Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

7.3.2Corneal epithelium/extracelllular matrix structure and repair

The corneal epithelium is composed of three types of cells: basal cells (one layer), wing cells (one to three layers), and squamous cells (three to four layers) – all of which tightly adhere to one another. The basal cells also form strong adhesion complexes with the underlying ECM and, ultimately, with Bowman’s layer.100 Only the basal cells have mitotic capabilities. Like all stratified epithelia in the body, the corneal epithelium is self-renewing; complete cellular turnover occurs every 5–7 days.101 Generally, after the basal cells undergo mitosis, the daughter cells begin to move upward toward terminal differentiation and eventual desquamation from the apical surface.

As part of its protective function, the corneal epithelium also has a strong wound-healing response. After corneal wounding, growth factor and cytokine levels increase in the tear fluid and stromal layers, and a provisional ECM is elaborated. Previous research has shown that epithelial recovery following an external injury is a complex process characterized by three overlapping phases.1 In the first phase, a single layer of epithelial cells at the wound margin becomes motile by forming cellular processes at the wound edge, releasing the hemidesmosomal attachments to the basement membrane and forming a provisional attachment complex called ‘focal contacts’. During this phase, the epithelial cells flatten and migrate as an intact sheet to cover the wound.

Adhesion of the migrating epithelial monolayer to the stroma is thought to be mediated by the glycoprotein FN.102 FN is normally not present beneath corneal epithelial cells but appears after injury and persists until migration is complete. FN contains both cell-specific binding sequences and a binding region for heparin sulfate and type IV collagen (basement membrane components).103 FN provides a temporary, subepithelial matrix on which the epithelial cells can migrate; this occurs in repetitive cycles, during which the cells cleave their attachments, advance, and then form new attachments. Recently, it has become known that a variety of other matrix components – such as LM-5, lumican, fibrin, and perlecan – are found in a temporary or

‘provisional’ matrix in response to wounding.104 A number of LM isoforms have been identified in the wounded cornea, indicating that, not only is LM important in the corneal epithelial cell–substrate adhesion complex, but it may play a role in cell migration during corneal wound healing.

In the second phase of corneal epithelial wound healing, cells distal to the original wound begin to proliferate to repopulate the wound area, and cell stratification and differentiation occur. In the third phase, the epithelium attaches to the basement membrane more firmly, via newly synthesized hemidesmosomes and associated type VII collagen containing anchoring filaments.105 The anchoring filaments pass through the basement membrane

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and are contiguous with anchoring fibrils that terminate as anchoring plaques in Bowman’s layer.

Integrins expressed on the corneal epithelial cell surface change during wound healing.106 Among the expressed integrins, α2β1, α3β1, and ανβ1 bind LM, collagen, and FN. Epithelial expression of β1 integrins has been shown to increase as FN in the wound increases and then decreases as wound healing becomes complete. α6β4 integrin is synthesized and redistributed in wound healing and binds LM-5, whereas LM-10 promotes reorganization of filamentous actin (F-actin) in corneal epithelial cells. The proteins LM

(isoforms 1, 5, and 10), k-LM, talin, integrin, and kalinin also play roles in the attachment of the epithelium to the stroma. Although much is known about the factors that make up the adhesion complexes, the precise sequence by which these adhesion complex components are assembled has not yet been fully elucidated. Certain growth factors, such as EGF,107 fibroblast growth factor (FGF)108 and transforming growth factor (TGF)-β1,109 enhance the rate of epithelial wound healing, and human EGF has been specifically shown to induce a dose-dependent increase in epithelial replication in the epithelial stem cells of the corneoscleral limbus.110

Extracellular gradients of growth factors, FN, or LM also may be important. The ECM contains a complex array of fixed charges, and the ionic charge of substrates has been shown to modulate corneal cell integrin expression, cell spreading, and cell motility. Thus, reorganization of cell surface receptors or extracellular molecules to induce a gradient of either receptor or ligand, respectively, is likely an initial event that activates cells asymmetrically, drives subsequent cytoskeletal reorganization, and directs cell migration.

