Download Barcelona-Cincinnati technique for limbal transplantation

ARTICLE
Barcelona-Cincinnati technique for limbal transplantation
Jose L. Güell, MD, PhD1,2,3; Merce Morral, MD, PhD2,4; Oscar Gris, MD, PhD2;
Daniel Elies, MD2; Felicidad Manero, MD2; Nerea Saenz, MD, PhD5
PURPOSE: To describe a new technique of allogenic limbal transplantation for total 28
limbal stem cell deficiencies (LSCD).
METHODS: The “Barcelona-Cincinnati” technique combines a cadaveric keratolimbal
allograft and transplantation of ex vivo expanded limbal cells on amniotic membrane (AM).
A 1 x 3-mm biopsy of limbal tissue was obtained from a living donor for ex vivo culture on
AM for 2-3 weeks. Two 120° corneoscleral rims were obtained from the cadaveric donor.
A 360° limbal peritomy was performed on the recipient eye, and the cadaveric crescents
were sutured on the superior and inferior limbus. Abnormal fibrovascular pannus and
epithelium were removed from the surface of the cornea and peripheral areas. A penetrating
keratoplasty was then performed. Finally, AM with ex vivo expanded limbal cells was
sutured over the cornea epithelial-side down. Postoperatively, topical steroids and systemic
immunosuppression with methylprednisolone and cyclosporine A were prescribed and, in
the absence of an immunological reaction, tapered off.
RESULTS: Two eyes of two patients with bilateral total LSCD were treated. Six years
postoperatively, Case 1 maintained a stable ocular surface. Case 2, however, presented
recurrence of LSCD 17 months after surgery.
CONCLUSION: Although prospective comparative studies including more patients and
a longer follow-up are required, the Barcelona-Cincinnati technique may be an effective
alternative for bilateral total LSCD while decreasing the risk for living donors.
J Emmetropia 2012; 3: 124-128
INTRODUCTION
Treatment of limbal stem cell deficiency (LSCD) is
challenging. In partial LSCD involving the visual axis,
sequential sector conjunctival epitheliectomy (SSCE)
is the treatment of choice.1 If fibrovascular pannus
covers the cornea, a sector limbal transplant may also
be required.
Submitted: 06/22/2012
Revised: 07/24/2012
Accepted: 07/26/2012
1
2
3
4
5
Director of the Cornea and Refractive Surgery Unit, Instituto
Microcirugia Ocular, Barcelona, Spain.
Instituto de Microcirugia Ocular (IMO), Barcelona, Spain.
Associate Professor of Ophthalmology at the “Universitat
Autonoma de Barcelona” (UAB), Barcelona, Spain.
Department of Cornea and Anterior Segment Diseases, and
Refractive Surgery of the Institut Clinic d’Oftalmologia, Hospital
Clinic i Provincial, Barcelona, Spain.
Hospital 12 de Octubre, Madrid. Spain.
Financial Disclosure: None of the authors has any financial or
proprietary interest in any material or method mentioned.
Corresponding Author: Jose L Güell, MD. Instituto Microcirugia
Ocular Barcelona, Spain. E-mail: [email protected]
124
© 2010 SECOIR
Sociedad Española de Cirugía Ocular Implanto-Refractiva
In total LSCD, transplantation of limbal tissue is the
mainstay. In bilateral cases, living donors are preferred
over cadaveric donors, as the fresh tissue contains a
higher number of stem cells and can be human leukocyte
antigen (HLA) matched, reducing the risk of rejection.2
Holland, et al. described the Cincinnati technique,
which combines a living-related conjunctival limbal
allograft (lr-CLAL) and a keratolimbal allograft (KLAL)
for LSCD associated with conjunctival deficiency.3 This
technique allowed transplantation of limbal stem cells
from two different sources, thereby increasing the density
of viable transplanted cells, and healthy conjunctiva,
which improves the local microenvironment conditions
by decreasing conjunctival inflammation and replacing
goblet cells.3 However, the potential risk of iatrogenic
LSCD in the living donor must be considered.4-6 To
decrease such risk, ex vivo expansion of limbus-derived
cells with or without a substrate (fibrin, collagen or
amnion), first described by Pellegrini et al. in 1997, is a
viable option for total, unilateral or bilateral LSCD.7-11
The surgical techniques for ex vivo expansion of limbal
stem cells and KLAL have been described.2,3,7,12-14 We
propose the “Barcelona-Cincinnati technique”, which
ISSN: 2171-4703
BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION
combines a cadaveric KLAL and ex-vivo expansion of
limbal stem cells on amniotic membrane (AM) from a
living donor, with the aim of increasing the number of
viable cells while decreasing the risk in the living donor.
