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

of reports on the transfer of conjunctival stem/progenitor epithelial cells as in-vitro-grown constructs. This work was excellently analysed in two recent reviews (Hatton and Rubin, 2005; Selvam et al., 2006), hence we will not further expand on the subject. The localization of the stem/progenitor cells in the conjunctiva proved to be more ambiguous when compared with that in the cornea. Forniceal, palpebral and mucocutaneous regions were all proposed as zones with enriched content of stem/progenitor cells. The human conjunctival goblet cells with proliferative capacity are notoriously difficult to grow in vitro (Shatos et al., 2003; Ang et al., 2004). The clinical success of the transplantation of conjunctival epithelial constructs is limited owing to a tendency of these cells not to differentiate into the corneal epithelial phenotype; therefore, the limbal constructs are much more effective in the therapy of OSDs. An interesting approach was to co-culture limbal and conjunctival epithelial cells on AM and use the resulting constructs in human patients (Sangwan et al., 2003).

8.4Corneal equivalents as replacements or study models

We should mention here that the developmental work for tissue-engineered corneal limbal epithelial constructs differs in its approaches and aims from the development of artificial corneas (Chirila et al., 1998; Duan et al., 2006; Ruberti et al., 2007; Sheardown and Griffith, 2008) or of tissue-engineered corneal equivalents (Griffith et al., 1999; Schneider et al., 1999; Germain et al., 2000; Orwin and Hubel, 2000; Germain et al., 2004; Duan et al., 2006; Ruberti et al., 2007; Sheardown and Griffith, 2008). The former are made from synthetic polymers and do not include biological components. While at least two models are US Food and Drug Administration (FDA)-approved for distribution and use, their implantation is generally associated with clinical complications. An artificial cornea is unsuitable for the reconstruction of the ocular surface, as its role is restricted to replacing an irreversibly damaged and opaque cornea where both the ocular surface and the underlying stroma and endothelium are damaged and there is no intent to induce a regenerative process. On the other hand, the tissue-engineered corneal equivalents as yet cannot fulfil the functional prerequisites to replace a damaged cornea, and their applications have been limited to in vitro studies. Although some experimental approaches are common to both epithelial constructs and corneal equivalents, the latter are confronted by some challenges that have so far prevented their clinical applications in human patients; such challenges include conservation of transparency, duplication of mechanical properties, reproduction of extracellular matrix components expression and of barrier–pump endothelial function, presence of neural elements and elimination of cell immortalization stage from the experimental protocols.

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As a final remark, while both artificial corneas and tissue–engineered corneal equivalents are aiming at the total replacement of a dysfunctional cornea and should be used when other procedures for reconstruction have failed or are not practicable, the limbal or conjunctival epithelial constructs are intended to restore the integrity and function of damaged sectors of the ocular surfaces only. Ultimately, as the efficacy of stem cell transplantation procedures improves, the future need for the previous two alternatives may diminish (Mannis, 2002). This issue is not always understood, a situation that leads to publications where regrettably the two topics have been intermixeds, resulting in significant confusion.

Some early research of corneal equivalents was not aimed at the treatment of OSDs, but contributed to a better understanding of corneal biology.

The three-layer constructs were the first corneal equivalents to be reported

(Minami et al., 1993), with the declared aim of producing an in vitro tool for investigating corneal pathophysiology. These equivalents were made by culturing bovine normal epithelial, stromal and endothelial cells in a matrix of collagen type I. The thickness of the final constructs was around 0.2 mm, and they had an epithelium stratified in four to five layers. In another development

(Zieske et al., 1994), three-layer corneal equivalents were made as a tool to study the influence of endothelium on the differentiation of epithelial cells.

They were prepared by casting a collagen gel containing animal or human keratocytes on the top of immortalized cultured murine corneal endothelial cells, and then seeding a suspension of animal or human epithelial cells on the top of the collagen layer. When such constructs were made without an endothelial layer, the epithelial cells did not express differentiation markers or basement membrane components. This is a fine example of using corneal equivalents to acquire further knowledge. One-layer constructs were made (Kahn et al., 1993) with the aim of providing an in vitro model for ocular toxicology studies. Human corneal epithelial cells immortalized by treatment with an SV40 hybrid virus were used in this study. The cells were able to synthesize corneal-specific keratins and to promote stratification, but many of the immortalized cell lines were still shedding free virus at the conclusion of experiments. Similar work was reported later by a Japanese group (ArakiSasaki et al., 1995), who created one-layer constructs for biological studies by using epithelial cells immortalized with an SV40-adenovirus recombinant vector, which supposedly eliminated the shedding of the virus. The resulting epithelial constructs had properties similar to those of normal epithelium.

