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

Stromal cells are subcultured in the same medium and can undergo more than nine passages. In this culture medium, stromal cells adopt a fibroblastic morphology after subculturing. Alternatively, human corneal keratocytes can be obtained after digestion of bare corneal stroma with collagenase (Carrier et al., 2008; Germain et al., 1999).

Keratocytes cultured in a protein-free culture medium adopt a dendritic morphology and maintain a differentiated phenotype. Keratan sulfate or keratokan protein expression is widely used as a marker for keratocyte differentiation (Beales et al., 1999; Long et al., 2000). Adding the growth factor fibroblast growth factor (FGF)-2 to the culture medium has been shown to upregulate keratan sulfate (Long et al., 2000). Insulin supplementation stimulates proliferation while maintaining keratocan expression (Musselmann et al., 2005). A recent study also showed that culturing keratocytes as substrate-independent spheroids preserved their differentiated phenotype and that addition of ascorbate-2 phosphate upregulated keratan sulfate protein levels (Funderburgh et al., 2008).

Culturing these cells in the presence of serum alters the keratocyte phenotype to an activated cell, stromal fibroblasts, mimicking wound healing. Corneal fibroblasts, cultured in the presence of ascorbic acid, secrete and organize

ECM which, when maintained in long-term cultures, can form sheets that can be assembled into three-dimenstional stromal substitutes (see also subsection

‘self-assembly approach’ in Section 6.3.2) (Carrier et al., 2008; Carrier et al., 2009; Ren et al., 2008).

6.2.3Corneal endothelial cells

Best anatomic site

In contrast to the epithelial side, where it is now largely accepted that epithelial corneal stem cells reside at the limbus, no clear evidence has demonstrated the presence and location of corneal endothelial stem/progenitor cells. Although they do not proliferate in vivo, it has been shown that corneal endothelial cells from both the central and peripheral areas retain potential proliferative capacity (Konomi et al., 2005). However, Joyce’s group (Mimura and Joyce,

2006) showed that human corneal endothelial cells from the peripheral area retain higher replication competence, regardless of donor age. Even though human corneal endothelial cells from the central area of corneas from older donors retained their replicative ability, the relative percentage of cells that were competent to replicate in vitro was significantly lower than in the periphery or in the central area of corneas from younger donors. This study was in accordance with a previous report demonstrating that corneal endothelial cells from the periphery undergo a higher number of population doublings than those from the center before reaching senescence (Bednarz et al., 1996)

Culture of endothelial cells from different species

Culture techniques and growth medium formulations for untransformed corneal endothelial cells have been previously developed for human (Bednarz et al., 2001; Engelmann et al., 1988; Engelmann and Friedl, 1989; Engelmann and Friedl, 1995; Joyce and Zhu, 2004; Zhu and Joyce, 2004) or animal (Giguere et al., 1982; Gospodarowicz et al., 1977; Lee et al., 1991; Proulx et al., 2007; Schultz et al., 1992; Woost et al., 1992a; Woost et al., 1992b) cells. Using human cells, the effects of numerous growth-promoting agents were tested, such as: epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), bovine pituitary extract (BPE) and endothelial cell growth factor (Samples et al., 1991); or ascorbic acid, insulin, selenium, transferrin, lipids and FGF (Engelmann and Friedl, 1995).

Joyce’s group (Joyce and Zhu, 2004; Zhu and Joyce, 2004) observed mitotic or morphologic changes in response to serum, EGF, NGF and BPE. Since animal models are required for pre-clinical studies, we have previously optimized the culture medium of porcine (Proulx et al., 2007) and feline (Audet et al., 2008) corneal endothelial cells. Human endothelial cell growth in our selected culture medium consistently generates cultures of small, polygonal-shaped cells (Fig. 6.3).

