Section: CME Category: Wound Care

Current Advances in Regenerative Medicine - Review of Published Level 1 Studies Utilizing Placental Based Allografts

Anthony Iorio, DPM, MPH, C Ped

Anthony Iorio, DPM, MPH discusses level 1 evidence from clinical trials on placenta based allografts, clinical results of healing ulcerations, as well as distinguishes between the available placenta based allografts available.

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Goals and Objectives
  1. Review levels of evidence and importance in wound care arena
  2. Report Level 1 Evidence clinical trials on placenta based allografts
  3. Relate the clinical results of healing ulcerations using evidenced based products
  4. Demonstrate through research that all products are not created equal
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  • CPME (Credits: 0.75)

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    Release Date: 05/14/2019 Expiration Date: 12/31/2020

  • Author
  • Anthony Iorio, DPM, MPH, C Ped

    Assistant Dean for Continuing Education
    Chairman, Department of Community Health and Medicine
    New York College of Podiatric Medicine
    New York, NY

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    Anthony Iorio has nothing to disclose.

  • Lecture Transcript
  • TAPE STARTS – [00:00]

    Male Speaker: So, we've asked Dr. Iorio to come and speak to us about current advances in regenerative medicine. Are PBAs created equally? Okay, I didn't know what PBAs were. So now we know it's Published Level One Study. So let's welcome Dr. Iorio to the podium.

    Anthony Iorio: Thank you very much. Great and good morning, everyone. And I would like to thank, Present for allowing me the opportunity today to present to you some evidence which I had put together.

    Years ago when we first came to these meetings, we noticed that there were plethora of different types of companies having placental-based Apligrafs and not knowing which one to use, which one should we use, which one should we consider to use and the story goes on.

    So what I'd like to do today is to look at regenerative medicine which in the future is going to probably be replacing the standard of care. It is gaining rapid and exploding evidence that these types of products when given in the right situation and in the right times can actually give us greater outcome at a more cost-effective and easy way to heal the wound.

    I have nothing to disclose on this particular lecture. This is a CME lecture, so I will be talking hopefully as generic as possible. But let me review with you some of the objectives to what I would like to accomplish in this morning. We are going to review levels of evidence because levels of evidence, as Dr. Frykberg had so elegantly had presented, is very important for the future of the positivity of our wound care products and the types of products we use in our arena which is gaining full respect and responsibility for allowing that to move on.


    We have some level one evidence on placental-based Apligrafs PBAs that I'm going to be sharing with you. There is about 14 of them but I have summarized all of them, so that you don't have to spend time just going through it all. And I will review briefly some of the strengths and some of the weaknesses of some of those studies. And I will share with you the results if they meet significant significance, if they don't meet statistical analysis, we will share with you some of the things in which they can do anecdotally.

    So let's go back and at the end you will decide which of those placental-based products you're comfortable using on a regular basis. But in order to understand what those tissues are and what role they play, we first have to understand the regenerative therapy cycle and not to bore you to death but to reeducate you that we know that we start with homeostasis and once that actually happens, we then deal with an inflammatory phase.

    It is that phase of the healing cycle that modulates inflammation. Inflammation can be good at times, it can also be not as good. And it needs to be regulated and monitored. And some of these products will actually do that because they are the modulators of inflammation and they come in and they shut down the MMPs and they activate the temps and this is how we can regulate it. Otherwise, the wound's going to transition into a chronic state for which we are all are obviously dealing with it and we all deal within a regular basis.

    Following that very short inflammatory stage of about two to five days, we then go in to the prolifera phase. And that prolifera phase can last for about two days up to about three weeks, less than a month. And as you can see, this is the area of the healing cycle that promotes good cellular proliferation, migration, and here angiogenesis will just come into play.

    It is the remodeling phase that when a wound heals and remains healed that last well over up to three weeks to the two years.


    And we still wanted to make sure that that phase is being well-replenished, but some of the activity of reducing scar tissue for which some of the products have actually done and have done so well in doing.

    So just one slide of where we were when we started to teach methodology back in the early 2000s and we find out that there are several levels of evidence. The level evidence five which correlates with some of those expert opinions we hear all the time and that's hearsay, “me telling you, you telling me and showing me a good study.” Moving forward, we look at case studies and case series, and even cohort studies.

