Simple limbal epithelial transplantation (SLET): A novel surgical technique for the treatment of unilateral limbal stem cell deficiency

Abstract

This study describes a novel surgical technique of limbal transplantation, which combines the benefits of existing techniques while avoiding their difficulties. Six patients with unilateral and total limbal stem cell deficiency following ocular surface burns underwent a single-stage procedure. A 2 × 2 mm strip of donor limbal tissue was obtained from the healthy eye and divided into eight to ten small pieces. After surgical preparation of the recipient ocular surface, these tiny limbal transplants were distributed evenly over an amniotic membrane placed on the cornea. After surgery, a completely epithelialised, avascular and stable corneal surface was seen in all recipient eyes by 6 weeks, and this was maintained at a mean ± SD follow-up of 9.2 ± 1.9 months. Visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/60 or better in four (66.6%) eyes, while none of the donor eyes developed any complications. This technique requires less donor tissue than previously used for conventional autografting and does not need a specialist laboratory for cell expansion. Although long-term results are awaited, this simple limbal epithelial transplantation promises to be an easy and effective technique for treating unilateral limbal stem cell deficiency following ocular burns.

Full text

Simple limbal epithelial transplantation (SLET):
a novel surgical technique for the treatment of
unilateral limbal stem cell deficiency
Virender S Sangwan,
1,2
Sayan Basu,
1,2
Sheila MacNeil,
3
Dorairajan Balasubramanian
4
ABSTRACT
This study describes a novel surgical technique of limbal
transplantation, which combines the benefits of existing
techniques while avoiding their difficulties. Six patients
with unilateral and total limbal stem cell deficiency
following ocular surface burns underwent a single-stage
procedure. A 232 mm strip of donor limbal tissue was
obtained from the healthy eye and divided into eight to
ten small pieces. After surgical preparation of the
recipient ocular surface, these tiny limbal transplants
were distributed evenly over an amniotic membrane
placed on the cornea. After surgery, a completely
epithelialised, avascular and stable corneal surface was
seen in all recipient eyes by 6 weeks, and this was
maintained at a mean6SD follow-up of
9.261.9 months. Visual acuity improved from worse
than 20/200 in all recipient eyes before surgery to 20/60
or better in four (66.6%) eyes, while none of the donor
eyes developed any complications. This technique
requires less donor tissue than previously used for
conventional autografting and does not need a specialist
laboratory for cell expansion. Although long-term results
are awaited, this simple limbal epithelial transplantation
promises to be an easy and effective technique for
treating unilateral limbal stem cell deficiency following
ocular burns.
Corneal blindness following ocular burns was once
considered incurable as it carried an extremely poor
prognosis for corneal transplantation.
1
Subsequent
discovery of epithelial stem cells at the limbal
region of the eye
23
demonstrated that ocular
surface disease was a manifestation of limbal
dysfunction and could possibly be reversed by
limbal stem cell transplantation.
4
As proof of this
principle, Kenyon and Tseng showed that
conjunctival limbal autografting (CLAU) success-
fully cured limbal stem cell deficiency (LSCD).
5
In
this technique, two donor lenticules, each
consisting of three clock hours of the limbus and
adjacent conjunctiva, were obtained from the donor
eye and transplanted on to the recipient ocular
surface. Rarely, the donor eyes developed LSCD.
6e9
To avoid this complication, Pellegrini et al devised
a technique called cultivated limbal epithelial
transplantation (CLET), wherein less than one
clock hour of donor limbus could be expanded ex
vivo into a transplantable epithelial sheet.
10
Currently, opinion is divided about which of the
two existing techniques of limbal transplantation is
a better surgical alternative. Both techniques are
effective in the long-term restoration of the
damaged ocular surface in eyes with LSCD,
4e16
and
the surgeon’s preference is often limited by
economic and logistic factors. Proponents of CLAU
consider ex vivo cultivation unnecessary and
expensive, while proponents of CLET consider
autografting technically challenging and risky. In
this study, we propose a novel simplified technique
of limbal transplantation, which combines the
benefits of both existing techniques while avoiding
the difficulties of either.
METHODS
Patients
Six adult patients with unilateral and total LSCD
(defined clinically as 360
o
absence of the limbal
palisades of Vogt, dull and irregular corneal epithe-
lium, superficial corneal vascularisation, persistent
epithelial defects or conjunctival overgrowth on the
corneal surface) following ocular surface burns and
no history of limbal transplantation were included
in this study.
