Lecture: Amniotic Membrane Transplant (AMT)

[Nidhi] I’m Dr. Nidhi Gupta and I am a senior consultant at the Cornea and the Ocular Surface Department. I also serve as a clinician scientist in the Stem Cell Lab at Dr. Shroff Charity Eye Hospital in Delhi.

The way our next hour is going to go is that we are going to start with trying to understand how did the amniotic membrane actually came to be useful in ocular surface disorders in ophthalmology. And then what it it’s unique structure and composition, and mechanism of action which makes it so useful as a biological membrane. Then we’ll go on to understand what is the process of procuring, processing, and preserving it so that it can be implanted into the eye and useful in our clinics in ophthalmology.

In the second half of my talk, I would be actually taking you into the OR and we’ll be discussing about principles of surgery, various techniques which are used while we’re implanting the amniotic membrane. And I’ve tried to put in a lot of cases, different case scenarios wherein I have used amniotic membrane and it’s very, very useful in those scenarios and various principles and surgical techniques have been used there. And I will show you the follow ups. And finally definitely all of this is loaded with the surgical videos.

This amniotic membrane actually is a Greek word which is derived from the word amnos which means the lamb. We all know it is the inner lining of the placental sac and usually it is discarded as a waste product. But if you look at the origin of this membrane, it is derived from the epiblast which is the outer cell mass and the fetus is derived from the inner cell mass.

Understanding its usage over time, its unique characteristic properties, it was taken up for use in the clinical use, mainly by skin specialists. And that is because it’s got a very, very unique structure, it is highly biocompatible. It is non immunogenic, it does not produce any immune reaction in the host when it is used as a graft. It has got very important biological functions so that at every stage of wound healing. It is easily available, very, very unlimited amount. And since it does not transfer any form of immune reaction then it has no ethical issues as well.

If you look at the history, earlier as I said, it was used by Dr. Davis in 1910 as a covering for the damaged skin burn patients. And later on it was used widely by physicians. But in ophthalmology it was only by de Rotth in 1940 when he used, for the first time, for treating cases wherein there was signal grade of symblepharon in burn patients. And he removed the symblepharon and used the amniotic membrane instead of the earlier use of the peritoneum. And he found remarkable results. Later on this membrane was not used for quite a lot of time, primarily because freshly prepared amniotic membrane was only required and there was a risk of transmission of infection. Until later on various protocols came in for its use and it’s only later that it came as a big bang in ophthalmology. And since then if you look at the papers, there are thousands of papers on amniotic membrane and it’s extremely beneficial on the ocular surface.

Looking at its structure, the amniotic membrane primarily consists of three layers. It has got an epithelial layer which is actually a single layer of cuboidal epithelial cells. Below it is the compact layer of the stroma which has got a layer of fibroblasts as well. And below the basement membrane is the stromal later and then comes the layers of the chorion. The chorion and the amnion are very closely adhered to each other. There is a middle layer, a fibroblast layer, between the amniotic and the chorionic layer.

The amniotic membrane is about 0.02-0.4mm thick and it’s got an epithelial layer as we discussed. The various other parts of it, the basement membrane and the stromal matrix are actually the ones which are useful as the biological products. The basement membrane consists of the collagen IV, the collagen VII, and the collagen V in abundance along with fibronectin and laminin. This membrane is very similar to the other basement membranes of the eye like the Bowman’s membrane, the Descemet’s membrane, and the lens capsule. They have very similar proteins. And the stromal matrix is very, very rich in the growth factors. And these are the growth factors which help in the growth of the epithelial cells.
If you look at the mechanism of action of the amniotic membrane, it basically facilitates every step of wound healing. It helps in epithelization. It not only promotes the epithelization, it maintains the epithelial phenotype, it is anti-inflammatory, it is anti-fibrotic, and it is anti-angiogenic. And all of this together reduces the pain as well. The direct mechanism of how it reduces pain is unknown. But however it’s understood that all of this together is acting as reduction of pain in the patient.

If we go on to understand that what are the factors by which it promotes the epithelization, then first and foremost comes the basement membrane. The basement membrane itself acts, as I said, is very similar to the basement membrane of the Bowmen, like the Descemet’s. It helps in migration of the epithelial cells of the host cornea. These cells can adhere to the basement membrane and they promote epithelial differentiation of these cells into the phenotype of that particular cornea. And prevents the epithelial apoptosis as well.

