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Clinical science
Amnion-assisted conjunctival epithelial redirection in limbal stem cell grafting
  1. Harminder S Dua,
  2. Ammar Miri,
  3. Mohamed S Elalfy,
  4. Anna Lencova,
  5. Dalia G Said
  1. Section of Academic Ophthalmology, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, Nottingham, UK
  1. Correspondence to Professor HS Dua, Department of Ophthalmology, B Floor, Eye ENT Centre, Queens Medical Centre, Derby Road, Nottingham NG7 2UH, UK; harminder.dua{at}nottingham.ac.uk

Abstract

Aims To develop a technique using amniotic membrane (AM) to prevent admixture of conjunctival epithelial cells and limbal explant-derived corneal epithelial cells in patients undergoing limbal stem cell transplantation. To compare this technique with the current method of ‘sequential sector conjunctival epitheliectomy’ (SSCE).

Methods 26 patients with total limbal stem cell deficiency who underwent ocular surface reconstruction with limbal stem cells transplantation were retrospectively studied. Patients were categorised into group A (11) in which AM was used to direct the conjunctival epithelial cells away from the corneal surface so that the latter could be covered by the limbal explant-derived epithelial cells—the procedure was termed ‘amnion-assisted conjunctival epithelial redirection’ (ACER)—and group B (15) in which the conjunctival epithelium was prevented from migrating on to the corneal surface by SSCE.

Results In nine eyes of group A, the conjunctival epithelium was successfully directed on to the AM, preventing admixture with limbal explant-derived corneal epithelial cells. The AM was removed or it came off spontaneously within 1–4 weeks. Patients treated with SSCE (group B) underwent two to four interventions until complete re-epithelialisation. 12 patients had pain or discomfort. 11 patients had conjunctival haemorrhage during SSCE. The cornea was epithelised from the limbal explant-derived epithelium in all patients.

Conclusions ACER is a viable option in limbal transplantation that reduces multiple patient visits, bleeding and pain that can be associated with SSCE.

  • Conjunctiva
  • Cornea
  • Stem Cells

https://doi.org/10.1136/bjophthalmol-2015-307935

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    Introduction

    Conjunctivalisation of the cornea is considered to be the hallmark of limbal stem cell deficiency (LSCD).1–4 Conjunctival epithelium migrates on to the corneal surface following injury such as chemical burns or during the course of chronic disease such as Stevens-Johnson syndrome, when the limbal ‘barrier’ is breached.1 ,2 ,5–8 Conjunctival epithelial encroachment on the cornea usually brings with it goblet cells and blood vessels.9 ,10 The surface remains irregular, shows late staining with fluorescein, is prone to recurrent erosions and, when it extends across the visual axis, results in poor vision.2 ,4 ,11 Following injury, when limbal involvement is sub-total, it is possible to monitor over time the re-epithelialisation of the ocular surface and prevent conjunctival encroachment on to the cornea. This is done by sequential sector conjunctival epitheliectomy (SSCE),12 ,13 wherein the conjunctival epithelial sheet that is migrating towards the limbus is mechanically brushed or scraped back in that sector, under topical anaesthesia usually at the slit lamp. This can be repeated every day or every other day as often as needed to allow the corneal and limbal surface to be covered by cells migrating from the remaining intact limbal epithelium.14 When the patient presents with part of the cornea already conjunctivalised, it is still possible to perform SSCE, removing the conjunctival epithelium on the cornea and keeping it at bay by repeat brushing or scraping peripheral to the limbus until the denuded surface is covered by limbus-derived corneal epithelium.4 ,12 ,14