8.5Naturally derived biomaterials as substrata for tissue-engineered epithelial constructs

substratum-free constructs, other materials were investigated for this purpose long before. In their quest to promote a suitable carrier, Tsai and Tseng cultured rabbit conjunctival epithelial cells on collagen type I or Matrigel™, and on a combination of these (Tsai and Tseng, 1988). It was found that both collagen and Matrigel™ promoted the growth and differentiation of the conjunctival cells leading to either monolayers (on collagen) or stratified sheets

(on Matrigel™). Matrigel™ is a commercially available synthetic basement membrane derived from the Engelbreth–Holm–Swarm mouse sarcoma tumour cell line, and was developed by BD Biosciences (San Jose, CA, USA) based on research carried out in several laboratories of the National Institutes of Health (NIH) in Bethesda, MD, USA (Kleinman et al., 1982; Kleinman et al., 1986). Tsai later reported the culture of human conjunctival epithelial cells on collagen type I (Tsai et al., 1994). When no other cells were included in the culture, the epithelial constructs were not stratified. In the presence of conjunctival fibroblasts or 3T3 cells, the constructs were multilayered and showed many characteristic epithelial features. We are not aware, however, of any therapeutic application of this research.

Other investigators also chose collagen as a substratum, but the approach was different. Animal (rabbit) corneal basal epithelial cells were obtained from biopsies and were cultured on crosslinked gelatine membranes or on the concave side of commercial collagen corneal shields (McCulley et al., 1991). The collagen cornea shields (Willoughby et al., 2002) contain mainly collagen type I and are manufactured from porcine sclera or bovine dermis. The shields are used for ocular surface protection following surgery or trauma, and for sustained administration of drugs. The degree of crosslinking, accomplished through ultraviolet (UV) light exposure, is variable and correlated to the intended duration of the device before dissolution. The mentioned study showed that following contact and 1 or 2 days of incubation, the cell layers grown on both substrata could be transferred on to rabbit denuded corneas or cryolathed stromal lenticules (both obtained from enucleated eyes). After removing the carriers, most of the cells remained attached to the stromal surface. In these experiments, the collagen shield was more suitable as a substratum in terms of growth rate and proliferation as compared with gelatin. The same concept was applied using primary cultures of human corneal epithelial cells, but the study was limited to collagen shields as substrata (He and McCulley, 1991). A variety of growth media were employed, and some of the collagen shields were coated with Matrigel™ or with collagen type IV. The coating enhanced cell attachment. However, the cells failed to reach confluence on the Matrigel™ layer, which is contrary to the results reported by Tsai and Tseng (see above). The multilayered cultures on the collagen type IV were successfully transferred on human denuded corneas (eye bank) through contact and 2–7 days of incubation. The adhesion was strong enough to withstand the removal of supporting collagen shields.

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Although this procedure would presumably simplify surgery, it appears that other investigators did not pursue the concept. About a decade later, the same group repeated the experiments, this time using human corneal limbal epithelial cells or human amniotic cells seeded on collagen shields (He et al., 1999). The substrata with cells were then transplanted in vivo on to the de-epithelialized corneas of 27 rabbits. For the first 2 days, the eyelids were kept sutured to maintain contact between cells and host stroma. The animals were monitored for 10 days. The procedure was successful in only ten eyes, as judged by the occurrence of re-epithelialization, formation of cell–substratum hemidesmosomes and confirmation of human-specific antigen presentation. This report suggested that the procedure of cell layers transplantation through cells–stroma contact is not exempted from failure and ultimately the surgery required may become as complex as in other cellular construct transplantations. However, the technique was recently resurrected as a procedure for transplantation of epithelial constructs (Di Girolamo et al., 2007), with the difference that, instead of collagen shields, contact lenses made of synthetic polymers were proposed. Based on the observed occurrence of epithelial growth on the contact lenses used as bandages after pterygium surgery, two brands of 30-day continuous wear siloxane hydrogel contact lenses – Focus® Night & Day™ (CIBA Vision) and Pure Vision™ (Bausch & Lomb) – have been investigated as substrata for corneal limbal epithelial cells obtained from explants and cultured in autologous serum. The materials in the two contact lenses, known as ‘lotrafilcon A’ and ‘balafilcon

A’, respectively, are quite different in structure and surface topography (Tighe, 2000; López-Alemany et al., 2002). Cell growth, assessed through the analysis of morphology, proliferative capacity and cytokeratin profile, was seen only on lotrafilcon. Since commercial contact lenses are designed to resist epithelial adherence, the validity of this approach is doubtful.