It also produces the rarest growth factors which are like the epidermal growth factors, the keratocyte growth factors, the hepatocyte growth factors. And inhibits the protease activity.
The other functions which are the anti-inflammatory, anti-angiogenic, anti-scarring, and anti-fibrotic, they all are actually working together. You cannot differentiate as to which factors cause which particularly, but together there are multiple reasons why the amniotic membrane is effective in all of the above functions. As I said it promotes cellular apoptosis of the inflammatory cells, it also down regulates the expression of CD80, CD86, the major histocompatibility complex inflammation. It induces the down regulation of transforming growth factors, it is also an anatomical barrier which keeps away the bare surfaces. It also has got the transcription proteins of tissue inhibitors of metalloproteinase.
Going on from the various layers to the various properties, let’s go to understand how it can be useful in clinics. What are the protocols of it to make it useful in clinics? It is procured from the gynecologists who are going to be doing it from mothers of consenting and planned cesarean sections. These mothers are tested negative for HIV, hepatitis, syphilis at one and six months are only the ones which can be donors for the amniotic membrane.
Usually if you’ll see, it comes like a huge placenta. That’s what they give, they give the placenta to the ophthalmologist who are having a collaboration them. And this placenta is then cleaned with BSS and a cocktail of antibiotics. And then the amnion and the chorion, the second picture shows here that the amnion and the chorion how they’re closely adherent, they’re actually dissected off with blunt dissection. And once you have the chorion which is separated, then it is completely washed off of all the blood and the heme and the clot which it has. And then finally the nitrocellulose paper’s put over this amniotic membrane, it is cut to various sizes depending on the clinicians usage.
And then they can be cryopreserved at -80 degree in the preservation media which is Dulbecco’s Modified Eagles medium, along with glycerol which helps to keep it for long. You can even keep it for about six months to one year in refrigerated state. And this is how the Dulbecco’s media looks like. It’s a pink colored media and that’s the shape and the size of the amniotic membrane. Whereas shapes and size can be cut off the nitrocellulose paper depending upon what’s the usage that you have.

Apart from the cryopreservation, which can be kept for a long time, the other things which are available, the other commercially available amniotic membrane, and I have no financial interest in these, are the various available companies which are there. They are basically freeze-drying or lyophilizating the amniotic membrane. What happens in them is they take the amniotic membrane, they cryopreserve it, then it undergoes a process of sublimation where it is dehydrated. And then gamma irradiated to remove any of the infections from it and finally it is preserved. But this comes like a paper thin membrane which is a dehydrated membrane and only when it becomes wet does it actually become wet and you can spread it on the surface. There are various companies which are available for the various types of freeze-dried amniotic membrane.
If you would compare, there is a question which has come up that comparing the fresh versus the ones which is cryopreserved versus the freeze dried versus the commercially available, which ones are the best? Definitely the fresh amniotic membrane has got maximum growth factors and it would be best for the ocular surface. However, it is not tested for infection, the risk of transmission of infection is high. It is nice that you have a cryopreserved one that can preserved also for longer and it is tested for any forms of infection. The comparison of it to the totally dry one, then definitely it’s to be understood that the more you freeze dry, the more you cryopreserve it and then dehydrate it, the more number of growth factors go down. Definitely the results would be best and most widely used is the cryopreserved amniotic membrane.
Going on from the procurement to processing. Let’s go to understand what are the various surgical techniques which are used to do the amniotic membrane? The basic principle is that you could either use it as an onlay or as an inlay technique or as a sandwich technique.

Onlay patch primarily means when the amniotic membrane is placed with the basement membrane down and where the stroma is, basement membrane down and the amniotic membrane acts as the temporary biological bandage. And it acts as a physical barrier to the surroundings. Then the epithelium starts to grow below the amniotic membrane and it helps to, it slowly degrades and gets removed. But the epithelium grows below this amniotic membrane. Basically this is an amniotic membrane with a basement membrane acts as a barrier against the outside environment and it’s only below which the epithelium grows in.
However you can also use it as an inlay patch wherein you have a large epithelial defect or a defect wherein you can put an amniotic membrane graft with the basement membrane up and the stroma side down, and the amniotic membrane acts as scaffold for the epithelial cells to grow it. And this tissue gets integrated as an inlay patch. And it acts as a substitute for this; it is called as a graft. It basically requires the basement membrane is up and you have the epithelial cells which start growing above it. That’s called an inlay patch where it can be used as a substitute for large epithelial defects.
You can also use it as a combination of techniques which is called the sandwich technique wherein you can actually fill in the graft, fill in the defect with an inlay graft and then you can put an onlay amniotic membrane to act like a bandage contact lens to prevent and behave as an anti-inflammatory and as a barrier function as well.

Let us go through this video and this video will help you understand this technique. This is a patient, I’ll just stop the video here. This is a patient wherein you can see there is a persistent epithelial defect. This was a grade four chemical burn and the patient was on medical therapy for a long time. And then at about three weeks of its presentation, the epithelial defect was not healing. And that’s where we’re going ahead and doing an amniotic membrane. What you see here is the nitrocellulose paper on which the amniotic membrane is spread out. And what we are trying to do is first to take forceps, which is a non tooth forceps, and trying to actually peel off the amniotic membrane, separate it from the nitrocellulose paper, and put it onto the ocular surface.

And once it’s put on the ocular surface you can spread it with the non tooth forceps, you try to spread it with the non tooth forceps and make it like a single layer, the choroid membrane is made into a single layer. And actually at this step, I’ve not done it here in the video, you can touch a bud and check which is the stroma side and which is the basement membrane side. The stromal side would have the chorionic fibers, a few white fibers which are sticky. Whereas the basement membrane side it is very, very, it is non sticky and it is clear membrane so you would not get any stickiness with your bud on that side.
Here since we are using as an onlay technique. What has happened here is that I’m trying to suture it now using a 10/0 nylon suture about 3-4mm away from the limbus. You could actually take these sutures at the limbus as well with vicryl also. I like nylon sutures primarily because they are more neat than the vicryl sutures and that’s why I’ve tried to take a continuous suture, a single suture which is going to go like a purse string around the limbus. This may cause some amount of bleeding because where your suture is passing is actually your episclera, it is passing through episclera. You can have some bleeding and that bleeding needs to be removed from below the amniotic membrane, otherwise your epithelialization can get delayed because of the bleeding which happens.