    But it doesn't become important until we hit the randomized control trial. That is where our level one evidence lies. And of course the systematic review which I will be doing today is a pseudo metaanalysis of the types of products used which will show and fall into that level one evidence or the systematic reviews containing randomized control or trials.

    So a little history about placental-based allografts, they're not really new. They have started about over a hundred years ago and they were first documented when they were used at Johns Hopkins Hospital for a treatment of dermal burns and dermal wounds. Then it went out into vogue and it went out for a while and then back in the '70s, it was regenerated literally. And it was retrieved and then we started to see the use of the product because of the way it was preserved, the type of storage and sterility that was needed and used and really, it wasn't that much all important – was that important should begin to utilize it in the real world.

    It wasn't until the late or mid '90s that the processing and preservation technologies delivered by certain companies started to become increased.


    And ophthalmology was probably one of the first types of disciplines to begin utilizing this placental-based allografts and use of it in their surgical procedures.

    Then we in around 2000 started to say, “Well if it works in all of those, why can't it work in wounds?” So the amnion-chorion grafts started to become increasingly popular and we started to look at the wound be it the acute and/or the chronic wound.

    And this is where we are today. As newer technologies bring us into the future, we're starting to look at now – well not only do we have the amnion and chorion, what about the umbilical cord? What about the fluid that's contained? What about placental tissue matrix? We're going to talk briefly about that because some has some level one evidence while others have none.

    So this is the placental tissue and why we use just the placental tissue in wound healing? Because it's immunologically privileged tissue. It modulates inflammation, it controls the inflammation during the time in which we call into play during the inflammatory and into their proliferative stage. It's full of nutrients. As we can see, these nutrients regulate the proteins, they also include growth – plethora of growth factors, chemokines, cytokines and other protease inhibitors. And it is easily available.

    The tissue has – is usually by a consented individual who is actually a donor tissue which is actually given and donated by a normal birth. And here we see the live normal consented birth for which the placental tissue is therefore serologically and micrologically tested after it is obtained. And then they processing of this is very individualized.

    The processing has a lot to do with the type of result we're going to get.


    Is it cryopreserved? Is it right? How is this presented? And obviously, the final result is going to be your placental-based allografts for the ones in which we're going to be speaking about today.

    Looking at a diagram of where this tissue is, you have the amnion layer which is the thinner layer against the fetus and you also had the chorion layer which is the thicker layer and that's against the uterus. We have very little literature – a level one evidence of literature on the placentalcology and matrix but we are beginning to look at the umbilical vessels.

    I believe we have one level one evidence I will review with you because of the stem cells and the blood supply that goes into that and where it's contained within this whole tissue. Amnion is a fluid and stem also play a role – limited role that it may be, it still needs more investigation in literature as you'll see.

    I'm going to just mention the intermediate level because the intermediate level to the amnion and chorion is that becomes and looked at, it really has a very minimal place in this type of arena. Because it contributes less than 5% of all the proteins that are needed. Also, it has a lot of, perhaps, contaminants. And then we say that some of the priority in the processing is when we meticulously process – we don't want to use too much of a pressure because that processing is what's going to remove the contaminants. But too much of a processing can also hurt you.

    So therefore, we think that the intermediate level is so thick, it has more contaminants and therefore the cycle to cleanse it is going to be a little bit more detrimental than the product is actually worth. So processing matters because this is how we look at it.


    We look at all the tissue cleansing and the preservation and its sterility are probably the things in which we really need to look at because it gives us, “Are these cells viable? Are they not viable? Are they intact, are they de-cellularized?"

    Cryopreserved versus dehydrated, what is the difference between the two? As some of the studies will show you, we will look into some of those studies to show that one may be different than the other. Is it frozen? Is it ambient? That's another thing we look for. How long does it live on the shelf and how long in a frozen situation can it be? Again, this all adds into you choosing the right path for your patient. And of course sterility, the type of sterility in which it's given.

    So this is not me, but this is Dr. Cobb. Dr. Cobb has come up with and saying that we have 250 proteins identified on the amnion/chorion layer. He is not saying that this is just the amnion or the chorion. This is the combination, amnion/chorion which comes together and he has nicely shown that there are 285 proteins chemokines and cytokines that are needed in order for the wound to heal.