Surgical technique
This was a single-stage procedure, performed by
a single surgeon (VS). Before the procedure, all
patients were administered peribulbar anaesthesia
in both eyes. In the donor eye, a 232 mm area was
marked centred on the superior limbus, the
conjunctiva was incised, and a sub-conjunctival
dissection was carried out until the limbus was
reached (figure 1A,B). A shallow dissection was
then carried out 1 mm into the clear cornea, and the
limbal tissue was excised (figure 1C) and placed in
balanced salt solution. In the recipient eye, a 360
o
peritomy was performed, and the vascular pannus
covering the cornea was removed (figure 1D,E and
online supplemental video). After cauterisation of
the bleeding points, human amniotic membrane
(hAM) graft was placed over the bared ocular
surface (figure 1F) and secured with fibrin glue
(TISSEEL Kit from Baxter AG, Vienna, Austria).
The excess membrane was trimmed and its edges
tucked under the surrounding conjunctival
margins. The donor tissue was then gently held
with Lim’s forceps and cut into eight to ten small
pieces with either Vannas scissors (figure 1G) or
a No 15 surgical blade. The small limbal transplants
were placed, epithelial side up, on the hAM and
distributed in a circular fashion around the centre of
the cornea, avoiding the visual axis (figure 1H). The
transplants were also fixed in place with fibrin glue.
A soft bandage contact lens was placed on the
recipient eye, and one drop of 2.5% povidone-iodine
was applied to both eyes, followed by overnight
patching.
<Additional materials are
published online only. To view
these files please visit the
journal online (http://bjo.bmj.
com/content/96/7.toc).
1
Cornea and Anterior Segment
Services, L V Prasad Eye
Institute, Hyderabad, India
2
Sudhakar and Sreekanth Ravi
Stem Cell Biology Laboratory,
L V Prasad Eye Institute,
Hyderabad, India
3
Department of Materials
Science and Engineering,
University of Sheffield, Sheffield,
UK
4
Champalimaud Translational
Centre for Eye Research,
Hyderabad Eye Research
Foundation, L V Prasad Eye
Institute, Hyderabad, India
Correspondence to
Dr Virender S Sangwan, L V
Prasad Eye Institute, Kallam Anji
Reddy Campus, Road No 2,
Banjara Hills, Hyderabad, India;
vsangwan@lvpei.org
All the authors (VS, SB, SM,
DB) contributed equally to the
study.
Accepted 17 January 2012
Published Online First
10 February 2012
Br J Ophthalmol 2012;96:931e934. doi:10.1136/bjophthalmol-2011-301164 931
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Postoperative care
All patients underwent comprehensive ophthalmic examination
of both eyes at all follow-up visits. Patients were seen on days 1,
7, 14, 42 and 90 and at 3-month intervals thereafter. Patients
were prescribed ciprofloxacin 0.3% eye drops (Cipla India,
Mumbai, India) four times a day for 1 week and prednisolone
acetate 1% eye drops (Alcon Laboratories Private Limited,
Bangalore, India) six times a day tapered weekly over 6 weeks in
both eyes. The bandage contact lens was removed from the
recipient eye on day 7, and carboxymethyl cellulose 0.5% eye
drops (Allergan India Private Limited, Bangalore, India) were
added in the recipient eye.
RESULTS
Demographic and clinical details of the six patients are provided
in table 1. A completely epithelialised, avascular and stable
corneal surface (figure 2 and online supplemental figure) was
achieved by 6 weeks and was maintained in all recipient eyes at
a mean6SD follow-up of 9.261.9 months. Best corrected visual
acuity improved from worse than 20/200 in all recipient eyes
before surgery to 20/40 or better in four (66.6%) eyes (table 1).
The fibrin glue on the ocular surface had disintegrated by the
end of the first postoperative week, and the hAM disintegrated
gradually over 6 weeks (figure 2). The transplants remained in
place and became progressively transparent over time, almost
completely disappearing by 6 months (figure 2 and online
supplemental figure). None of the donor eyes developed any
complications. The donor site epithelialised in all six eyes within
14 days, and none of the donor eyes showed a decrease in best
corrected visual acuity or developed conjunctival overgrowth on
the donor site, filamentary keratitis or LSCD.
DISCUSSION
The only study directly comparing the two existing techniques
of limbal transplantation found that conventional limbal
transplantation resulted in delayed corneal epithelialisation,
prolonged ocular surface inflammation, and significantly greater
scarring than CLET.