I’ll just fast forward this video to show you how the purse string suture is passed and then tied at one end and the extra membrane is cut off. The bleeding is all cleaned up from the ocular surface, and you can once you clean up the bleeding by pressing with your forceps then you can put a bandage contact lens. You may or may not choose to do a tarsa long depending upon what is the indication of your patient. That’s the onlay technique of amniotic membrane.
Let’s go on to the second video wherein I’m actually discussing the inlay technique of amniotic membrane. I’ll go back to the picture of this patient, start the video. This is a patient of Stevens-Johnson syndrome which presented in the clinics with descemetocele. You can notice that there is a lot of aspirazation on the ocular surface. That’s where you can see the descemetocele and there is inflammation of the ocular surface as well. What we are going to do here is we are going to take the amniotic membrane and I’m trying to cut a very small piece of amniotic membrane which can fit into this defect. And use the fibrin glue here to first fashion my amniotic membrane, use the fibrin glue, and replace this small defect with the amniotic membrane. In an inlay you’re actually using a small graft inside this defect that you have.
And once I have done that, that’s where I’m transplanting and pressing onto, it sticks very well with the amniotic membrane. You can use multiple layers as well. Fashioning the amniotic membrane the same size and use multiple layers depending upon the depth of your defect. And then I’m combining it. I’m doing the sandwich technique, actually combining it with an onlay amniotic membrane seeing the inflammation on the ocular surface here. We have actually done onlay along amniotic membrane with it. Towards the end we will be removing all the bleeding here and put a BCL onto the ocular surface and this patient really healed well. We’ll see some examples in the cases where we have discussed.
That was the inlay and the onlay technique and the sandwich technique of the amniotic membrane on the ocular surface. From here let’s go on to discuss some of the case examples wherein we have used these techniques.
This is a patient, this was a 20-year-old patient who had a thermal burn. And this is an acute state, he almost presented about 2 weeks of thermal burn. You can actually see the large area of the epithelial defect, not only the epithelial defect there is an inferior area of ischemia which you can notice from about four to six to eight clock hours, that’s the area where there is scleral ischemia as well. There is involvement of the lid burn as well in this patient.
This video which I’m showing is not of this patient but what I wanted to show here is that in acute burn, this is a very similar child who had come. A lyme injury wherein you can see that there is a lot of limbal ischemia here as well as conjunctiva necrosis secondary to the acute chemical burn. What we are doing now is first we are trying to remove all the necrosed epithelium, which is from the cornea, as well as a few, you’ll notice there is a very, very thick necrosed conjunctival epithelium which has become thickened and necrosed. We are trying to remove all of the necrosed epithelium. And once we have removed the necrosed epithelium we’ll be doing combining a Tenonplasty procedure wherein we’ll be pulling up Tenons from all areas in the periphery, from there the muscles to get it close to these areas of ischemia. And suture it with a 10/0 nylon. That’s where I’m pulling in all the Tenons, I’m dissecting it off in the periphery and trying to pull in to actually heal, to treat the necrosis of the sclera which has ischemia of the sclera. I’m suturing it with a 10/0 nylon sutures. I have used fibrin glue as well in this patient which helps me to reduce the number of sutures, and makes the procedure quicker in such patients.

Once I have done a Tenonplasty 360 degree for this patient, as you can notice here I’ve put an amniotic membrane onlay on this and I’ll be suturing this with a 10/0 nylon suture and put a BCL on the top of it. That’s about this patient wherein you can use it in the acute state.
This is how the post op looks like wherein you have a purse string suture and only amniotic membrane. This amniotic membrane in such cases where there is so much inflammation can undergo degradation and you may have to replace it after one or two weeks, as much possible, until you have a reduction, until your surface epithelializes in case it gets degraded earlier than epithelialization.
This patient later on over the next six months, did extremely well. And we didn’t require a second procedure and the ocular surface became quiet and there was no further requirement, only medical management was maintained. The patient gained 6/9 vision over months.
The second indication where an acute state amniotic membrane is extensively used in ocular surfaces is the acute Stevens-Johnson syndrome. Here this is a patient wherein you may have to actually be seeing the patient, an ophthalmologist may be called in an ICU. And we are seeing here at the bedside, this is actually one of the patients in which I was called at the multispeciality hospital wherein this child was suffering from acute Stevens-Johnson syndrome. And you can notice in both the eyes there is a lot of conjunctivitis that you can see. There is an early lid keratinization which is coming up. And what you can see is actually an epithelial defect in one of the eyes.
The technique which is used here is a bedside amniotic membrane can be done, depending upon the systemic situation and how you are placed for this patient. You can either do, if possible, under GA, you can actually do a little bit of amniotic membrane. But the more important point in this technique is that the amniotic membrane should be spread anterior to the lid margin and goes down onto the ocular surface, spreads onto the ocular surface, and comes out through the lower lid anterior to the lid margin. You have to actually be covering the gray line in these patients on both the upper and the lower lid to prevent the further lid keratinization which can be prevented with using of this amniotic membrane. And also it reduces the inflammation and heals the epithelial defect, apart from the medical management, of course, which goes along.
If you are not able to do this suturing, you can actually take a prokera which is an amniotic membrane on a round ring which can be just placed onto the ocular surface. This really helps in reducing a long term chronic sequelae in these patients of Stevens-Johnson syndrome.