    Another important application of SSCE is in association with autologous limbal transplants and living-related limbal transplants. In these situations, donor limbus explants are transplanted to the recipient eye usually at the 12 and 6 o'clock positions (some transplant it at the three and nine o'clock positions) after removing the fibrovascular pannus from the affected cornea.2 ,4 ,15 ,16 A large area of the recipient limbus between the explants both nasally and temporally is left uncovered. Ideally the limbal explant-derived epithelium migrates circumferentially along the recipient limbus12 ,14 from above and below. However, often conjunctival epithelium from the peritomised conjunctiva migrates comparatively rapidly and crosses the limbus to cover the cornea. This results in conjunctivalisation of the cornea. SSCE is used successfully to prevent this.4 ,12 ,14 Limitations of SSCE in these circumstances are that it can cause bleeding from surface vessels, can be painful and requires the patient to stay in hospital or make multiple trips for observation and treatment. We devised an approach, using amniotic membrane (AM), to redirect conjunctival epithelium on to the membrane allowing the corneal surface to be covered by explant-derived epithelium, thus completely avoiding admixture of limbal/corneal and conjunctival epithelium and obviating the need for SSCE. This procedure has been termed amnion-assisted conjunctival epithelial redirection (ACER). A description of the technique and results of our first cohort of patients are presented herein.

    Material and methods

    Twenty-six patients with total LSCD who underwent ocular surface reconstruction with limbal stem cells transplantation (LSCTs) were included in this retrospective study. Patients were categorised into group A (11 patients) (nine men and two women) in the age range from 6 to 72 years with a mean age of 42 years who underwent autologous or living-related limbal stem cell transplantation with ACER for total LSCD and group B (15 patients) (nine men and five women) in the age range from 18 to 72 years with a mean age of 43 years who underwent autologous or living-related LSCT and SSCE for total LSCD. Patients' history, cause of LSCD, laterality (unilateral or bilateral) and clinical findings from slit lamp microscopy and in vivo confocal microscopy (IVCM) of limbus (to confirm LSCD) were ascertained and recorded. Chemical burns were the most common cause for LSCD in both groups, followed by aniridia. Patient details are given in table 1.

    View this table:
    Table 1

    Clinical details of patients undergoing limbal stem cell transplant with amnion-assisted conjunctival epithelial redirection (ACER) (group A) and patients undergoing limbal stem cell transplant without ACER (group B)

    Previous surgery in group A included amniotic membrane transplantation (AMT) in three patients and botulinum toxin-induced ptosis in one patient. Two patients had glaucoma, one of whom had previous Ahmed valve surgery. One patient had mucous membrane transplantation with punctual cautery. One patient had a penetrating corneal graft which had failed with persistent epithelial defect. One patient had excision of a conjunctival melanoma. Previous surgery in group B included AMT in three patients. Two patients had mucous membrane transplantation. Four patients had a penetrating corneal graft which had failed with persistent epithelial defect. One patient had excision of a conjunctival intraepithelial neoplasia. One patient had entropion repair.

    Surgical technique for ACER: Under local or general anaesthesia a 360° peritomy of the conjunctiva was done and the conjunctiva recessed for 2–3 mm. The fibrovascular pannus on the cornea was dissected and peeled off (figure 1A, B) and bleeding vessels were cauterised with a bipolar cautery. Deep corneal vessels were cauterised by fine needle diathermy (Force FX Electrosurgical Generator, Valleylab Electrosurgery Products, Covidien, Colorado, USA). Harvesting of donor limbal grafts was identical for autologous and living-related tissue (In group A, six eyes had autolimbal grafts and five eyes had living-related allolimbal grafts. In group B, eight eyes had autolimbal grafts and seven eyes had living-related allolimbal grafts). Two clock hours of limbus, 0.5–0.75 mm of peripheral cornea and 3 mm of adjacent conjunctiva were marked superiorly and inferiorly. The conjunctiva was incised and dissected from the fornix end towards the limbus. The marked area of limbus and peripheral cornea was lamellar dissected (approximately 100–150 µ) with a bevel-up mini crescent knife (#MCB12, MANI, Tochigi, Japan) and the harvested tissue was kept moist on a wet sponge. Inclusion of the peripheral cornea is a subtle but important modification4 to the techniques of conjunctival limbal autograft and living-related keratolimbal allograft that have been well described.17 The explants were sutured to the 12 and 6 o'clock positions of the recipient eye with 10-0 nylon sutures (#8065208001, Alcon, Surrey, UK). The donor conjunctiva was sutured to the recessed recipient conjunctiva (figure 1C, D). In three cases where the exposed corneal stroma after removal of the fibrovascular pannus was deemed irregular, an AM graft (inlay) of 8 mm diameter was sutured on the cornea with a running 10-0′nylon suture. The use of the inner membrane was to afford a suitable substrate for the cells to grow and adhere to, that is, act as a graft (inlay). It is part of the ocular surface reconstructive procedure and specifically not a part of ACER. In all cases in group A, another piece of AM (onlay or patch, also termed outer membrane when two membranes were used) was fashioned freehand and placed on the corneal surface. The edge of the membrane (5 mm) was tucked under the recessed peritomised conjunctival edge, nasally and temporally between either ends of the superior and inferior limbal explants. The membrane was glued to the sclera and the conjunctiva glued on to the membrane with fibrin glue (Tisseel, Baxter Healthcare, Deerfield, Illinois, USA). Superiorly and inferiorly where AM could not be tucked under the conjunctiva, which was sutured to the donor conjunctiva, two radial incisions were made on either side of the explant and excess AM was excised (figure 1E).