Another proposed substratum material was a collagen–glycosaminoglycan (CG) copolymer, initially developed for skin regeneration (Yannas et al., 1989). It is made by the coprecipitation of bovine collagen type I and shark cartilage chondroitin 6-sulfate. The material has been evaluated as a graft for the regeneration of experimentally injured rabbit conjunctiva (Hsu et al., 2000). The grafts clearly inhibited scarring and induced the formation of a tissue resembling normal conjunctival stroma. However, no reports are available on the use of CG substrata for ocular surface reconstruction in human patients.

In Italy, De Luca’s group continued their work and treated 18 human patients with limbal stem cell deficiency in one eye by grafting autologous limbal stem cells constructs (Rama et al., 2001). This time, the cell from limbal biopsies were cultured on a layer of commercial fibrin sealant (Tissucol™, Baxter-

Immuno, Austria), which was prepared by mixing solutions of thrombin and fibrinogen. The resulting fibrin is a biodegradable and biocompatible material,

although there are biosafety issues associated with its use (Eyrich et al., 2006).

The grafts were implanted attached to the fibrin substratum. The restoration of the ocular surface was successful in 14 patients, where within 1 month the surface was covered with a transparent epithelium. The ocular surfaces were stable on follow-up between 1 and 2 years postoperatively, and three patients underwent successful penetrating keratoplasty about 1 year after the limbal transplantation. Schwab’s group suggested new directions in the application of fibrin by growing human corneal limbal epithelial cells within a matrix of fibrin gel (Han et al., 2002). In this study, both fibrinogen and thrombin components were prepared in a specialized machine, the CryoSeal FS System (ThermoGenesis Corp., Rancho Cordova, CA, USA). Cultured human corneal epithelial cells were added to the thrombin solution, and then mixed with the fibrinogen solution to form a fibrin gel with the cells embedded throughout. The cells proliferated within the gel matrix, showing normal growth kinetics. Despite all these promising results, there has been no subsequent use of fibrin reported in the literature with the exception of a recent animal study (Luengo Gimeno et al., 2007). Severe limbal stem cell deficiency was experimentally induced in rabbit eyes by inflicting alkali burns in one eye, and limbal biopsies were harvested from the contralateral eye.

Cells were grown on fibrin (Tissucol™) and the autografts were implanted about 3 weeks from the start of culture. The ocular surface was restored completely within 12 months, with a transparent, stratified epithelium covering the cornea. In the same study, for the first time platelet-rich plasma

(PRP) was used as a substratum for limbal epithelial cells, and showed some advantages (e.g. elasticity and transparency) over fibrin. PRP is an autologous product consisting of a small volume of plasma containing a large number of platelets, which can be prepared from the blood of the patients, and is biodegradable and biocompatible. Its use in transplantations is increasingly advocated (Yazawa et al., 2003; Luengo Gimeno et al., 2006).

Although introduced slightly later than most of the materials discussed above, AM prevails as a substratum for ex vivo expanded epithelial stem/ progenitor cells. A thorough analysis of the human clinical trials that have used ex vivo expanded epithelial cells (Schwab et al., 2006) indicated that of 20 studies (involving 275 patients) published between 1996 and 2005, AM was used in 16 studies as the substratum for cells, an estimate largely confirmed in a subsequently published review (Shortt et al., 2007). The first reports on the use of AM as a substratum came from Schwab’s team at the University of California at Davis (Schwab, 1999; Schwab et al., 2000). In the first study (Schwab, 1999), 19 patients were involved. With the exception of 2 patients where sibling allogeneic limbal epithelial constructs were transplanted, all other patients received autologous constructs. A variety of substrata were used in the constructs including corneal stroma, collagen type I, soft contact lenses, collagen shields and AM; the latter was used in