Other situations which are very commonly present in our OPDs are the persistent epithelial defects. That could be secondary to a post PK patient wherein you have an epithelial defect on the graft which is not healing with medical management. It could be also a neurotrophic ulcer, it could be a corneal infective keratitis which has become now non-infective and there’s a persistent epithelial defect. And not actually very resistant to your usual medical management. In those situations you can do an onlay amniotic membrane. Like in this patient, there was a superior almost 30% of the cornea was involved in this persistent epithelial defect following a very, very complicated cataract surgery wherein the patient’s epithelium was just not healing. And this was a diabetic patient as well, although controlled. Wherein we did an only amniotic membrane and it healed very well in two to three weeks time. This is the igress, the most common indication apart from chemical burns wherein an onlay or an inlay a combination could be done for a persistent epithelial defect.
There are patients of bullous keratopathy wherein you’re not planning a DSEK or a DMEK and has a very poor prognosis for any form of transplantation. And it’s an end stage disorder when the patient is coming with pain, then you could think of doing an only amniotic membrane. It helps them heal with the pain as well. The pain also comes down with these patients.
This is one more indication wherein you could have patients with band keratopathy. Where you do an EDTA removal of the band keratopathy and to heal the ocular surface you could combine an EDTA along with onlay amniotic membrane. Heals very, very well in these patients. Also reduces the post op pain which otherwise could be a problem post EDTA.

Let’s see some examples wherein the amniotic membrane has been used by an inlay technique. I guess a lot of questions had come about the use of the amniotic membrane in pterygium surgery. In primary pterygium if you have a small pterygium involving a few clock hours, then only a conjunctival autograft may be sufficient. However, in patients in which you have an extensive pterygium involving a lot of clock hours or you have a bi-headed pterygium then you could combine your conjunctival autograft by this technique that you can have a large amniotic membrane graft covering the whole of the defect that you have produced. And use a small conjunctival autograft over this using a fibrin glue which could prevent the recurrence as well. But the primary use of an amniotic membrane in the primary pterygium would not produce results as good as a conjunctival autograft. That’s where in pterygium you could be using an extensive pterygium, you could be combining it with a conjunctival autograft.
This is a very interesting patient. This is a patient wherein there was a descemetocele. This is a patient of Stevens-Johnson syndrome where you can see the upper and the lower lid keratinization and that’s definitely responsible for the descemetocele which has happened. Here we did, apart from treating the lids, we did a multilayered amniotic membrane and it’s healed very beautifully and reduced down the inflammation also from the ocular surface.
One more place where I have used this as a dermoid excision. And instead of using a patch graft of corneal tissue, a multilayer amniotic membrane grafting has been done in this patient, and it really, really substitutes the tissue very well and produces very less astigmatism. In these patients also you could actually go ahead and use a multilayered amniotic membrane substitutes the corneal tissue and the defect can be healed.
This is a patient of peripheral ulcerative keratitis wherein an inlay amniotic membrane has been done along with an onlay has been done along with the excision of the peripheral rim of the conjunctiva and then an onlay has been done for this. And healed very well with an inlay and an onlay.
From here, I guess I’ll be sharing a very long case history of this child and take you from acute to chronic usage in chemical burn of amniotic membrane graft. This is a grade five chemical burn which came as a two weeks of chuna injury. And you can see all of the ocular surface, the cornea and the conjunctival epithelium is gone. What we did was in the acute stage, two to three times we had to do an amniotic membrane grafting in this child. And later on at six months it healed with almost a total LSCD, limbal stem cell deficiency with a superior symblepharon.
And six months later to that, we went ahead and we did the procedure of limbal stem cell transplantation. That’s called, what we are doing very extensively now is the simple limbal epithelial transplantation where you really do not require a lab to grow your limbal biopsy.

In this child I’ll just quickly show you the procedure. This is where we are taking the biopsy from the good eye, from the other eye of the patient which is healthy, about 2mm biopsy is being taken from the good eye of this patient. And then on the other eye, once the biopsy is taken from the good eye, that’s the limbus we are trying to harvest from the patient’s superior limbus, 2mm of the biopsy. And then we go into the diseased eye wherein a 360 degree peritomy is done about 4mm away from the limbus, and the pannus is removed from the ocular surface from the cornea. Once the pannus is removed over from the cornea, then an onlay AMG is done. With the fibrin glue and it is stuck in the periphery below the conjunctiva, 360 degree, you can put a fibrin glue, tuck it inside. And once that has been done you can put the limbal biopsy about 8-12 pieces of limbal biopsy, that’s where we’re putting the limbal biopsy onto the ocular surface in the paracentral cornea, away from the limbus as well about 2-3mm inside the limbus. And this works very, very well as an in vivo method of a stem cell transplantation.
This is extremely producing very, very good results. And that’s the child. Now post operatively you can see we have a very long follow up of many, many patients of SLET and they do very, very well. Amniotic membrane actually is helpful in both acute and in stem cell transplantation with SLET as well.