    So let's take a look at some of the – what we want to speak together on today. This brings us to do we want to use amnion? Do we just want to use the amnion/chorion, the combined layer? Do we just want to use chorion? Do we just want to use umbilical cord and amniotic fluid and/or de-cellularized matrix? So these are some of the things in which we're going to show you where the evidence lies.

    As we see that the highest grade is given in the umbilical cord and the amnion/chorion layer, we like it because it's a thin membrane. It micronized the thicker – and it's also good with the cytokines, the collagen and the hyaluronic acid to a certain degree. The thicker membrane again is the umbilical cord with similar types of likings towards the cytokines homogenous to the acid.

    And then the literature becomes a little bit less as we go down.


    But you can see here that the placental-based Apligrafs, the composition is there. We just need to know what we want, where we want, and how we want to do it. Remembering that the amnion layer is the thinner of the two layers, continue 20% of the growth practice as opposed to the chorion layer which is 80%. So a combined amnion/chorion giving you the 100% and that's exactly what you want.

    And, here, you can actually see that the dehydrated amnion/chorion layer has just that. So in the literature, this is the only one that we can actually see where the collagen is plentiful, the elastin, laminin and all the other attributes to make this the extracellular matrix firm and to the point is there.

    So we also look at the placental-based Apligrafs and what we find when we look at the placental-based Apligrafs, we know that they come in an area which is the amnionic membrane, also comes micronized and we'll show you one or two cases because I have put personal cases along with the literature to show you that the literature actually is favorable in the results. And the umbilical cord, it gets less and less as we go on. We do not have any studies in the placental-based matrix at this time. However, we're still looking to see all of those.

    So here, this is where it actually begins. These are the level one studies of the placental-based Apligrafs as of February 19th. We did systematic approach review with somewhat of a meta-analysis of the studies that are out there. And when we find that with the amino Apligrafs, the amnion – sorry, the amnion Apligrafs, that's the single layer, we only found three papers that have shown that.


    And when we look at the healing rates from just that alone, we see that the healing rates rate from everywhere from, again, to heal which is usually based on 12 weeks, 33% to 62%. When we move down to the combined amnion/chorion allograft, that's the dehydrated type, we find that that has the most amount of papers that have been written on that and there are eight level 1 papers which we will review and show you briefly some of the strengths and the weaknesses and the outcomes of each.

    But, as you can see, this, the non-diabetic foot ulcers, what it also does to venous leg ulcers as well with the four-week as a backup as standard of care and following the wound up to an included 12 weeks. We found one paper on the umbilical cord allografts. I'll share that with you and that is good for diabetic foot ulcers particularly the thicker type. And then we also have the micronized ones. The micronized ones, not so much using the diabetic ulcers even though I have showed and will show you one, but it's also shown to reduce pain and to decrease the amount of inflammation because, remember, placental-based are considered anti-inflammatory and can be used and you will see in some of the studies or limited studies on plantar fascia.

    So let's get into the first topic. And here we have Dr. Lovy in 2014, this is where he used amnion alone Apligraf. He had some positive results in two of his outcomes. It was the healing at 12 weeks which was 62% with a viable cryopreserved opposed to 12% of the standard of care. And the medium time to heal was 42 days with the cryopreserved versus 69 days of the standard of care.

    The study had some strengths. It was a prospected blinded study. It was also a multicenter study despite the fact that we do not know the number of sites in which we used publicly and he have a large enrollment, about 97 patients.


    The weaknesses though as we've shown before, he allowed the wounds to heal after one week and did not have a run-in period. So, therefore, these could have been easier wounds to heal for which we interpret or we expect that to be.

    The second one would be the study by Snyder back in 2016 and his amnion only Apligraf with a prospective, randomized, multi-sector, controlled evaluation of the use of dehydrated amnion membrane allograft compared to the standard of care for the closure of chronic diabetic foot wounds. In that study, we have a two-part study, the intent to treat and the per protocol shows that at six weeks, you get at least the 33% to a 45% healing rate respectfully versus 0% in the standard of care. His mean average of grafts were about maybe 4.3 that he used.

