Section: CME Category: Wound Care

Angiogenesis at the Crossroads of Regenerative Wound Therapy

William Li, MD

William Li, MD focuses on regenerative wound therapy and the science and evidence behind its use.

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Goals and Objectives
  1. Understand how angiogenesis plays a role in regeneration
  2. Discuss regenerative wound therapy today
  3. Understand dHACM and its use for wound healing
  4. Discuss the future of regenerative therapies
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  • CPME (Credits: 1)

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  • Author
  • William Li, MD

    President and Medical Director
    The Angiogenesis Foundation
    Cambridge, MA

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    William Li William W. Li, MD has disclosed that he receives Grant/Research Support from Genentech, is a Consultant to Baxter and serves on the Johnson & Johnson Speaker's Bureau. He will be discussing the investigational use of CO34 autologous stem cells and NV1FGF.

  • Lecture Transcript
  • Male Speaker: Well, thank you, Dr. Freiburg [phonetic] and welcome everybody. It’s a pleasure to be here. And when I was asked by my friend to speak, I remember past meetings coming up to Phoenix at Desert Foot. And each time, it looks like the meeting is growing in terms of its audience, and also in terms of the exhibit hall, more people coming to showcase what technologies are available. And it’s really because of that that I’ve chosen this theme, which is angiogenesis of the crossroads regenerative therapy. You just heard Dr. Anderson [phonetic] talk about angiogenesis and how to image it so I’m not going to speak to that topic. But you don’t have to go very far just outside of this room to see these terms, regeneration, regenerative therapy, angiogenesis to recognize how important this is. So I thought I would bring to you not a discussion of products, per se, but really a framework for thinking about how science and research allows us as clinicians to be able to distinguish between the different products from a differentiation perspective and ask the questions, how do these things work? And where might we go in the future? So, I don’t need to actually tell a group of woe dealing [phonetic] folks how important angiogenesis is other than to state that the capillaries and granulation tissue connect to the rest of the vasculature, which comprises of a 60,000-mile network of channels, conduits in the body. And so, really we’re talking about a systemic organ system, the circulation, and not just a local one. But what happens systemically for example in a diabetic or in somebody with cardiovascular disease is definitely going to impact on the small tissues and the fine tissues. And also what pharmacology people are taking is also going to impact that. And as well as with diet and nutrition is going on systemically is going to impact on the local microenvironment. So a lot of different factors can cause problems with angiogenesis. This is a close-up view just showing from upper left to lower right, that’s a venule. And you can see all the small capillary branches that are growing the venule. Angiogenesis tends to start on the venular side of the circulation because it’s a low-flow side. On the arterial side, its intense jets of blood actually make it difficult to form vessels in an architecturally stable way. And so venular angiogenesis seems to be where it all begins. Now, this process of blood vessel growth is often referred universally as important in reproduction and tissue repair. And this is just a picture in the uterus every month in the female reproductive age. The generation of the endometrium, the lining of the uterus is angiogenesis. And if there is a fertilized egg by sperm and implantation, then that will wind up turning eventually into the placenta, which is angiogenesis of sorts, which then generates the fetus, which requires more angiogenesis in order to be able to develop and pattern the organs within the womb for the growing fetus. And so you can kind of see this, it’s the gift of life that keeps on giving and this idea of conduits that support life is incredibly important. Those of us who have been working in the angiogenesis and microvascular space have been asking, what is locked inside the secret of the capillaries, of the cells that compose the capillaries, the cells that are associated with the capillaries, and the tissue units for which the capillaries comprise? What are the molecules? What are the substances? What are the signaling pathways? And that’s really what I’m going to try to share with you, some of the latest research that give us some insight into that question. This is a picture of wound granulation but from underneath the wound. If you’ll actually cut a hole and let it heal up and granulate in, and then cut a flap around the wound and look underneath it, this is what the carpet looks like underneath a healed wound. And you can see in the center of the circle, hundreds of convergent capillaries growing in to heal that wound. So this is kind of a view sort of the antipodal view of granulation that you don’t normally see. Normally, we see it from the top and we’re just looking at an eagle’s eye view. By the way, what Dr. Anderson [phonetic] was showing earlier is really a deeper peer at these types of vessels and even a little bit deeper than that. So, we’re really now beginning to even think clinically how to peer beyond what we see on the surface, which is what we call a flyby. The other issue in terms of angiogenesis is its connection to regeneration. Now, when I was in grade school and in high school taking my biology class, the axiom of my biology teacher is that starfish can regenerate, salamanders can regenerate, but people can’t regenerate.