    Figure 1
    Figure 1

    Diagrammatic representation showing the amnion-assisted conjunctival epithelial redirection (ACER) technique: (A) an eye with total limbal stem cell deficiency; (B) 360° peritomy and recession of the conjunctiva; (C) the donor limbal (auto or living-related) grafts are sutured in place superiorly and inferiorly; (D) an amniotic membrane (AM) graft (inlay) is sutured in place; (E) the outer AM is large enough to cover the cornea and adjacent sclera and be tucked under the edge of the recessed conjunctiva between the ends of the limbal explants. The edge of the membrane adjacent to the explants is trimmed and hangs free. The edges of the outer membrane and the recessed conjunctiva are held in place with fibrin glue.

    Postoperatively, all eyes were treated with preservative-free dexamethasone (Minims Dexamethasone sodium phosphate 0.1% w/v Eye drops solution, Bausch & Lomb UK, Kingston-upon-Thames, Surrey, UK) four times a day for 1 month, then twice a day for another month. The frequency of instillation was adjusted up or down depending on the degree of inflammation. Preservative-free chloramphenicol (Minims Chloramphenicol 0.5%, Eye drops solution, Bausch & Lomb UK, Kingston-upon-Thames, Surrey, UK) eye drops, four times a day for 1 month were also administered. Group A patients were followed at day 1 and weekly for 4–5 weeks and at two monthly intervals thereafter for 6 months. Group B patients were followed daily until the epithelialisation of the cornea was complete. All of them needed SSCE every 1–2 days. This was done at the slit lamp, under topical anaesthesia with crescent knife (#MCB12, MANI, Tochigi, Japan) and a dry cellulose sponge (WECK-CEL, Beaver-Visitec International, Waltham, Massachusetts, USA). All patients in group B were examined weekly after complete corneal epithelialisation for the first month and at two monthly intervals thereafter for 6 months. Thereafter, all patients (groups A and B) were followed at 6-monthly intervals for 18 months. However, some of them continued to attend annually for review for up to 8 years (table 1). Patients undergoing living-related limbal transplants were also given oral tacrolimus (Prograf Astellas Pharma US, Northbrook, Illinois, USA), 1 mg twice a day and monitored monthly for liver and kidney function, blood pressure and blood sugar and serum tacrolimus trough levels.

    In all group A patients, the onlay membrane (patch or outer membrane when two membranes were used) was removed at week 4 if it had not already come away. This was done at the slit lamp under topical anaesthesia. The onlay membrane was found to adhere to the episclera in the bare area created by recession of the peritomised conjunctiva. When the outer membrane came away, it did so along the sclerocorneal junction where it was not adherent to the underlying limbus/cornea. When it had to be removed, one blade of Vannas scissors was pushed through the unattached membrane, which was then cut along the limbus. The junction of the attached (adherent) and unattached (non-adherent) membrane could be ascertained by lifting the blade of the scissors anteriorly, thus tenting the unattached membrane and incising along the junction with the attached part of the membrane. Thus, the denuded sclera remained covered by AM with overlying conjunctiva-derived epithelial cells. The corneal surface was assessed for epithelialisation with fluorescein stain and slit lamp photographs were taken.

