Journal Reading

Limbal Stem-Cell Therapy and Long-Term

Corneal Regeneration

Paolo Rama, M.D., Stanislav Matuska, M.D., Giorgio Paganoni, M.D.,

Alessandra Spinelli, M.D., Michele De Luca, M.D., and Graziella Pellegrini, Ph.D.

Pembimbing :
Prof. Dr. dr. Winarto, DMM, Sp.MK, Sp.M (K)

dr. Lady Pranodia Sukmawidowati

dr. Riskha Pangestika
dr. Dea Prita Caesarita
Background
Corneal renewal and repair are mediated by stem cells
of the limbus, the narrow zone between the cornea and
the bulbar conjunctiva

Ocular burns may destroy the limbus, causing limbal

stem-cell deficiency

The long-term clinical results of cell therapy in patients

with burn-related corneal destruction associated with
limbal stem-cell deficiency, a highly disabling ocular
disease.
Background

A clear cornea is essential to visual acuity

and depends on stromal avascularity and
epithelial integrity

Corneal renewal and repair are mediated

by stem cells of the limbus, the narrow zone
between the cornea and the bulbar
conjunctiva
 limbal
Ocular destroy
stem-cell
burns the limbus deficiency

-Neovascularization
- Chronic
inflammation
- Stromal scarring,
with corneal opacity
and loss of vision
cornea acquires
an epithelium
through the
invasion of bulbar
conjunctival cells
 Allogeneic corneal transplantation (keratoplasty)
restores transparency temporarily & the
conjunctival cells begin to invade and resurface
the cornea.

To prevent this invasion is to restore the limbus by

grafting of limbal fragments obtained from the
uninjured eye.
 Keratinocytes

Holoclones Meroclones Paraclones

 The finding : human limbal cell cultures contain
holoclones led to the first therapeutic use of such
cultures in the regeneration of cornea epithelium.

Related studies :
They have been limited because of the comparatively
small numbers of cases,
the relatively short follow-up
the heterogeneity among causes of the disorders and
among the sources of transplants and cell cultures.
 Patients & Methods
112 patients with severe or total, unilateral or partial
burn-related lateral limbal stem-cell deficiency
were included.
LSCD was divided in three grades :
i. Moderate conjunctivalization was sectorial
with less than 2 quadrants involved (>180) and
did not reach the central cornea
ii. Severe recurrent or persistent epithelial
defects, peripheral corneal conjunctivalization
involved more than two quadrants (<180) and
central corneal opacification was present
iii. Total the cornea was completely vascularized
and opacified
 Moderate
Severe

Total
Patients & Methods

Disorders of eyelids and conjunctiva were

treated beforehand.
Inflammation negative prognostic
factor treated before surgery
Eye examination included visual acuity,
tonometry, slit-lamp examination, fundus,
ecography, Schirmer test and
photographs.
Patients & Methods

Cell Culture and Treatment

Autologous limbal stem cells were obtained
from a biopsy specimen (1 to 2 mm2) taken
from the patients contralateral eye
The cells were cultured on fibrin and Clinical-
gradecertified, lethally irradiated 3T3-J2
cells from a master cell bank were used as
feeder cells.
Primary limbal keratinocytes were cultivated
on plastic dishes containing a feeder-layer
 Subconfluent primary cultures were trypsinized
and plated at a cell density of 1.5104/cm2 on a
circular fibrin substrate (3 cm diameter) in the
presence of feeder-layer.
At confluence, fibrincultured epithelial sheets
were washed in Dulbeccos minimum essential
medium (DMEM), placed in sterile holders for
contact lenses filled with DMEM and transferred
at room temperature to the hospital.
Patients & Methods

Cell Culture and Treatment

Colony forming efficiency assay was performed
Limbal biopsies were processed within 30 hours of
removal from the limbus.
Primary cultures and preparation of fibrincultured
grafts required 1416 days.
Grafts were applied within 2436 hours after their
transfer in the contact lens holder.
Patients & Methods

Surgical and post-operative procedures

Limbal biopsies and grafting procedures
were performed under topical
(oxybuprocaine 0.4%) and local-regional
anesthesia in 110 cases and general
anesthesia in 2 cases.
The surgical procedure for grafting the
cultures was performed.
Briefly, a 360 limbal peritomy was
performed and the fibrovascular corneal
pannus carefully removed.
 Patients & Methods