From SLET, let’s go on with patients where it has been used for symblepharon release and fornix formation. This is a child which had a grade three symblepharon, very, very extensive. We did a stepwise surgery wherein using the amniotic membrane, we first did the symblepharon release and fornix formation. Then a SLET was done and then later one it was followed by a penetrating keratoplasty and a cataract surgery and the child is doing extremely well.
Also it is used in conjunctival tumors. And this is a patient of OSSN wherein a large OSSN has been excised from the ocular surface. You can notice here that a large conjunctival defect and a corneal defect is produced post excision of this OSSN. And here conjunctival patch/graft was put in the defect which was produced and combined with an onlay amniotic membrane it heals very well. It actually can substitute the conjunctival defects.
This is an example when it has been used as a sandwich technique. This is a patient which has presented two months later after corneal melt. And you can see there’s a descemetocele, there is a corneal inflammation, and there is a large epithelial defect as well. We combine the inlay with an onlay and actually multilayered inlay with an onlay. So the multilayer inlay actually helps to close the defect and the descemetocele and the onlay helped to cover up hole of the ocular surface. And it helped to heal the patient. Later on the patient went ahead we did a DALK for this patient and did very well.
It can also be used to relieve the pain in the end stage disorder, reduce inflammation. This was a patient where it was sent from someone else as a referral. The child had a chemical burn, a stem cell transplantation was attempted, it had a total melt, we did a therapeutic PK. And later on we combined with an amniotic membrane grafting to quiet in the eye along with a Gunderson flap in this child. In end stage disorder also to relieve the pain and inflammation, it can be used as an onlay technique.
Going on from clinics, I think we all know that amniotic membrane acts as a very, very good substitute, aware in it. It is used in cultivated stem cell transplantation also. All the CLET is now not so much used as in SLET, but CLET is where you can actually use it in ex vivo growing the cells in ex vivo as well. That is one place where it is used as well.
That’s about from the clinics wherein we use extensively a lot of amniotic membrane in various indications.
Some very few contraindications wherein the amniotic membrane should not be used would be cases wherein it is a severely dry eye, a keratinized surface, where it is just going to fall off and will not be taken up in these situations. In simple cases of dry eye, it can help you to reduce the inflammation and improve the ocular surface, however in patients of severe dry eye it does not really act. And patients in which there is poor blink mechanism or like I said, if there is a place wherein you have an ischemia, you would actually, like we saw the case where there was a scleral ischemia, you would have to first given some form of vascularization through that tissue before you can help allow it to be epithelizing using an amniotic membrane.

Some of the very, very few complications reported, although very, very minor complications could be hematoma and you would have to actually remove that blood from below the amniotic membrane for it to act on the ocular surface. That could be there. There could be a premature degradation if there is a lot of inflammation on the ocular surface, you may have to do it again. There could be a microbial infection, very much possible. You’d have to remove the amniotic membrane and use the antibiotics to treat it. And there could be drug deposits on it if you’re combining by using a lot of drops apart from the lubricants. That’s about the complications in the amniotic membrane grafting.
Something which I wanted to bring up was the use of the amniotic membrane along with varying of it into forming into a much useful either a gel, a powder, et cetera. Amniotic membrane extracts is something which is the new field which is coming up where the biological properties of the amniotic membrane are used. And these extracts are combined with various other combinations of various other components to make it more useful in various other places. Not only in ophthalmology, but also in systemic disorders it is being used. That’s what it’s about, the amniotic membrane extracts which can be useful in various other situations. It’s something I’m not going to be talking about much but that’s the future, probably of amniotic membrane.
Coming towards the end of my talk. I’ll just conclude to say that amniotic membrane grafting has biological properties which help in every stage of wound healing. Cryopreserved is the most widely used for surface reconstructions. You could use an inlay or an onlay or a sandwich technique depending upon what is the clinical case that you’re dealing with. But it is extensively and very, very important in chemical burns and it is now used a lot in stem cell transplantation with SLET. I think it’s a technique which is going to go a long way.
I want to thank my team at Shroff, that’s the clinical team on the top. We have a great, amazing team with which I work. And the team at the clinical scientists and the stem cell lab. It’s a hundred year hospital and you’re welcome any time to be a part of our team. Thank you all and I will be taking up questions now.

The first question is from Deshaun, thank you Deshaun for your question. And your question is that are there other alternative options to amniotic membrane transplantation? In places where the harvesting of amniotic membrane is less practiced, what is the advice? I think the most common indication wherein an amniotic membrane is used is either in acute chemical burn or in pterygium. In pterygium, like I said, if it’s a primary pterygium you can take the conjunctival autograft. If it’s a large pterygium you can think of taking some part of the conjunctiva autograft from the other eye as well, or other area of the ocular surface from the same eye. If you’re wanting a large conjunctival autograft. And if in acute chemical burn, probably you would have to use eye drops if you do not have an amniotic membrane. Maybe the extracts and you could think of using if not, cryopreserved, then the ones which I showed the lyophilized ones. Maybe the ones which are very commercially available of this and you could think of using those in your clinical practice. They are widely used as commercial membranes. I hope I’ve answered your question.
I’ll go on to the next question and that says is there an optimum age of an amniotic membrane graft which facilitates its clinical usefulness? I guess the question meant is there a particular period of time till which it acts? Like I said at -80 degree you could preserve it for a very, very long time, about six months to a year. And its growth factors are preserved. It’s shown that growth factors are preserved and it acts very well for a very long time. I guess that’s not a problem, you could use it for a very long duration.