    The primary outcome measure was prevention of conjunctival epithelium from encroaching on the corneal surface in both groups but without the need for SSCE in group A patients. The secondary outcome measure was complete epithelialisation of the cornea with donor limbus-derived epithelium and alleviation of preoperative symptoms. Local ethics committee/audit approval was obtained and the study conformed to the tenets of the Declaration of Helsinki.

    Results

    The diagnosis of LSCD was made on the basis of clinical examination and IVCM as described before.9 ,10 ,18 In group A, the outer membrane succeeded to divert the conjunctival epithelium from growing on the cornea in nine patients. In two patients the membrane fell the next day and SSCE had to be performed. Final best-corrected visual acuity (BCVA) was variable and ranged from perception of hand movements to 6/6.

    The conjunctival epithelium from the recessed conjunctiva could be clearly seen to migrate on the outer AM nasally and temporally as illustrated in the sequential images from preoperative status (figure 2A, B), immediate postoperative on day 1 (figure 2C, D) to final follow-up (figure 2E–J). There was no epithelial growth on the ‘outer’ AM superiorly and inferiorly (figure 2E week 2 and figure 2F week 3) as the recipient conjunctiva at these sites was sutured to the donor conjunctiva. In cases where the onlay/outer membrane stayed on till week 3 to week 4 follow-up visit, it was found to be completely covered with conjunctival epithelial cells (figure 3A, B). Removal of the outer membrane at this time point revealed a completely epithelialised corneal surface (figure 2G, H and figure 3C). In most eyes the outer membrane started to come off within 1–3 weeks. In vivo confocal microscopy corroborated the clinical evaluation of the presence of hyper-reflective conjunctival epithelial cells on the outer membrane and of corneal epithelial cells, dark cells with bright borders, on the cornea after removal of the outer membrane (figure 4).

    Figure 2
    Figure 2

    Slit lamp photomicrographs (diffuse illumination, without and with fluorescein stain) showing (A and B) preoperative images of the conjunctivalised vascularised cornea with persistent epithelial defects; (C and D) 1 day postoperatively. The outer amniotic membrane (AM) adjacent to the superior explant is seen to hang loose. (E) image showing conjunctival epithelium being redirected on to the amniotic membrane nasally and temporally at week 2 and (F) image showing further progression of conjunctival epithelium on the amniotic membrane nasally and temporally at week 3. The outer membrane is seen to be progressively covered with conjunctiva-derived epithelial cells. Conjunctival cell migration is seen nasally and temporally only between the superior and inferior explants. There is no migration superiorly and inferiorly where the host conjunctiva is sutured to the explant conjunctiva, which prevents host conjunctival cells from migrating on to the outer membrane. (G and H) The outer membrane is seen coming off revealing a completely epithelised (from autologous limbal explants) corneal surface beneath. (I and J) Images of the treated eye at last follow-up, 1 year postoperatively.

    Figure 3
    Figure 3

    (A and B) The outer membrane is completely epithelised with conjunctiva-derived epithelium. (C) Image of the underlying corneal surface, epithelised by limbal explant-derived cells seen on removal of the outer membrane. A focal area of recurrence of the superficial fibrovascular panus is seen infero-nasally but did not affect the visual axis.

    Figure 4
    Figure 4

    In vivo confocal microscopy images of (A) conjunctival epithelium on the outer membrane in amnion-assisted conjunctival epithelial redirection (ACER) showing hyper-reflective cell bodies of different sizes and with dark and ill-defined cell boundaries; (B) the amnion stroma seen under the conjunctival cells. This gives a homogenous acellular appearance. (C) Keratocytes in the anterior stroma are visible posterior to the amnion stroma. This indicates that the conjunctival epithelial cells were growing on the amnion. (D) Limbal explant-derived corneal epithelium on the corneal surface after removal of the outer membrane showing a fairly uniform cell size, dark cell bodies and hyper-reflective cell margins.

