PRMRP 2012 Investigator Vignette

Title: Corneal Protection for Burn Patients

Investigator: Irene Kochevar, PhD; Massachusetts General Hospital

The goal of our research is to develop material that can be used to protect the eyes of burn patients that have a lot of scarring around their eyes that prevents the eyes from blinking. This leads to drying of their eyes, damage of the-to the corneas, and eventually opacification of the cornea so they can't see at all.

This is a collaboration between my group at the Mass General Hospital and Dr. Johnson who is an Army corneal specialist down at the Institute of Surgical Research in San Antonio. The problem that Dr. Johnson posed was is there some way we can put something on this eye during this period that the patient is undergoing skin grafts and other procedures that would protect the cornea.

One of the things that has been tried before is human amniotic membrane. But what happens is that these kinds of eyes are so inflamed, they're so irritated and red and running and there are lots of tears-those tears contain enzymes that break down the proteins in the amniotic membrane so that the-these membranes, which cost you know several hundred dollars each, had to be changed each day.

So we had been crosslinking proteins for-in tissue for a number of different projects and hypothesized that if we crosslinked the proteins in this HAM, human amniotic membrane, it would produce a degradation-resistant protective and healing material for these inflamed corneas. So that was our hypothesis.

So we looked at these different criteria and asked which of the crosslinking methods that we know about would best protect the proteins in this amniotic membrane from enzymatic degradation. And this shows collagen fibers, these long squiggly lines. These little pink dots are the crosslinking agent; I've shown them sort of associated with the collagen there. And the idea is you-you take the amniotic membrane, add a reagent. And what has happened while it's sitting in the solution that you form crosslinks between the different molecules of the collagen. And it's protecting so when you have these crosslinks, the big enzyme molecule can't come in and maybe try to cut it so we're trying to block the access of the enzyme to the sites on these molecules that would be chopped up.

We use five different methods and two of them are chemical methods for crosslinking. One is called carbodiimide and one is called genipin. And then we use three physical methods. Two of them are in the category called photosensitization. And then there is germicidal light. This has been used to crosslink other membranes and so that's why we tried it.

And then what we did is we took the membrane that we had treated and we tested with an enzyme that is similar to the enzymes that are in the tears of inflamed eyes and our test was to say after we've crosslinked this membrane does it still dissolve when we put an enzyme on it.

We tried all the different methods I mentioned and two of them turned out to be better than the others, and they are the two chemical methods, the carbodiimide and the genipin.

And then this chart summarizes all the different agents that we've tested. One hundred percent inhibition would be ideal; we don't want the enzymes to degrade the membrane at all.

The other thing we did is-reasoned that suppose we put several layers of amnion together. This might also protect the amnion from degradation by the enzyme, just by being thicker. And so we made bi-layer and tri-layer membranes, but we found out that the tri-layer amnion when it's crosslinked is also very resistant to degradation.

Second question we asked was are these beneficial factors that are in the amniotic membrane still available after crosslinking. It looks like the crosslinking methods I'm showing here are not going to be good for preserving these growth factors in the membrane. However, some of the other ones such as Rose Bengal or riboflavin or UVC might be good on this criterion. So we're still looking for the ideal conditions for crosslinking that also preserve these pro-healing factors that are found in amniotic membrane.

And then the third criterion is whether after crossing does this amnion drape very nicely over the cornea. What we measured is how much pressure could be produced within the anterior chamber before fluid in the chamber started leaking out underneath the membrane, We found that some of the products that we produce are flexible enough that they conform very nicely to the surface of the cornea and can be used for this application. It varies from agent to agent. But when we used the tri-layer amnion and tried to crosslink it and put it on the cornea, it was much too stiff. It was almost like a little board. It never really bonded well at all.

Where we are right now-we have products that can be tested in Dr. Johnson's animal model. We sent him some materials already. He's starting to put these on the animal model and seeing whether they will really help. That's a real challenge and we're real eager to hear what the results are from those studies. After that if that works through, then I think it's-to patients.