The next question to us says how many times can you use AMG in case of a persistent corneal epithelial defect with corneal perforation? Can we use a glue, or a Tenons, or a perforation with? In corneal, the cases that I had shown to you in descemetocele, you can use in large descemetoceles also, you can use multilayered amniotic membrane transplantation about 4-5mm. Epithelial defects you can use easily a multilatyered amniotic membrane transplantation. In cases, yes, you can combine it with glue like I showed in the cases. You can combine it with a Tenons patch graft as well. However in cases where there is a perforation, amniotic membrane alone may not help. And then you could either decide to use either a glue or you could decide to use a patch graft, that would be more appropriate. Because the tensile strength of amniotic membrane is not very great. It would give way in a patient wherein you have a perforation, so that would not be appropriate.
We have a question which says, how will you prevent the dry amnion from slipping out of the bandage contact lens? Do we need to suture it if we have no fibrin glue? Yes, we need to suture it. You can use, like I said, a 10/0 nylon suture or a vicryl suture. You can either take a purse string suture along the amniotic membrane or you could take up in with multiple sutures. So it falls flat onto the ocular surface. I hope that answers your question.
Can we use a patient’s own Tenons to fill the defect? Yes, we can use a patient’s own Tenons to fill the defect. That’s called a Tenons patch graft, it’s widely mentioned in literature and it’s very, very useful and very, very effective. You could go ahead and do a Tenons patch graft.
We have a question which says, when we use a single layer of amniotic, we have to put the epithelium down to what’s the corneal epithelium? I guess I’ve answered the question very extensively. My presentation where I’ve told you that there is an inlay and an onlay technique and a sandwich technique. Depending upon what are you using for, you can decide to use the basement membrane up or basement membrane down. I hope I have answered that question while I was presenting.
Is there any difference in amniotic membrane transplant insertion, epithelial side up or down for corneal epithelial defect? Yes, I answered that as well in my presentation. If you’re using it with the basement membrane up, then the epithelium is going to grow above it. That is more useful in cases where you have a crater. However if you’re using as an onlay patch and the basement membrane is up and the stromal side is down, the epithelium would be sliding on both the sides to cover the defect. Only the growth factors are acting on the surface. It is not utilizing the basement membrane of the amniotic. That’s where you have to make the decision whether you want to use it with the basement membrane up or the basement membrane down.

There’s a question by Dr. Major, where I can find all the process to prepare my own amniotic membrane? My country doesn’t have it. There is a lot of literature about this, Dr. Major, you can if you want you can contact me on my email ID. We can send you the exact protocol that we follow. But these steps which I showed are exactly the steps for the amniotic membrane preparation. And you can have a collaboration with the practicing gynecologist, you can take their amniotic placenta from their cesarean section. And one placenta prepares a lot of amniotic membrane which may last for three to six months for you. And the methodology is mentioned in literature. You could also email us, we can send you through our eye bank what is what we are following.
The next question comes from Dr. Eyer, and she wants to know, in veterinary ophthalmology, I use fresh amniotic membrane graft for dermoid. Any special advantage over preserved one. Yes, fresh is the one which has got maximum of growth factors. The only issue with it is that you procure so much amount of the placenta and you only use it for one patient. That’s not good. That’s why to have a cryopreserved one wherein one placenta can be used to prepare so many of the amniotic, and can be kept for later use as well, is why cryopreserved is used worldwide. It’s definitely better to have anything which is fresh which will have maximum growth factors, but you could actually use one placenta for many patients if you cryopreserve it.

We have a question here which says, when to decide whether it is a single layer or multilayer? Especially when using dry amniotic membrane? It will just depend upon the amount of defect that you have. In a descemetocele you could decide to use multilayers. Where you have to substitute the corneal tissue, there you would require a multilayered. Wherein where you do not need to substitute corneal tissue, then you do not use multilayered. Like I showed, 2-3 patients I’ve shown in my presentation when we have used multilayered and sandwich technique for amniotic membrane.
Dr. Mittel wants to know Placentrex gel is already available in market use for healing. Can it be used on ophthalmic field too for purpose of healing? I’m not aware of this gel, Mr. Mittel, I would not be able to answer this question. I’ll go on to the next one.
In chemical injury do you prefer to do the amniotic membrane transplant immediately or delayed? That’s a very important question. Whether to do an amniotic membrane transplant early or late in a chemical burn. If it’s a grade 2-3 chemical burn, you could wait for the medical management for the epithelial defect to heal. However it’s grade four or above, it’s best to use AMT as soon as possible for these patients because they have a long term impact if you’re not using an amniotic membrane transplantation. There have been a lot of randomized controlled trial wherein they have used AMT versus the medical management. Wherein they found that in grade four and above, definitely amniotic membrane transplant is going to go a long way. In grade four and below, maybe you could wait for the medical management and decide to do AMT a little later if the epithelial defect does not heal.
What is the final destination of amniotic membrane epithelium in case of inlay and sandwich technique, is there any risk of epithelial ingrowth? No, there is no risk of epithelial ingrowth because epithelium is a devitalized epithelium the moment you process it, there is no epithelial cells which remain. They do not have any immunogenic properties or they do not ingrow. There is no risk of having any type of epithelial complications because the devitalized membrane.
The next question is there, in which media is preserved? Usually the stracks of the amniotic. It’s the Dulbecco’s media with the glycerol. In which the AMT is in cryopreserved amniotic membrane.
Next question is there any indication of AMT in infectious keratitis? Yes, the literature has mentioned the use of AMT in infectious keratitis as well. And it is used in patients wherein you have an infected keratitis you could go ahead and look for literature a bit about this. Because it does inhibit a few of the organisms as well.
The next says, if both patient’s eyes affected, where amniotic membrane to be used come from? In both patient’s eyes affected, you can use two amniotic membranes. If your patient has a bilateral injury, then you can use a larger amniotic membrane, you could divide it into two and use for both the eyes. I guess that’s answered.
We have Dr. Bramen asking us what is your experience of the AMT in corneal perforation, do you use alone or with Tenons patch graft? Large perforation or small? I did show this and I’ve answered that in previous question with one of the attendees. We just answered that question.

