Jeffrey Frenchman, DPM focuses on the molecular composition of the extracellular matrix as well as the properties of amniotic membrane. Dr Frenchman also discusses the surgical applications for the amniotic membranes and provides case studies to support its use.
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Release Date: 03/16/2018 Expiration Date: 12/31/2018
Jeffrey Frenchman, DPM
Director of Limb Preservation
Atlanta VA Medical Center
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Male Speaker: We have two guests this morning that are going to tag team this symposium on Advances in Amniotic Membrane Therapies in Podiatric Medicine and Surgery and we have two popular speakers for you. First will be Dr. Jeffrey Frenchman from Atlanta VA and then my friend Chuck Zolan from Romanoff Virginia, both have authored several pieces on the use of these advanced wound products. And I would like to hear what they have to say, so we are going to see perspectives from two different people on applications of this new unique amniotic products. So let’s first welcome Dr. Jeff Frenchman.
Dr. Jeffrey Frenchman: Good morning, thank you for coming out so early. What I am going to talk to you about this afternoon, this morning is the benefits of dehydrated human amnion-chorion membrane, and how it works in the surgical and in the advanced wound care arena. As you can see that’s my disclaimer, we will go through a couple of slides, this is my disclosure information. The learning objectives, hopefully you are going to be able to walk away with understanding some of the history of amniotic membrane. I will able to describe several methods of how it is actually prepared and understanding of the molecular composition of the extra cellular matrix. And then be able to describe some of the properties there and the I am going to go through a few case studies before turning it over to Dr. Zolan to go over some of the level one evidence with you. These are my references, so let’s just jump right in to all the fun stuff.
So when you look at amniotic membrane no matter what your belief system is, let’s just say it’s been around for an extremely long time. You go back to 1910 with Davis, he attempted to graft pieces of amniotic sack onto granulating tissue. You move forward in to 1940 with Derolf and they began to use it in the ophthalmology space, that’s where a lot of the literature originally developed out of it. And it’s good in that area because it’s anti-inflammatory, anti-scarring. Then you move into the 1980s, it began to go out of phase for a variety of reason primarily because of the infectious disease HIV AIDS. And then in the early ‘90s, late 1990s early 2000s you began to see a resurgence of the product in a cryopreserved form. There are multiple methods of actual preservation and preparation of the product, the first being frozen followed by freeze-dried, irradiated and then something called stabilized amniotic membrane or SAM which is primarily used in experimental animals followed by dehydrated membrane. And that’s where we are going to focus primarily on this morning for you.
So dehydrated human amnion-chorion membrane also known as dHACM is a 361 ACTP product. It’s minimally manipulated, meaning that the processing is not all through the relevant natural characteristic. It’s designed to be used for re-construction, repair or replacement also known as homologous use. Before we get into some of this, I think, it is important to understand extra-cellular matrix itself in both acute and in a chronic wound. So when you look at the ECM, it is the largest component of normal skin and it is gel like matrix. It is produced by the cells that surround it, what’s important with this is understanding something called dynamic reciprocity. So that is important that the extra-cellular matrix gets to interact with the cells and the cells get to interact with the extra-cellular matrix, helping to guide the process that goes on to go on to repair and re-generation of tissue.
In the acute wound, the provisional matrix itself contains fibrin fibronectin and it provides a scaffolding in order to direct cells in to that side of injury. And the cells are then stimulated and up-regulated in order to proliferate, differentiate and synthesize new extra-cellular matrix. And then we have the problem in a chronic wound where there are increased levels of inflammatory cells and not to go through that whole cascade with you but we know once it gets into the inflammatory phase, we have nutrophiles that come in followed by macro-phases and those macro-phases wind up converting over to help with fibroblast production. There is elevated levels of protease activity in that chronic wound and what that tends to do is it degrades the extra-cellular matrix components, growth factors and also receptors.
So how I would like to look at it is, this, all of us drive around our neighborhood and we see that one house that has perfectly manicured lawn. The grass is very green, it is very dense, it is very robust.
