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

et al., 1999a; von Recum et al., 1999b). RPE cells from various sources (chicken, post-mortem human, or D407 line) were able to attach and grow on this substratum, and stable cell sheets could be detached and manipulated. Importantly, the RPE cells maintained a normal retinoid metabolism, a function usually lost during cell culture. Owing to the chemistry of the surface of the copolymer, growth factors could be easily immobilized on to the surface, a treatment that improved the proliferation of RPE cells. Other types of RPE cells (rat, ARPE-19) showed similar behaviour when the substratum was the structurally simpler homopolymer, poly(NIPAAm) (Abe et al., 2006; Kubota et al., 2006).

Although both biodegradable and thermoresponsive polymers have been the synthetic materials most studied as substrata for RPE cells, there is no record of any in vivo or clinical application so far.

There have also been episodic reports of individual polymers evaluated as potential substrata for RPE cell constructs. For instance, membranes made from a proprietary synthetic hydrogel based on methacrylamide (Organogel Canada, Quebec), coated with poly(d-lysine) and fibronectin, were used as substrata for human RPE cells in vitro (Singh et al., 2001). Although the growth and behaviour of cells was deemed as successful as that seen on lens capsule substrata, we are not aware of any further developments involving this hydrogel. A group at the University of Liverpool (UK) reported the in vitro evaluation of a series of commercial polyurethanes (Williams et al., 2005) and of a commercial silicone elastomer (Krishna et al., 2007), which were plasma-treated for the enhancement of cell adhesion. Although the RPE cells readily proliferated and the assays showed the maintenance of the main cellular functions, it is to be noted that these polymers are not biodegradable and consequently they would be retained indefinitely after transplantation. Nevertheless, the authors perceived this as an advantage over the biodegradable substrata because of the potential release of toxic breakdown products from the latter.

Some advanced materials based on the allotropic forms of carbon have been contemplated as substrata for RPE cell growth. Such a material was ‘bucky paper’ (sometimes worded as ‘buckypaper’). Bucky paper is a member of the fullerenes, which are molecular structures composed entirely of carbon and include spherical, cylindrical, and planar molecules – known, respectively, as buckyballs, carbon nanotubes (CNTs), and graphenes. Bucky paper is an entangled aggregate of CNTs held together as a planar film. Conventionally, the thickness of bucky paper is between 50 and 500 μm. This material can be generated by a variety of methods based on the dispersion of CNTs (using surfactants, acid oxidation, etc.) followed by filtration. Recently, a method (‘frit filtration’) has been established (Whitby et al., 2008) that avoids some of the disadvantages associated with the previous methods. Although there are contradictory reports on the biocompatibility of CNTs (Zanello et al.,

402 Biomaterials and regenerative medicine in ophthalmology

2006), bucky paper has attracted attention as a potential substratum for cell growth because of its inertness, adjustable thickness and porosity, and mechanical properties. Collaborative work at Stanford University and NASA Ames Research Center proved that bucky paper can function as a substratum for RPE cells (Leng et al., 2003; Loftus et al., 2006). Both human RPE cells (ARPE-19) and IPE cells (harvested from rabbit eyes) were cultured successfully on sheets of bucky paper (50–100 μm in thickness) in serumsupplemented media, although the IPE cells did not from a uniform layer. The in vivo biocompatibility of bucky paper was investigated by implantation of sheets into the subretinal space of rabbits and was followed-up for 1 month. The bucky paper sheet was easy to handle and the material was well tolerated in the subretinal space. The issue of the non-biodegradability of bucky paper was not mentioned by these investigators. We are not aware of any continuation of this work.

15.6.3Preliminary evaluation of silk fibroin as a substratum for retinal pigment epithelium cells

We have recently proposed and evaluated a protein isolated from natural silk as a substratum material for the growth of RPE cells (Kwan et al., 2007).

Silk proteins belong to the group of fibrous proteins, which also includes collagens, elastins, and myosins. There is an enormous range of silks, which are produced predominantly by the larvae of insects from the order

Lepidoptera (i.e. moths and butterflies) and by spiders (Araneae). We have focused our attention on the silk produced by the domesticated silkworm (Bombyx mori), which is basically constituted from fibroin and sericin.

