Cultivated autologous limbal epithelial transplantation for symptomatic bullous keratopathy

Abstract

We report the clinical outcome of cultivated autologous limbal epithelial transplantation for symptomatic bullous keratopathy in an 87-year-old female patient presenting with recurrent corneal erosion and intractable ocular pain. The patient refused corneal transplantation, and transplantation of a cultivated autologous limbal epithelial sheet was carried out to free her of the symptoms associated with chronic pseudophakic bullous keratopathy. Both integrity of transplanted epithelium and relief from intolerable ocular pain were achieved and maintained immediately after surgery. No complications were noted in either the transplanted eye or donor eye during the 43-month follow-up period. Our findings support that patients with bullous keratopathy not consenting to procedures to improve visual acuity in whom the primary aim is relief of symptoms may benefit from cultivated autologous limbal epithelial transplantation.

BACKGROUND

Abnormalities in corneal epithelial adhesion resulting in the formation of blisters or bullae have been associated with long-standing bullous keratopathy. Such blisters or bullae may rupture causing intolerable ocular pain and recurrent corneal erosion. The replacement of endothelium by penetrating keratoplasty or endothelial keratoplasty is the definitive treatment for bullous keratopathy. However, in some patients where keratoplasty is refused or where there is limited visual potential there are a number of alternatives, including application of autologous serum,¹ bandage contact lenses, patching of the eye, anterior stromal punctures,^2,3 excimer laser phototherapeutic keratectomy and conjunctival flap. However, all of these options have been associated with persistent epithelial problems that may result in corneal infection or recurrence of blister formation.

Transplantation of human amniotic membrane has been reported to be successful in the restoration of corneal epithelial adhesion resulting in prevention of recurrent corneal erosion and relief from pain.^4–6 However, as epithelial healing on amniotic membrane may take several weeks to complete, corneal infection or deposition of calcium can occur. Amniotic membrane is also used as a substrate for the cultivation of limbal epithelial cells. The transplantation of cultivated epithelial sheets has been reported for the treatment of corneal surface disorders and offers the advantage of immediate epithelialisation following surgery.^7,8 The present report describes a novel and first-time application of cultivated autologous limbal epithelial transplantation as an alternative treatment for symptomatic bullous keratopathy.

CASE PRESENTATION

The patient was an 87-year-old woman presenting with a 3-year history of intractable ocular pain due to long-standing pseudophakic bullous keratopathy in the right eye. She had undergone secondary implantation of an anterior chamber intraocular lens following intracapsular cataract extraction and anterior vitrectomy. Recurrent erosion developed 3 years after surgery. Although a bandage contact lens, autologous serum eye drops and ointment were applied at a local hospital, corneal erosion recurred and pain persisted. At her initial examination at our hospital, diffuse corneal oedema and tilting of the intraocular lens were noted (fig 1). Best-corrected visual acuities were 20/2000 in the right eye and 20/20 in the left eye. Intraocular pressure was normal in both eyes. The left eye was pseudophakic with normal limbal structure. She refused penetrate keratoplasty for visual recovery and cultivated autologous limbal epithelial transplantation was carried out to relieve her symptoms. Written informed consent was obtained before surgery. The ethics committee of Tokyo Dental College approved the use of amniotic membrane in ocular surface surgery and cultivated limbal epithelial transplantation.

Figure 1.

Pre-operative slit-lamp photograph (A) and fluorescein staining (B) of bullous keratopathy with blister formation (arrow).

TREATMENT

Preparation of limbal epithelial cell sheets was performed as previously described.⁹ Briefly, an approximately 2×2 mm sheet of limbal tissue was obtained from the fellow eye and cultured on denuded amniotic membrane with mitomycin C-treated 3T3 fibroblasts for 2 weeks. With written informed consent in accordance with the Declaration of Helsinki, amniotic membrane was harvested from mothers seronegative for hepatitis B, C and syphilis at the time of caesarean section. The epithelial sheet was submerged in medium until confluence and cultured at the air-liquid interface for the final 2 days.