When we look at a wound that itself, that’s like the earth lying underneath it. And a lot of times it is in very hostile environment, I mean there is lots of rocks in there, there is high hydrogen levels in the soil and no matter what type of sod you lay over that, it is either going to die off or there’s going to be spots fit that are going to die off. So when we look at it being able to differentiate between engineered tissue, looking allographs and looking at the zenographs, primarily we are going to focus on today it is going to be the allographs and being able to differentiate between a decellularized product verses a product that actually has cells in it that are bio-active. And that is what dehydrated human amnion-chorion membrane has in it. So when look at the extra-cellular matrix itself, it is composed of polysaccharides, water and collagen proteins.
It helps to regulate cellular function via cell adhesions, it helps to lubricate the cells around it and also provides the transport system for both waste and nutrients in and out. There are two main classes of extra-cellular matrix molecules and they are both secreted by fibroblast and by epidermal cells. The fiber structural proteins are going to provide your strength and your resilience to the actual tissue itself. So depending upon where you are in the body, depends on the level of ECM that you actually have, so in kidney for example high levels of parenchymal cells, low levels of ECM. When you get in to tendon structures, there is a much higher level of ECM in there. So within those fiber structural proteins are going to be your collagen, your elastin and your laminin.
Then we look at proteoglycans that helps provide the cushion to the cells in the ECM itself. So you want to have cushion for there. Otherwise, what we are doing is we are going to compress those cells and we are going to cause apoptosis and cell death. The primary proteoglycans that you are going to look at are going to be dermatan sulfate and your hyaluronan. Let’s delve a little bit deeper into the ECM itself. So when you look at the molecular composition and you look at collagen, those are the largest class of fibrous proteins that you are going to have in your ECM. Primarily type-1 collagen, 80% to 85%, you have type-2 collagen which is roughly 8% to 11% and then type-4 collagen in association with laminin and the proteoglycans go on to form that basement membrane.
You have elastin which helps to provide stretching and recoil to the tissue itself and then the laminin are very important because they form a bridge between the cells and the basement membrane itself. And they help to influence differentiation migration and adhesions that is very important. When you look at differentiating between a cellular ECM versus a decellularized product, it is important that we have cells in that that we are able to take down in to that hostile environment, and help to re-modulate the wound bed itself. So when you look at that hostile wound environment and it is stuck in that prolonged inflammatory phase, you are looking at having higher levels of nutrophiles in there, higher levels of micro-phases in there in that initial phase and what happens is those cells undergo apoptosis and when they undergo apoptosis they release proteases into the wound bed and degrade the matrix itself. So what we want to do is to be able to go back in there and not knock inflammation out totally because you need it but be able to down regulate it so we are able to go into more an acute healing phase and be able to regenerate the tissue that we need in order to close those wounds. Proteoglycans play an important role in the signaling between the cells and then fibronectin is very important from the stand point that there is multiple binding sites for the collagen, fiber and molecules as well as heparan sulfate proteoglycans. And it is important from fibronectin that allows for the different ECM molecules in order to link together and bind with other cells. So that binding to the integrin receptor becomes important because it stimulates the signaling pathways and promotes the cell attachment, migration and differentiation.
We all know that in a chronic wound state itself there are higher levels of MMPs in there, there are lower levels of growth factor and the receptors in the integrins themselves are defective. And what we need to do is to take those just like the earlier analogy looking at the sod and as I stated you are going to get some brown spots, maybe parts are not going to take when you are able to feed that, fertilize and you are able to feed it water and you are able to feed it the other things that you need more likely now you are going to have more robust growth in-growth of that sod down in to the underlying earth.