METHODS
All patients were fully informed of the details
and possible risks of the specific procedure. Written
informed consent to perform the surgical procedure was
obtained from all patients before surgery in accordance
with the Declaration of Helsinki, and the study was
approved by the ethics committee of our institution,
Instituto de Microcirugia Ocular, and the Universitat
Autonoma de Barcelona. All eyes were operated by the
same surgeon (JLG).
The “Barcelona-Cincinnati technique” combines
ex vivo expansion of limbal stem cells on amniotic
membrane (AM) from a living donor, and a cadaveric
KLAL. In these two cases, penetrating keratoplasty (PK)
was also performed as a single-stage procedure.
125
Preparation of cadaveric donor tissue and the recipient
eye, and placement of donor tissue
For KLAL, the posterior half of each hemisection
of the corneoscleral rim was removed on the cadaveric
donor. A 360° limbal peritomy was performed on the
recipient eye. Abnormal fibrovascular pannus and
epithelium were removed from the cornea and peripheral
areas. The cadaveric donor crescents were placed with the
donor corneal edge overlying the recipient superior and
inferior limbus with interrupted 10-0 Nylon sutures at
the corneal margin, and interrupted 9-0 Vycril sutures
at the conjunctival margin.2,14
Penetrating keratoplasty
PK was then performed as a single-stage procedure
using the same donor as for the KLAL. An 8.5-mm
Hessburg-Barron punch was used to obtain the corneal
graft, and an 8-mm trephine was performed on the
recipient´s eye. The corneal graft was then sutured with
16 interrupted 10-0 Nylon sutures. The epithelium of
the donor was not removed.
Ex vivo expansion of limbal stem cells
Briefly, a 1 × 3-mm biopsy of limbal tissue was
obtained for ex vivo culture. The superior limbus is
preferred as it is thought to contain a higher density of
stem cells. The obtained tissue was placed with Ham’s
F12 medium containing 50 µg/ml gentamicin and 1.25
µg/ml amphotericin B until it was processed. Limbal
tissue was exposed for 5 min to Dispase II (1.2 U/ml
in Mg2+ and Ca2+ free Hank’s balanced salt solution
HBSS) at 37 °C under humidified 5% CO2. The
explants were then cultured in DMEM medium, which
is a 1:1 mixture of DMEM and Ham’s F12 medium
containing 5 ng/ml epithelial growth factor (EGF), 5
mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml sodium
selenite, 0.5 mg/ml hydrocortisone, 30 ng/ml cholera
toxin A, 0.5% dimethylsulfoxide (DMSO), 50 mg/
ml gentamicin, 1.25 mg/ml amphotericin B and 5%
autologous serum, at 37ºC under 5% CO2 and 95%
humidity. The medium was renewed every 2-3 days.
For allogeneic related transplantation, donor serum was
used and for allogeneic non-related transplantation,
ABO tested blood bank serum was employed. The
limbal epithelial cell explants were plated onto the center
of the basement membrane side of the AM denuded
of epithelium. The extent of the limbal epithelium
outgrowth, which exhibits a compact uniform cell layer,
was monitored with a phase contrast microscope until it
covered an area of 2-3 cm in diameter (2-3 weeks later).
Bacteriological testing, including mycoplasma and
Gram´s test, was performed to assess microorganism
contamination.12
Implantation of ex vivo expanded limbal stem cells
After the KLAL and PK were completed, the AM
containing ex vivo expanded limbal cells was sutured
epithelial-side down with a running 10-0 Nylon suture
so that the cultivated cells were in direct contact with
the ocular surface. The biopsy specimen was left located
in the limbal area (Figure 1). Finally, subconjunctival
methylprednisolone was injected. Mechanical
protection was achieved by placing a bandage contact
lens over the graft at the end of surgery, leaving it in situ
until complete reabsorption of the AM.
Postoperative regimen
The immediate postoperative topical regimen was:
tobramycin and dexamethasone (Tobradex® Alcon,
El Masnou, Barcelona, Spain) drops five times per
day, timolol 0.5% (Cusimolol 0.5%®, Alcon) twice a
day (BID), and chloramphenicol and dexamethasone
ointment (Oftalmolosa Cusi De Icol®, Alcon) at
bedtime. Additionally, systemic immunosuppression
was achieved with methylprednisolone (Urbason®,
Sanofi Aventis, Barcelona, Spain; 0.5 mg/kg/day) and
cyclosporine A (Sandimmun®, Novartis SA, Barcelona,
Spain; 5 mg/kg/12 hours, maintaining blood levels of
100-400 ng/ml). Patients were monitored for potential
side effects, especially nephrotoxicity. Blood tests and
blood pressure were checked on a weekly basis for
the first month, every 2-3 weeks for the second and
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BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION
Figure 1. The “Barcelona-Cincinnati technique”. (A) The cadaveric donor crescents are sutured superiorly and inferiorly. Interrupted 10-0
Nylon sutures are used to secure the graft to the recipient´s cornea, and interrupted 9-0 Vycril sutures are used to secure the graft to the
recipient´s conjunctiva and epiesclera. (B) Ex vivo expanded limbal stem cells on amniotic membrane (AM). The AM is sutured epithelial-side
down, with the expanded cells contacting the ocular surface. (Arrows: (1) segmental cadaveric donor keratolimbal section; (2) AM with ex vivo
expanded limbal stem cells; (3) Original biopsy from a living-related donor).