How about the effectivity in treating infectious keratitis? We just answered that question.
What is your opinion on using amniotic membrane compared with mitomycin C for extensive pterygium to prevent recurrence? First of all in primary pterygium, if you do not have a very extensive pterygium, then you can use a conjunctival autograft, that works extremely well. If you have a very extensive pterygium, then I have not combined it with mitomycin C. What I have done is I’ve used a large amniotic membrane like what I showed in my presentation and done a conjunctival autograft above it. I’ve really not combined mitomycin C and it’s worked very well in my hands without using mitomycin C. The only place where I would use mitomycin C are in patients of chemical burn wherein I’m trying to deconstruct a large symblepharon. There’s where recurrence of the symblepharon is prevented with the use of the mitomycin C.

What about amniotic membrane eye drops? Yes, there is mention of the amniotic membrane eye drops as well, I do not have any personal experience, but the literature does talk about the amniotic membrane eye drops being effective.
The next question is, is there any difference in final outcome if you do the conjunctival peritomy and suture the amniotic membrane with sclera or without peritomy and suturing it through the conjunctiva? I think the question would have to be combined with what is the pathology that we’re talking about? If we do the conjunctival peritomy and suture with sclera or without peritomy sutures, it will all depend upon what is it that we’re talking about with Dr. Khaled, what is the medical situation in for which you have put this question? Because if you’re doing a SLET, then you’re doing a simple limbal epithelial transplantation, then you’ll be actually removing most of the pannus and the fibrous tissue. So you’ll have a large epithelial defect. In that you’ll have to put an onlay amniotic membrane, tuck it in the periphery with the fibrin glue, and then do your stem cell transplantation. Without, the amniotic membrane will only stick with glue in areas of epithelial defect. I hope that principle is clear, that amniotic membrane will not stick on an epithelialized surface. In cases where it’s epithelialized, you’ll have to pass sutures.
If you have an epithelial defect on the corneal, if that’s your question, if there’s an epithelial defect on the cornea, then you can do two things. Either you can do an onlay amniotic membrane and suture it and the epithelial defect will heal below. The second thing which you can do is that you can create a periotome and then you can tuck in this with the glue outside. Probably I have answered your question.
The next question. In SLET do you do two layers of AMT under the limbal stem cell and other above? No, there is most of the SLET that we have done we do only one layer of AMT. We use the fibrin glue below that to stick the amniotic membrane and we put the limbal biopsies above it. There is a group which does the, and literature does mention, the two layered AMT, but we have not done it. But yes the literature mentions two layer AMT as well. The only concern that I would be having in doing a double layered AMT is the basement membrane sometimes get integrated with the corneal. You may have a higher haze in these patients post operatively and that may reduce the vision of the patient eventually. I would want to do only one layer of the amniotic membrane with the glue and put the biopsies and put a BCL above it. That would, in my hands, work very well.

Dr. Desai wants to ask where can one get a cryopreserved amniotic? There are a lot of eye banks, a lot of centers like ours in India, I’m sure they have people all over who have joined. There are lots of eye banks who do give cryopreserved AMG so from whom you can procure the cryopreserved membrane. The technique of preparing it in your own center is we’ve described it in our presentation as well.
The next question is, is there any specific concentration of AMG in cases of OSSN? In cases of OSSN, primarily are only trying to cover the defect. There is no risk of any other risk that I can think of. You only have to be covering the epithelial defect that you’d be producing onto the conjunctiva corneal with your amniotic membrane graft.
The next question is that so the epithelial side is the shiny side of the amniotic membrane? Yes, you’re right. Epithelium, I would just want to say that again, that it’s a de-epithelialized amniotic membrane that is available in the market. You have the basement membrane which is the shiny one which does not stick with the bud. And it is the stromal side, if you look at the histopathology which I described. The stromal side which is called the chorion below it, that’s the sticky side. Yes, the shiny side is the basement membrane side, the sticky side is the stromal side which sticks to your bud. That’s how you identify the basement membrane up or basement membrane down.
The question that we have next is if we don’t have fibrin glue, can we do the SLET with other alternative or not possible? It is possible if you do not have fibrin glue. The only difficulty you would have is you could either do the other procedures of limbal stem cell transplantation that is CLAU. That is one procedure wherein you could avoid a fibrin glue, that’s what you can do.