The glycosaminoglycans are strongly hydrophilic, they allow the ECM to withstand a lot of compression forces. Hyaluronic acid is the primary component of the ECM. It is found in high concentrations both in damaged tissue and in growing tissue and helps to stimulate cytokine production through macro-phases and in turn helps to promote angiogenesis. Frans Christian made a very good statement in one of their papers, what they said was the advantage of the ECM in its native state is the presence of all attending growth factors and inhibitors in relative amounts that exist in nature and most importantly in their native 3D ultrastructure.
And that is very important because what we want to do is we are able to come in and replace the tissue that is damaged by these higher levels of MMPs, by the defective growth factors and by defective receptors. So some of the properties of amniotic membrane itself is that it is anti-inflammatory, anti-scarring in nature and it is angioganic, it helps to promote reepithelialization. It is also an immune-privileged tissue, meaning that there is little to know HLA ABC antigens and the B2 micro-globulin and up to date there has been no rejection of the tissue itself.
When we look at fresh amnion chorion versus the dehydrated amnion chorion can see by the charts in front of you is that the growth factors, cytokines, inter loops that are in there are relatively same concentrations within the fresh and within the dehydrated version. If you look at the TIMS also same in the fresh and in the dehydrated human amnion chorionic membrane, It is important because we spoke about having high levels of MMPs. No matter what products you lay down in to that wound bed itself, if you are not adjusting for that, it is going to ultimately degrade the ECM itself. And then when we look at the relative growth factors in differentiating between the amnion layer and differentiating between the chorion layer, the large majority of growth factors are located in the chorion layer. So there are a multitude of products that are out there, I don’t believe that there is one product that is going to close every single wound but when I look at my patient population understanding that I have a high levels of diabetics that I am treating and understanding that their immune compromise and knowing that they are going to have decreased growth factors and they are going to have higher levels of MMPs within their wound bed and they are going to have defective receptors, what I want to be able to do is defeat that wound or defeat that grass, everything that I can give it. So when I am looking at choosing one product over another I am choosing a product that is not decellularized, choosing a product that actually has cells in it. Although they are not living cells, the cells are bio-active so when they get deployed down in to the wound bed they start to be able to signal the body and the surrounding tissue that, ‘hey something’s wrong in here’ we begin to bring in the native cells that are in there, begin to up-regulate them and that allows for the migration proliferation and differentiation.
So that’s really good so we go over some of the science within which inside a very briefly go over the differentiation between the de-cellularised versus a cellular product. And what I would like to do now is to go over some of the case studies with you, Zolan, utilized product in various places. So as far as dehydrated human amnion chorion tissue technologies, it comes in either a membrane itself or you can get a micronized version. It has a wider range of application one of them is going to be in the surgical wound space, it is going to be in chronic wounds, it will be in acute wounds and it will also be in disposed medicine arena.
What we are going to focus on, today, is going to be surgical application and in the wounds. When I first started using dehydrated human amnion chorion membrane it was only in the wound space and I saw such very good results with it that I started to expand that and take it in to the surgical arena as well. For one of the reasons is because it is anti-inflammatory and anti-scarring, when we first started using it we were primarily using it in space of doing a simple bunionectomy. And what I was impressed with post-operatively when they were coming back is that they had decreased inflammation and as a result of that they had decreased pain. And I was getting a better scar post-operatively. So there are a couple of techniques that I will do with it, one of them being is that actually lay the product in between subcutaneous tissue and the tissues above that and then suture it in. When I started to move towards more now is actually wrapping the product around the tendon itself, if you go back to a few minutes ago when I told you that depending upon the tissue that you are actually working on depends on the level of ECM in that tissue. And in tendons there are higher levels of ECM, what we are finding with this is one of the big complications is when we do surgery they sometimes get adhesion or they get scaring down and when the patient comes back we tell him, ‘Hey, you know, we will put a little bit of steroids in there’ we try to break-up some of those adhesions and hope for the best with it. What I am finding doing this is by wrapping it with membrane is that we are getting decreased adhesions therefore decreased scaring.
