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

6.6 Macroscopic aspect of the wound in the hTECWH model. hTECWH immediately (a), (c) and 2 days (b), (d) after wounding with a 6-mm punch biopsy. Two days after wounding, the reepithelialization progressing from the wound margin toward the

center can be observed macroscopically (arrows). Note that in panels

(c) and (d), the angle of the camera and the omission of the flash allowed us to visualize properly the extent of re-epithelialization from the surrounding epithelium. (Taken from Carrier et al. 2008 with permission.)

cell should get into. For instance, wound healing of any of the eye structures not only requires the formation of scar tissues but also the restoration and maintenance of the tissue’s integrity, such as transparency for the cornea.

In order to heal properly, the damaged corneal epithelium or any given epithelial tissue such as that of the skin, cells must first release themselves from the basement membrane through hemidesmosomes dissociation, and then reorganize their cell–substrate contacts to allow migration (Crosson, 1989). As hemidesmosomes are disassembled, important basement membrane remodeling is occurring that is chiefly characterized by the massive secretion of FN which is then used by the epithelial cells bordering the injured area as a provisional matrix over which they can migrate (Berman et al., 1983; Gipson et al., 1993). Expression of this provisional FN matrix peaks at between 3 and 12 hours following damage to the corneal epithelium and starts disappearing 1 week later (Kang et al., 1999; Murakami et al., 1992). As FN staining progressively diminishes, secretion of LM increases to reach maximal expression 1 week after corneal damage (Murakami et al., 1992).

These clinical findings suggest that FN might promote cell migration and proliferation in response to tissue injury whereas LM would signal exactly the opposite by restricting both these properties and forcing the cells to either progress into growth arrest or differentiate (Gaudreault et al., 2007; Gingras et al., 2009). The primary function of both FN and LM is in cell–matrix attachment, but many additional biological activities – including promotion of cell migration, wound repair and ECM-mediated cell-signaling events – have been demonstrated (Kurpakus et al., 1999; Malinda and Kleinman, 1996). As with FN, tenascin (TN), a large hexameric protein from the ECM whose expression has been correlated with development and wound healing, was shown to accumulate beneath the migrating epithelial cells 3 days after damage to the corneal epithelium in a mouse wound debridement model (Stepp and

Zhu, 1997). TN accumulation in the corneal basement membrane reached a peak 6 days after injury and then progressively decreased to undetectable levels as it is normally absent from the normal, unwounded cornea. Unlike FN, LM and TN, collagen type IV, also a major basement membrane component (Nakayasu et al., 1986; Philipp et al., 2003; Zimmermann et al., 1986), disappears during the early steps of the wounding process until the denuded area is completely covered, and then sequentially reappears beneath the newly produced epithelium (Ljubimov et al., 1998). Major remodeling of the ECM, affecting nearly all of its constituents, therefore occurs suddenly over a very restricted period of time during corneal wound healing.

The role of integrins in corneal wound healing

Integrins are widely expressed, glycosylated, heterodimeric transmembrane adhesion receptors made up of non-covalently bound α and β subunits that link the ECM to the cell’s cytoskeleton. They can promote either cell–ECM or cell–cell interactions (Hynes, 1992; Ruoslahti, 1996). To date, 18 α- and 8 β-subunits that can heterodimerize into the 24 known integrins have been reported (Clark and Brugge, 1995; Hynes, 1987; Hynes, 1992; Plow et al., 2000). The rapid changes in the composition of the ECM that occur during the wound-healing process also translate into similar changes in the expression of many integrin subunits at the cell surface of corneal epithelial cells, which have been reported to express the integrin subunits α2, α3, α4, α5, α6, αv, α9, β1, β4 and β5 (Huttenlocher et al., 1995; Latvala et al., 1995; Lauweryns et al., 1991; Lauweryns et al., 1993; Maldonado and Furcht, 1995; Paallysaho et al., 1992; Stepp et al., 1993; Stepp et al., 1995; Tervo et al., 1991; Tuori et al., 1996; Vorkauf et al., 1995) (also reviewed in Stepp, 2006 and Vigneault et al., 2007). Indeed, the massive increase in

FN secretion that typically characterizes this process was postulated to be coordinated with the expression of the α4 subunit (Lauweryns et al., 1991). Cell surface expression of α4 has also been suggested to increase during