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7 cases. Surgical procedures were carried out between 1994 and 1998 and were followed up for durations between 2 and 24 months. At the time of reporting, 5 cases were unsuccessful, partially successful or undetermined. Only 1 unsuccessful outcome was reported with the AM. In a subsequent study (Schwab et al., 2000), 14 patients received autologous or allogeneic grafts grown on AM, and were followed up for between 6 and 19 months. The treatment was successful in 10 patients. In these studies, successful clinical outcome was determined by restoration or improvement of patient’s vision, re-epithelialization and non-recurrence of the original OSD. In a study carried out in Taiwan (Tsai et al., 2000), 6 patients were treated with autologous limbal constructs on AM substratum, and monitored for 12–18 months. In all patients the vision improved and there were no recurrent problems.

Recently, sutureless AM transplantation has been developed and assessed in the rabbit eye, using adhesive materials instead of sutures. Either fibrin glue (Sekiyama et al., 2007) or a novel ‘chemically defined bioadhesive’

(Takaoka et al., 2008) have been evaluated as adhesives. The latter adhesive was prepared by the chemical reaction of aldehyde-functionalized dextran with ε-poly(l-lysine) (Nakajima et al., 2007).

In spite of favourable clinical outcomes generally reported with AM as a substratum for the epithelial constructs, the procedure is affected by inherent difficulties in the growth and maintenance of the cells on AMs (Kinoshita and

Nakamura, 2005), and by the drawbacks mentioned previously (see Section 8.2). In addition, some investigators obtained quite disappointing clinical results that led them with conclude that there is no advantage in using this procedure when compared with other limbal transplantation techniques or with transplantation of AM alone (Shimazaki et al., 2002).

8.6Synthetic biomaterials as substrata for tissue-engineered epithelial constructs

The first use of an artificial material in the reconstruction of the ocular surface should be rather regarded as a singular episode, as the material was not intended as a substratum for cellular constructs but as a substitute for the autologous mucosal membrane grafts in the reconstruction of the socket. Such membranes, harvested from the buccal or nasal regions, have been in occasional surgical use for the past four decades and they still have proponents, although their collection involves additional surgical procedures. In this study (Levin and Dutton, 1990), patients who lost an eye as a result of previous malignancies, cicatricial pemphigoid or congenital conditions, and could not maintain their artificial socket implants because of severe damage to conjunctiva, were treated by grafting 0.1-mm-thick sheets of polytetrafluoroethylene, supplied as Gore-Tex® (Gore & Associates, Flagstaff, AZ, USA). Within 2 weeks, the residual conjunctival epithelium grew

beneath the Gore-Tex® membranes. In some instances, the polymer graft was removed to reveal complete epithelialization. Ultimately, being an opaque, hydrophobic and non-biodegradable material, polytetrafluoroethylene did not have any future in the field of ocular surface reconstruction.

Closer to our time, perhaps prompted by the drawbacks perceived with using AM, some investigators contemplated artificial substrata for the creation of limbal or conjunctival epithelial constructs. As shown below, a range of synthetic polymers were studied in vitro and in experimental animals, although not always with the purpose of creating limbal or conjunctival epithelial constructs.

As the epitome of biodegradable polymers, the lactone-based polymers, with the polylactides and polyglycolides as their prominent representatives, attracted some attention as potential substratum materials. In one study (Lee et al., 2003), poly(lactide-co-glycolide) (PLGA) porous scaffolds were modified by treatment with collagen or hyaluronic acid, or mixed with particles of AM, or subjected to combinations of these treatments. In vitro, both corneal epithelial cells (commercial line) and human corneal stromal fibroblasts

(obtained from biopsies) attached well to the scaffolds and proliferated throughout the cross-section. The PLGA scaffolds modified with collagen and hyaluronic acid were then used in vivo as grafts on rabbit eyes where conjunctival wounds were experimentally created. After 4 weeks, the wound contraction and scar formation were much less in the grafted than in the ungrafted eyes. In a more recent study (Zorlutuna et al., 2006), a commercial polylactide (Resomer® LR 708) was combined with poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), a natural biodegradable polymer. Membranes and porous scaffolds were both produced from this polymer mixture. The membranes were seeded with retinal pigment epithelial (RPE) cells, and the scaffolds were seeded with 3T3 fibroblasts. The RPE cells generated a stratified epithelium, while the 3T3 fibroblasts colonized the scaffolds and deposited neocollagen type I, prompting the authors to conclude that this polymer combination can function as a substratum material for corneal reconstruction, although is not clear why RPE cells were employed instead of corneal epithelial cells. Fibrous constructs made of another polymer from this class, poly(glycolic acid), were also reported as suitable scaffolds for the growing of corneal stromal cells (Hu et al., 2005).