    In two eyes, though complete epithelialisation of the cornea was initially achieved, persistent epithelial defects were seen on subsequent follow-up visits, which had to be conservatively managed with topical lubrication and serum drops. In one eye, the membrane had fallen off on day 1 and SSCE was carried out to ensure that complete epithelial cover from donor explants was achieved. All eyes maintained normal corneal epithelial cover on the last follow-up visit (figure 2I, J; and figure 3C).

    In group B, SSCE was needed in all patients every 1–2 days during the first 10–14 days until epithelialisation was complete. The minimum number of SSCE interventions was two and the maximum was six (mean=4). Eight patients presented with pain and four presented with discomfort after the effect of topical anaesthesia had worn off. Eleven cases had subconjunctival haemorrhage during one or more SSCE interventions. In all patients the cornea was epithelialised from the limbal explant-derived epithelium. One patient developed a persistent corneal epithelial defect after initial epithelialisation. Final BCVA was variable and ranged from hand movements to 6/6.

    Discussion

    Ocular surface wound-healing with a desirable outcome and successful ocular surface reconstruction following injury or disease depend to a considerable extent on the amount of surviving conjunctival epithelium.19 The conjunctiva can be both friend and foe. When there is total corneal and limbal epithelial denudation, working on the principle that any epithelial cover is better than no epithelial cover,4 ,20 conjunctival epithelial migration on to the cornea is welcomed and often encouraged. Epithelial cover of the cornea reduces the risk of melts and perforation. Ex-vivo expanded sheets of conjunctival epithelium on AM have been used to reconstruct the corneal surface.21 Despite this, when there is any surviving limbal epithelium, the ideal outcome is achieved when the corneal surface is re-epithelialised by limbus-derived corneal epithelial cells.

    When autologous or living-related limbal transplants are used for ocular surface reconstruction following total limbal deficiency, it is desirable that corneal epithelialisation is achieved from the transplanted limbal explant-derived cells. The limitation is the amount of donor limbus tissue that can be harvested from the other normal eye or from a living relative. This means that large areas of the limbus are left uncovered between explants. This allows easy access to conjunctival epithelium to migrate on to the denuded corneal surface. Attempts to close the gaps and hold back the conjunctival epithelium have been made by transplanting two pieces, each of four clock hours of cadaver limbus, on the exposed recipient limbus on either side of the other explants—the Barcelona-Cincinnati technique.22 This has the advantage of affording 360° of limbal cover to the recipient cornea but offsets the advantage of autologous and human leucocyte antigen -matched living-related transplantation by the introduction of (unmatched) cadaver tissue that is more at risk of rejection. Simple limbal epithelial transplant (SLET) described by Sangwan et al23 and more comprehensively by Basu et al24 is another effective technique in ocular surface reconstruction. In this technique too, a 360° peritomy of the conjunctiva is performed and pieces of limbal tissue are attached with fibrin glue on the surface of an AM sheet (inlay/graft), which in turn is glued on to the corneal surface. The authors have not commented on the migration of conjunctival epithelium on to the amnion carrying the limbal explants. It is likely that the ACER technique would benefit SLET as well by keeping the conjunctival epithelium away until the surface is re-epithelialised with cells derived from the limbal explants.