    And there’s a wealth of information, even textbooks written, that really try to underscore how regeneration is something that we’ve lost the ability as humans. But in fact, that’s false and the textbook on human regeneration is being written as we speak. If you think about it, our hair regenerates and our skin regenerates. Our gut lining regenerates everyday. Oral mucosa regenerates after you injure it, if you have bite something sharp or cut the mucosa on your mouth. Nerves regenerate. We know exactly how fast it regenerates and we even know some of the things that proteins that help them regenerate. And the liver of course is an amazing regenerative organ, as well as the lungs. And the other thing is that as tissues regenerate, what we do know is that regeneration is usually accompanied by angiogenesis. And this is actually a picture of a lung. This is all vascular bed of the alveoli. You can see the little air pockets scattered throughout. And on the left side of that yellow line is normal lung. On the right side, that entire piece of lung was actually, there was a partial pneumonectomy and that lung tissue’s grown back. And look at all the new capillaries that have actually grown back to regenerate that lining and so angiogenesis and regeneration often go hand in hand. Question is what are the links and signals that should make that happen? And what is the implication of that for wound healing? So, again, every regenerating cell, tissue requires a blood supply and that’s why this link between angiogenesis, delivering oxygen, nutrients, survival factors, regenerative factors really makes sense when you talk about tissue regeneration in any context. This is a bit of an update from my organization, the Angiogenesis Foundation, on the complexity of the steps in angiogenesis. And in fact, we’ve oversimplified it here. But just to give you a sense, in the old days, we showed a simple loop and said, there are these 10 growth factors and we’re done. But now we know that there are literally hundreds of steps. And if you drill down even further, we can get down into the cell pathways in each one. They all take place when we’re actually doing wound healing. And many of them actually are also involved with regeneration. And this area in the green circle shows regenerative stem cells that are mobilized from the bone marrow by an angiogenic stimulus or growth factor, let’s just call it VEGF but there are other growth factor, mast or stem cell growth factor, other ones as well. When they are released in the wound area by hypoxia, injury, inflammation, among the local responses that occur in terms of sprouting, differentiation, tube formation, things I’m showing on here, there’s also the proteins get into the bloodstream. They circulate all over the body and they call out stem cells like bees coming out of a beehive. And those stem cells circulate but they seem to know how to home in directly into the site where they’re needed. And this idea of homing induced by soluble factors, signals that mobilize stem cells is really important because it connects one of the connectors for an angiogenesis and regeneration. In fact, you can think about these soluble factors as magnets that actually attract stem cells to the site of action. The concept of the stem cell magnet is something I’ve been talking about for a few years. If you have a magnet in place and you’re attracting stem cells, what’s happening at the local level is the upregulation within the vessel wall of protein signals. I’m not going to talk about all the different biochemistry about each one. But if the green is the stem cell, literally it’s like Velcro. They’ll be molecular Velcro being expressed. So that cell becomes recruited and attracted into the wall of the vessel. Once it’s in place, it turns on other growth factors and releases them right into that wound bed. So again, think about this as a Trojan horse so that the injury attracts stem cells like a magnet, the stem cells integrate into the area where the wound is. Once they integrate, they then open up like a Trojan horse to release other growth factors, so it’s a cascade literally a domino effect that actually takes place. One of the questions is, and we don’t know the answer to this. How long do these cells have to stick around in order to be able to have function? Now, if you’re getting to the debate of live cell, dead cell, stem cell versus non-stem cell, the argument is that these stem cells don’t stick around very long. The scientific question is how long do they need to stick around? We don’t know that. And that’s really the answer. That’s really the next set of questions to ask because it may turn out that some of these Trojan horse released factors will kick in the next set of actions into place where the cell itself is no longer needed beyond a few days. Now we do know that even a regular acute injury will mobilize some of these progenitor stem cells.