7.3.3Corneal epithelial cell repair and growth ligands

Elucidation of signaling events and the underlying biological factors that produce them is key to understanding the fundamental elements required to produce an ECM-like environment. Identification of these important moieties for corneal epithelial cells has been substantial to date. LM and FN are multifunctional ECM proteins that play a central role in cell adhesion and migration. Expression of integrin receptors for both of these proteins has been shown to occur within hours of the ligands being detected in the matrix.37 LM is a major component of the intact basal lamina and has been shown to enhance epithelial cell adhesion and spreading on surfaces. Twelve isoforms of LM have been identified; however, only three isoforms – LM-1, LM-5, and LM-10 – have been consistently found within the basement membrane of the cornea.111, 112 In unwounded corneas, expression of lumican is limited to stromal keratocytes. The expressed lumican molecules are glycanated with keratin sulfate. In healing corneal wounds, a non-glycanated form of

lumican is produced and has been shown to significantly enhance epithelial cell migration and proliferation.113

FN, seen on the surface of healing epithelial wounds, is an insoluble glycoprotein dimer (unlike the soluble disulfide linked dimer) found in plasma. The primary transcript of FN is alternatively spliced and, therefore, different isoforms, such as EDA-, EDB-, CS1-, and a further isoform, OncFn, are expressed by alternative glycosylation.114 Exogenous FN has been shown to promote the healing of corneal epithelial wounds experimentally

in vivo and in vitro, and the healing of persistent corneal epithelial defects in humans.115 FN is widely expressed in the cornea and EDA-FN emerges

during wound healing.116 In addition, studies have concluded that the cell adhesion properties of FN can be generated using only peptide fragments of the molecule.103 FN peptide sequences, RGD (the central cell binding domain) and PHSRN (from the ninth type II repeating unit), when co- localized, have been found to enhance the cell-adhesive activity of RGD.68 Fibronection adhesion peptide (FAP), the carboxy-terminal heparin binding and cell adhesion-promoting domain, from within the FN molecule, has been shown to promote attachment and spreading of several cultured cell lines including corneal epithelial cells.117, 118 YIGSR is a peptide sequence from the LM A chain that has also demonstrated the ability to promote cell attachment and spreading in corneal epithelial cells.119

7.3.4Engineering surfaces for corneal epithelial growth and health

A number of different surface modification techniques have been investigated in the attempt to foster productive biological interactions between the modified elastomer/hydrogel surface and the corneal epithelial cell membrane. Franco et al.120 evaluated the interactive effects of different surface functionalities with corneal epithelial cells using self-assembled monolayers of alkanethiols and

alkylsiloxanes. In addition, several investigators have used plasma modification of various surfaces including silicone rubber121–123 and polyvinyl alcohol124

to improve the corneal epithelial cell response with limited success.

Adsorption and direct covalent attachment of ECM proteins such as FN and LM, as well as collagens types I and IV, to various surfaces have also been investigated. Hydrogels pre-adsorbed with types I and IV collagen showed a more rapid rate of cell growth than those coated with either FN or LM.125, 126 Adsorption of FN to a collagen-modified surface resulted in further acceleration of corneal cell attachment.127, 128 Collagen-modified corneal inlays also maintained a healthy, differentiated, and stratified epithelium with focal attachments for over 6 weeks in feline corneas.129 More recent studies with polyvinyl alcohol substrates modified with collagen and amniotic membrane also resulted in stratified corneal epithelium over finite time periods.130, 131

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However, collagen-modified surfaces have been shown to be susceptible to collagenase activity over time. The ability of these modified surfaces to exist over the lifetime of the implant is not clear.

Cell adhesion peptides – including RGD, YIGSR, and a novel collagen peptoid Gly-Pro-nLeu – directly attached to surfaces have also been studied,

but these peptide/peptoid modifications have produced only modest increases in the rate of epithelialization in vitro.38, 132–135 However, direct attachment of

YIGSR throughout a collagen matrix increased not only epithelialization rates and produced some stratification of the epithelium but also neurite in-growth after 6 weeks of implantation in porcine corneas.119 In general, although a few replacement materials with either coating and/or direct attachment of biological factors (both proteins and peptides) to their surfaces have had some short-term success in maintaining a somewhat normal epithelial layer,

long-term analysis (up to 8 years) has shown that the density of the epithelial layer is not maintained.135–139