third month, and every two months thereafter. In the
absence of an immunological reaction, corticosteroids
were discontinued 1 to 2 months after surgery.
Low-dose cyclosporine (25-50 mg/day) was maintained
for at least 12 months, and then discontinued if possible.
RESULTS
Two cases were treated. Case 1 presented with total
LSCD secondary to congenital aniridia. A limbal biopsy
was taken from the patient’s partially HLA-I-compatible
sister, and was expanded ex vivo on AM. Three weeks
later, penetrating keratoplasty, cadaveric KLAL, and
implantation of ex vivo expanded limbal stem cells were
performed. The postoperative regimen included: topical
combined tobramycin and dexamethasone drops five
times per day, timolol 0.5% BID, and chloramphenicol
and dexamethasone ointment at bedtime for a month.
Additionally, systemic methylprednisolone and
cyclosporine A were prescribed for a month. Three
weeks postoperatively, a clear corneal graft and localized
inflammation on the donor tissue were observed.
Topical and systemic steroids were slowly tapered
off, but low-dose cyclosporine (25-50 mg/day) was
maintained for 12 months and then discontinued. Six
years postoperatively, visual acuity (VA) had improved
to 20/200, limited by terminal glaucoma, and LSCD
had completely resolved (Figure 2).
Case 2 presented with bilateral total LSCD secondary
to acute chemical injury. A limbal biopsy was taken from
the patient’s HLA-I-compatible mother. Three weeks
later, penetrating keratoplasty, cadaveric KLAL, and
implantation of ex vivo expanded cells were performed.
The same postoperative immunosuppressive regime was
followed, and three weeks postoperatively visual acuity
Figure 2. (A) Clinical photograph of a total limbal stem cell
deficiency in the right eye of a patient with congenital anirida.
The fellow eye was eviscerated. 360° corneal neovascularization
and total corneal clouding can be observed. (B) The conjunctival
epithelium appears irregular, with areas of fluorescein pooling
and fine neovessels. No epithelial defect is seen. Visual acuity
was hand motion. (C) Penetrating keratoplasty, keratolimbal
allograft (KLAL) from a cadaveric donor and implantation of ex
vivo expanded limbal stem cells were performed. Clinical photograph showing the 3-week postoperative appearance. A clear
corneal graft and localized inflammation on the donor tissue can
be observed. (D-F) Clinical photograph 1 (D) and 6 years (E-F)
after surgery. A clear corneal graft with regular epithelium and
no inflammation on the KLAL area are seen. Visual acuity was
20/200, limited by terminal glaucoma.
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BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION
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Figure 3. (A) Clinical photograph of a total limbal stem cell deficiency secondary to acute chemical ocular burn. The fellow eye was also
affected. 360° corneal neovascularization and total corneal clouding can be observed. A central epithelial defect is seen. Visual acuity was
counting fingers at 6 feet. (B-C) Penetrating keratoplasty, keratolimbal allograft (KLAL) from a cadaveric donor, and implantation of ex
vivo expanded limbal stem cells were performed. Clinical photograph showing the one-month postoperative appearance. A clear corneal
graft and localized inflammation on the donor tissue can be observed. The corneal epithelium is smooth. Visual acuity was 20/200. (D-E)
Clinical photograph 17 months after surgery. A subtotal epithelial defect is seen, suggesting graft failure and recurrence of the limbal stem cell
deficiency. No inflammation on the KLAL area is seen. Visual acuity decreased to counting fingers at 6 feet.
(VA) improved from counting fingers to 20/200 with
a clear corneal graft. Seventeen months postoperatively,
however, VA had decreased to counting fingers, and a
subtotal epithelial defect suggested recurrence of the
LSCD and failure of the limbal graft. The patient was
advised to undergo repeat keratolimbal transplantation,
but declined (Figure 3).
