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Cover Story
cornea
ocular regeneration
Ocular surface diseases and disorders including chemical or burn injuries, Stevens-Johnson
syndrome and neurotrophic keratopathy continue to be major challenges
by Sean Henahan
“
Now we have seen
much progress
and have a better
understanding of
the pathogenesis of
corneal disorders,
which is leading to
some sophisticated
therapeutic modalities
Shigeru Kinoshita MD, PhD
“
Patients receiving
E-PRP for chronic
epithelial defects and
for corneal ulcers did
exceedingly well
Jorge Alio MD, PhD
EUROTIMES | Volume 17 | Issue 3
W
ith several new stem
cell strategies and tissue
engineering techniques now
close to clinical utility, and
numerous innovative biotech approaches
in the pipeline, research groups around the
world report being tantalisingly close to the
goal of producing effective new treatment
options while solving the limitations of
current approaches.
“When I began residency some 36 years
ago we had very little knowledge of the
pathogenesis of devastating ocular surface
disorder such as Mooren’s ulcer, StevensJohnson syndrome, and meibomitis-related
severe keratoconjunctivitis. Now we have
seen much progress and have a better
understanding of the pathogenesis of
corneal disorders, which is leading to some
sophisticated therapeutic modalities,” said
Shigeru Kinoshita MD, PhD, professor and
chair, Department of Ophthalmology, Kyoto
Prefectural University of Medicine, Japan,
in a keynote lecture at the 2nd Asia Cornea
Society conference in Kyoto.
Ocular surface reconstruction using
regenerative medical approaches and
tissue engineering aims to restore the
anatomic and physiologic ocular surface,
and ultimately to prevent recurrence of the
ocular surface pathology.
Japanese corneal specialists pioneered
some of the key approaches in the treatment
of ocular surface disease, such as cultivated
mucosal epithelial cell transplantation. Dr
Kinoshita, a leader in the field, continues
to be involved in some of the most leadingedge research.
Dr Kinoshita and colleagues are also
interested in producing human corneal
endothelial cell regeneration through
implantation of cultivated corneal
endothelial cells. His lab is currently looking
at three types of therapies to treat corneal
endothelial disease: cultivated corneal
endothelial cell sheet transplantation,
cultivated corneal endothelial cell injection
therapy, and eye drops for promoting
corneal endothelial cell proliferation and
migration.
He has reported promising results in
animal experiments with transplantation
of corneal endothelial cell sheet on type 1
collagen. This produced high endothelial
cell density with long-lasting clear corneas.
“This research showed that the cells
could easily migrate out of the transplanted
cultivated cell sheet and proliferate. They
migrated to peripheral cornea, eventually
creating endothelial cells on the entire
cornea,” he told a session of the XXIX
Congress of the ESCRS.
Problems with sloughing of the cell
sheet led to the search for an alternative
way to deliver the cells. Animal
experiments using human DSAEK flaps
demonstrated that it was possible to
transfer cultivated cells into the anterior
chamber in this manner, with transparent
corneas as far out as eight months.
Dr Kinoshita and colleagues are also
evaluating direct injection of cultivated
corneal endothelial cells. He believes this
would ultimately prove a better approach
than transplanting cultivated cornea
endothelial cell sheets. Several researcher
groups are now working on this.
While doing this research Dr Kinoshita
and colleagues, notably Drs Naoki
Okamura, Noriko Koizumi and Morio
Ueno, discovered an interesting molecule,
ROCK (rho kinase) inhibitor (Y027632).
This molecule enhances corneal endothelial
cell survival, promotes cellular attachment
and proliferation, and inhibits apoptosis.
Ocular regeneration with an eye
drop The combination of direct cell
injection with ROCK inhibitor offers an
intriguing potential treatment of ocular
surface disease. In a recent landmark
publication (N Okamura et al., Br J
Courtesy of Shigeru Kinoshita MD, PhD
4
Courtesy of Bruce Jackson MD
5
A slit lamp photo and OCT image of the corneal lamellar graft at 24 months in a patient treated with keratoconus by Dr Per Fagerholm
Ophthalmol doi:10.1136/bjo.2010.194571),
Dr Kinoshita and colleagues report
research demonstrating that ROCK
inhibitor promoted and enhanced corneal
endothelial wound healing both in vitro and
in vivo. This was accomplished not with
cultivated cell sheet transplantation, but
rather through the use of an eye drop. This
suggests that in the not too distant future it
may be possible to regenerate the corneal
endothelial surface in some conditions with
a simple eye drop administration.
“We are quite confident about this kind
of procedure. Cell injection combined
with ROCK inhibitor, which we are calling
‘advanced cell therapy’, offers a distinct
advantage over other approaches, as it
enables cultivated corneal endothelial cell
transplantation by a minimally invasive
surgery,” said Dr Kinoshita.
Dr Kinoshita said he is preparing a
clinical study protocol to test this approach
in humans. It may take some years to gain
approval according to Japanese government
guidelines for clinical research using
somatic stem cells, he noted.
