), time to 95% recovery (440.6+/-255.8 and 445.4+/-186.7 minutes) (P = .93), and percentage recovery per hour (38.1%+/-10.9% and 36.1%+/-9.6%) (P = .40). Subjects followed up after 1 year of dorzolamide use did not differ significantly in values of endothelial cell density, percentage recovery per hour, or time to 95% recovery from those obtained a year before. One subject developed persistent corneal edema after his stress test in the eye treated with dorzolamide. CONCLUSION There is no significant difference in the recovery from induced corneal edema after either a short-term or 1-year use of dorzolamide in patients with glaucoma or ocular hypertension with a normal corneal endothelium. One patient had persistent corneal edema after the stress test was performed on the dorzolamide-treated eye.

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.

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.

On-eye dehydration of proclear, resolution 55G and acuvue contact lenses

In order to compare the extent of dehydration of Proclear, Resolution 55G and Acuvue contact lenses overtime, 13 patients wore each lens during 4, 6 and 8 h. Three observers, initially shown to have a high inter-observer reliability, measured water content, with a soft contact lens refractometer before and after each period of wear. Absolute dehydration of Proclear and Resolution 55G lenses were similar after 4, 6 and 8 h of wear, but were smaller than for Acuvue lenses (1.2% compared to 5.7% after 8 h). The loss of lens mass following dehydration of Resolution 55G lenses, calculated with the change in lens relative water content was found to correlate very well with an in vitro study of the same material. Lens dehydration should be taken into account by clinicians when making their choice of soft contact lens.

Comparing the extent of protein build-up on several disposable lenses by two spectrophotometric methods

This study aims to quantify levels of protein build-up on high-water ionic (Type IV) disposable contact lenses worn by patients, by two spectrophotometric methods, previously validated only with conventional lenses. Forty lenses were analyzed and the related results were compared. The Ninhydrine-Lysozyme method and the Coomassie Blue Albumin one agreed in identifying the most spoiled lenses. Etafilcon A attracted significantly more protein than the other materials tested, which were in the following decreasing order of protein spoilation: Ocuflcon D, Ocufilcon A and Vasurfilcon A. These similar findings suggest that it is possible to use the above mentioned analysis methods for an accurate evaluation of the contamination of several materials of disposable/frequent replacement lenses as for conventional lenses.