I spoke to couple of my colleagues and they were doing an anti-cervical spine fusions and they would pull the esophagus and trachea off to the side and then go back and close it, one of the big complications associated with that is going to be tethering and the patient ends up with dysphasia. So I said, ‘hey, that’s pretty interesting, that’s not happening in them, let’s see what we can do in this space’. And then again just laying membrane over now what I do actually is physically wrap tendon itself.
So I said okay, so we are getting some good results there, what else can we wind up doing this and what space can I take and kind of push the envelope a little bit further with it. One of the big problems when we are doing Achilles tendon surgery especially with ruptures is that paratenon is destroyed and when you go back and close it all down they want to put a lot of scarring in that region and want to having decreased ankle joint range of motions. So in this case here what we have decided to do is freshen up the edges, the band still intact, the plantaris tendon, bring the edges together, these are typical suture technique and then go in behind that and lay membrane down. One thing I like about the dehydrated version of the membrane is that it is a little bit easier to handle at least in my hands. We are able to lay it down, actually make notches in to the graft itself and then be able to take it and lay it on the inferior side of the tendon. And then just take a little bit of saline, re-moisten it and close down over the structure itself.
Then I said okay, not in acute rupture but let me see what I can start to do when we are going to take him in and go in do Achilles tendon lengthening. When you have those ruptures a lot of time the paratenon is destroyed and we know that paratenon is important from the stand point is that the tendons wrapped around and lost for that gliding mechanism back and forth. So in here this individual had a previous wound that we wanted to go in and close during a motor vehicle accident and he had decrease range of motion within his ankle, very, very tight Achilles tendon. So we went ahead and did an hope procedure for him and then laid graft down over notching at the actually fit in to where we made our mix within the tendon itself and then taking paratenon coming up and over the grafting material itself and then going on for closure.
What I was very impressed with him again, decreased inflammation, decreased pain and very good healing of that scar. We know a lot of patients that we unfortunately have to operate on are diabetic and a lot of times we wind up with postoperative wound dehiscence with that. So again looking back at the Achilles tendon structure itself is an individual had a GSW to the back of his leg, had shrapnel in there. We went in and tried to remove some of that shrapnel, closed him up, had a pretty good result. And then he went and walked all over it, ruptured the tendon apart again, and went on to put a pretty significant wound dehiscence, and we will call that Day Zero. What we did immediately with him is we started to use dehydrated human amnion chorion membrane within the membrane the micronized version and within 38 days under negative pressure we were able to generate a tremendous amount of new tissue growth in there. One thing I will do with tendon, especially the ones that I know are not going to be functional is to take an 11 blade and nick in to the tendon and take the micronized version of it and actually pipe it down in there and it will help promote granulation tissue up and through the tendon. We know it can be hard to sometimes close over the structures and then we move on here you can see that, that is going to be the piping technique which I am going to go over again with you very briefly in a moment.
And then showing when I have the deeper tunneling type wound where I can’t necessarily get membrane down in there. I will either make the micronized version of the particular in to a paste and lay it down in there and or pipe it down in to the location itself. This is just showing a membrane going down in that smaller wound by day 76 and then we have foreclosure of that wound site and then, this was taken around day 96 and here we are probably a few days ago. And one thing I want you to notice is that there is increased pigmentation around the surgical site itself, when I get into the VOU case you are going to see that even more prominently. This is an individual who underwent a partial first ray amputation even though we put a drain in there. He had vancomycin impregnated beads and he wound up dehiscing proximally and along that central incision site.
So in this case to go over with that piping technique would be as actually took the micronized of that particulate, took it out and put it in to a sterile specimen cup, re-hydrated with saline and then what I will do is I will take a 20 gauge IV cap needle, pull the needle out and use the sheaf, put it in to a 3CC syringe and actually take it and fill it down in to that void again. Some of the spaces are a little more technically challenging to get material down in there to the membrane and be able to deploy it against the side of the wound, to be in direct contact with the wound bed, I find this is a very effective method to get that and be able to get those growth factors, and get those cytokines and get those chemokines down in there and help accelerate the wound closure than simply just using Steri-Strips, and then closure of the wound site itself.