Practical application of a new
emerging therapy Stevens-Johnson
syndrome, persistent epithelial defects,
dormant corneal ulcers, and chronic severe
dry eye are some of the most common, and
most challenging ocular surface disorders.
Jorge Alio MD, PhD, chair, Department of
Ophthalmology, Alicante Ophthalmology
Institute, believes that biological activation
of the ocular surface by platelet rich plasma
(E-PRP) could prove a useful new way to
approach these problems.
Blood-derived products have been used
for a long time in ophthalmology. Blood
derived products have demonstrated their
capacity to enhance healing and stimulate the
regeneration of different tissues. This healing
effect is attributed to the growth factors and
bioactive proteins in the blood.
“Autologous platelet rich plasma (E-PRP)
is easy to prepare. It is an amber coloured
and slightly turbid blood fraction obtained
after centrifugation of uncoagulated total
blood with sodium citrate. It is very rich in
platelets, growth factors and clotting proteins.
It is different from autologous serum because
in that case the blood has previously been
coagulated, so platelets are not present in the
fraction. Platelets contain great reservoirs of
growth factors that enhance proliferation and
EUROTIMES | Volume 17 | Issue 3
wound healing. In particular, growth factors
enhance ocular surface healing, and induce
mesenchymal and epithelial cells to migrate
and proliferate, restoring the ocular surface,”
he told the XXIX ESCRS Congress.
Dr Alio reported treating more than 1000
patients in the last five years with autologous
platelet rich plasma (E-PRP). Depending on
the situation, he uses either a topical eye drop
or a solid form of the blood product.
He has used the drops for medical
treatment of ocular surface disorders
including persistent epithelial defects,
dormant corneal ulcers, corneal trauma,
immunological disorders, chronic severe
dry eye and post-LASIK ocular surface
syndrome.
A review of cases showed for example
that 77 per cent of epithelial defect patients
got good or excellent results following
treatment with E-PRP drops. The eye drops
also benefited patients with dormant corneal
ulcers not responsive to conventional
therapy, with 50 per cent healing completely
and 42 per cent showing improvement.
“Patients receiving E-PRP for chronic
epithelial defects and for corneal ulcers
did exceedingly well. The drops promoted
healing, improved the anatomy and
symptoms, closing the wound, and restoring
visual function in most cases. This appears
to be a very promising and practical
approach,” he said.
The drop regimen also improved the
conjunctival and corneal surface in cases
of micropunctate keratitis, decreasing
inflammation, and promoting wound
healing, lubricate. A study of patients with
severe dry eye showed that at three months
89 per cent had improvement in subjective
symptoms, and 86 per cent had significant
decreases in hyperaemia, he reported.
Ocular surface syndrome post-LASIK can
be very distressful for patients and refractive
surgeons, he noted. It is characterised
by symptoms of dry eye, micropunctate
keratitis, decreased and unstable tear film
and decreased visual acuity. Of neurotrophic
aetiology, it is a long-lasting problem that is
difficult to treat, he commented.
Treatment with platelet rich plasmin
shows promise in the treatment of that
condition as well. In a study of 32 eyes
treated for four to six weeks, half of the
patients had significant or very significant
improvement in symptoms. Most had
improvements in vision. Staining showed
that the underlying neurotrophic problems
improved, Dr Alio said.
He reserves a solid form of E-PRP as an
adjuvant in surgical procedures for corneal
and ocular surface reconstruction. It can
be used with amniotic or autologous fibrin
membrane transplantation in perforated
eyes, or in those at high risk for perforation.
“In our hands E-PRP is a reliable and
an effective therapeutic tool to enhance
epithelial wound healing in ocular surface
disease. Platelet rich plasmin provides a
high concentration of essential growth
factors and cell adhesion molecules by
concentrating platelets in a small volume of
plasma. It has a major role in wound healing
and enhances physiological processes at the
site of injury or surgery,” he said.
The E-PRP is made from patients blood
an hour before the procedure. Nothing
needs to be added, and it requires no special
preparation. This means it can easily be
used in any practice, he added.
Improving limbal cell
transplantation One goal of
ocular surface reconstruction strategies
is repairing limbal epithelial stem cell
deficiency, a problem common to many
disorders including thermal or chemical
injury, Stevens-Johnson Syndrome,
microbial keratitis, and contact lens
keratopathy. Symptoms of limbal epithelial
stem cell deficiency include epithelial haze,
sub-epithelial vascularisation and
epithelial defects.
Limbal epithelial deficiency can be
treated with limbal epithelial cell culture
for transplantation, derived from human
donor corneas. This can be accomplished
with autologous grafting in unilateral
disease. Bilateral disease is currently treated
with allogeneic grafting, although other
approaches are in the early stages.
Alternative approaches for unilateral
disease now under investigation involve
culturing autologous tissue from oral
mucosa, nasal mucosa, and other sites. This
research field is very active, motivated by the
need to overcome inherent immunogenic
problems associated with allogeneic grafting.
