PATHOGENESIS OF CORNEAL DISORDERS

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 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 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.

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.

Biosynthetic corneal substitute

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.

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.

Gene therapy with a contact lens

Looking farther ahead, the future of 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.