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

6.1Introduction

6.1.1The cornea

The transparent cornea is the main lens and the curved window of the human eye (Fatt and Weissman, 1992). The cornea is not vascularized except at its extreme periphery, the limbus, which is the transitional zone between the transparent cornea and the opaque sclera. The avascular nature of the cornea contributes to both the immune privilege of the anterior segment and to corneal transparency, but requires the circulation of the clear aqueous humor in the anterior chamber of the eye to supply it with essential elements and to evacuate metabolic wastes. The cornea is composed of three main layers, each with its resident cells. From outside to inside the eye: the corneal epithelium, which represents about 10% of the corneal thickness; the corneal stroma; and the corneal endothelium (Maurice, 1984) (Fig. 6.1).

The stratified, non-keratinized, squamous corneal epithelium acts as a barrier to protect corneal transparency. Deepest into the epithelium, basal cylindrical cells resting on the basal lamina (Maurice, 1984) are responsible in part for the renewal of epithelial cells: during mitosis (Fatt and Weissman,

Corneal limbus

Ep

S & E

Cornea

En S

Corneal limbus

(a)

Stroma and endothelium

(c)

6.1 Anatomy and histology of the human cornea. (a) Side and frontal view of the cornea and the eye to show the epithelium (Ep), the stroma (S) and the endothelium (En). S & E is the back half of the corneal stroma with the endothelial layer, as represented in detail by the drawing and picture in panels (b) and (c). Panel (d) is a diagram of the corneal stroma representing a few corneal lamellae, each containing parallel collagen fibrils as well as corneal fibroblasts or keratocytes, represented by flat cells between these lamellae. (e) Drawing of the central corneal epithelium that shows the superficial, wing and basal cells of the anterior epithelium resting on Bowman’s membrane and anterior stroma. (Panels (b), (c), (d) and (e) are reproduced courtesy of Elsevier and were originally published in Hogan et al., 1971.)

1992), the daughter cells migrate towards the corneal surface and progressively flatten to become polygonal cells in the two or three intermediate epithelial cell layers and then differentiate into very flat cells in the most superficial couple of layers. With their continuous tight junctions, these superficial cells form a barrier between the tears and the cornea. They eventually desquamate into the tear film (Tripathi and Tripathi, 1984). It was initially believed that

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cell division of the basal cells accounted for the replacement of the whole corneal epithelium during the entire life of an individual. However, it was realized 25 years ago that this process alone was unable to account for the epithelial rate of desquamation, and that such a calculation required the contribution of the limbic stem cells (Thoft and Friend, 1983).

At the limbus, the corneal epithelium gradually thickens and becomes the conjunctival epithelium. The corneal epithelial stem cells are located deep into the limbic zone. Unlike basal cells, stem cells are undifferentiated and have an unlimited capacity for renewal (Cotsarelis et al., 1989; Dua and Azuara-Blanco, 2000; Kruse, 1994; Schermer et al., 1986). They also form a barrier to invasion of the cornea by cells from the conjunctival epithelium. Each cell is produced by successive divisions of the limbal epithelial cells.

The first division from a stem cell is irreversible and asymmetric. It gives rise to a stem cell and a mid-differentiated cell (Schermer et al., 1986), with the following characteristics: it is more sensitive to apoptosis than its mother cell, it has a limited potential for division, and migrates in the basal layer toward the center of the corneal epithelium in a centripetal movement (Thoft and Friend, 1983). After several rounds of cell division, cells reduce their contact with the basement membrane, move towards the epithelial surface, and enter terminal differentiation, initiating the expression of keratins 3 and 12 before desquamation and loss into the tear film (Dua et al., 2003). In the central cornea, the daughter cells from the basal cell mitosis desquamate into the tear film after a period of 7 (Hanna and O’Brien, 1960) to 14 days (Haddad, 2000).

The emergence of a new cell occurs before the old cell is removed (Klyce and Beuerman, 1998) in order to preserve the regularity of the corneal surface and the optical stability. The mitotic activity, which shows a rate of mitosis of 10–15% per day in the basal cell layer, allows the epithelium to ensure the tightness of the tissue (Edelhauser and Ubels, 2003). Experimentally, the mitotic activity of the limbic epithelium is higher than that of the epithelium at the periphery of the cornea, which is itself larger than the center.

The corneal epithelium lies on its basement membrane, which rests over the Bowman’s membrane, a less organized form of corneal stroma. The corneal stroma represents 90% of the corneal thickness (Maurice, 1984). It is a pile of 300 superposed lamellae in the central cornea and 500 in the periphery (Hamada et al., 1972). The lamellae extend from one end of the limbus to the other. Collagen molecules that form collagen fibrils are aligned parallel to each other within one lamella as well as to the corneal surface

(Komai and Ushiki, 1991). The fibrils within adjacent lamellae are oriented at various angles (Klyce and Beuerman, 1998; Radner et al., 1998). The angle between collagen fibers from two contiguous lamellae varies, having a mean of 60°. This arrangement not only provides extreme resistance to a tissue as thin as the corneal stroma, but also accounts for corneal transparency.