6.3Corneal tissue reconstruction

6.3.1Reconstruction of the anterior cornea

Amniotic membranes

Limbic cells can be grown over a support of amniotic membrane in the presence or absence of feeder cells (Dua et al., 2004). Limbal cells cultured on plastic in the presence of a feeder layer retain great potential for proliferation and regeneration suggesting the conservation of stem cells (Lindberg et al., 1993). When limbic human cells are cultured on amniotic membranes, they retain their native characteristics (Du et al., 2003; Hernandez Galindo et al., 2003), and present, compared with the corresponding cells grown on

(b)

6.3 Effect of different culture media formulations on cell number, size and morphology of porcine corneal endothelial cells (PCECs). Representative results of the additive effect of 50 μg/ml bovine pituitary extract (50-BPE), 0.08% chondroitin sulfate (0.08CDS) and 20 μg/ml ascorbic acid (20AA) on (a) mean cell size and (b) cell number at day 4 on PCECs grown in Opti-MEM I supplemented with 0, 4 or 8% fetal bovine serum (FBS), and compared with the classic Dulbecco’s modified Eagle’s medium (DMEM) 20% FBS (diagonal dashed bar; mean ± standard deviation). The asterisks indicate a significance of p < 0.001 compared with DMEM 20% FBS (cell number and cell size). Lower panels show the morphology of PCECs grown in (c) DMEM 20% FBS or (d) the selected medium, consisting of Opti-MEM I supplemented with 8% FBS, 50 μg/ml BPE, 0.08% chondroitin sulfate and 20 μg/ml ascorbic acid. Cells were left in culture 1 week passed confluence to assess cell morphology of postconfluent cultures. The scale bar is equal to 100 μm. Note that in panel (c), endothelial cells are elongated and of different size, whereas in panel (d) they are small and cuboidal and have a morphology more characteristic of native cells. (Taken from Proulx et al. (2007) with permission.)

plastic, smaller proportions of K3+ and Cx43+ cells and larger proportions of cells positive for the transcription factor p63 or its isotype Np63 (Du et al., 2003; Hernandez Galindo et al., 2003; Hernandez Galindo et al., 2003).

This transcription factor, expressed in basal cells of stratified epithelia, is crucial for the development and differentiation of epithelia. An homologue of the tumor-suppressor p53, p63, is localized in limbal basal epithelial cells of the cornea. The p63 isotype Np63 has been proposed as a marker of corneal stem cells (Pellegrini et al., 2001). For clinical applications regarding transplantation of epithelial cells seeded on amniotic membranes, see Section 6.5.1.

Fibrin gels

Fibrin gels, prepared from proteins that cause blood coagulation, have long been used clinically to prevent bleeding and promote wound healing. Their mechanical compliance as well as their biological features make fibrin gels a good carrier for cell transplantation, and their use has been reported for skin (Pellegrini et al., 1999b) and cornea (Rama et al., 2001). Limbal epithelial cells can easily be grown on fibrin gels (Rama et al., 2001; Talbot et al., 2006). The phenotype of rabbit limbal epithelial cells attached on fibrin gels and the negative immunostaining for both K3 and K4 suggest a low differentiation status, as the only cells with this phenotype in situ are the basal cells from the limbus where stem cells are located (Fig. 6.4).

In vivo experimental applications: transplantation of a reconstructed corneal epithelium

In order to assess whether cultured limbal epithelial cells are able to reform a corneal epithelium, the cultured cells seeded on a fibrin gel were

Merged

(c)

6.4 Keratin 3 staining of rabbit limbal epithelial cells cultured on a fibrin gel in vitro. (a) Phase contrast of confluent rabbit limbal epithelial cells (RLECs) cultured on fibrin gel. (b) Keratin 3 (K3)

immunostaining of the same confluent RLECs cultured on fibrin gel.

(c) A merger of the phase contrast (a) and K3 immunostaining (b). At this step, the RLECs cultured on fibrin gels were ready for grafting. Scale bar, 50 μm. (Taken from Talbot et al. (2006) with permission.)

autologously grafted on to a denuded conjunctivalized cornea of a limbal stem cell deficiency rabbit model (Talbot et al., 2006). Results show that 1 month post-transplantation, neither goblet cells nor MUC5AC staining were observed in the central cornea of experimental animals, as opposed to untreated control rabbits that had goblet cells and stained positive for MUC5AC over the whole cornea in all epithelial layers. Therefore, the grafted epithelium most certainly adheres to the bare cornea, and the grafted epithelial cells proliferate and regenerate the corneal epithelium. For clinical applications regarding transplantation of human limbal epithelial cells, see subsection on

‘Fibrin gels’ in Section 6.5.1.