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cell migration (Clark, 1990; Stepp et al., 1993). Although expression of α4 has recently been found to be modulated by cell density in primary cultures of rabbit corneal epithelial cells (Zaniolo et al., 2004), yet no in vivo data support a role for this integrin in re-epithelialization of the damaged corneal epithelium. On the other hand, the integrin α5β1 was shown to be present during corneal wound healing after radial keratectomy (Garana et al., 1992). As expression of α5β1 was shown to increase in corneal fibroblasts grown on three-dimensional collagen gels when FN was also present, Liu and coworkers postulated that FN then actively participates in the corneal fibroblast-mediated contraction of the collagen gel (Liu et al., 2006). The use of a debridement wound-healing model in mouse cornea showed that expression of both α6 and α9, as well as that of β4, is closely associated with wound repair, resulting in a significant increase in the level of expression of these integrins at both the mRNA and protein levels (Stepp and Zhu,

1997). Appearance and disappearance of the integrin subunits are thus well coordinated with the changes in the secretion of the ECM components during wound healing of the cornea. The complete closure of the wound typically coincides with the diminution of FN secretion and the beginning of LM accumulation beneath the leading edge. The signal transduction pathway that is then activated upon binding of the α6β1 and α6β4 integrins to their ligand LM is expected to trigger growth regulatory signals, most probably negative ones, totally distinct from those resulting from the binding of FN to the α5β1 integrin (Gaudreault et al., 2007; Gingras et al., 2003; Vigneault et al., 2007).

Three-dimensional tissue-engineered human cornea as a model for studying integrin genes expression during corneal wound healing

As it is often exposed to injuries, the cornea has become a particularly attractive tissue for studying wound healing. Indeed, corneal wounds account for a large proportion of all visual disabilities (approximately 37%) and medical consultations (estimated to be about 23%) for ocular problems in North America (Reim et al., 1997). Because of their close association with the ECM components, integrins act as sensors that can alter the transcriptome of a given adherent cell in response to any changes that may occur in its outside environment. This is ensured by the activation of one, or a few, of the signal transduction pathways that integrins use to transmit these environmental changes down to the nucleus of the cell (Juliano, 2002; Lee and Juliano, 2004). However, most of these studies, if not all of them, have been conducted in either transformed or primary cultured cells that are grown as monolayers on a plastic tissue culture support. For instance, primary culturing of corneal epithelial cells (from either human or rabbit) at varying cell densities as monolayers on tissue culture plates proved to be

a very informative, and a particularly practical in vitro model for studying integrin gene expression in conditions (such as subconfluence) that closely resemble those of corneal epithelial cells during healing (Audet et al., 1994; Gaudreault et al., 2007; Gingras et al., 2003; Larouche et al., 2000; Vigneault et al., 2007; Zaniolo et al., 2004; Zaniolo et al., 2006). However, despite their relative ease of use, monolayers of primary cultured cells also have their limitations as they lack a properly organized basement membrane and are therefore unable to adopt the appropriate behavior that is typical of their corresponding intact tissues.

Considering the limitations linked to the use of cell monolayers, the tissue-engineered corneal substitute described in subsection ‘Self-assembly approach.’ in Section 6.3.2 is therefore viewed as an outstanding tool for gene expression studies and can be used to study the expression of integrins and other participating genes during wound healing. Furthermore, tissue- engineered human corneas also render possible the study of the influence exerted by human stromal keratocytes on integrin gene expression as interaction between these two types of cells is now recognized as an important factor in corneal wound healing (Daniels and Khaw, 2000; Mishima et al., 1998; Nakamura et al., 2002) (also reviewed in Lim et al. (2003)). Indeed, signaling from the stromal keratocytes was suggested to play a significant role in the growth and proliferative response of the corneal epithelium (Wilson, 1998; Wilson et al., 1999). Recently, the use of the self-assembly approach of tissue engineering using both epithelial cells and fibroblasts originating from either the cornea or the skin shed new light on the role of fibroblast in the homeostasis of the epithelium (Carrier et al., 2009). Quite remarkably, these experiments revealed that the tissue origin of the fibroblasts that are used to generate the stroma on which the epithelial cells are deposited does indeed dictate the stratification and differentiation properties of these epithelial cells. Besides examining the expression of endogenous integrins in the epithelial cells, the use of tissue-engineered human corneal substitutes is also particularly attractive in that it can be exploited as a tool to virtually dissect the regulatory sequences (both the promoter and 5′-flanking region) of any given integrin gene (for instance, those of the α5 and α6 genes) and define precisely how they behave functionally when introduced into corneal epithelial cells that are re-allowed to grow and expand on the keratocytescontaining collagen matrix. By exploiting lentiviral technology to ensure a high level of stable transduction and expression of integrin gene promoters (for reviews see Kafri, 2004, and Stevenson, 2002) in corneal epithelial cells prior to their seeding on the reconstructed corneal stroma, one can easily bypass the limitations of commonly used transfection procedures such as low efficiency of transfection and transient expression of the recombinant construct. The use of tissue-engineered human corneas constructed using recombinant lentivirus-infected corneal epithelial cells will render possible

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the direct survey of integrin gene promoter activity into the completely stratified lentivirus-infected epithelium in response to wounds produced with a biopsy punch (Carrier et al., 2008), a type of analysis that otherwise could not be conducted in intact human corneas.