There is much interest in using the silkworm silk as a biomaterial (Altman et al., 2003; Hakimi et al., 2007; Wang et al., 2006). In fact, this silk has a long record of use as surgical sutures, in spite of frequent inflammatory response in the eye (Moore and Aronson, 1969; Salthouse et al., 1977; Soong and Kenyon, 1984), which was attributed to the allergenic activity of sericin. By removing the sericin, this problem can be avoided (Altman et al.,

2003), although reportedly fibroin itself may trigger delayed hypersensitivity

(Kurosaki et al., 1999).

In our project, we evaluated a membrane based on a silkworm silk fibroin (Bombyx mori silk fibroin, henceforth BMSF), and assessed the feasibility of using such a tissue as a scaffold for growing a monolayer of RPE cells.

We believe that one of the features that makes silk fibroin attractive as a substratum for the tissue-engineered RPE constructs is its ability to degrade in the presence of enzymes at a rate that suits the ideal duration for such an application. In addition, silk membrane is a flexible and pliable material, and is mechanically strong even as a very thin film. This physical consistency makes it a potential material for carrying RPE cells into the subretinal space.

In a pilot study, we evaluated BMSF as a substratum for RPE culture both alone or coated with a selection of ECM proteins, and with or without serum. The ARPE-19 cell line was seeded on to tissue culture plastic (TCP), BMSF membrane alone, or BMSF membrane coated with laminin, vitronectin, fibronectin, a laminin–vitronectin–fibronectin combination (LVF), or collagen type IV. Samples were cultured in media containing foetal bovine serum

(FBS) for 72 hours, then fixed and stained with nuclear stain Hoechst, and the cells attached were counted. Experiments were repeated with serum-starved ARPE-19 cells, which were seeded on to the different substrata, and cultured under serum-free conditions for 24 hours. The results showed that ARPE-19 cell growth on the BMSF membrane demonstrated no statistical difference (P > 0.05) when compared with TCP in FBS-containing culture conditions. The ARPE-19 cell count on the BMSF membrane alone in serum-free culture conditions was 50% of that on standard TCP in FBS (P = 0.01). However, the cell counts on BMSF membranes coated with ECM proteins surpassed unmodified BMSF membrane alone (vitronectin > collagen IV > fibronectin

> LVF > laminin). Furthermore, cell attachment on BMSF membranes coated with vitronectin or collagen IV in serum-free conditions was found to be comparable with that seen in medium containing FBS on TCP (Kwan et al., 2007). We are looking further into the characteristics of RPE cells growing on BMSF and different conditions that may enhance their cellular expression and survival.

15.7Conclusions and future trends

In conclusion, there is a need to identify the ideal substratum for RPE cell growth and potential RPE transplant as current clinical therapies are inadequate for curing the different presentations of AMD. Although there have been some advances in the development of potential substrata for RPE cell growth and maintenance, it remains unclear as to what represents the best substratum (e.g. material), the most suitable vascular components (e.g. underlying vascular scaffold), the best microenvironment (e.g. combination of growth factors), the ideal cell type (e.g. RPCs vs genetically modified cells), and the most appropriate timing of the surgery. All these factors pose a great challenge to both scientists and clinicians in finding a cure for AMD, but if this enigma could be solved it would make a tremendous impact on the lives of AMD sufferers in the future.

15.8Acknowledgements

We would like to thank Drs Damien Harkin, Zeke Barnard, and Zainuddin for providing their expertise in cell culture and production of silk fibroin membranes. We are also grateful for support from the Prevent Blindness

404 Biomaterials and regenerative medicine in ophthalmology

Foundation through Viertels’ Vision, Queensland, Australia. Funding for the project through an ORIA/Vision Australia research grant is also acknowledged.

