OCULAR SURFACE RECONSTRUCTION

Images courtesy of Centre for Sight http://www.centreforsight.com

Treatments for conditions like limbal stem cell deficiency, epithelial defects and neurotrophic keratitis have been raised to a new level by the advances in biotechnology that now permeate all fields of medical practice.

The technologies include ex vivo stem cell expansion techniques and new pharmacological agents designed to enhance corneal and epithelial healing. But barriers remain to their wider accessibility to the patients who need them and some treatments may require further clinical validation before they are adopted into mainstream ophthalmic practice.

Ex vivo stem cell expansion

The use of limbal epithelial stem cells culture for eyes with severe limbal stem cell deficiency is based on the identification of the limbus as the epithelium’s stem-cell repository and on the success achieved with keratolimbal grafts and transplantation of a small portion of the limbus from the fellow eye.

Ex vivo expanded stem cell transplants have the advantage that they enable a large amount of tissue to be cultured from a tiny biopsy from the patient’s fellow eye or from a donor eye. Studies have shown that cultivated limbal stem-cell grafts give good and stable long-term clinical results, even in eyes with severely damaged ocular surfaces and total limbal stem cell deficiency.

The surgical procedure is not difficult for an experienced anterior segment surgeon. The technique of isolating stem cells from a limbal biopsy specimens and expanding them into sheets of cells requires specialised laboratories.

In an interview with EuroTimes, Sheraz Daya MD, FACS, FRCS (Ed), UK, noted that different centres use slightly different techniques for insuring that stem cells cultures retain their stem cell properties and that the cell sheets are easily transferred to the patient’s eye. Therefore, as yet there is no single standard technique but rather a parallel evolution of techniques with a lot of crossover of methodologies.

“There seems to have been a plateau in the development of this technique, however there are still some questions that need to be answered. In terms of standardisation, as there has been an exchange in technology, there are ‘lineages’ of techniques which include culturing cells on amniotic membrane as a carrier, a contact lens carrier and free cell culture and transfer of sheets directly to the eye – our preference and learnt from deLuca and Pellegrini in 1997 and modified by using an amniotic membrane to retain the stem cell sheet.

“Other carriers are being evaluated and things look promising in terms of 3D printing and the use of a variety of biopolymers. As usual, funding research is an issue, with fortunately so few patients requiring this type of treatment it is hard to make convincing arguments to the industry, and much development will continue in university research laboratories,” he added.

Apart from the biotechnological aspects of these approaches, Dr Daya emphasised that the host eye environment must be rendered as hospitable as possible if the epithelial stem-cell sheets graft is to be successful.

“What requires more evaluation is how to optimise success of the ex-vivo technique. There are a large number of variables with each patient being quite unique. There is much to consider when deciding how to proceed. We demonstrated that conjunctival inflammation can be a major problem and this must be controlled prior to proceeding. Ocular surface restoration in terms of lid closure, elimination of keratinisation and the creation of decent fornices is also of vital importance and even after surface restoration, this requires constant maintenance along with repeat procedures,” he said.

It is very useful to have to hand the skills and expertise of a good oculoplastic surgeon who understands the need for good lid closure.

One unresolved question is whether immunosuppression is needed in the long term with expanded stem cell allografts. Dr Daya noted that his findings suggest donor epithelial cells are replaced by host epithelial cells over time, which would make long-term immunosuppression unnecessary.

Ultimately, the greatest challenge may be to make the use of expanded limbal stem cell technology more accessible to patients, Dr Daya noted.

“As these are tissue engineering techniques, they need to be performed in accredited laboratories with dedicated staff, with ‘Designated Individuals’ overseeing and ensuring regulations are met. The cost is quite prohibitive and it would be a waste of resources having this duplicated in too many institutions. Supply of tissue to other corneal surgeons from central laboratories should be the goal and research into transport mechanisms is necessary,” he said.

Topical treatments

For epithelial defects and corneal healing problems, biological agents such as autologous serum platelet-enriched plasma and amniotic membrane transplants have proved very useful. Now there are also biotechnological agents designed to enhance the natural healing process in a less cumbersome way.

Among them is Cacicol® (Laboratoires Théa), a structural analogue of the heparan sulphate glycosaminoglycans, which has been commercially available for clinical use in most European countries since last year. The compound is intended for the management of chronic corneal wound healing, such as persistent epithelial defects, neurotrophic keratopathy and persistent anterior corneal dystrophies with associated pain.

“The heparan mimetic stimulates extracellular matrix healing process, and is a possible alternative therapy to heavy and invasive treatments such as autologous serum or amniotic membrane transplantation. Applied topically to a damaged corneal surface it penetrates into the damaged extracellular matrix, where it substitutes the endogenous heparin sulphate and binds to collagen and fibronectin. This mechanism protects the corneal matrix growth factors and cytokines against degradation,” said Ewa Mrukwa-Kominek MD, PhD, Silesian University of Medicine, Katowice, Poland.

She cited a study in which 80 per cent of patients with persistent epithelial defect or corneal ulcers resistant to usual treatments had a rapid decrease in pain and a reduction in the area of ulcerated ocular surface after instillation of Cacicol eye drops. Another study showed that in a small group of patients with neurotrophic keratitis, 72 per cent had a complete healing of the ulcer, after receiving the agent for a mean period of nine weeks.