    In one study in granulation tissue, up to 25% of the endothelial or vascular cells originated from the bone marrow. So, this is again a big question that’s out there, is what percents of reproducing proliferating cells in the wound are actually stem cells. We don’t really know that and some people suggest as little as 1%, some say as high as 25%. Answer is probably somewhere in between. But the really interesting question is, if these are acting as Trojan horses, what can we do to make more of them come into play? What are they releasing and what does it do to the tissue? Because we want better quality healing, we don’t want a scar if we can avoid it. I think that’s very much wound healing in the 20th century. 21st century wound healing is what can we attract to the area, release and have a biological effect that has clinical impact that creates better quality healing? So that’s really what I’m going to talk about. Before I do that, let me just say that regeneration is sort of an area that it’s hard to go to a wound or tissue repair or surgical medium without seeing a lot of companies racing to develop regenerative products. But in fact, there is a bigger national call to action here and that’s the Department of Health and Human Services released a few years ago a report, a vision report for 2020. So this is a white paper that came out of an NIH-HHS meeting, in which they basically described what the vision is for the future. And what they described the national vision for regeneration is that there’s multiple ways to get at this. For one thing is that we can actually use therapies that prompt the body, we could put something into the body that call out stem cells and regenerative tissue. Or we can put some tissue engineered implants into the tissue in order to be able to directly manufacture outside of the body something that will be useful on the body. Or we can actually just transplant healthy tissue, graft healthy tissue in order to be able to affect transplantation. So if you were a cardiologist or if you are a neurologist, you might sort of say this is all in the future. But for us in the wound healing world, we’re already doing this. This has actually been happening in the wound care space for well over a decade. And so many people are now referring to the wound care field really as the tip of the spear, the pioneering front, the benchmark for how regenerative therapies can actually be developed. But I’m putting this framework for you to understand that there’s no one way to win at this game. In fact, there’s a lot of different ways and it’s not just one or two companies doing it with one or two investigators, but really there’s a national call to action and we’re going to get there. So, this is a billion-dollar industry estimated by the government, in the private sector, more that $4 billion dollars invested. They’re looking at the fully developed field to be worth $100 billion. They’re not asking who’s going to pay for that but it shows you how broad it is and $500 billion globally over the next 20 years. There’s a federal initiative of regenerative medicine that involves many different national agencies, including FDA, and NASA, and NIST, Department of Commerce, and NSF. And the FDA obviously has established divisions that actually are beginning to tackle some of the questions. The Office of Cellular Tissue and Gene Therapies, the OCTGTs are actually starting to take a look at the claims that people are pursuing that their technology is regenerative. And so one of the questions that gets asked is, well, how do you know and what is the evidence? And so, this idea that we can’t get away from evidence and good research really is the backbone to the success of this field. And there are milestones to create regenerative medicine as a future standard of care. I think even in the wound field, you can start seeing this technology shift to more sophisticated products that can aim towards manipulating the tissue environment more towards regeneration and scar formation today as part of the new standard for advanced wound care. What about clinical development? If you go to the national database, and you just type in regeneration, you can find, and this is from yesterday, 161 open clinical trials of different types of regenerative therapies that are being studied. So again, this is a locomotive that’s actually already left the station and there’s a lot of new ways of doing regeneration. It’s just not what you’re seeing in the exhibit that literally is what is actually happening in the modern frontiers of medicine that we’re all participating in. I’m not going spend too much time talking about companies other than to show you this graph that over time, more and more companies are coming into the space.