Investigations into the use of spacers or tethers to increase the long-term corneal epithelial cell health over hydrogel materials have been reported. These include studies to develop and test a wet chemistry method to covalently bond PEG-tethered ECM proteins and/or peptides on to the surface of a hydrogel.140, 141 The resultant hydrogels were biologically active and possessed physical characteristics similar to the natural cornea. Analysis of the corneal epithelial cell response to six different tether-modified hydrogels showed that, while little to no cell growth occurred on plain (unmodified) hydrogels, and only a maximum confluence of 20% occurred on protein/ peptide-coated hydrogels, three types of tether-modified hydrogels (LM, an FN adhesion peptide sequence (FAP), and FN/LM (1:1)) all achieved 100% confluence at the end of the same culture period.142 However, the rate of cell confluence was considerably different for the three different tether-modified hydrogels. LM-only-tethered surfaces initially had significantly less cell growth than the surfaces with FAP-only or FN/LM. However, during the last third of the culture period, the rate of epithelial cell confluence over the LM-only modified surfaces surpassed that of the FN/LM surface. This result indicates that FN or a ligand within the FN molecule is a key component for early epithelial cell adhesion and replication in cell culture. The relative strength of the cellular attachment to the tether-modified hydrogel surfaces was determined with jet impingement. Epithelial cells grown on LMand

FAP-tethered hydrogel surfaces attached with significantly higher adhesion strengths than any other type of tether-modified hydrogel.143 There are several possible explanations for the significantly lower adhesion strength to the FN-modified surfaces: (1) the modified surfaces presented too little FN to provide the ligand surface concentration necessary for the formation of stable epithelial cell receptor–ligand bonds; (b) the percentage of FN-bound molecules bound to the surface with the appropriate receptor binding sites

exposed to the epithelial cells was inadequate for strong cell adhesion; and

(c) the tethering technique presented competitive ligand sites on the FN molecule that have a down-regulating effect on epithelial cell adhesion. Further studies are needed to clarify these issues. Addition of PEG-tethered EGF to allylamine-activated polydimethylsiloxane (PDMS) by other investigators also showed increased epithelial cell coverage rates as compared with unmodified controls in both serum-free and EGF-free medium studies.144 Overall these studies support the hypothesis that tethering proteins and peptides increases their ability to interact with cell membrane receptors and, therefore, only very small amounts (<0.1 μg/cm2) of proteins and/or peptides are necessary to elicit a cellular response.

In addition to which biological factors are added to the surface and by what method, the spatial density of the addition is important. The natural substrate has a variety of biological factors spatially distributed for cell signaling. Therefore, the optimal biomimetic surface for engineering the cell response would have a nanopatterned distribution of biological factors over its surface. There are three main methods for providing reactive groups on the surface for addition of the tethered moieties including (a) monomer pendant groups within the base polymer;140 (b) addition of reactive groups (acetaldehyde or allylamine) or reactive polymers through plasma addition;144 and addition of reactive groups through photopatterning.145 The ability to specifically populate a surface with combinations of signaling moieties can require extensive blocking chemistry unless specific patterning techniques are used. Spatially controlled patterning of the surface, by masking during the plasma polymerization, for increased corneal epithelial outgrowth recently showed preferential binding of collagen I and cell growth on those areas.146 However, the areas of photopatterning in this study were very large (13 mm) in relation to cell size. Continued research into developing nano-scale patterning methods for the spatial addition of specific cell-activating moieties will be important in furthering developments in this area.

Engineering of corneal epithelial cell attachment and growth should focus not only on the structure of the normal epithelial ECM but also on how the natural cornea heals a de-epithelialized stroma. Knowledge of the important cell signaling events and the biological factors that produce them provides the researcher with the fundamental elements needed to produce an ECM-like environment. Engineering a surface to mimic the natural corneal epithelial ECM and basement membrane sufficiently involves populating the surface with enough cellular signals (proteins/peptides) to initiate cell attachment, migration, differentiation, and stratification and then maintain the stratified epithelium over time. As more detailed information on the specific isoforms of proteins and functions of peptide sequences becomes available, more advanced surfaces can be developed. In addition to further refinement of the type of tethered moieties, optimization of their exact