6.3.2Reconstruction of the corneal stroma

Collagen gels

The unique properties of the cornea require that an engineered corneal stroma must be transparent and strong, and have an appropriate curvature. Since the corneal stroma is mostly made out of type 1 collagen (Klyce and Beuerman, 1998) many investigators have produced a corneal stroma by mixing cultured keratocytes into a collagen gel. The many drawbacks of this technique include, for instance: the important contraction that the collagen gel undergoes (Germain and Auger, 1995; Germain et al., 2000; Germain et

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al., 2004; Reichl and Muller-Goymann, 2003; Reichl et al., 2004; Tegtmeyer et al., 2001); the poor stability and strength of the matrix as well as its rapid degradation (Kondo et al., 2008). The type of collagen used to produce the matrix also influences the end result, bovine collagen gels being softer and more fragile than rat tail tendon collagen gels (Doillon et al., 2003), although all of them remain weak.

Hydrogels

In order to stabilize the collagen matrix, different cross-linking agents can be added to the collagen solution. The composition, cross-linking and configuration of collagen matrices were all shown to influence the optical and biochemical properties of stromal equivalents (Crabb and Hubel, 2008).

Stromal substitutes, made from cross-linked porcine collagen or from recombinant collagen, have been shown to sustain epithelial and nerve regeneration both in vitro and in vivo (Dravida et al., 2008; Lagali et al., 2008; Liu et al., 2008b; Liu et al., 2009; McLaughlin et al., 2008; Merrett et al., 2008; Rafat et al., 2008). Biosynthetic corneal substitutes fabricated using recombinant human collagen have the advantage of lowering the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens. They have recently been reported to be biocompatible and to promote epithelial, stromal and nerve regeneration in phase 1 human clinical trials (Griffith et al., 2009).

Keratocytes or stromal fibroblasts can be photoencapsulated into hydrogels

(Garagorri et al., 2008). Depending on the intended goal, the surface of the hydrogel can be modified to promote (Zainuddin et al., 2008) or reduce cell attachment (Rafat et al., 2009), or even to gradually release growth factors (Princz and Sheardown, 2008) or drugs (Liu et al., 2008a), which could help in the healing or the long-term implant success of the grafted stromal equivalent.

Self-assembly approach

The self-assembly approach constitutes a truly new concept for tissue engineering. We originally designed it to reconstruct tissues as similar as possible to their physiological in vivo counterparts. In this approach, cells produce their own ECM and organize it into a structured three-dimensional network, without adding exogenous collagen or synthetic material (Auger et al., 2002). Combined with the use of adequate cell types, the self-assembly approach results in the production of complex tissues with the expected histological, mechanical and functional properties as has been previously demonstrated by our group for blood vessels and skin (L’Heureux et al., 1998; Michel et al., 1999). The vascularization system can also be reconstructed as

demonstrated by the formation of a capillary-like network in our skin construct. This new technology has recently given rise to very promising results for the reconstruction of human corneas (stroma and epithelium) in that corneal substitutes made out of corneal fibroblasts and corneal epithelial cells yielded a translucent tissue comparable to a native cornea, and dermal substitutes made out of dermal fibroblasts and dermal epithelial cells yielded an opaque tissue

(Carrier et al., 2008; Carrier et al., 2009). In these tissue-engineered corneas, the stromal cells produced a dense matrix and the epithelial–mesenchymal interactions led to the formation of a complete basement membrane with all the expected ultrastructural components (lamina lucida, lamina densa, hemidesmosomes). Moreover, a new technology allowed the formation of oriented consecutive collagen lamellae (see Section 6.6).

6.3.3Reconstruction of the posterior cornea

For many years, researchers have evaluated the feasibility of reconstructing a corneal endothelium from cultured corneal endothelial cells seeded on a carrier for eventual transplantation in humans. Different carriers have been proposed, such as hydrogels (Doillon et al., 2003; Griffith et al., 1999; Mimura et al., 2004b Mohay et al., 1994; Mohay et al., 1997), vitrigel

(Koizumi et al., 2007), thin carriers such as Descemet’s membranes (Lange et al., 1993), amniotic membranes, (Ishino et al., 2004; Wencan et al., 2007) or gelatin membranes (Jumblatt et al., 1980; Lai et al., 2007; McCulley et al., 1980; Schwartz and McCulley, 1981), as well as living or devitalized native corneas (see following two subsections).