    And that’s good news because companies are able to invest the money, to do the research, run the clinical studies, to generate the evidence that ultimately will give us those tools in the toolbox. Now, if you take a look at what the top three current applications are in terms of products and clinical development, cardiovascular, wound healing and bone, orthopedics, tissue repair across a broad area and obviously fixing the heart are some of the most attractive areas today. So it’s back to our own neighborhood, our own sandboxes, the wound healing field. From our perspective, obviously we want to have better healing, faster healing, but the quality of healing is something that is really, really important to address. And I think that I’d encourage any of you here that are involved with research or using regenerative products, the early sort of the first generation of these things, to think about the question, is the effect that we’re aiming for or getting leading to better quality healing? And that means that we need more endpoints, different endpoints than just simply closing the wound. Or closing up the wound with epithelium is important, but that’s basically like just putting sod on your lawn when there’s a hole on it. We want to actually understand what’s going on underneath. Now why is that important in terms of thinking about tissue repair? Well, if you’re in cosmetics, there’s no room for actually having poorer quality healing. You want perfect quality healing and you want better quality healing than you started with. If you’re in reconstruction for plastic and reconstructive surgery, some of these really audacious, daring face transplants, skull reconstructions, terrible trauma, massive tumor removals and then reconstructing the face that involve a team of 20 people, you also want really good quality healing. You don’t want a big ball of a scar forming in something reconstructed in someone’s head. Surgical dehiscence, something that unfortunately happens more commonly than we would like but this happens in a compromised, diabetic, obese population that require abdominal surgeries. And so how do we actually prevent that from happening? And at what point do we do treatment? By the time you get a situation like this, the horse is already out of the barn and hopefully, that patient is not going to get infected and it inevitably looks like a scar will form. Is there something we could have done at the time of closure in order to be able to prevent this kind of thing from happening? Same thing, bowel anastomosis, another area where dehiscence occurs, burns another area. You don’t want to have just your entire hand reepithelialized with a giant scar glove. We want form and function. It becomes really important these questions are asked. And in orthopedics, when you think about sports medicine but then you think about an aging population, we want form and function to be normal and age appropriate as you’re aging but it doesn’t mean that we have to degenerate as we get older. How do we regenerate these tissues so you can have the best possible function in your 60s, in your 70s, in your 80s? And for some of you here that are in your 40s, I’m sure that you already have aches or pains that only herald things that need to be replaced later on. So how do we get around that problem? And can we fix hips and feet and knees and backs? That’s the interesting question. So now, I’m going to land back in our space, which is the chronic wound space. And I hope I’ve given you a little bit of a broad sweeping view of regeneration and how angiogenesis plays a role. If you take a look at these wounds, you would say they all need better blood supply. And so how do we grow them? Well, 15 year ago, we said, “Oh, let’s take a recombinant growth factor and slap it on there and we’re done.” Well, we now know that single growth factors are unlikely to be able to achieve what we need. And in fact, growth factors alone are probably not able to do what we need. And that of course is in parallel with this argument of just what is good wound care and what is good standard wound care. So there’s a lot of heavy duty questions that are asked about this. But in the meetings like this, we like to think about what’s the future. And so the question is, what can we do that would stimulate angiogenesis and regeneration? And is there any science that can allow us to establish that so it gets beyond marketing claims? And I think that’s one of the biggest problems that we clinicians face in the wound care field is that there is a richness of product offerings out there with a lot of different statements about products that make it confusing for us to know where we are. And that’s where regenerative wound therapy is today. This is a picture from Cirque du Soleil. You see all these really interesting actors all dressed up. And like individually or as a group, it’s fascinating to look at. And that’s really where we are. All these products that we have today are mesmerizing but it’s really hard to know how they are different from one another.