For cultivated autologous limbal epithelial transplantation, local anaesthesia with sub-Tenon injection of 2% lidocaine was applied and an 8.0 mm trephine was used to create an annular and partial-thickness keratectomy incision 0.2 mm in depth. Loose epithelium in the central cornea was removed and an 8 mm diameter cultivated limbal epithelial sheet was secured to the edge of the annular and partial-thickness keratectomy incision with a single 10–0 nylon running suture. A soft contact lens was applied to protect the transplanted epithelium. Topical steroid (0.1% dexamethasone, Santen Pharmaceutical Co, Osaka, Japan) and antibiotics (levofroxacin, Santen Pharmaceuticals) were used for 4 months after surgery.

OUTCOME AND FOLLOW-UP

Immediate corneal epithelialisation and relief from intolerable pain associated with bullous keratopathy was observed after cultivated autologous limbal epithelial transplantation. The integrity of the transplanted epithelium was maintained throughout the 43-month follow-up period (fig 2). Postoperative best-corrected visual acuity was 20/2000. No complications such as infection, necrosis, or rejection of amniotic membrane were observed in either the transplanted eye or donor site during the follow-up period.

Figure 2.

Post-operative slit-lamp photographs at 3 months (A) and fluorescein staining at 9 months (B) show stable integrity of transplanted epithelium with no staining or recurrent blister formation.

DISCUSSION

The results indicate the viability of cultivated autologous limbal epithelial transplantation as an alternative treatment for symptomatic bullous keratopathy offering relief from intolerable pain and restoration of epithelial integrity. Transplantation of amniotic membrane for symptomatic bullous keratopathy has been reported.^4–6 Amniotic membrane provides not only substrates for epithelium, but also functions to prevent inflammation, angiogenesis and scarring; thus, enhancing rapid wound healing in epithelium.¹⁰ Although epithelialisation in many cases was completed approximately 2 weeks after surgery,^4–6 delays in epithelialisation can develop with completion sometimes taking as long as 6 weeks. In addition, deposition of calcium resulting from delayed epithelialisation causing chronic foreign body sensation can occur. The advantage of cultivated autologous limbal epithelial transplantation over amniotic membrane transplantation alone is fast recovery. In this case, the patient’s priority was relief from intolerable ocular pain, which is why cultivated autologous limbal epithelial transplantation was preferred over amniotic membrane transplantation. Prompt epithelialisation can reduce the risk of infection or deposition of calcium on the cornea. It is also particularly beneficial in cases where follow-up may be difficult—for example, in elderly patients.

In addition to fast recovery, cultivated autologous limbal epithelial transplantation can also manage the subclinical limbal stem cell deficiency associated with long-standing bullous leratopathy.¹¹ In countries with a shortage of donor cornea supply, like Japan, patients may have to wait several years before being able to undergo corneal transplantation. Repeated corneal erosion may cause exhaustion of limbal stem cells leading to limbal stem cell deficiency. Although impression cytology was not carried out in this case, we believe that the long-standing bullous keratopathy with superficial vascularisation and lack of visualisation of Palisades of Vogt in slit-lamp examination in this patient indicated possible subclinical limbal stem cell deficiency.

However, the use of cultivated limbal epithelial transplantation has some drawbacks, including the time-consuming and laborious laboratory procedures it requires such as preparation and cultivation of epithelial cells. We also have to be concerned about the potential risk of inducing stem cell deficiency in the donor eye by harvesting limbal tissue.

The current report describes the application of cultivated autologous limbal epithelial transplantation for long-standing bullous keratopathy for the first time in the literature. Although only one case was discussed in this report, we believe that our results suggest that cultivated autologous limbal epithelial transplantation offers a viable alternative in the treatment of bullous keratopathy from the viewpoint of prompt post-surgical epithelialisation.

LEARNING POINTS

• The patients with long-standing bullous keratopathy can have subclinical limbal stem cell deficiency.

• Although the transplantation of amniotic membrane is a very effective treatment for symptomatic bullous keratopathy, it has got limitations in treating patients with limbal stem cell deficiency.

• Cultivated autologous limbal epithelial transplantation is an alternative treatment for symptomatic bullous keratopathy offering immediate relief from intolerable pain and restoration of corneal epithelial integrity.