Surgical and post-operative procedures

The fibrin-cultured epithelial sheet was placed on
the prepared corneal wound bed spanning the
limbus about 2-3 mm, to reduce competition with
conjunctival ingrowth.
The conjunctiva was then sutured over the
peripheral fibrin sheet with 8.0 vicryl sutures in order
to protect the border of the sheet and help it to
adhere on the surface.
The eyelids were kept closed with Steri-Strip and
patched for one week.
Patients & Methods
Surgical and post-operative procedures
During the first 2 weeks after grafting,
topical treatment was avoided to prevent
mechanical damage when opening the
eyelids and secondary toxic effects while
cells were engrafting.
Systemic post-operative therapy was
administered from the day of the surgery:
doxycycline (100 mg bid) or Amoxicillin
(500 mg tid), as antibiotic prophylaxis,
were administered for two weeks;
Patients & Methods

prednisone (25 mg) and ranitidine (150 mg)

doses were gradually reduced and stopped
within one month.
Topical preservative-free dexamethasone
(0.15 % TID) was started at day 14 and topical
ofloxacin (0.3%) was administered only in case
of incomplete reepithelialization.
Frequent use of preservative-free lubricants
was advocated.
Patients & Methods

Surgical and post-operative procedures

Patients were seen at day 1, 3, 14 and at month
1, 3, 6, 12.
Follow-up was then performed at least one a
year for the entire folllow-up. Each visit included
history, recording of symptoms and complete
eye examination.
Patients & Methods

After at least 1 year follow-up, standard lamellar

or penetrating keratoplasty or phototherapeutic
excimer laser keratectomy (depending on the
depth of the stromal involvement) were
performed on 46 patients considered to have
been successfully treated, but whose visual
acuity was still impaired because of stromal
scarring.
Patients & Methods

Surgical and post-operative procedures

Penetrating keratoplasty-associated donor
cornea rejection was graded as
i. Mild (epithelial rejection or endothelial
precipitates with clear graft);
ii. Moderate (endothelial preciptates and
edema of the graft with visible iris details);
iii. Severe (diffuse edema with non visible iris
details).
Patients & Methods

Surgical and post-operative procedures

Mild and moderate rejection was treated with
topical dexamethasone (0.2%), every 4 and 1
hour(s), respectively. The regimen was tapered
depending on clinical amelioration.
For severe rejection, systemic
methylprednisolone was added (1 gr I.V. The first
day, 1mg/Kg per os during the following 4 days).
Patients & Methods
Follow-up and Assessment of Outcome
The outcome of treatment was assessed at 1 year. The
treatment was considered:
Successful if all symptoms disappeared and a
transparent, avascular, and stable corneal surface
was restored
Partially successful if most symptoms disappeared but
superficial neovascularization recurred, even if it was
not as extensive as at the time of admission.
Treatment failure was defined as the presence of
symptoms, recurrent epithelial defects, pannus, and
inflammation at 1 year.
Result
 Characteristics of the patients
Amount : studied 113 eyes from 112 patient
Age : The mean (SD) age of the patients was 46.514.4
years (range, 14 to 80)
Sex : 78.6% of them were men
Case : The limbal stem-cell deficiency ranged from severe
(in 35.4% of the patients) to total (in 64.6%). The most
common causes of limbal stem-cell deficiency (in 97.3% of
cases) were chemical burns (83 alkali, 16 acid, and 4 other)
and thermal burns (7)
Patients had unilateral (87.5%) or bilateral (12.5%) burns;
only two patients had bilateral severe limbal stem-cell defi-
ciency
Two patients had a bacterial infection and one patient had
undergone orbit irradiation.
 Most of the eyes (84%) had been treated previously with
surgery (in some cases multiple surgeries), but all surgical
treatments had failed, probably because of an
insufficient
Number of residual limbal stem cells to regenerate
corneal epithelium. Twenty-eight patients had
undergone a single unsuccessful keratoplasty, and 20
had undergone multiple unsuccessful ker-atoplasties.
Period

A total of 125 cultures were grafted in 113

eyes (112 patients) between July 1998 and
December 2007.
with up to 10 years of follow-up
 Complication
none
Intraoperative
Complication

Hemorrhage
Postoperative Residual Fibrin
Inflammation
Adverse Blepharitis with Epithelial Involvement
Events Herpetic Keratitis
 112 Patients