Transparency arises from destructive interference that neutralizes reflections

at the surface of the tissue; the relatively constant fibril diameter and density of the stroma act as a grating (Maurice, 1957). Laminin (LM), fibronectin

(FN) and glycoaminoglycans (GAGs) are also found in the corneal stroma.

The GAGs, which are located in the interfibrillar space, have a very strong tendency to attract water: under normal circumstances, the epithelium and the endothelium can maintain stromal hydration at 77% of water per corneal weight (Maurice, 1984). However, when these cell layers are compromised, the cornea swells. A significant edema disturbs the regular stromal arrangement and leads to loss of transparency and corneal blindness.

The keratocytes, dormant fibroblasts, are the most abundant stromal cells. From their cell bodies, which lie between lamellae of collagen fibrils (Klyce and Beuerman, 1998), emerge long cytoplasmic filaments that form a three- dimensional network of interconnected cells possibly involved in the process of healing and nutrition of the stroma (Watsky, 1995). The keratocytes closely regulate the synthesis and degradation of the extracellular matrix (ECM) with the synthesis of enzymes (please consult Maurice (1984)). During stromal trauma, the keratocytes lose their connections in order to repair the injury and become activated fibroblasts involved in the synthesis of procollagen and GAGs (Ham, 1974). The corneal stroma also contains other cells, especially during inflammation: dendritic cells, macrophages, lymphocytes as well as polymorphonuclear leucocytes.

The non-vascular corneal endothelium is a monolayer of cells. Their apical side with its discontinuous tight junctions (macula occludens) (Barry et al., 1995; Petroll et al., 1999) faces the aqueous humor contained within the anterior chamber (Hogan et al., 1971). This leaky barrier allows for a paracellular passive influx of water that contributes to supply all corneal layers with essential nutrients (Fatt and Weissman, 1992). The cornea can maintain its hydration and transparency only if this passive water influx is opposed by an active water outflux of the same magnitude. This concept is referred to as the ‘pump and leak hypothesis’ (Maurice, 1984). The ionic pumps from the corneal endothelium regulate corneal hydration through Na+-, HCO3– - and Cl–- dependent mechanisms. When the endothelium becomes unable to sustain this important function, because of insufficient cell density or disease, the cornea loses its transparency. Because the endothelium is unable to undergo mitosis in vivo, the endothelial cell density decreases steadily with age from 0.3 to 0.6% annually (Bourne et al., 1997; Murphy et al., 1984). This cell loss is usually compensated by the spreading and thinning of the remaining cells.

6.1.2Clinical problems and progress in regenerative medicine

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(Barron, 1998), with a rise to 39 391 in 2007 (http://www.restoresight.org/ newsroom/newsroom.htm). On the other hand, the pool of donors is bound to decrease as a result of the popularity of refractive surgery and eventual improvements in selection criteria and in donor matching.

According to two studies of the keratoplasties conducted in Toronto from 1964 to 1997 (Maeno et al., 2000), and in Philadelphia between 2001 and 2005 (Ghosheh et al., 2008), the two groups of diseases for which keratoplasty was most frequently indicated were: (a) primary or secondary endotheliopathy, and (b) keratoconus. Whereas endothelial dysfunction causes edema and corneal blindness, keratoconus is characterized by a progressive dystrophic ectasia, which ultimately causes corneal thinning, scarring and deformation of corneal surfaces with complex optical problems. Both of these corneal disorders as well as their management are reviewed in textbooks (Kaufman et al., 1998; Krachmer et al., 2005).

Most corneal allogeneic transplantation involves a full thickness graft of a donor cornea from a deceased patient, a surgery called penetrating keratoplasty (PK) (Barron, 1998). Lamellar keratoplasty, a now rarely used type of procedure, involves a partial replacement of the cornea that is limited to the anterior cornea (epithelium with some stroma) (Hamilton and Wood,

1998). Recent improvements in surgical tools and techniques allow for the replacement of the endothelium with a thin supporting layer of stroma. This delicate procedure, called either Descemet stripping automated endothelial keratoplasty (DSAEK) or deep lamellar endothelial keratoplasty (DLEK), has much fewer side effects compared with PK for treating endotheliopathy

(Price and Price, 2007; Terry, 2007) and this explains why it has become very popular among corneal surgeons.