In summary, tissue-engineered human corneal substitutes as a tool for studying gene expression will most certainly contribute to bring integrins to the forefront of components required to ensure proper healing of the injured cornea. As the understanding of the potential roles and implications of each integrin gene increases, the possibility of modifying the transcription of each of these subunits becomes quite interesting. Promising new technologies that directly affect the outcomes of a gene product are being developed and these represent potential therapeutic means to address wound healing directly. Interesting advances in the use of small interfering RNAs (siRNAs) are being proposed as a promising way to knockout the transcript of any chosen gene, but they could also be used against any transcription factors that regulate a particular gene (such as those encoding integrin subunits) to alleviate a repressionnal state that could be induced by a repressor on the core promoter of an integrin gene, for instance. Lentivirus-mediated RNA interference therapy is also considered to be a promising method for efficiently delivering stable expression and control of gene expression (reviewed in

Morris and Rossi (2006)). Stably altering specific integrin expression in corneal epithelial cells may prove useful for improving the growth properties and stability of the tissue-engineered corneas, thereby reducing the risks of scar formation upon grafting on patients afflicted with corneal diseases.

6.5Clinical applications

In ophthalmology, the most successful clinical application of tissue-engineering techniques has been in the treatment of patients with limbal stem cell deficiency (Pellegrini et al., 2008; Shortt et al., 2007). In this procedure, limbal cells that have been sampled and expanded in culture are autologously or heterologously grafted to patients on a support material (a polymer, an amniotic membrane or a gel). When a fibro-vascular outgrowth is present, it is necessary to remove it before transplanting corneal stem cells on a support of amniotic membrane or fibrin gel. An autologous limbal graft is considered in cases of unilateral lesions (Coster, 1998). In this case, a limbal biopsy as small as 1 mm2 proved sufficient to cover the entire corneal surface (Pellegrini et al., 2008). However, limbal sampling bears the potential risk of causing limbal stem cell deficiency in the healthy contralateral eye (Chen and Tseng,

1991), unless cells are expanded in culture prior to transplantation. Limbal allograft with systemic immunosuppression is another option; however, the risk of significant side effects from long-term immunosuppression is a major drawback of this technique.

6.5.1Cultured epithelium grafting

Amniotic membrane

The amniotic membrane has been used as a biological dressing or as a substrate for epithelial growth in the management of various ocular surface conditions (Bouchard and John, 2004). The amnion abounds in cytokines that have antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties; in addition the amnion allows, as fetal skin does, wound healing with reduced scar formation (for review, see Dua et al., 2004). Specifically, the amnion is used to: (a) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or (b) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. For this last condition, the amniotic membrane is used with a limbal transplantation (Gomes et al., 2003).

The limbal corneal and conjunctival cells may also be cultured separately on amniotic membrane before being transplanted to patients with eye burns. As burns destroy conjunctival and corneal stem cells, the addition of these two cell types stabilizes the ocular surface and prepares it for possible penetrating keratoplasty after the initial reconstruction (Sangwan et al., 2003).

The amnion is also used to treat disorders of the eyelid and bulbar conjunctiva, and corneal diseases, and as a replacement tissue used in the management of corneal or scleral ulcers (Bouchard and John, 2004). It is indicated for treating persistent epithelial defects due to a neurotrophic keratopathy, bullous or exposure keratopathy, or during atopic keratoconjunctivitis and severe ocular pemphigoid (Bouchard and John, 2004). The use of amnion in patients suffering from Stevens–Johnson syndrome has produced mixed results (Gomes et al., 2003; Shimazaki et al., 2002). Finally, after a trauma, the amniotic membrane has been used as an alternative dressing to a bandage contact lens in order to reduce the friction between the eyelid and the injured surface epithelium and pain during blinking (Baum, 2002).

Fibrin gels

Pellegrini and coworkers reported the use of fibrin gels as a substrate for supporting cultured epithelial cells for the treatment of patients with severe skin burns (Pellegrini et al., 1999b), and later for patients suffering from limbal stem cell deficiency (Rama et al., 2001). The fibrin gel acts as a substrate that helps to support the cultured epithelial cells before and during grafting. In addition, its adhesive properties help to glue the sheet of epithelial cells to the underlying stroma. In contrast to the amniotic membrane, which lasts 10 days after grafting, the fibrin gel is degraded very rapidly within 24 hours without any deleterious effect on the survival of the cultured epithelial

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cells (Nakamura et al., 2003). Fibrin is the only substrate for which the maintenance of stem cells and long-term proliferation of limbal cells have been demonstrated (Pellegrini et al., 2008; Rama et al., 2001).