    And that’s really why science and clinical evidence, scientific research and clinical research is so important for us to be able to have a deep dive. We need to define regeneration. We need to be able to differentiate. I mean, all of us here who went to medical school, at one point, we were trained in the scientific method, we were taught how to create a hypothesis, how to frame the questions, and then how to actually deliberately look through ways to address each of those hypotheses, ruling out the ones that don’t fit, and critically analyzing the results of the ones that actually do, and then putting together a summary. So, what I’d like to do really with the remainder of my time is to just give you an example of how a solid scientific approach gives us the ability to take a look at regeneration and angiogenesis in a different way. So how do we look for regenerative factors, right? So there’s a lot of places you can look, the bone marrow I mentioned. There are sources outside of the body and there’s a lot of stem cells you can go for. But I’m an internist and so I think about the entire lifespan and one of the areas that we recognized a long time ago in the angiogenesis space is what I started with, a picture of the reproductive system. And it turns out that the pregnant mom with a developing fetus and all of the components then including the placenta is probably one of the richest depots for regenerative factor, because that’s actually what’s happening over the course of nine months. So, let me just show you a little bit of research that’s going on there. And obviously, you know that there is the placenta which is the connection between mom and baby. It’s an outcome of a type of specialized reproductive angiogenesis. And this connection is then modified and then as a fetus forms, you have coverings and layers like the amniotic membrane that is composed of amnion and chorion that itself is packed with proteins, growth factors, cytokines, regenerative factors. And although most of us until a few years ago probably forgot more than we learned about amniotic membrane, the reality is this was an airbag for the baby, for the fetus. It was a protective lining to prevent bacteria and infection. It was an immunoprivileged barrier and also is inflammatory. And now we know that it’s also regenerative because when you apply molecular biology techniques, you find out there’s so much going on in there. So, how do you actually study this? Well, you basically get donor placentas and you can stick them in a tray and then if you know your anatomy, you make an incision and you literally can peel off the membrane and then you can clean it. And at this point, this is kind of like the cut point for doing research in this area. You could put it onto a mouse, you could put it into processing, you do all kinds of other things with it. But the fact of the matter is that the source from the placenta is very much the same. I’m making this point about where things are the same up to a certain point because it’s very important if you go out into the exhibit hall, you start seeing lots of different choices out there and products can be confusing. But we need to always understand where are the fundamentals and at what point do the starting of the source material start to ramify? So this is all pretty much common denominator stuff. And then you can process the tissue. And today, because of the regulatory channels that amniotic membrane therapies have undergone, you can put it pretty much on a wound. And what the tissue does is it releases those biological factors into the wound bed, alright? So that’s literally what we’ve been hearing now for several years and there’s clinical data that supports the efficacy and the effectiveness of this. I’m going to show you a bit of the clinical research data. But I want to challenge you, dare you to ask even more difficult questions, which is, alright, I’m able to suspend this belief that this will actually do something good to the wound. How do we actually prove that? And what’s in it that actually allows us to get down to that level? In other words, what’s known about the factors that are delivered into the wound itself? So, this is where I say, I’m a clinician, I’m a scientist, and also I’ve spent 25 years looking at innovation, you can’t get away from good science if you want something that’s reproducible and if you want something that’s credible at the end of the day, not only for clinicians but also to payers as well. And ultimately as patients get more engaged into their care, all they have to do is click on the internet to take a look at the research data themselves. And so, the level of transparency of scientific data is really increasing and that’s where following the research is important.


    So I’m just going to give you an example. Again, there’s a lot of different tissues I kind of chose. I’ll just give you an example because amniotic membrane therapy is very hot right now in wound care and how a scientist might look at this. Going back as early as 2002, there’s been studies looking at human amnion and choriodecidua as expressing angiogenic and neurotrophic factors, okay? So more than a decade ago, people, researchers, and laboratories were doing the analyses inside this tissue and finding these growth factors. Well, that’s very exciting. And then the question is, where do they come from, because the amniotic membrane has got multiple components to it? It’s polar, so meaning that there’s an epithelial side, there’s an amnion side and a chorion side. That’s the way that Mother Nature engineered it. And so how do you actually figure out what’s inside there? So I want to actually just give you a case study. Again, this is not a product pitch, this is a case study, sort of as I would lecture to a university or a medical school or a research community. I’m giving you the starting point that we’re looking at. So amnion and chorion is the whole package. It’s the top lining, the chorion underneath it. I have no idea what’s in there so let’s take a look at one that’s actually available to be researched on. There’s a processing for one version of this called dHACM that’s very shelf-stable and it’s been processed in a way that is non-manipulative or minimally manipulative and it allows it to be cleaned and dehydrated. It’s got very low bioburden and it’s sterilized. So it’s actually a good reagent for research purposes to look at. In some of the work that was done initially, what was found was the one growth that we had in 1998, which was becaplermin, recombinant human platelet-derived growth factor that everybody got excited about, but there is 57 different growth factors and chemokines that were found in an initial kind of a sweep, a research sweep of this. You take the tissue, you let it release the factors into a solution, and then you use different techniques to pick up the proteins and identify them against a big database of growth factors and cytokines to see which ones are there. So this is pretty impressive to look at 57 growth factors, exciting. And those are all published. But I like to give you an update now. And so this is where research keeps moving forward. Those 57 which are just published a few years ago have now been updated and there’s fourfold. So the same platform now has 226 growth factors and chemokines in this dHACM. Now, I can’t speak to what are the other amniotic products because there hasn’t been the same type of research done. But when you actually get the whole shebang, the top layer and the bottom layer, the amnion and the chorion, so I’m asking this kind of question as a researcher in thinking what does Mother Nature have, there are at least 226. There probably will be more if we were to go back and do an even deeper dive. So, what are these factors doing in utero with the developing fetus? That’s an incredibly interesting and important question because if we crack the code on that, it might allow us to actually have a better idea of how we could actually use it for tissue repair. Not just for more chronic wound healing, but what about cardiac repair? What about nerve repair? What about bone repair? So many interesting things we might be able to do with the source material, but if we don’t do the research, we won’t actually know. Now, the other thing is that taking the same source material, the dHACM, we found that you can actually induce wound angiogenesis. And this is a paper that we published last year, showing that you can actually induce cells to migrate and penetrate in a wound into the dHACM implant and that we can quantify using different types of molecular probes, the blood vessels seen here in green, the lighting up fluorescent blood vessels growing in. It’s quantifiable, we have a rate, we can actually show how it achieves tissue normalization. This kind of research would go really nicely with that fluorescence microangiography that Dr. Anderson showed so we can then show in a dynamic imaging, a parallel experiment to then show it’s actually happening as the vessels are growing. Again, research tools at our disposal that we can apply so we know exactly what’s going on. By the way, this is what would be happening in oncology or cardiology or neurosurgery. Most other fields of medicine, in order to be able to take a look at innovation, this is the type of research that actually happens. Now the other thing that was done that I think is really interesting, and I’ll leave it to you to go look up the research papers is that this dHACM, again, the whole shebang, amnion and chorion dehydrated, actually mediates stem cell behavior.


