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

were removed soon after grafting. For healing purposes, the operated corneas were covered with soft contact lenses. The animals were monitored for 6 months. Corneal transparency was restored and the grafts remained stable. There is a mention at the end of this study that four human patients were grafted with allogeneic or autologous limbal epithelial sheets, and grafts were stable after 2–6 months. Favourable results in rabbits were reported with sheets of oral mucosal epithelial cells (Hayashida et al., 2005). Reconstruction of the ocular surface in four human patients using sheets of autologous oral mucosal epithelial cells was also reported (Nishida et al., 2004b). Corneal transparency was restored and maintained over a mean follow-up period of 14 months. Visual acuity improved remarkably and the eyes were free of complications. It is worth mentioning that the cell sheet engineering approach was also used successfully by the same group to prepare sheets of human corneal endothelial cells (Ide et al., 2006; Sumide et al., 2006).

The cell sheet strategy offers some advantages over other procedures for the reconstruction of the ocular surface. For instance, cells can be harvested without using proteolytic enzymes. The avoidance of using biodegradable polymers as substrata which are transplanted together with the cell sheet is also regarded as an advantage (Yang et al., 2006). However, there is still a need for a support in order to transfer the cell sheet on to the cornea. As for using oral mucosal epithelial cells, longer-term results in larger-groups are needed to prove the validity of the procedure, as this phenotype is unlikely to differentiate into the corneal phenotype.

8.7.2Thermoresponsive gel matrix

The block copolymers of poly(N-isopropylacrylamide-co-butyl methacrylate) and poly(ethylene glycol) behave like thermoresponsive polymers able to undergo sol-gel transitions at ambient temperatures (Yoshioka et al., 1994a; Yoshioka et al., 1994b; Yoshioka et al., 1994c). One such polymer, developed as Mebiol gel® (Mebiol Inc., Kanagawa, Japan), dissolves in water at temperatures below 20 ºC to generate solutions but becomes hydrophobic at temperatures above 20 ºC to form water-insoluble gels. It offers the possibility of embedding growing cells either by mixing them with the solution at low temperatures or by placing them on a solid gel substratum and then covering it with the solution. The temperature is then raised to the routine incubation value of 37 ºC, when the matrix solidifies and the cells become embedded within it. A variety of cell types have been cultured successfully in a Mebiol gel® matrix, and recently the culture of human corneal limbal epithelial cells has also been reported (Sudha et al., 2006). In this study, cells from donor tissue biopsies were grown between two plates of solidified gel. The cells proliferated profusely and by the tenth day migrated out of the gel matrix to form an external layer. Various assays indicated that the cells expressed

230 Biomaterials and regenerative medicine in ophthalmology

presumed limbal stem cell association markers, transient amplifying cell markers and corneal differentiation markers. Mebiol gel® remains an interesting alternative as a substratum for limbal epithelial constructs, although the matrix is not biodegradable.

8.8Preliminary evaluation of silk fibroin as a substratum for human limbal epithelial cells

8.8.1Background

We have recently proposed and evaluated a silk protein as a potential substratum material in the development of limbal epithelial constructs (Chirila et al., 2007; Chirila et al., 2008). Silks are natural high-molecular-weight polypeptide composites belonging to the group of fibrous proteins – which also includes collagens, elastins, keratins and myosins – and are characterized by highly repetitive amino acid sequences leading to significant homogeneity of their secondary structure and remarkable mechanical properties and functional performance. Silks are produced mainly by the larvae of certain species in the class Insecta, including the order Lepidoptera (moths and butterflies), and by species in the class Arachnida, prominently the order

Araneae (spiders). There is a great variation in the chemical composition, structure and properties of the silks between species. It was estimated (Kaplan et al., 1994) that perhaps only 0.1% of the silks presently known have been characterized in any detail. However, the silk produced by the domesticated silkworm (Bombyx mori) has been widely investigated. Silkworm silk fibres are constituted of core fibrous proteins (fibroins), which are held together by coats of glue-like proteins (sericins). The secondary structure of fibroin consists in planar β-pleated sheets packed in an anti-parallel fashion (Marsh et al., 1955). There has been an increasing interest in the use of silks, especially of silkworm silk, as biomaterials (Minoura et al., 1990; Altman et al., 2003; Gobin et al., 2006; Wang et al., 2006; Bettinger et al., 2007; Hakimi et al., 2007). Silkworm silk has a long record of use as surgical sutures, although these did elicit an inflammatory response in the eye (Moore and Aronson,

1969; Salthouse et al., 1977; Soong and Kenyon, 1984; Altman et al., 2003), which was attributed to the allergenic activity of sericins. By removing the sericin component, this problem is usually avoided (Santin et al., 1999; Altman et al., 2003; Panilaitis et al., 2003; Meinel et al., 2005), although there are some concerns (Kurosaki et al., 1999) that in rare instances fibroin too may cause delayed hypersensitivity.

We have investigated the growth and morphology of human limbal epithelial cell cultures, established from donor tissue, after seeding on to

Bombyx mori silk fibroin (BMSF) membranes. In our most recent study

(Chirila et al., 2008), the cells were cultured in the absence of feeder cells,

and using a commercial serum-free culture medium. Comparison was made with cultures grown on tissue culture grade plastic. This was the first time that corneal limbal epithelial cells have been cultured on BMSF, although a significant number of other cell types have been grown on it, as recently reviewed (Wang et al., 2006; Chirila et al., 2008). BMSF proved to be a suitable substratum for the marrow mesenchymal stem cells in the tissue engineering of ligament, cartilage or bone (Wang et al., 2006).

