(6bi) Engineering of the Corneal Epithelium

Every year, two million people worldwide suffer from corneal blindness.  While the traditional therapy (the use of corneal transplants) is highly successful (72% retention after five years), the number of donors is insufficient, especially on developing countries.  Besides this, corneal transplants are not suitable for some pathologies such as dry eye syndrome or alkali burns.  These facts highlight the need to develop suitable artificial corneas.  However, the artificial corneas currently on the market suffer from integration problems such as extrusion, infection, leakage or epithelial downgrowth.  In general, these devices address the integration to the host only through the stroma of the cornea (the middle portion of the cornea), therefore we propose that their success rate would improve if we promote the formation and maintenance of a healthy corneal epithelium, which is a highly specialized stratified tissue with a very high regenerating potential that covers the cornea and acts as a barrier to protect from mechanical injury and infection.

The healthy functioning of the corneal epithelium depends mainly on three attributes:

1)      Barrier properties, dependent on the formation of tight junctions between cells.  The corneal epithelium must have impermeable characteristics to prevent the hydration of the stroma (corneal edema) and the penetration of pathogens.

2)      Adherence of the basal cells to the underlying basement membrane.  The basement membrane of the corneal epithelium is a laminar structure that provides desmosomal attachments to the corneal epithelium.  The physicochemical characteristics of the basement membrane of the cornea have been demonstrated to affect the adhesion, shape, migration, differentiation and proliferation of the corneal epithelial cells.

3)      Hydration.  The cornea has the unique characteristic of being an avascular tissue.  The nourishment of the corneal epithelium is achieved mainly through the adequate formation of a tear film covering the ocular surface.

The epithelialization of corneal prosthetics must replicate the process of epithelial wound healing, which occurs in four steps:  1) Lag phase, where cells reorganize their attachments to adjacent cells and basement membrane; 2) Migration phase, where the leading edge of the wound migrates toward the center of the defect; 3) Differentiation phase, where epithelial cells proliferate and stratify, and 4) Maintenance phase, where basement membrane components are synthesized, assembled and remodeled.  Since the basement membrane of the corneal epithelium is the microenvironment providing cues to the epithelial cells, it is fundamental to have a surface that supports cells adhesion, is permeable to nutrients and metabolites, and presents physical and biochemical cues to the cells.  Therefore, we propose that by engineering an artificial basement membrane of the corneal epithelium we can promote the epithelialization of corneal prosthetics.

The design of an artificial basement membrane must incorporate three different types of signals: 1) Biochemical:  ligand-receptor interactions; 2) Mechanical: cells respond to the stiffness the substrate by modifying the cytoskeletal elements, and 3) Topographical:  The 3-D topography of the extracellular matrix affect the behavior of the cells.  We developed a hydrogel substrate with controlled compliance that can be functionalized with specific biomolecules.  This substrate can be molded with topographic features ranging from the micro to the nanoscale that can mimic the rich topography found in the native basement membrane.

By using nonfouling hydrogel substrates, such as poly (ethylene glycol), functionalized with specific molecules such as the cell-adhesive peptide RGD, we were able to successfully isolate the confounding influence of nonspecific protein adsorption from the medium onto the surfaces from other soluble factors and provided evidence that nanoscale topography is a fundamental biomimetic cue impacting cell phenotype.  Furthermore, our biomimetic topographic substrates improved the rates of healing due to increased migration of the wound border towards the center of an in vitro wound.  These substrates may be fabricated as laminates and be incorporated to the surface of artificial corneas to increase their success rate.

However, as mentioned earlier, the maintenance of a healthy corneal epithelium depends on the nourishment of the corneal epithelial cells through the aqueous tears; and some pathologies of the ocular surface such as the dry eye syndrome impedes the formation of a film that hydrates the epithelial cells.

Dry eye syndromes are a heterogeneous group of pathologies of the ocular surface, affecting up to 20% of the adult population.  In spite of its name, dry eye are not exclusively due to drying or lack of aqueous tears, but also to insufficient wettability of the epithelial surface. 

The tear film is a stratified structure, forming three principal phases: mucous phase, aqueous phase and lipid phase.  Traditionally, the treatment of dry eye is focused on replacing the aqueous tears with the use of eye drops or reducing the evaporation by treating the lipid layer of the tears.  However, the interaction between the different components and phases of the tear film is largely understudied.  We hypothesize that those surface properties are relevant for the stability of the tear film and propose the engineering of the epithelial surface to modify its wetting properties and improve the retention of the fluid on the ocular surface.

The wettability of the ocular surface has traditionally been attributed to the existence of a thick glycocalyx covering the cell surface, composed mainly of membrane-associated mucins and other glycosylated proteins.  However, the dependence of the surface properties of the corneal epithelium on the expression of membrane-associated mucins has never been characterized.   We have investigated the influence of the glycocalyx on the surface properties of the ocular epithelium by inducing the expression of mucins on the apical surface of corneal epithelial cells.  We measured the contact angle of the cell surfaces using a two fluid method, with phosphate buffer saline as the medium and perfluorocarbons (perfluorodecalin, perfluorooctane or tetradecafluorohexane) as the testing droplets, and found a significant increase of the contact angle hysteresis when ocular mucins were expressed on the surface.  This contact angle hysteresis is most likely due to the differential expression of mucins on the cell surface (chemical heterogeneity), where some cells show low expression of surface glycans and other cells show high surface glycosylation.  This high contact angle hysteresis is closely related to adhesion of the droplets onto the surface, impeding the growth of dry patches and may be key for the stability of the tear film.

Although our current aim is to improve the outcomes of artificial corneas through the application of chemical engineering principles; the general approaches and efforts used in our research may also be used to enhance the integration of other medical devices, improve the outcomes of wound healing, or develop novel therapies to treat dry mucosal syndromes.