“In all these cases, a clear improvement of corneal healing, demonstrated by a decrease in the ulcerated ocular surface or complete tissue regeneration, was observed within a few weeks with Cacicol therapy at a dosage of one-two drops a week to one-two drops every two days. No local or systemic side effects were observed. Patients are very satisfied
with this treatment,” Prof Mrukwa-Kominek said.

Nerve Growth Factor

Another new topical agent for ocular surface regeneration is recombinant human nerve growth factor (rhNGF, Dompé). Preliminary results from the phase I REPARO trial showed that most patients receiving the agent achieved healing of neurotrophic ulcers resulting from various causes.

The randomised, double-masked study included 18 patients. Fourteen received rhNGF at concentrations of 10mg/ml or 20mg/ml and the remaining four patients received vehicle eye drops. All patients were instructed to instil their drops six times a day for eight weeks.

Overall, the corneal lesions completely healed in 73 per cent of eyes, and around a third of eyes had improvements in their Schirmer and corneal sensitivity scores. Visual acuity was decreased in two patients, and improved in 11 at the end of treatment. Treatment was well tolerated.

The REPARO study group, headed by Prof Stefano Bonini at the BioMedico Campus of Rome, are currently conducting a phase II trial with rhNGH involving 39 centres in nine European countries and a parallel phase study is also under way in the US. In addition, Dompé are conducting clinical trials evaluating the efficacy of rhNGH in the treatment of dry eye, retinitis pigmentosa and glaucoma.

Dr Daya told EuroTimes that he has as yet no experience with Cacicol, but that he regards rhNGF as very exciting, although it is very difficult to acquire at this point.

“Platelet rich growth factors (PRGF, BTI Technology Institute, Spain) are also very promising. We have to date used 100 per cent plasma, however there is good evidence that the platelet fraction of plasma along with inactivation of complement provides a high concentration of growth factors that may be a great adjuvant in the context of ocular surface restoration and ex-vivo stem cell allograft transplantation,” he said.

Return of the biosynthetic cornea

One of the great unmet challenges in the treatment of corneal disease is maintaining the supply of donor corneal material. One potential solution for that may be a biosynthetic cornea, and that is a technology that is now entering its second generation. The cell-free constructs offer the prospect of eliminating the risk of infection from donor to recipient as well as the risk of immune rejection, which accounts for the majority of the 10 per cent of grafts that are lost within two years of transplantation.

At the ESCRS Winter Meeting in Rome in 2008, Per Fagerholm MD of Linköping University, Sweden, presented the first case series of patients ever to be implanted with a biosynthetic cornea composed of human recombinant type III collagen crosslinked with water soluble carbodimides. The implant was developed in a collaborative effort between Dr Fagerholm’s team in Sweden and the Eye Institute in Ottawa, Canada, under the direction of May Griffith PhD. Prof Griffith is now professor in regenerative medicine at Linköping University, Sweden.

The series included 10 patients. In all cases, the epithelial surface barrier was established by two months, innervation of the implant was rapid and complete, and colonisation with host keratocytes was good but slower than in donor grafts. At 24 months’ follow-up, the biosynthetic implants remained stably integrated and avascular, and there were no cases of immune rejection.

However, the results also showed that there was room for improvement. In a couple of patients there were problems with re-epithelialization, which in one case resulted in corneal melting. In addition, spectacle-corrected visual acuity at two years' follow-up had a mean value of only 20/110.

“The results in the first 10 human implants were good, but not perfect. What we found was that some of the corneas became slightly opacified in different ways and we traced that back to the suture technique we used. We had overlying sutures and that seems to prevent the epithelium from resurfacing properly. And inflammation in the beginning causes the production of local enzymes and inflammatory cells, so that had some effect on the implant,” Dr Fagerholm told EuroTimes in a recent interview.

Dr Fagerholm and his associates in Ottawa suspended further clinical testing until they could develop and test new biomaterials. To make the biomaterial better able to resist enzymatic degradation and sturdy enough to be secured with a single-stitch suture technique, it has been strengthened using a double crosslinking technique.

He noted that he and his associates have completed animal testing with the new material and are now seeking approval to commence testing it in humans.

“Experiments with pigs tell us that the new implants work. You can suture them and they are otherwise as good as those with the older material. In March this year we sent our application to our MPA, which corresponds to the FDA in America, to be allowed to implant this construct and we expect to get everything in order this summer,” he added.

Their application concerns not only the use of the new material, but also attempts at optimising the re-epithelialization through the use of limbal stem-cell sheets. Dr Fagerholm added that they will also be soon seeking approval for clinical testing with other new biomaterials.

“There are more alternatives popping up, human collagen isn't the only one. We’re also testing pig cornea collagen and artificial collagen. We have many options to test, some are further ahead than others, we're going to apply for clinical testing with those also within this year,” Dr Fagerholm said.

Sheraz Daya: sdaya@centreforsight.com

Ewa Mrukwa-Kominek:
emrowka@poczta.onet.pl

Per Fagerholm: per.fagerholm@liu.se