Very quickly go over some acute chronic diabetic foot and venous leg ulcers. This individual has a motorized wheel chair, instead of keeping on that little toe least function he likes to keep it on the rapid function and he likes to run his feet in to everywhere where he goes. In this case he decided to run it in to his TV stand and you can see here full thickness all the way down to tendons.
So I decided to take a little bit of membrane, lay it down in to the wound itself just take a sterile strip to help hold that down to reanastomose the edges, we call that day zero, when he returned by day 5 we had a significant amount of closure over the lateral side of the wound. By day 12 further closure and by day 19, so no matter what I am doing the main thing that I have focus on is improving quality of life and getting those wounds to close as rapidly as possible. And by feeding it and giving it the fertilizer and giving it the cells with extra cellular matrix and allowing it to react with the tissue within our body helps it to accelerate that overall healing process. So there is really a chip shot in the healing, initially I thought yes but then as you do this long enough you realize that nothing is really that easy.
So this individual here, we've all treated simple onychomycosis. He had a fungal toe nail. Cited to remove that toe nail, the vascular assessment was not adequate and we wound up the wound though that wasn’t closing. So what I did in that case with him, with the product was to take that membrane and just bring those skin edges up all the way where the bone was and because of his inflow his options to really to have a distal signs procedure done or a first time BJ just articulation. I offered him to be able to go in and just saucerize out the bone itself, I think a lot of us do a really good job of externally offloading we also need to look at internally offloading these wounds. So when you have that hammer toe that’s when you have a wound over the dorsal IPJ. Unless you really go in there and decompress the internal pubic content, you can get it close but there's a higher recurrence rate with it. So in this case we wound up going and taking off the superior side of that bone. This is five days out. That’s actually exposed distal phalanx there. And then immediately starting using membrane technology, when I use that technology actually fenestrate the graft and go with negative pressure over that.
And then by day 19, by day 28 and then at day 55 you can see, if you, in some of the earlier photos you can see re-pigmentation back in to that area there. Again just another post-op case, this is a chronic wound on the bottom of his foot, we went in, toe came off, took the metatarsal head out and by day 21 increased closure, by day 58 and then by day 71 and he received one more graft after that. And then he wind up closing off, we are going to close off on this case here, this individual early had a BK on one side, he presented to us, they offered him on BK on the contra-lateral limb, the wound size was initial about 400 square centimeters. So we did with this individual was debride him in the clinic where we are going to pay more attention to with this case, this is going to be a re-pigmentation back in to the tissue.
A lot of times with VOUs when we treat them they wind up having deep pigmented tissue especially in the African-American population. So debriding wound you can see increased wound margin, reepithelialization, and then by day 72 you can see there is deep pigmentation already growing up in to the wound side itself. This is just Z-ox a lot of times we can’t get in there and actually debride the wound or get him in to ultrasonic debriding units, this is something you can spike a bag of saline and turn it on and you have got instant power washer. And then again just showing membrane laying down over, here is using Adaptic Touch. A lot of time I use an Unna boot over it in order to bring zinc into the wound side. Zinc helps to promote re-epithelialization and then using a multi-layer compression bandage and here we are with that wound. You can see its re-pigmentation and still some de-pigmentation in that area, we are going to follow up on him and you can see that increasing re-pigmentation. So when you take this potentially it translated to doing other type of procedures, you can help cosmetically and improve quality of life.
With that I am going to close on, visualize what you want to happen because that is what creates the world that makes you go beyond what you think is possible. On that note no matter when you go out there and you are going to choose one of the most important thing is to seek evidence and you want evidence that is going to support the product that we are using. So I encourage you all to go and find that data, seek it out and on that note what I would like to do is turn it over to Dr. Zolan, he is going to go over the level one evidence regarding de-hydrated human amnion chorion membrane. Thank you all very much.