Another synthetic polymer investigated experimentally as a substratum for growing corneal cells was a polyurethane. In this study (Liliensiek et al., 2006), a transparent polyurethane, commercially available as an optical adhesive (NOA61™, from Norland Products, Cranbury, NJ, USA), was modified by creating surface nanoscale topographic features. The attachment and growth of three types of human corneal cells (SV40-transformed epithelial cells, primary epithelial cells and primary fibroblast) were investigated in cultures. Topographic features (e.g. grooves, ridges) below 1 μm in size

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inhibited significantly the proliferation of all categories of cells. While this study may contribute to the vast field of research regarding the interactions between surface characteristics and cell attachment, it is of less relevance to epithelial constructs. The polymer in the study is available as a liquid that is curable by UV exposure and contains some toxic ingredients. As clearly indicated by the supplier (https://www.norlandprod.com/adhesives/NOA%20 61.html), such optical adhesives are strictly designed for binding ‘to glass surfaces, metals, fiberglass and glass filled plastics’, and prolonged contact with skin and contact with the eyes should be avoided. The investigators’ assertion that this adhesive material has ‘limited toxicity to corneal epithelial cells’ is totally unsubstantiated and misleading; in fact, the optical adhesives do not have to be biocompatible as they are designated for use outside the body.

A research project was recently carried out in the laboratories of Singapore’s

National Eye Centre and Eye Research Institute, with the precisely defined aim of assessing a synthetic polymer, poly(ε-caprolactone) (PCL), as a potential substratum for conjunctival epithelial constructs (Ang et al., 2006). PCL is a degradable material that is approved by the FDA for medical use. In the study, biaxially stretched PCL membranes were prepared to a thickness of around 6 μm. Some of the membranes were also treated chemically to enhance the hydrophilicity of their surface. Rabbit conjunctival epithelial cells were then cultured both as monolayers and as explants (the latter in a serum-free medium) on the PCL membranes, which successfully supported their attachment and proliferation leading to confluent stratified epithelial sheets. In parallel experiments it was found that the goblet cell densities on PCL and AM were not statistically different. Interestingly, the cell proliferation and stratification were greater on the PCL membranes with enhanced hydrophilicity. This finding is at odds with the well-documented trend of cells growing at interfaces of variable hydrophilicity: the more hydrophilic the surface, the fewer attached cells. It is also at odds with the principle of the strategy presented in the next section. The conjunctival epithelial constructs of this study were implanted subcutaneously in immune- deficient mice, and explanted after 1 week. The histopathological analysis revealed the formation of multilayered epithelia over the PCL membranes. There was no mention about the biodegradability of the PCL substrata, most likely because of the too short residence time. No evaluation in human eyes has yet been reported.

Poly(vinyl alcohol) (PVA) is a synthetic polymer that can be obtained with an enormous range of properties thanks to its indirect synthesis by the hydrolysis of poly(vinyl acetate), which allows variable degrees of hydrolysis and a variety of chemical and physical crosslinking methods. Being non-toxic and hydrophilic, with good mucoadhesive properties, PVA has received much attention as a biomaterial. In the early 1990s, Yoshito

Ikada’s group at Kyoto University developed PVA hydrogels intended for artificial corneas (Chirila et al., 2005). In order to promote epithelialization, the hydrogels’ surface was modified by immobilization of extracellular matrix components (collagen, fibronectin or adhesion peptides). After successful in vitro corneal epithelial cell growth experiments, the collagen-immobilized PVA was used for in vivo experiments in rabbit corneas. Unfortunately, the placement on to the cornea and the implantation into the cornea were both associated with severe postoperative complications. PVA was abandoned as a keratoprosthetic material and the research group’s interest was re-directed to polyurethanes (Chirila et al., 2005). However, 15 years later, another team used collagen-immobilized PVA (henceforth COL-PVA), prepared following Ikada’s methodology, as a substratum for cells (Miyashita et al., 2006). Although this research was aimed at developing an epithelializable keratoprosthesis, it is worth mentioning it here since the cells seeded and grown on the PVA substratum were corneal limbal epithelial cells of either human or animal origin. The cells were cultured in the presence of 3T3 feeder cells, and they generated stratified epithelial layers displaying the characteristics of a corneal epithelium. The study also included the intralamellar grafting of epithelialized COL-PVA. While the grafts were easy to handle and suture, and showed mechanical properties similar to donor tissue grafts,