17
Other proposed advantages of CLET over
CLAU include greater safety for the donor eye, as it needs less
tissue, and better chances of long-term tissue regeneration, as
the number of stem cells transplanted is possibly increased by ex
vivo cultivation. However, the cost of establishing and main-
taining a stem cell laboratory is prohibitively high. Miri et al
estimated in 2010 that, in the UK, a single CLET procedure cost
approximately £10 300 (or about V12 000).
18
Despite having
successfully performed over 500 CLET procedures for the
treatment of LSCD since 2001,
15 19
we felt that, in reality, CLET
has become restricted to only a handful of advanced centres
worldwide because of these limitations. Similarly, CLAU has
also failed to garner universal acceptance because of the
Figure 1 Clinical photographs showing the surgical technique of limbal biopsy from the donor eye and transplantation of the limbal tissue on the
recipient eye. (A) A 232 mm area is marked across the superior limbus of the donor eye. (B) A sub-conjunctival dissection is carried out 1 mm into the
clear cornea. (C) The limbal tissue is excised. (D, E) A peritomy is performed and the fibrovascular pannus is excised from the recipient ocular surface.
(F) A human amniotic membrane graft is placed on the bare ocular surface and secured to it with fibrin glue. (G, H) The donor limbal tissue is cut into
eight to ten small pieces and secured to the amniotic membrane overlying the cornea with fibrin glue.
Table 1 Preoperative clinical characteristics and postoperative outcomes of patients with unilateral and total limbal stem cell deficiency following
ocular surface burns who underwent simple limbal epithelial transplantation (SLET)
Case Age Sex Eye
Aetiology
of LSCD
Duration
(months)
Prior ocular
surgery
Baseline
BCVA Symble-pharon PED Conjunti-valisation
Follow-up
duration
(months)
Final
outcome
Final
BCVA
1 21 M OS Blast 7 AMG CF at 1 m Yes No Yes 11 Success 20/30
2 20 M OS Alkali 48 AMG HM Yes No Yes 9 Success 20/200
3 20 M OD Alkali 6 AMG CF at 1 m No No Yes 12 Success 20/20
4 17 M OS Alkali 4 AMG CF at 1 m No No Yes 8 Success 20/40
5 35 F OS Alkali 11 AMG CF at 1 m Yes Yes Yes 7.5 Success 20/100
6 22 M OD Acid 6 AMG PL No No Yes 7.5 Success 20/30
AMG, amniotic membrane grafting; BCVA, best corrected visual acuity; CF, counting fingers; F, female; HM, hand motions; LSCD, limbal stem cell deficiency; M, male; OD, right eye; OS, left
eye; PED, persistent epithelial defect; PL, perception of light.
932 Br J Ophthalmol 2012;96:931e934. doi:10.1136/bjophthalmol-2011-301164
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perceived risk of donor site complications. Even Tseng and
colleagues who pioneered the classical technique
4
tried using
smaller single two-clock-hour-long lenticules to treat total
LSCD, terming the new technique mini-CLAU.
20
This study
begins to address the challenge of determining how little of the
limbus can be clinically effective given a suitable underlying
substrate. The results from this study, at least, suggest that less
than one clock hour of donor limbal tissue may be sufficient.
Table 2 summarises the comparison of this new technique,
which we termed simple limbal epithelial transplantation
(SLET), with CLAU and CLET. SLET incorporates the advantages
of CLAU by being a single-stage procedure, easily affordable,
and not requiring sophisticated laboratory support while
retaining the benefit of CLET, using minimal donor tissue. The
surgical technique of SLET is relatively straightforward, and our
results should be easily replicated by other ocular surface
surgeons. Before the study, we were concerned whether the
limbal transplants, consisting of epithelium with a thin slice of
underlying stroma, would remain opaque over time. Rather
surprisingly we found that they almost completely disappeared
by 6 months. To explain this, we hypothesise that, as the
epithelial cells grow out of the islands of stem cell containing
limbal transplants and form a confluent and stratified corneal
epithelial layer by 6 weeks, the stromal element undergoes
remodelling and is gradually incorporated into the underlying
cornea by 6 months. We plan to study this proposed mechanism
in greater detail in subsequent cases by performing in vivo
confocal microscopy and from the histopathology of corneal
buttons obtained from eyes needing keratoplasty surgery.
Further refinement of the surgical technique may include
avoiding the hAM or by replacing the hAM with biodegradable
synthetic polymer scaffolds to support culture of corneal
epithelial cells.
