Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants

Abstract

Currently, damage to the ocular surface can be repaired by transferring laboratory cultured limbal epithelial cells (LECs) to the cornea using donor human amniotic membrane as the cell carrier. We describe the development of a synthetic biodegradable membrane of Poly d,l-lactide-co-glycolide (PLGA) with a 50:50 ratio of lactide and glycolide for the delivery of both isolated LECs and of cells grown out from limbal tissue explants. Both isolated LECs and limbal explants produced confluent limbal cultures within 2 weeks of culture on the membranes without the need for fibroblast feeder layers. Outgrowth of cells from explants was promoted by the inclusion of fibrin. Membranes with cells on them broke down predictably within 4–6 weeks in vitro and the breakdown was faster for a lower molecular weight (MW) (44 kg/mol) rather than a higher MW (153 kg/mol) PLGA. Membranes could be reproducibly produced, sterilised with gamma irradiation and stored dry at −20 °C for at least 12 months, and the ability to support cell outgrowth from explants was retained. We demonstrate transfer of cells (both isolated LECs and of cells grown out from limbal explants) from the membranes to an ex vivo rabbit cornea model. Characterisations of the cells by immunohistochemistry showed both differentiated and stem cell populations. A synthetic membrane combined with limbal explants in theatre would avoid the need for tissue banked human amniotic membrane and also avoid the need for specialist laboratory facilities for LEC expansion making this more accessible to many more surgeons and patients.

Introduction

The overall aim of this work is to simplify and make safer and more accessible the methodology for transplanting limbal epithelial cells (LECs) to resurface and regenerate the cornea for patients who have lost vision due to limbal stem cell deficiency (LSCD). Currently there are only a small number of specialist ophthalmic centres worldwide (we estimate less than 10 on the basis of published results) who have expertise in laboratory expansion of LECs and of transplanting these cells to the cornea using either fibrin as a substrate [1] or human amniotic membrane (hAM) [2], [3].

An attractive alternative to using cultured cells, as was recently demonstrated [4], is outgrowth of cells from limbal explants in situ (on amniotic membrane) obviating the need for expansion of LECs in the laboratory at least for patients with unilateral LSCD.

Accordingly our specific aims are to develop a synthetic, sterilised, rapidly degrading membrane that can be readily available to use as an alternative to the hAM and to explore regeneration of the corneal epithelium both using laboratory cultured cells and outgrowth of cells from small limbal explants. A combination of off-the-shelf synthetic membrane and culture of limbal epithelial cells in situ could make this technique available to many more surgeons and hence patients than can currently access treatment for LSCD only through specialist centres.

It is necessary to review the current clinical situation to set the context for this study. Damage to or loss of the LECs of the eye can lead to extensive scar tissue formation often with vascularisation, an unstable epithelium and considerable pain [5], [6].

Full thickness corneal transplantation (i.e. penetrating keratoplasty) can temporarily restore vision in patients with LSCD but eventually fails due to insufficient or lack of recipient limbal stem cells necessary for sustained regeneration of the corneal epithelium [7], [8]. To replace the loss of stem cells, the culture of LECs in the laboratory and their subsequent transfer was established 16 years ago [9] and this is now a treatment option available to patients in some specialised centres around the world [2], [4], [9]. Culture of cells from the contralateral unaffected eye is undertaken wherever possible (under regulated laboratory conditions approved by the appropriate regulatory bodies for each country) or from donor eyes when no autologous cells are available [10]. The latter patients then require long term immunosuppression.

At its simplest if the loss of LECs is unilateral, then a biopsy can be taken from the healthy contralateral eye, cells expanded in the laboratory and then transplanted to the affected eye following removal of the scarred tissue. The condition of the eye post removal of the scarred tissue is such that cultured cells placed directly on it would be unlikely to survive; hence cells are by preference placed onto a degradable substrate prior to transplantation. Of the various substrates that have been explored, the hAM is the one most commonly used in clinical transplantation. A recent report by our group (reviewing 200 patients) found a 76% success rate of LEC transplantation on the amniotic membrane after 1 year, dropping to 68% after 4 years [11].

There are likely to be many reasons why some patients continue to do well after several years and others do not including the initial aetiology of the condition which may dictate the extent of the damage to the corneal stroma and the likelihood of recurrence of the condition. In addition, donor variability in the quality and processing of the fresh hAM may also contribute to the success of the treatment. Certainly the rate of breakdown of the amniotic membrane is not consistent between different recipients.

For all of these reasons including the need to establish tissue banks for processing the hAM, several groups are investigating alternatives to this material with both synthetic and natural materials being explored. These include fibrin glue [1], collagen membranes [12], [13], thermo-sensitive substrates [14] and synthetic polymer membranes [15], [16]. Of these only hAM and fibrin are currently routinely used as carriers for cell transfer in the clinic for corneal regeneration.

The desirable characteristics of a membrane for delivering LECs are that it provides secure attachment for the cells and supports their proliferation and migration. Additionally, it must be as low risk for clinical use as possible, i.e. ideally it must be sterilised using an acceptable sterilisation methodology. It must be capable of being fixed to the eye post culture with cells and for it to be successful it needs to degrade within a few weeks leaving LECs securely attached to the underlying cornea without eliciting an immune response. Thus we seek a membrane that will degrade predictably within a few weeks, support LEC attachment and subsequently deliver these cells securely attached to the cornea and which is capable of being produced, sterilised and stored reproducibly.

We selected poly(d,l-lactide-co-glycolide) (PLGA) for this purpose because it is biodegradable and non-cytotoxic, FDA approved and has been used for many years in products such as dissolvable sutures [17]. Such sutures are currently used in ocular surgeries and by varying the ratio of lactide to glycolide it is also possible to predict the degradation rates of these membranes in vivo [18].

In this study we examined the ability of these synthetic membranes to support the growth of isolated LECs, both in the presence and absence of feeder cells, and also its ability to support outgrowth of LEC from intact limbal tissue explants placed directly on the membranes. We looked at the rate of breakdown of the membranes with and without cells in vitro. We confirmed that both isolated cells and cells outgrown from limbal explants would transfer from these membranes as they broke down onto an ex vivo rabbit cornea model. Finally, we looked at the impact of γ-irradiation on the physical characteristics of the membrane and its breakdown in the presence of cells and also the ability of these membranes to be stored in the presence of desiccant for long term storage.