    The strengths of the study, perspective randomized study, was a multicenter study using eight sites, and it had had good intent to treat analysis. The weakness again, they allowed the 30% to heal within two weeks, no run-in period but the small sample size also made it somewhat weak. And the last of the amnion allografts which is the amnion one case was a multicenter, randomized single-blind comparing the efficacy of viable cryopreserved placental membrane to human fibroblast-derived dermal substitute to the treatment of the chronic diabetic wound.

    Here, the results are showing that at nine weeks, 48% of the patients that have the viable cryopreserved versus 38% for the human fibroblast-devised and the number of grafts used was 5.4 versus 4.4.

    And the strengths, single-blind study, it was a comparative effectiveness with the tissue which is the dermal-derived substitute, the fibroblast-derived dermal substitute that was there in multiple sites.


    The weaknesses, obviously, it was lacking standard of care. It was a relatively small sample size and there was no run-in period over that time.

    Moving on to the one in which we have the most amount of information and that's the one amnion/chorion, dehydrated human amnion/chorion. We have eight papers that made the level of evidence. The first one back in 23 is Dr. Zelen who did the prospective, randomized comparative parallel study on the amnion membrane and in wound graft in the management of diabetic foot ulcerations. His findings where the healing rates that the rate to heal at four weeks, six weeks were 77% to 92% respectfully as compared to the standard of care. In the meantime, to healing was about 2.5 weeks for his control.

    The study strengths are – it is prospective, it's a randomized, controlled trial. And his weaknesses are at this particular case, it's a single site and obviously is a small sample size and he, too, had the lack of the two-week period at the run-in period. But then he has done another paper and he – a year later, 2013, he evaluated the dehydrated human amniotic membrane allografts in patients with diabetic foot wounds and these were the hearty humans because this is the crossover study and these crossover studies, those challenging patients who failed at the beginning are now put in to this study – have been in to this study and it shows that those patients that had been healing at the 12-week period who had not been previously healed now 91% healed.

    So the strength of this study was shown that these are challenging patients. These were the ones that were not healed previously that are now healed and now they have gone on to healing at 91%. The weakness of this study, again, is single center and only has 11 and it’s a retrospective study as well. The allograft by Zelen – he did another one which was the prospective RCT follow-up manuscript that showed that diabetic foot wounds and 94.4 healed within 12 weeks and the long-term study when those people who have had others that came back also showed a significant improvement in that – those wounds were to heal. The weaknesses of this study – single center, retrospective and a small sample size.

    And last – I think I did this one already. Excuse me. Yeah, okay. So those were these five studies that I have shown him and as far as the healing rates, as well as those that are hard to heal and those in which he – if I can go back, there was one there that actually – should I use this weekly or should I use it twice a month. We showed that in this particular comparison study, he showed that really, there was no major significant change.

    However, putting this product on weekly surpasses that than putting it on bi-weekly. The healing time obviously was not reduced as much as we would see. So therefore, a single would be better – single weekly would be better than twice a month. And again, a single size and a small patient size made this the weakness.

    This slide by the Di Domenico also shows the results of diabetic foot ulceration.


    He showed that the healing time is 6 weeks and 12 weeks. And the mean graph is 3.8. He showed when compared to the standard of care, 70% healed with the dehydrated human amnion/chorion membrane as opposed to 85% in the standard of care.

    Here, his strengths were a randomized clinical trial and he used intent to treat analysis. It was a multi-center. However, his weakness was a small size and the high rate of centering at 12 weeks due to some of the people that have withdrawn.

    And lastly, in this one, we have the patient with a title bound who used the study where he showed the efficacy of the dehydrated human amnion compared to perspective multiple randomized trial of 110 patients. They healed at the 12-week period, using both the intent to treat and also the protocol 81% to 70% of the time. And the follow-up at 16 weeks, which a month larger than the 12-week find that he had 95% as opposed to 86%.

    So in this study, the strengths are it's a prospective multi-study, however, the weakness is obviously, were very different types and not consistent in the type of setting and offloading and debriding. So, that needed to be evaluated again.

    So, see, these are some of the live wounds that show human amnion/chorion living toward up what it says. Here is a wound that has cleaned – has healed on the lower right at week six. This is the patient who similarly had the ulcer sub-second or sub-first, goes on to uneventful full closer which mean re-epithelialization, no drainage on the dressing, and others. This had closed in 28 weeks. This was a post debridement and cleansing and utilization of the amnion/chorion layer.