21
As this is a pilot study, the sample size is small and the follow-
up is limited. However, as most failures after limbal trans-
plantation usually occur within the first 6 months,
13 15
the
results of this study are truly encouraging. Validation of these
initial results in a larger number of patients followed for longer
periods of time, possibly on a multicentre basis, is now required.
This could significantly simplify the treatment of LSCD,
offering a simple approach, easily replicable by practising corneal
surgeons, which could benefit many more patients worldwide.
Contributors The corresponding author states that authorship credit of this
manuscript was based on: (1) substantial contributions to conception and design,
acquisition of data, or analysis and interpretation of data; (2) drafting the article or
revising it critically for important intellectual content; and (3) final approval of the
version to be published. All listed authors met conditions 1, 2 and 3. All persons
designated as authors qualify for authorship, and all those who qualify are listed. Each
author has participated sufficiently in the work to take public responsibility for
appropriate portions of the content.
Funding This work was funded by the Department of Biotechnology, New Delhi, India;
Champalimaud Foundation, Lisbon; Portugal, and the Hyderabad Eye Research
Figure 2 Serial clinical photographs of recipient eyes of two different patients before and after undergoing simple limbal epithelial transplantation
(SLET). (A) A 20-year-old college student (case 3) with a history of lime injury to the left eye presented with a conjunctivalised corneal surface,
symblepharon in the superior fornix, and visual acuity of counting fingers at 3 m. (B) Six weeks, (C) 3 months and (D) 6 months after SLET, the corneal
clarity gradually improved. The visual acuity at 6 months after SLET was 20/20. (E) A 22-year-old car mechanic (case 6) with a history of acid injury to
the right eye presented with a completely conjunctivalised cornea and visual acuity of perception of light. (F) One day after surgery the cornea and
transplants were covered with fibrin glue. (G) One week after surgery, the corneal clarity markedly improved, revealing the location of the limbal
transplants. Haemorrhage under the disintegrating amniotic membrane graft was also seen at this point. (H) Six months after surgery the cornea was
clear and avascular, the opacities at the site of the transplants had almost completely disappeared, and the visual acuity had improved to 20/30.
Table 2 Comparison of the current technique with existing techniques
of autologous limbal transplantation for treatment of unilateral limbal
stem cell deficiency
Features SLET CLET CLAU
Stages Single Two Single
Gap between stages None 2 weeks None
Donor tissue size in mm
(clock hours)
2(<1) 2 (<1) 10e20 (3e6)
Need for donor conjunctiva No No Yes
Need for stem cell laboratory No Yes No
Need for hAM Yes Yes No
Location of transplant All over cornea All over cornea At the limbus
Time to epithelialisation 4e6 weeks 0 4e6 weeks
Repeatable from same
donor eye
Yes Yes No
Donor eye LSCD No No
15
Yes
6e9
Long-term success Awaited 50e100%
10e15
77e100%
5e918
Complications in
recipient eye
None None None
Cost* in INR 12 000 96 000 12 000
Cost* in GBP 155 1246 155
*Current charges at L V Prasad Eye Institute, Hyderabad, India for the surgical and
laboratory procedures, excluding anaesthesia charges and room rent (additional 20e30% of
surgery costs).
CLAU, conjunctival limbal autograft; CLET, cultivated limbal epithelial transplantation ; GBP,
Great Britain pounds; hAM, human amniotic membrane; INR, Indian rupees; LSCD, limbal
stem cell deficiency; SLET, simple limbal epithelial transplantation.
Br J Ophthalmol 2012;96:931e934. doi:10.1136/bjophthalmol-2011-301164 933
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Foundation, Hyderabad, India. None of these had any role in the design, collection,
analysis and interpretation of the data.
Competing interests None.
Patient consent Obtained.
Ethics approval The study was approved prospectively by the Institutional Review
Board (IRB) and the Institute Committee for Stem Cell Research and Therapy
(ICSCRT), L V Prasad Eye Institute, Hyderabad, India. The study followed the tenets of
the Declaration of Helsinki, and informed written consent was obtained from all
patients enrolled in this study.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The authors are committed to make materials, data and
associated protocols promptly available to others without preconditions.
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doi: 10.1136/bjophthalmol-2011-301164
10, 2012 2012 96: 931-934 originally published online FebruaryBr J Ophthalmol
Virender S Sangwan, Sayan Basu, Sheila MacNeil, et al.
deficiency
treatment of unilateral limbal stem cell
(SLET): a novel surgical technique for the
Simple limbal epithelial transplantation
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