Is there any indication of intraocular use of amniotic membrane? That’s a tricky question because intraocular use of amniotic membrane has been described in retinal disorders as well. I have specifically not covered in my talk primarily because I am a cornea surgeon, I have no personal experience. But yes, it has been described in covering the macular holes, used for covering the macular holes and treating macular holes.
Dr. Desai has a question. Dry amniotic membrane graft is very fragile and thin and is not so useful as they are commented. Therefore please advice sourcing of cryopreserved AMG. Also does one have to be registered with HOTA to use AMG in the hospital? The HOTA registration is not required to use amniotic graft in your hospital. As I said, you can procure it from the eye banks which provide amniotic membrane graft. In India at Shoff’s, we do supply the cryopreserved amniotic membrane graft. People from other country will have to find out the resource wherein the cryopreserved one is supplied from.
Can you kindly explain in some detail the surgical steps in using the amniotic membrane in bullous keratopathy, is that the last resort? Would it be incompatible to the eye for any of the further? This question I answered in my presentation, Dr. Zemodine. That is if you’re using an amniotic membrane transplant in a patient with PBK, then it has to be in those cases wherein it is an end stage disorder where you’re not planning to do any visual rehabilitation. The technique that you can use is to remove the whole of the diseased epithelium, and you can use a fibrin glue to stick an amniotic membrane over it. Or you can even do an onlay amniotic membrane to epithelialize the surface below. Either of the two would be useful in these patients, it reduces their pain, it reduces their discomfort. But definitely this is an eye where you’re not looking for vision in future.
We have Dr. Briggs who’s asked us are there any other concerns for transmissible infections aside hepatitis, HIV, and syphilis? Not really. Most of these membranes when they are sent out for usage, they are tested for most of the infections. There is no risk of having any transmission of infections.

Dr. Bukari has a question that if fibrin glue is not available what would be the approach for the inlay technique? You could suture the graft but it would be difficult to do a multilayer. But yes, you can. What you can do is you can take the 10/0 nylon suture and suture in that defect your amniotic membrane. And then you can maybe, if required, put a BCL or you can put an onlay amniotic membrane and a BCL depending on is the indication for which you’re doing an inlay technique. Yes, that means you can take sutures for doing an inlay in these patients.
If the amniotic membrane transplant is fresh, how long can we preserve in the standard conditions? Fresh would not stand for too long. You can only use it for some time and then you’ll have to either look for a cryopreserved one or a lyophilized one.
What form of amniotic membrane is best for SLET? Cryopreserved is the most extensive use for SLET and that I guess would be the best one which was available as compared to the lyophilized one. This one is the better one because as I mentioned the more you’re going to preserve it, the lesser the growth factors would be. And in a patient where you’re doing SLET, you’re doing a limbal stem cell transplantation, you want to do the best for the patient in the first go.
How long, Dr. Chanasala has a question, how long does it usually take for the degradation of amniotic especially when we are supposed to counsel patients for visual potential? Usually one amniotic transplant would get degraded into 2-3 weeks. That’s the time period it gets degraded. And if it’s a highly inflamed surface, it can get degraded earlier.
We have some thanksgiving from some of our viewers, thank you all for being a part along with me.

And there is a question which says, what are the post AMG therapy and antibiotics and steroids for how long do we give? In patients where you’re doing amniotic membrane transplantation then if it’s an inflamed surface, you would definitely have to combine it with steroid therapy and that could be 4-6 times in the day. And an antibiotic therapy until your epithelial defect has healed. Especially if you’re using a BCL, then you would have to use at least four times an antibiotic eye drop. Two weeks, as I said, the amniotic membrane will get degraded. And by that time depending upon what is your ocular surface state you could taper your steroids and stop your antibiotics therapy if the epithelial defect has healed.

Enlarged pterygium, does the conjunctiva autograft come above the amniotic graft or below it? It comes above it. Dr. Zymodine, it comes above it. In my presentation, the amniotic membrane graft which is substituting the large conjunctival defect is below. And over it you can put a small conjunctival autograft. That’s how you do it.
Can amniotic membrane be used in case of corneal bacterial ulcers? I’ve already answered this question.

Then I have the next question is that in SLET is the limbal biopsy sutured to the AMG before BCL? Kalpana, we do not suture it, we cut the biopsy of 2mm into about 10-14 pieces and we place it in the paracentral area of the cornea beyond the pupillary margin and inside the limbus. Midway between the pupillary margin and inside the limbus in equal distance and we put fibrin glue. It is stuck with the fibrin glue, you won’t be able to suture it.
What is your personal experience with dehydrated? I do not really have a personal experience with dehydrated amniotic membrane. But with what literature says and what discussions we’ve been having, the dehydrated one is papery thin and it’s difficult to handle it. Definitely cryopreserved is the one which I use and that’s where I would be able to answer the questions.

In both eye of severe chemical burn, how do you procure the limbal cells for SLET? Calponin patients wherein you have a bilateral chemical injury and the patient has got a bilateral limbal stem cell deficiency, you would have to do an aloe stem cell transplantation.
I hope this was something which was useful and I enjoyed learning and sharing along with all the viewers and thanks for joining us. And thanks to host Cybersight for inviting me for this webinar.