6.6Future trends

6.6.1Stromal cell/collagen orientation

In the corneal stroma, the specific arrangement of collagen fibers is important for transparency and strength. Various methods of cell and ECM component alignment have been described and have recently been applied to corneal fibroblasts in culture. Corneal fibroblasts, as with many other cell types, will follow a specific orientation if cultured on microgrooved substrates

(Crabb and Hubel, 2008; Guillemette et al., 2009; Teixeira et al., 2004; Vrana et al., 2007) or on magnetically aligned collagen scaffold (Torbet et al., 2007), a process called ‘contact guidance’. Some local cell alignment over small areas has also been reported using cells grown over disorganized collagen substrates (Ren et al., 2008) or without scaffolds (Du et al., 2007;

Guo et al., 2007). The resulting aligned fibroblasts then secrete GAGs and aligned collagen fibrils over small surface areas. Furthermore, fibril size and interfibrillar spacing can be modified by adding epithelial cells (Builles et al., 2007a; Builles et al., 2007b).

A thermoplastic elastomer engraved with a depth of 1 μm and a grating period ranging from 1 to 4 μm was used to cultivate corneal fibroblasts

(Guillemette et al., 2009). Results show that cells grew aligned to the grooves and that cell–cell interactions were possible over the entire sample, leading to ECM secretion and organization following cell orientation. The second overlying cell layer was also oriented with a mean angle shift of 53 ± 8° relative to the first corneal fibroblast layer (Fig. 6.7(a) and (b)). This cell organization reflects the physiological organization within a human corneal stroma. Both cells and the cell-secreted ECM (mainly type I collagen) layer were highly organized. Transmission electron microscopy was used to visualize the regularly spaced rows of lines alternating with dots in the microstructured samples, indicating that the stromal sheets comprised lamellae of aligned collagen fibers shifting from plane to plane (Fig. 6.7(c) and (d)).

Furthermore, results showed that oriented self-assembled stromal sheets exhibited a better transparency compared with the same nonstructured tissue.

In contrast, tissues produced from skin fibroblasts did not show organization of the second overlaying cell layer.

Thus, stromal cells and corneal fibroblasts retain the potential to organize into a multilayered and aligned stromal tissue in vitro. Taking advantage of this ability using various cell culture techniques may ultimately lead to higher quality tissue-engineered corneal stromas.

Cell

TEM

6.7 Cell and ECM alignment imaging. Actin filaments immunofluorescence staining of corneal fibroblasts grown on control (a) and oriented (b) substrates. Cells that have been cultured on control substrates have a random orientation compared with cells grown over microgrooved substrates where the cell shift in between layers can be observed. Transmission electronic microscopy (TEM) images of the corneal stroma. TEM images of control (c) and oriented (d) corneal stroma that have been cultured on microgrooved substrates, dots represents collagen fibres perpendicular to the plane and striated lines represents angled collagen fibres, they do not run longitudinally on the plane since they have about a 53° shift between each layer. Scale bars. (a) and (b), 50 μm; (c) and (d), 1 μm.

6.6.2Future clinical applications

The fibrin support that has been used mainly for treating limbal stem cell deficiency could eventually be used to grow the endothelium in vitro and could be coupled with posterior lamellar keratoplasty such as DLEK or DSEAK in order to treat endotheliopathies.

6.7Sources of futher information and advice

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reviews: Shortt et al. (2007), McLaughlin et al. (2009), Shah et al. (2008) and Ruberti and Zieske, (2008) and Griffith et al. (2009). For an in-depth review of the integrins, see Vigeault et al. (2007). More information on tissue engineering and regenerative medicine can be found at www.loex.qc.ca.

6.8Acknowledgements

The authors would like to thank Elsevier, Molecular Vision and Investigative Ophthalmology and Visual Science for their permission to use previously published figures. This work was supported by the Canadian Institutes of

Health Research (CIHR), Ottawa, Canada (L.G., S.L.G., F.A.A.), the Fonds de recherche en santé du Québec (FRSQ) Research in Vision Health Network, Montreal, Canada (L.G., S.L.G., C.G.J., F.A.A.), and the Centre de recherche FRSQ du CHA de Québec (L.G., F.A.A.). S.P. holds a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC), Ottawa, Canada. L.G. is the recipient of a Canadian Research Chair from the CIHR in Stem Cells and Tissue Engineering. The authors would like to thank Alexandre Deschambeault, Caroline Audet, Alain Lapante, and the members of the LOEX who have participated in these studies.

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