8.8.2Experiments and results

The protocol to isolate fibroin from silk cocoons included the removal of sericin in hot sodium carbonate solutions, dissolution of raw fibroin in a concentrated solution of lithium bromide, filtration and dialysis against water. The membranes were obtained by the evaporation of water at room temperature, followed by stabilization with concentrated aqueous methanol.

The resulting dry films of BMSF are remarkably transparent, smooth and easy to handle with forceps in a similar fashion to pieces of cellophane.

Upon addition to the culture medium, the BMSF membranes remain flat and acquire a certain degree of flexibility.

Cultures of human limbal epithelial cells were established from donor corneoscleral rims discarded after routine keratoplasty. Primary cultures were grown in a commercial serum-free medium designed to promote growth of corneal epithelial progenitor cells (PCT Corneal Epithelium Medium, Chemicon Australia, Boronia, Victoria, Australia), referred to as CnT-20 medium. A quantitative comparison of the cell growth on BMSF and on tissue culture plastic was performed by seeding freshly passaged cells on to either substrate at a density of 3000 cells/cm2. The total number of cells after

3 days in culture was counted in a phase-contrast fluorescent microscope, after staining the cell nuclei.

Despite some degree of variation between cell donors, the growth of cells on BMSF was found to be not significantly different from that displayed by the cells grown on tissue culture plastic (Fig. 8.1).

8.8.3Discussion

We have demonstrated a similar growth of human corneal limbal epithelial cells on BMSF material and on tissue culture plastic. Importantly, this result was obtained under serum-free conditions thus encouraging the development of protocols that avoid the use of animalor donor human-derived products. The transparency of BMSF membranes is an obvious advantage for a biomaterial targeted for the reconstruction of the ocular surface. Another advantage of using the BMSF membranes is the ability of fibroin to biodegrade slowly in a biological environment (Altman et al., 2003; Wang et al., 2008). Owing

to its defined chemical structure at the surface, BMSF can be modified to bind growth factors, defence peptides and other bioactive agents that may be beneficial in the incorporation and maintenance of the epithelial constructs.

We believe that the BMSF membrane might therefore provide a valuable alternative to the AM. Nevertheless, a comparative study of cultures grown on BMSF and AM, including the identification of cellular phenotype, has yet to be performed.

The mechanism of cell attachment to BMSF in the absence of serum proteins remains an intriguing issue. Some studies (Minoura et al., 1995a; Hakimi et al., 2005) have shown that the adhesion of certain cells to BMSF was less than that observed on membranes made from the wild silkworm (Antheraea pernyi) silk fibroin. This was explained (Minoura et al., 1995a) by the presence of the adhesion ligand peptide sequence arginine–glycine–aspartic acid

(RGD) in the structure of wild silk fibroin, and it was tentatively suggested that the abundant presence of arginine alone in BMSF may be sufficient for

imparting cell-adhesive properties. It was alternatively surmised (Minoura et al., 1995b; Gotoh et al., 1998) that such properties could also be a result of the electrostatic interactions between cell surface (with a net negative charge due to the glycocalyx) and the positively charged primary amine residues on the surface of BMSF. If this is the case, the cell adhesion to BMSF is a non-specific process. More recently, two peptide residues located near the N-terminus of the fibroin heavy chain that apparently enhanced the growth of fibroblasts were isolated from BMSF and identified as a decapeptide and an octapeptide (Yamada et al., 2004). The assumption that these sequences may be hitherto unknown cell adhesion ligands has been neither substantiated nor confirmed so far.

8.9Conclusions

OSDs have the potential to cause partial or complete limbal deficiency and result in pain and blindness. The search for a remedy to these diseases is vitally important, but such a remedy has not been forthcoming, despite an increased scientific emphasis for more than 30 years. Initially, this was because of the relative ignorance of the pathogenic processes. However, even with scientific investigation and understanding of the anatomical, physiological, and biochemical pathways of corneal limbal stem cells, progress in the search for these remedies has been slow and frustrating. Techniques for the ex vivo expansion and grafting of cultured epithelial sheets using cells harvested from the eye or even the oropharynx have not yet addressed the long-term needs of these patients. Perhaps this is because there has been little attention paid to the role of the stroma and its resident cells, which is immediately adjacent to the overlying epithelium. The extracellular matrix and its cellular components have a substantial, if poorly understood, effect on the regenerating epithelium. It is unlikely that an answer to the replacement of the surface lining will come without some acknowledgement of the importance of the underlying substratum. Communication between epithelial and stromal elements is vital for the normal function of the ocular surface and thus the grafting of healthy epithelial cells on to a damaged or diseased stroma is unlikely to produce successful outcomes. Whether the inner third lining of the cornea, the endothelium, will also – in the fullness of time – be shown to participate directly in the creation of a healthy ocular surface, remains to be seen.

Gazing into the crystal ball of medical science, one can only presume that the rehabilitation of the ocular surface will require a stable and mature epithelial layer together with stem/progenitor cells for replacing the constantly shed mature cells, and an active substratum. What the requirements of the substratum will be needs even more intense crystal gazing, but ultimately it will depend less on its composition and origin, i.e. either naturally derived

234 Biomaterials and regenerative medicine in ophthalmology

materials or synthetic polymers, and more on its ability to support and activate an overlying epithelium with the necessary biochemical messages that promote, stabilize and characterize this epithelium. We believe that composite cultures of epithelial and stromal cells, including keratocytes and neuronal cells, will need to be developed in conjunction with new or modified biomaterials as substrata.

8.10Acknowledgements

Support is acknowledged from the Prevent Blindness Foundation through Viertel’s Vision, Queensland, Australia, and from Research to Prevent Blindness, New York, USA. We are grateful to Dr Edeline Wentrup-Byrne for her careful reading of the manuscript and for her helpful suggestions.

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