    The introduction of SSCE has made it possible to restrict the migration of conjunctival epithelial cells on to the cornea following conjunctival-limbal-allograft or autograft transplantation. One major disadvantage of SSCE is that the patient needs to be monitored closely on a daily basis and the procedure is to be carried out every day or every other day as demonstrated in this study. ACER offers the surgeon the option of using a well-tested and tried biological tissue to achieve the objective of keeping the conjunctival epithelium away from the corneal surface without the increased risk of immune rejection. In the context of autolimbal and living-related limbal grafts, it addresses the disadvantages of SSCE by eliminating the need for multiple visits and avoiding the discomfort and bleeding created by repeated brushing off of the unwanted epithelium. In this study we were able to achieve this objective successfully in 9 of 11 cases. In two cases the outer membrane fell off very soon. This complication was subsequently avoided by ensuring that the outer membrane was tailored such that at least 5 mm of amnion was covered by the peritomised conjunctiva. Sutures can be used instead of glue or together with glue to secure amnion and conjunctiva together, thereby reducing the possibility of the outer membrane falling off prematurely. AM has been used by several groups either as a graft (inlay)24–26 or combined as a graft and patch (inlay and onlay)27 ,28 in association with limbal transplantation. In all reported studies, when the amnion was applied as a patch (onlay, single membrane or outer membrane of two membranes), it was used to ‘protect’ the corneal surface and limbal explants. The edges of the membrane were not tucked under the peritomised conjunctiva but sutured to the episclera and edge of the peritomised conjunctiva. By ensuring that the ‘onlay’ membrane or ‘patch’ is overlapped by the recessed edge of the peritomised conjunctiva nasally and temporally, conjunctival cells can be completely excluded from mixing with the limbal explant-derived epithelial cells as we have described. The migration of cells from the periphery to the outer membrane nasally and temporally and the lack of such migration from above and below where the host conjunctiva is sutured to the explant conjunctiva (which is expected due to contact inhibition) indicates that the cells growing on the outer membrane were conjunctiva-derived. Furthermore, the IVCM demonstration of conjunctival phenotype cells on amnion stroma, which in turn is resting on corneal stroma, adds to the evidence supporting the redirection of the ‘unwanted’ conjunctival epithelial cells on to the outer membrane.

    One potential disadvantage is that it is difficult to clinically monitor the outgrowth from the limbal explants under AM. The transparency of AM can vary; thus, accurate observation of the progress of the process of re-epithelialisation of the cornea beneath the membrane is not possible. However, IVCM can be used to visualise cells on the cornea posterior to the outer membrane as previously described.29 Amnion not covered with (conjunctival) epithelial cells stains with fluorescein just as denuded cornea, making it more difficult to visualise the corneal surface beneath. Epithelial growth from the conjunctiva occurs at a rapid pace compared with that from the limbal explants. This can be delayed by the use of fibrin glue to attach the conjunctiva on to AM. Fibrin glue is known to mechanically interfere with the migration of cells, which starts only when the glue has lysed.30 In most cases the redirecting sheet of amnion begins to fall off between week 1 and 3 by which time the cornea is substantially resurfaced by limbal explant-derived epithelium, making ingress of conjunctival epithelial cells much less likely due to contact inhibition. If the amnion has not spontaneously come off, it can be removed by the slit lamp under topical anaesthesia around 3–4 weeks.

    ACER is thus a useful option to be considered in the management of patients undergoing limbal transplantation for LSCD. It obviates the need of SSCE and possible associated adverse events of bleeding, pain and repeated patient visits. The same outcome can be achieved by the Barcelona-Cincinnati technique22 and transplantation of an ex-vivo expanded sheet of limbal epithelial cells. However, the former introduces additional antigen load and increases of immune-mediated rejection, while the latter is more expensive by several orders of magnitude. On the other hand, ACER can complement SLET, which itself is a promising and relatively new technique.

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    Footnotes

    • Contributors HSD and DGS were responsible for the concept, design, data acquisition and interpretation and surgery. AM and MSE undertook the literature review and also participated in data acquisition. AL and DGS helped with the images. HSD wrote the manuscript and all authors suggested changes and approved the final version.

    • Funding Elizabeth C King Trust.

    • Competing interests HSD receives personal fees from Allergan, Thea, Santen, Nicox and Dompe and has shares in GlaxoSmithKline Beecham. None of the other authors have any competing interest to declare.

    • Ethics approval Nottingham Research Ethics/Audit Committee.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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