The adaptation of techniques for cell culture of cells grown over a backing support (see also Section 6.3.3) with the advent of posterior lamellar surgery could open up efficient ways to treat patients with less rejection and better optical outcome compared with PK, while possibly allowing more than one recipient to be treated with a single donor cornea (Ishino et al., 2004; Wencan et al., 2007).

Tissue engineering methods have already been very successful in treating patients with limbal stem cell deficiency. The treatment of this condition is usually not successful with PK, as prognosis is not good for patients treated with PK alone (without a graft of limbal cells) (Whitson et al., 1999). This is because the diameter of the corneal graft is insufficiently large to also include the stem cells (located at the limbus) from the donor’s cornea, and these are necessary to treat corneal conjunctivalization. During this process, the absence of stem cells allows conjunctival epithelial cells to migrate over the limbus and invade the cornea. Neovascularization extends from the limbus and covers the whole cornea. Goblet cells, and their specific mucin product MUC5AC, are then present over a keratin 4 (K4)-expressing corneal

epithelium. In normal eyes in situ, goblet cells and MUC5AC, as well as K4, are only associated with the conjunctiva (Meller et al., 2002; Puangsricharern and Tseng, 1995, Tsai et al., 1990). Limbal stem cell deficiency is either hereditary or acquired after disease or following a thermal or a chemical burn. Such a change in phenotype leads to loss of corneal transparency and visual disability. In the clinic, tissue engineering of the epithelium coupled with the classical treatment of limbal graft has been used to treat successfully patients with limbal stem cell deficiency (Pellegrini et al., 2008; Shortt et al., 2007). These treatments are covered in Section 6.5.

6.2Cell source

Reconstructing a functional tissue in vitro first requires high-quality cells, like stem cells that have the property of self-renewal. Corneal stem cells, and progenitor cells that are already committed to a cell lineage, may be more appropriate for corneal tissue reconstruction, because such cells differentiate to produce their tissue of origin and do not have a tendency to produce multiple cell lineage such as multipotent stem cells. Thus, for many years, investigators have searched for the presence of corneal epithelial, stromal and endothelial stem/progenitor cells. Once located, cells have to be isolated and grown under appropriate conditions (substratum, culture medium) so that the tissue reconstructed with the cultured cells maintains functional and regenerative capacities.

6.2.1Corneal epithelial cells

Best anatomic site

As discussed in Section 6.1.1, epithelial stem cells are located at the limbus. It is interesting to note that cells isolated from the limbus grow much better in culture than cells from the central cornea, suggesting that stem cells are preserved in culture. Cells from the central cornea have a short lifetime in culture as they can barely be subcultured. In contrast, limbal cells can be subcultured for several passages (up to seven passages depending on the donor). Moreover, cells isolated from the central cornea present morphological characteristics consistent with their low proliferative potential. They form small colonies containing large cells with a low nucleus to cytoplasm ratio, indicating that they are differentiated. In contrast, limbal cells produce large colonies constituted of small cells that possess a high nucleus to cytoplasm ratio. These characteristics are typical of less differentiated cells.

Within the limbus, a higher colony forming efficiency is attributed to cells that have been isolated from the superior and temporal quadrants compared with cells from nasal or inferior limbus (Deschambeault et al.,

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2002; Pellegrini et al., 1999a) or from limbal regions containing crypts and focal stromal projections, mostly present in the superior and inferior corneal limbal quadrants (Shortt et al., 2007) suggesting that biopsies should preferably be taken from the superior quadrant. The preferential location of stem cells in the superior quadrants is consistent with the better protection against environmental and mechanical injuries provided by the eyelids in this region.

Culture of corneal epithelial cells

The use of the enzyme dispase for separating the epithelium from deeper tissues yields pure cultures without any contamination by stromal fibroblasts (Gipson and Grill, 1982). With this technique, epithelial cells may be detached from the basement membrane in order to detach a viable sheet of epithelium (Espana et al., 2003) from which cells are isolated and cultures established (Germain et al., 2000). The addition of a feeder layer is advantageous since large colonies can be grown from single cells under these conditions (Germain et al., 2000; Green and Barrandon, 1988; Pellegrini et al., 1999a).