    Similarly, this hyper granulation – granular tissue closed within a reasonable time as well as the extension over complex wounds which can be used over bones or soft tissue, which also can be used after doing, again, the good wound care and using adjunctive placental-based products. Post-amp, it can be used because you can close it within a reasonable time and my colleagues who have done other work that shared their findings with me also show that the diabetic foot wound and the diabetic ulceration closes appropriately within the guidelines.

    Now, this also came up because this is a good area where we can cover over a tendon or a ligament because it does decrease the amount of inflammation and it strengthens the tissue, prevents scar tissue. So this can used surgically – Achilles tendon. This is a longus – a peroneus longus tendon repair and also a cheilectomy over the head of the first metatarsal, as well as other surgical wounds.

    So we have seen that the amnion/chorion layer seems to have the robust amount of literature that can be safe and efficacious in using at a good period of time. I briefly want to touch upon two more of the remaining five minutes but I do want to tell you that allograft is also used in venous leg ulcers.

    So for the venous legs studies, Bianke in 2017 did the study where his healing rates, 60% using the dehydrated human amnion/chorion versus the standard of care which is your four-ply compression dressing. Similarly, at 16 weeks it showed an increase. So what the study is actually is showing to us is – actually, it’s showing that this can be used as an adjunct to compression therapy because the wound will heal in a more appropriate period of time as opposed to just compression of care. Here are some of the examples in which we show venous leg ulcers.


    Using the dehydrated amnion/chorion with both and then, again, in three months we can see within twelve weeks it heals on the lower right. Again, these are just types of wounds that it’s indicated for and can be used and sharing from our colleagues on venous leg ulcers and venous stasis ulcers, the prevention of the early closure in getting the wounds to close and to heal are greatly exacerbated.

    And, lastly, I want to talk about the umbilical cord allograph – we only have one but we show and these are the ones for thicker wounds is that the title back in 2018 most recently used this and he found that there had been some improvement by using 81% versus 54% and similarly when he did his twelve week study in an intent to treat and you can read on the slides the strengths and the weaknesses there and the success rate for which he had.

    A little bit too soon to tell but these are studies that have made it to the level one and that are going on to the other. This is the use of the allograft umbilical cord for which you can see to the right has gone on to almost healed. There seems to another week or two that needs to heal it.

    And lastly, I want to talk about the micronized amnion/chorion injectable. We do know that there are studies by Zelen. Zelen did a prospective randomized study for which he injected – plantar fasciitis. Just to let you know, more evidence is needed. This was given to a point where he had injected the heels with a lidocaine or marcaine and then he 0.5 and 1.5 respectfully of the dehydrated micronized. And what he found was that here has been an improvement in the decrease in the amount of inflammation. However, we know that more studies need to be done. And lastly, Kazel study had shown the same thing where he used the scoring – similar scoring to show that there is a decrease. And the amount of inflammation and that a treatment of chronic plantar fasciitis can be treated as an alternative with type of therapy.


    So this is what it looks like and this is how it’s applied and this is how it is done. As you can use for a diabetic foot ulcerations as well. So the micronized can be used as anti-inflammatory and it can also be used to stimulate around the periphery of an ulceration to move it along after you do a good reduction debridement and then subsequent studies – subsequent types of tissues can be utilized to help close the wound.

    So, in conclusion, what I would like to discuss with you hopefully, we discuss the following. That we know that there’s strong level of evidence that uses amnion and chorion layer allografts in supporting the safety and the efficacy of treating diabetic and venous leg ulcers. I hope we have shown the published clinical literature might suggest that there are higher rates in diabetic foot when we use those two together and more so with the combination as opposed to the single layer which gives minimal amount of results.

    And the umbilical cord, something that we need to keep our eyes open and more data is needed. It is something that is up and coming. We need to understand the safety and efficacy and the use of that. And, of course, the quality of wound care impacts the factors in which we successfully treat. These products are not meant to be used solo. They are meant to be used when we do adequate – obviously, meeting the standard of care, excellent debridement, revascularization, as well as antibiotic is necessary and proper offloading.

    With that success, we can then move on to a very safe treatment where our placental membranes and allografts can only continue to serve us and the patient well. Thank you very much.

    TAPE ENDS - [30:11]