Amniotic membrane, a useful treatment in
its own right in ocular surgery, is currently
the primary substrate for limbal epithelial cell
transplantation, but several alternatives now
in the pipeline may help overcome some of
the limitations of amniotic membrane.
“In our hands E-PRP
is a reliable and an
effective therapeutic tool
to enhance epithelial
wound healing in ocular
surface disease”
Jorge Alio MD, PhD
“
The ultimate goal
is a biosynthetic
mimic that restores
refractive function
with optical clarity,
and resists enzymatic
degradation
Bruce Jackson MD
contacts
6
Cover Story
cornea
Long-Term Results of
Allogeneic Cultivated Corneal Epithelial Transplantation
Shigeru Kinoshita – [email protected]
Jorge Alió – [email protected]
Bruce Jackson – [email protected]
Akira Murakami – [email protected]
“There are still a lot of problems to solve. But we have
transfected a gene using this approach, which is an
important first step”
Courtesy of Shigeru Kinoshita MD, PhD
Akira Murakami MD
Autologous Cultivated Oral Mucosal Epithelial Transplantation
19y
Male
SJS
61y
Female
SJS
Nakamura T, Takeda K Am J Ophth, in press
Future View of Corneal Endothelial Tissue Engineering
Don’t miss Eye on Travel, see page 39
EUROTIMES | Volume 17 | Issue 3
Amniotic membrane has a number
of positive characteristics as a substrate,
particularly its anti-inflammatory and
anti-angiogenic properties. However,
disadvantages include the biological
variability between donors and even within
donors. Moreover, it lacks the transparency
of an ideal substrate.
The next step will be to strengthen
these implants and make them more
resistant to enzymes. The researchers are
now investigating additional applications
including imbedding the transplants with
nanoparticles for the delivery of drugs,
and creating collagen implants suitable for
refractive correction.
Biosynthetic corneal substitute
Gene therapy with a contact
lens Looking farther ahead, the future of
Speaking at the same meeting, Bruce
Jackson MD, University of Ottawa Eye
Institute, Ottawa, Canada, described a
promising research approach that could
allow the use of recombinant collagen for
corneal substitution.
In addition to helping cope with the
chronic shortage of human corneal
donors, a safe source of synthetic implant
material could reduce concerns about
disease transmission and might be useful
when donor grafting is not appropriate,
such as in cases of previously failed grafts,
autoimmune diseases, and alkali burns,
he noted.
“The ultimate goal is a biosynthetic
mimic that restores refractive function
with optical clarity, and resists enzymatic
degradation. It could be sutured, would be
biocompatible, would allow regeneration
of host tissue, be non-toxic, with no
inflammation or immunogenic problems,”
he said.
Recombinant type 3 human collagen
might prove an ideal scaffold to recreate
the natural corneal microenvironment,
allowing for the colonisation of the
central cornea by keratocytes. Type III
human collagen, which is found in the
skin, rather than type II collagen, which is
found in the cornea, appears to be a better
choice because of its superior mechanical
properties and lower cost.
Dr Jackson’s institution is involved in
collaborative research with Per Fagerholm
MD, University Hospital, Linköping,
Sweden, to evaluate the clinical potential
of this approach. He cited a recent
clinical study in which 10 patients, nine
with keratoconus and one with a deep
corneal scar, underwent anterior lamellar
keratoplasty and implantation of the
biosynthetic cornea. At 24 months, the
biosynthetic implants remained stably
integrated and avascular, with evidence
of nerve ingrowth. The epithelial
surface barrier was re-established and
remained stable.
corneal disease treatment is likely to include
gene therapy. The successful treatment of
humans with a rare form of retinal disease,
Leber’s congenital amaurosis, with gene
therapy, suggests that treatment of corneal
disease may not be that far in the future.
Leber’s congenital amaurosis was a
particularly good candidate for gene
therapy because its pathogenesis was
associated with a very small number
of genes, and the gene defect was well
understood.
Akira Murakami MD, professor of
ophthalmology, Juntendo University School
of Medicine, Tokyo, Japan, has identified a
potential candidate corneal disease for gene
therapy, and a potential treatment.
Gelatinous drop-like corneal
degeneration (GDLD) is a rare refractory
corneal disease that generates sub-epithelial
deposition of amyloid. Mutations in the
TACSTD2 gene have been identified in
Japanese families with GDLD, leading
researchers to believe it may be amenable to
gene therapy.
Presenting at the 2nd Asia Cornea
Society conference in Kyoto, Dr Murakami
noted that he and his team wanted to use
a non viral vector approach to deliver the
corrected gene. He reported the transfer
of the TACSTD2 gene into the corneal
epithelial cells encapsulated in a hydrogel
contact lens. In animal studies the team
reported being able to successfully
introduce the gene into epithelial cells, and
to record expression of the relevant protein.
“There are still a lot of problems to
solve. But we have transfected a gene using
this approach, which is an important first
step. We believe that some day the use of
phosphate groups containing hydrogel as
a gene transfer device could be applied to
non-invasive novel therapeutic method for
GDLD,” said Dr Murakami.