Improved cultures with a feeder layer through stabilization of transcription factors

Human corneal epithelial cells reach senescence quickly, even in the primary culture (Lindberg et al., 1993), and culturing them along with a lethally irradiated fibroblast feeder layer (such as i3T3) proved to be a major step forward as such mesenchymal cells not only provide an environment that limits invasion of the culture surface by contaminating living fibroblasts but also considerably improve both the cell morphological and growth properties (Pellegrini et al., 1999a; Rheinwald and Green, 1975). Experiments that we recently conducted with both human skin keratinocytes and human corneal epithelial cells revealed that besides considerably improving the ability to sustain cell passages, the presence of a feeder layer also preserves the morphological and growth properties that typically characterize undifferentiated cells. Although we demonstrated that both secreted factors and cell–cell interactions are important for maintaining these feeder layer-mediated cell properties (Masson-Gadais et al., 2006), the exact mechanism by which i3T3 improves cell behavior remains elusive. Recently, we reasoned that one possible way in which i3T3 may delay tissue-cultured cells reaching senescence is by altering either the expression or DNA binding of nuclear transcription factors which regulate the expression of a large array of genes in the human genome. Cell migration and proliferation, as observed with human corneal epithelial cells co-cultured along with i3T3, is dependent on cell cycle-related gene expression, the latter being modulated by the

action of transcription factors that act either positively or negatively on the transcription of these genes. GC-boxes and related motifs are regulatory elements very frequently observed in the promoters and 5′-flanking sequences of many, if not all mRNA-encoding genes. The positive transcription factor

Sp1 was one of the very first transcription factors to be identified and cloned by virtue of its ability to bind GC-rich motifs. Similarly, members from the nuclear factor I (NFI) family of transcription factors were also found to regulate a large number of human genes (although not as many as for Sp1) by interacting with a GC-rich motif (5′-TGGA/C(N)5GCCAA-3′) (Roulet et al., 2000; Roulet et al., 2002) distinct from that recognized by Sp1 (5′- GGGGCGGGG-3′) (Dynan et al., 1985) and present in the promoter of the regulated target genes. Both Sp1 and NFI have been shown to play a critical role during cell cycle progression by acting on key regulators such as the cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) that also participates in regulating apoptosis, senescence and differentiation besides a function in cell cycle modulation (Opitz and Rustgi, 2000; Ouellet et al., 2006; Watanabe et al., 1998). By exploiting the use of primary cultured skin keratinocytes grown with or without a feeder layer, we indeed provided evidence that i3T3 dramatically improves both the expression and DNA binding of the transcription factors Sp1 and Sp3 ((Masson-Gadais et al., 2006), see also Fig. 6.2). The process by which i3T3 acts on the properties of these transcription factors is believed to reside, at least for Sp1, in stabilizing them through post-translational modifications such as glycosylation which may prevent them from being degraded by the proteasome, as has been proposed for Sp1 (Bouwman and Philipsen, 2002; Han and Kudlow, 1997; Majumdar et al., 2003). Preserving expression of these transcription factors through an as yet unclear feeder layer-mediated influence will most certainly affect genes whose respective products are either related to the cell cycle or encode cell structural proteins, such as keratins and membrane-bound receptors, that are required for cell adhesion, migration and differentiation (Gaudreault et al., 2003; Masson-Gadais et al., 2006). In turn, improving these transcription factor properties is expected to also improve the quality of the epithelial layer overlaying the corneal stroma in the reconstructed corneas.

6.2.2Corneal stromal keratocytes

Best anatomic site

In 2005, keratocyte progenitors were identified in adult bovine corneal stromas

(Funderburgh et al., 2005). In this particular study, the authors reported that 3% of freshly isolated bovine stromal cells exhibited clonal growth. Using sphere-forming assays, stromal precursors from mouse (Yoshida et al., 2005), rabbit (Mimura et al., 2008) and human (Yamagami et al.,

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(b)

U

(a)

6.2 Expression and DNA binding of the transcription factors Sp1 and Sp3 isolated from skin keratinocytes co-cultured with i3T3. (a) Electrophoretic mobility shift assay (EMSA) analyses of Sp1 binding in human skin keratinocytes co-cultured with i3T3. Crude nuclear proteins (5 μg) from skin keratinocytes cultured from skin biopsies of 16- (KAd16), 26-(KAd26) and 29-(KAd29) year-old human donors and grown either alone (–) or in the presence of (+) i3T3 were incubated with a 5′ end-labeled, double-stranded oligonucleotide bearing the high affinity binding site for the transcription factor Sp1, either alone or in the presence of a 250-fold molar excess of unlabeled competitor oligonucleotides bearing target sites for either Sp1 or

NFI. The formation of DNA–protein complexes was then examined by EMSA through a 6% native polyacrylamide gel. The position of the DNA–protein complexes corresponding to the transcription factors Sp1 and Sp3 is indicated along with that of the free probe (U). P, labeled probe with no proteins. (b) Western blot analyses of Sp1

and Sp3 in human skin keratinocytes co-cultured with (+) or without

(–) i3T3. Approximately 30 μg of proteins from each of the nuclear extracts used in panel (a) were examined in Western blot using polyclonal antibodies directed against the transcription factors Sp1 and Sp3. As a normalization control, the membrane was also blotted using a monoclonal antibody against human actin.