DISCUSSION
Disorders of the ocular surface resulting in both
limbal stem cell and conjunctival deficiency are
extremely severe and challenging. Total LSCD will
require transplantation of a population of autologous
or allogeneic limbal epithelial stem cells if a stable
corneal epithelial phenotype is to be regained.2 The
main limitations of limbal stem cell transplantation are:
(1) The need for a high density of viable transplanted
cells; (2) Maximal cell compatibility (HLA I-II, and
ABO matching)15-17; (3) The need for local and systemic
immunosuppression; and (4) Local microenvironment
conditions as physiological as possible.
Autologous limbal transplants are preferred in
unilateral cases to avoid the risk of immune rejection
and the side effects of systemic immunosuppression.18
Living donors are also preferred over cadaveric donors,
as the tissue is fresh and presents a higher density of
limbal epithelial stem cells. However, removal of limbus
from a living donor or the contralateral healthy eye is not
without risk, and partial stem cell deficiency following
limbal biopsy for conjunctival limbal autografts
(CLAU) or allografts in previously normal eyes has been
described.4-6 Ex vivo expansion of limbus-derived cells
constitutes an excellent alternative. Biopsies from livingrelated donors have been shown to be more reliable in
terms of successful establishment of cultures than those
obtained from cadaveric corneas.19
A progressive loss of donor cells in the recipient eye
over time has been described, and is considered the
main cause of KLAL failure, with a dramatic decrease
in graft survival rate over a 2-year period.20,21 The aim
of the “Barcelona-Cincinnati” technique is to provide
a greater amount of viable limbal stem cells by using
two different sources: KLAL and ex vivo expanded cells.
Compared to CLAU and lr-CLAL, the small-sized
biopsy minimizes the risk of iatrogenic stem cell failure
in the donor eye, and enables another biopsy to be taken
if the first fails. However, as no healthy conjunctiva is
transplanted, improvement of local microenvironment
conditions by reducing conjunctival inflammation and
replacing goblet cells is limited.
One of the main concerns when two different donors
are combined is the theoretical increased risk of immune
rejection related to the additional immune stimulation.
Compared to CLAU, ex vivo cultured limbal epithelial
cell grafts, especially if a cadaveric donor graft is not
required, present a reduced risk of rejection due to
the absence of antigen-presenting Langerhan´s cells.13
As a direct relationship between ocular surface stem
cell transplantation failure and immune rejection has
been reported, it is critical that patients are closely
monitored for rejection and for compliance with
immunosuppressive treatment.22 Although we used
two different donors (cadaveric for the KLAL and PK,
and living-related for ex vivo expansion), we did not
introduce any changes in our standard postoperative
immunosuppressive regimen. The question of whether
case 2 would have succeeded if immunosuppression had
been raised remains.
Ex vivo expansion from limbal biopsies provides
a mixed cell population, which includes limbal stem
cells, corneal and conjunctival epithelial cells. Although
techniques to check the quality of the cell culture before
it is transplanted have evolved considerably in recent
years, they were not available at the time we treated these
patients. Therefore, the actual proportion of putative
stem cells was unclear.7,11,23
One of the main challenges when interpreting the
results is to establish whether using a combined approach
(e.g. KLAL and ex vivo expansion) is better than using
one of the techniques alone. The relative benefit of each
cell source can be determined by polymerase chain
reaction (PCR) genotyping for DNA microsatellites
on the corneal epithelial cells obtained by impression
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BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION
cytology postoperatively, as long as a blood sample from
the cadaveric donor was obtained.9,24 A limitation of our
study is that we did not perform such analysis.
Several studies have suggested that a two-stage
approach of autologous cultivated limbal epithelial
transplantation followed by PK successfully restores
ocular surface stability and vision, and that a single-stage
approach is associated with poorer clinical outcomes
and should be avoided.24,25 However, in our experience,
we have found similar results with both approaches,
although we prefer the one-stage technique as it has two
clear advantages: (1) the patient undergoes only one
surgical procedure; and (2) the same donor is used for PK
and KLAL, which reduces the antigenic load (Personal
communication, Güell JL, Gris O. Keratolimbal
Allograft Combined with Penetrating Keratoplasty in
Severe Chemical injuries. The Cornea Society/Eye Bank
Association of America. AAO. America Academy of
Ophthalmology. Las Vegas 11-14 November, 2006). This
finding has also been reported in other studies.26
In conclusion, the Barcelona-Cincinnati technique
represents an encouraging preliminary experience
but the limited follow-up, the small number of eyes
treated and the lack of a control group, does not allow
any conclusion to be drawn. Continued stem cell,
pharmacological and surgical research is essential to
improve our capacity to treat these major disorders.
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JOURNAL OF EMMETROPIA - VOL 3, JULY-SEPTEMBER
First author:
Jose Luis Güell, MD
Instituto de Microcirugia Ocular (IMO)
Barcelona, Spain