113 eyes
6 patients
exclude

107 eyes
1 year
follow up

Success (73) Partial Failure (16)

Success (18)

11 eyes
regrafted

Partial
Success (82) Failure (11)
Success (14)
Univariate logistic regression

Age

The cause of limbal stem

cell deficiency

Previous surgical procedure

The severity of damage

Failure Culturing and graft-transport
were complication
associa
ted Postoperative complication
with
inflammation
Multivariate Logistic Regression Analysis

Graft Transport

Culturing

Postoperative
Complications
 Graftable Limbal Culture
the percentage of colony forming cells is inadequate as an indication of
the percentage of stem cells, because it is not possible to visually identify
holoclones
quantitative immunodetection of p63, a marker of holoclones, is
straightforward and can be performed before grafting
Clinical result according to culture status, Cultures that contained more
than 3% p63-bright cells led to successful corneal epithelial regeneration
in 78% of the eyes.
The percentage of p63-bright cells in the culture was positively associated
with the clinical outcome of limbal stemcell grafting. These data are
consistent with the finding that the human limbus contains at least 5%
stem cells.
Restoration of Visual Acuity
The regeneration of normal avascular corneal epi-
thelium was associated with amelioration of
symptoms (burning, pain, and photophobia) in all
pa-tients whose grafts were considered successful.
Normal vision was restored in only those patients with
undamaged corneal stroma. To improve the visual
acuity of eyes with stromal scarring, we performed
corrective surgical procedures 12 to 24 months after
grafting the limbal stem-cell cultures:
Histologic examination of the central cornea
removed at the time of keratoplasty showed that the
regenerated epithelium expressed keratin 12 (a
marker of corneal epithelium) but not keratin 19 (a
marker of conjunctival epithelium)
 Discussion
Cultures of limbal cells provide an adequate
long-term source of transplant tissue for the
treatment of corneal damage due to burns.
A minimum of approximately 3000 stem cells,
detected as p63-bright holocloneforming cells,
was required to achieve clinical success.
On the basis of this observation, it is speculated
that corneal regeneration cannot be ascribed
to a nonspecific stimulatory effect of epithelial
cultures, fibrin, or surgical manipulation on
spared residual limbal cells.
Discussion

Retention of holoclones also requires

appropriate substrates for the cultivation of cells;
both fibrin and plastic have been shown to
preserve holoclone-forming cells
It is observed that the association between
the percentage of p63-bright cells and clinical
success was never related to a nonspecific
stimulatory effect of the transplanted culture on
resident stem cells.
 Discussion

In conclusion, this study shows that in

patients with limbal stem-cell deficiency
who received cultured limbal stem-cell
grafts for corneal transplantation, the
clinical results were successful at up to 10
years (at a median of 2 years) in more than
75% of the patients treated.
Cultures of limbal stem cells thus represent a
source of cells for transplantation in the
treatment of burn-induced destruction of
the human cornea.
Critical Appraisal
 TITLE

Too long / short ? No, the title consist of 7 words

Illustrate the observed variables ? Yes (supplementary appendix)

Non standard abbreviation ? No

Any corresponding author and

No
email ?
 ABSTRACT
Consists of 4 parts:
background, method, result, Yes
and conclusion
Any keywords ? No
Do the abstract is wholly
Yes
appropriate ?
AIM & BENEFIT OF THE RESEARCH
Was the aim explained ? Yes

Was the benefit explained ? Yes

 METHODS
Is there any research design ? No

Population & samples ? No

Inclusion-Exclusion criteria ? No
Sampling & sample size formulation
No
?
Did the subject selection is
Yes
appropriate?
Is there any bias ?
Yes
 METHODS
Did the measurement blind ? No
Is there any bias on procedure, means, and
Yes
subject obedience ?

Is there any explanation about independent

No
& dependent variables ?

Is there any operational definition ? No

Is there any ethical clearance consent ? No

Data analysis ? Yes

 RESULTS
Any drop out ? Yes
Is there any subject characteristic
No
table ?
Is there any aim for the results? Yes

Cultures of limbal stem cells represent a

What is the main result of the source of cells for transplantation in the
research ? treatment of destruction of the human
cornea due to burns.

DISCUSSION
Did the result suits the theory ? Yes
Is there any comparison with
Yes
another research ?
 CONCLUSION

Could it be applied in chosen

sample, reachable and target Yes, but it needs further research
population ?

Could this research be applied

Applicable
for patients ?