Seeding cultured endothelial cells on living native stromas

The native corneal stroma is an ideal carrier primarily because it is transparent, has the right curvature, is naturally biocompatible and is mechanically stable. For these reasons, many investigators have used fresh native corneas as carriers for in vitro studies (Aboalchamat et al., 1999; Amano, 2003; Amano et al., 2005; Bohnke et al., 1999; Chen et al., 2001; Engelmann et al., 1999; Gospodarowicz and Greenburg, 1979) or for transplantation in rats (Mimura et al., 2004a; Tchah, 1992), mice (Joo et al., 2000), rabbits (Jumblatt et al., 1978), cats (Bahn et al., 1982; Gospodarowicz et al.,

1979b) and monkeys (Insler and Lopez, 1986; Insler and Lopez, 1991a; Insler and Lopez, 1991b). Descemet’s membrane was denuded of its endothelium using a cotton-swab. Cultured allogeneic or xenogeneic corneal endothelial cells were then seeded on top and cultured for times varying from 30 minutes (Joo et al., 2000) to 7 days (Tchah, 1992). However, the high risk of contamination of the endothelium by the proliferating native epithelium of the living carrier impedes the use of prolonged periods of culture with fresh corneas.

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Seeding cultured endothelial cells on devitalized native stromas

An alternative to fresh native corneas is the use of devitalized frozen corneas as carriers (Amano et al., 2008; Bohnke et al., 1999; Engelmann et al., 1999; Proulx et al., 2009a). Various methods have been reported for the initial removal of the native corneal endothelium. Most groups denude the

Descemet’s membrane mechanically using a cotton-swab (Alvarado et al., 1981; Amano, 2003; Bahn et al., 1982; Bohnke et al., 1999; Engelmann et al., 1999; Gospodarowicz and Greenburg, 1979; Gospodarowicz et al., 1979a; Gospodarowicz et al., 1979b; Insler and Lopez, 1986; Insler and Lopez, 1991a;

Insler and Lopez, 1991b; Joo et al., 2000; Jumblatt et al., 1978; Mimura et al., 2004a; Tchah, 1992). Other studies have reported denuding Descemet’s membrane chemically using ammonium hydroxide (Bohnke et al., 1999; Chen et al., 2001; Engelmann et al., 1999) or physically using one freeze–thaw cycle (Bohnke et al., 1999; Engelmann et al., 1999). In their comparative study of these three techniques, Engelman et al. found that the best method was denuding Descemet’s membrane using one freeze (–80 °C)–thaw cycle

(Engelmann et al., 1999), as endothelial cells did not adhere well on to corneas treated chemically with ammonium hydroxide, and mechanical debridement with a cotton-swab left abundant residual components on the

Descemet’s membrane. We used the freeze–thaw technique to denude the Descemet’s membrane and also found good preservation of that membrane after three cycles (Proulx et al., 2009a).

We previously reported the production of a tissue-engineered corneal endothelium reconstructed from cultured feline corneal endothelial cells seeded on a devitalized stromal carrier (Proulx et al., 2009a). Native cells of the carrier were eliminated through three freeze–thaw cycles. Devitalization eliminates any potential risk of contamination of the seeded endothelial cells by epithelial cells or keratocytes, while maintaining normal corneal shape and transparency. The destruction of all native cells by the three freeze–thaw cycles is also expected to reduce the immunogenicity of the carrier (Hori and Niederkorn, 2007; Quantock et al., 2005). Most of all, corneas traditionally discarded because of the poor quality of their epithelium or endothelium could eventually be used and even kept frozen until needed, thus increasing tissue availability and decreasing wastage.

In vivo experimental applications: transplantation of a reconstructed corneal endothelium

Our laboratory has demonstrated that these cultured endothelial cells seeded on a devitalized stromal carrier can recover an active pump function and restore and maintain normal corneal thickness as well as crystal clear transparency over a 7-day observation period after transplantation (Proulx et al., 2009b).

Assessment of the long-term functional outcome in the feline model will be the next necessary step in the development of this bioengineered living tissue, thereby representing a very promising approach for the treatment of endothelial dysfunctions.