‘the sutures became loose after a few days due to inflammation, causing the epithelium to detach from the polymer surface’ (Miyashita et al., 2006). In order to improve the outcome, the same team later developed a substratum where AM was glued to COL-PVA using a tissue adhesive based on citric acid (Uchino et al., 2007). In vitro, rabbit limbal epithelial cells generated a stratified epithelium. In a comparative study in vivo using rabbits, AM-PVA and COL-PVA were implanted in pockets created on the cornea. All corneas transplanted with COL-PVA lost epithelium by 2 weeks, while those with AM-PVA showed partial or complete epithelialization in all eyes. Clearly, the better outcome was due entirely to the presence of AM, which probably would have happened even in the absence of PVA. However, it is difficult to fathom how an artificial cornea incorporating an AM can be made into a commercial product.

8.7Strategies based on thermoresponsive polymers

8.7.1The ‘cell sheet engineering’ approach

This approach is the result of work carried out over the last two decades by Teruo Okano’s group at Tokyo Women’s Medical University, and is based on the existence of polymers able to display thermoresponsitivity as they possess a so-called ‘lower critical solution temperature (LCST)’. The

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phenomenon and substances displaying it, including polymers, have been known for a long time (Freeman and Rowlinson, 1960; Patterson, 1969; Taylor and Cerankowski, 1975). The most investigated polymer showing LCST is poly(N-isopropylacrylamide) (PIPAAm) (Heskins and Guillet, 1968; Fujishige et al., 1989; Kubota et al., 1990; Takata et al., 2002; Kara and Pekcan, 2003), which was also chosen by Okano for this application (Yamada et al., 1990; Okano et al., 1993; Okano et al., 1995; Nishida, 2003; Yang et al., 2006). In principle, at temperatures above 32 °C (which is the polymer’s LCST), PIPAAm is hydrophobic and therefore can support the attachment, spreading and growth of cells. The hydrophobic behaviour is due to particularities in the structure of the polymer and to the hydrogen bond interactions between amide groups and water molecules. At temperatures above LCST, water is partially displaced from the macromolecular coil, the hydrogen bonds involving water are weakened and the hydrophobic interactions between polymer segments become dominant, resulting in a compact (‘collapsed’) conformation of the macromolecular chains that does not allow further water penetration. The routine cell incubation temperature (37 °C) is well above LCST, thus assuring normal growth of cells on the PIPAAm surfaces in their hydrophobic state. When the temperature is lowered below LCST, the polymer surface turns hydrophilic, as the hydrogen bonding between the hydrophilic segments and water molecules becomes dominant and leads to an extended conformation of the macromolecules. As soon as the surface turns hydrophilic and consequently swells in the aqueous medium to become hydrated, the cell sheet detaches completely because of the very poor propensity of cells in general to attach to hydrated surfaces.

The confluent cell layer can be harvested as a single uninterrupted sheet; it was shown (Yamato et al., 2001) that normal cell–cell junctions and the extracellular matrix are maintained in the sheets obtained by this technique. Cell sheet engineering has been applied so far in ocular surface reconstruction and myocardial tissue engineering, as well as in alternative therapies such as endoscopic transplantation for treating cancers of the gastrointestinal tract, development of tracheal prostheses and healing enhancement after laser refractive surgery (Yang et al., 2006).

Application of the cell sheet engineering concept to the reconstruction of the ocular surface is a result of the collaboration between Nishida (at Osaka University Medical School) and Okano’s team. In a preliminary study (Nishida et al., 2004a), human and rabbit corneal limbal epithelial cells were cultured on tissue culture dishes that were coated with a layer of PIPAAm by electron beam irradiation. Cells were cultured in the presence of 3T3 feeder cells (growth arrested with mitomycin C) at 37 °C for 2 weeks, and then harvested at 20 °C. Rabbit cell sheets were transplanted in rabbits with experimentally induced limbal stem cell deficiencies. The sheets were transferred while placed on poly(vinylidene difluoride) membranes, which