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16

Hydrogel sealants for wound repair in ophthalmic surgery

M. Wathier and M. W. Grinstaff, Boston

University, USA

Abstract: Each year, 15 million individuals worldwide seek treatment for the repair of ocular wounds. New hydrogel sealants – crosslinked polymer matrices that possess a large fraction of water by weight – have been successfully used to repair a variety of different types of trauma and

surgically induced ocular wounds. This chapter recounts the recent advances in the development of hydrogel materials and the ongoing work to design the ideal ophthalmic sealant.

Key words: hydrogel, ocular wounds, ophthalmic sealant, biomaterials, dendrimers.

16.1Introduction

Today, the use of sutures is the most common method to repair ocular wounds; however, this technique has many drawbacks due to its application and properties. Indeed, among the drawbacks, this technique induces new trauma and new sites for infection, inflammation, and vascularization, while often leading to uneven healing. Furthermore, it is a time-consuming procedure and requires technical skill. Within the last 8 years, there has been significant research into the development of new alternatives, with some notable successes. The expansion of material engineering and macromolecular sciences has enabled the development of new hydrogel sealants – crosslinked polymer matrices that possess a large fraction of water by weight – that have been successfully used to repair a variety of different types of trauma and surgically induced ocular wounds. Herein, we recount these recent accomplishments in the development of hydrogel materials and the ongoing work to design the ideal ophthalmic sealant.

16.2Background and clinical needs

Each year more than 15 million individuals worldwide seek treatment for the repair of ocular wounds. Of these ocular wounds, there are numerous ophthalmic conditions and procedures that result in corneal wounds, including corneal ulcers, lacerations, perforations, transplants (0.1 million), incisions for cataract removal and intraocular lens (IOL) implantation (11 million),

412 Biomaterials and regenerative medicine in ophthalmology

and laser-assisted in situ keratomileusis (LASIK) (3 million). Today, the standard procedure for repairing ocular injuries involves using nylon sutures. Unfortunately, the use of these sutures constitutes an invasive surgical procedure and gives rise to a host of other complications. In addition to the potential for further trauma to corneal tissue (to include corneal inflammation and vascularization), the site itself is often more prone to infection. Instances of uneven healing and even astigmatism have also been reported. These drawbacks in and of themselves are problematic, and when one also considers that sutures may loosen or break postoperatively, or that they require removal after surgery, the need to find viable alternatives becomes all the more pressing.

Therefore, the need for alternative methods to repair ocular wounds is of current basic and clinical interest. Of the methods being explored, the use of polymeric adhesives or sealants has attracted significant attention. Polymers that adhere to tissue are of clinical value for applications where surgical procedures using sutures are not effective. It is for this reason that tissue sealants are an attractive alternative to sutures. It is hypothesized that the negative effects largely associated with sutures can be minimized by the use of tissue sealants. The goal of tissue sealant therapy is to provide immediate restoration of structural integrity and to prevent further tissue thinning. In fact, there is a precedent for the use of glues in ophthalmology, particularly in the management of corneal perforations. Previous ophthalmic sealants based on synthetic and/or natural polymers – such as cyanoacrylates, fibrin, and modified chondroitin sulfate–aldehyde systems – are not ideal for a number of reasons (including lack of biocompatibility, degradation, expensive cost, limitations in sealing only small ocular wounds, and/or complex methods of application). A new trend in ophthalmic sealant development is the use of hydrogels for repairing corneal perforations, and thus these hydrogel sealants must meet a number of requirements. Ideally, they should: (a) be biocompatible; (b) adhere to the moist corneal surface; (c) have a suitable polymerization time to set the polymer on the wound; (d) restore quickly the intraocular pressure (IOP); (e) possess a degradation time that matches the normal healing process; (f) be more elastic than corneal tissue, so as to disfavor formation of an astigmatism during healing; (g) be transparent and soft, making them the ideal adhesive sealant; and (h) be easily delivered to the tissue site.

The following sections describe the major uses of natural and synthetic hydrogel-based adhesives (see Fig. 16.1 for examples) in ophthalmology with an analysis of their uses in amniotic membrane transplants, scleral lacerations, corneal wounds, corneal transplants, and retinal detachments.