6.3.4Reconstruction of a complete cornea with all three corneal cell types

Reconstructing a complete cornea that mimics the structure and function of the normal tissue is a major bioengineering challenge. An approach that has been previously used by many investigators is a step-by-step procedure consisting of first culturing corneal endothelial cells on a culture insert, then pouring over either a collagen or a fibrin–agarose gel (cross-linked or not) embedded with stromal fibroblasts and, finally, culturing corneal epithelial cells on top of the polymerized gel (Alaminos et al., 2006; Doillon et al., 2003; Griffith et al., 1999; Reichl and Muller-Goymann, 2003; Reichl et al., 2004; Tegtmeyer et al., 2001; Zieske et al., 1994). Using an ouabain test assay, Griffith et al. (1999) have shown that their immortalized endothelial cells seeded on a collagen-chondroitin sulfate hydrogel were actively pumping fluid out of their stromal substitute.

More recently, a collagen–chondroitin sulfate foam was used to reconstruct a complete cornea. The scaffold was first seeded with stromal keratocytes and then successively with epithelial and endothelial cells (Vrana et al., 2008). Other than demonstrating the feasibility of reconstructing a complete corneal equivalent containing all three cell types, two studies have shown that adding endothelial cells to the epithelial/stroma construct had an effect on epithelial cell differentiation and basement membrane quality. Indeed, in a rabbit model, constructs with endothelial cells showed a more defined LM and collagen VII staining and an improved epithelial differentiation (Zieske et al., 1994). Using a human model, this influence was shown to be mediated through soluble factors secreted from the underlying corneal endothelium (Orwin and Hubel, 2000).

6.4In vitro experimental applications

6.4.1Re-epithelialization in a three-dimensional woundhealing model

Because of its anatomical localization, the cornea is more likely to be injured due to exposure to abrasive forces and occasional mechanical trauma, leading to wounding and scarring. Scarring of the corneal surface can result in the loss of transparency and even blindness. Many of these wound-healing problems are associated with the inability to reorganize a complete and mature smooth

168 Biomaterials and regenerative medicine in ophthalmology

epithelium. Therefore, several models of wound healing have been developed in order to better understand the corneal mechanisms of re-epithelialization (Boisjoly et al., 1993; Grant et al., 1992; Maldonado and Furcht, 1995; Nelson et al., 1990; Simmons et al., 1987). However, the in vitro models of wound healing using cell monolayers lack the epithelial–mesenchymal interactions and are limited by the absence of multiple epithelial cell layers. Corneal wound healing has also been studied using human ex vivo organ culture models (Chuck et al., 2001; Collin et al., 1995; Foreman et al.,

1996; Lin and Boehnke, 1997; Hardarson et al., 2004; Tanelian and Bisla, 1992; Zagon et al., 2000). However, the availability of normal human donor corneas is limited and results can be influenced by factors such as variable delays between death and reception (Zhao et al., 2003), reduction in epithelial cell layers, incomplete epithelium and stromal edema (Richard et al., 1991; Tanelian and Bisla, 1992; Van Horn et al., 1975).

The in vivo animal models of corneal wound healing using rabbits, rodents and horses (Brazzell et al., 1991 Burling et al., 2000; Kim et al., 2001; Zieske et al., 2001) are difficult to extrapolate to humans because, in contrast to humans, animal corneas have been shown to possess many stem cells in their central cornea, certainly affecting their response to wound healing (Majo et al., 2008). Thus, there is a need for a new human three-dimensional corneal wound-healing model comprising both a well-differentiated epithelium and living fibroblasts. Using the previously described self-assembly approach (see subsection ‘Self-assembly approach’ in Section 6.3.2), we developed a fully human three-dimensional and completely biological anterior cornea, comprising both living fibroblasts and epithelial cells, thus allowing epithelial–mesenchymal interactions (Carrier et al., 2008). In this in vitro model, human corneal epithelial cells migrate over a natural ECM (Fig. 6.5). This model also allows the study of a single parameter at a time as a result of controlled and reproducible in vitro conditions. Results showed that, during re-epithelialization, epithelial cell migration followed a consistent wave-like pattern (Fig. 6.6) (Carrier et al., 2008) similar to that reported for human corneal wound healing in vivo (Dua and Forrester, 1987). It also allowed quantification of the re-epithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. Therefore, this model offers a tool to compare and evaluate, under standard conditions, the effects of various exogenous factors on the rate and quality of re-epithelialization of the cornea.

This completely biological three-dimensional model sounds very promising for further studies on the mechanisms involved in the corneal reepithelialization process, such as investigation of the expression, distribution and characterization of the role of growth factors, their receptors and extracellular matrix proteins.