    So we we’re able to prove, that in fact, this is an actual bona fide stem cell magnet. So if you take a mouse that is normal, a white mouse and you sew it together with a fluorescent mouse, the green fluorescent mouse genetically programs that the stem cells are all green. You sew their skin together and the capillaries that healed the skin will connect the circulation, and then you make a wound on the white mouse and you actually put some dHACM on the white mouse and then look for green. You can actually see the stem cells being mobilized in crossing the threshold from the green mouse to the white mouse, and those green fluorescent stem cells integrate into the dHACM. That’s the first bona fide demonstration of actual true regeneration. It crossed the threshold and that should immobilize from even another animal and it got in there. And that was very exciting, we published in the general surgically research, and then subsequently, we looked at other types of stem cells as well. Some Mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, as you know there’s a big long list of different types of stem cells. And because what is coming out of the amniotic membrane to the dHACM actually is not the cell, it’s really the soluble materials that are used to bathe developing fetus, the interesting thing is to study and isolate in fashion what they do to stem cells. You know, they got to do something because they’re actually helping to contribute to the development of fetus. And now we’re beginning to profile that and showing at least for this particular product that was studied, this technology that you can mobilize bone marrow-derived stem cells, you can actually stimulate the migration, proliferation, and gene expression, cytokine gene expression and mesenchymal stem cells, adipose stem cells, adipose stem cells, hematopoietic stem cells, you can study the markers, you can quantify the proliferation, and I think that sets into motion what we’re able to do going into the future to think about this whole space. Now, one of the interesting things that was also done and published recently is thinking about, can we reactivate, restore, reprogram, reboot defective cells in diabetes? So, here in a meeting like this, we’re talking about the diabetic foot and all the problems, mechanical problems with the diabetic foot. And there are systemic problems, it turns out the stem cells are also defective. Diabetics have stem cells whose performance is not as good as individuals who don’t have diabetes. We’re just beginning to really understand the differences in that, there are a fewer number. They are activated less, they mobilize less, the whom more poorly. And so the research on dHACM was second to the next level, which is to isolates stem cells from adipose-derived stem cells from patients with Type 1 and Type 2 diabetes, and expose them to dHACM, and to say what actually happens. You can actually see, from this study there was a reactivation or rebooting an engagement of the defective stem cells that’s induced by dHACM. So that’s really interesting as well. So maybe there’s a way of trying to do more in a defective baseline population to get the stem cells to perform better. That’s really where this type of research leads us to ask the next questions and the question is how do we actually show that in the clinic. Now, before I showed you some of the clinical results, the thing about amniotic membrane is I get confused when I listen to people to talking about their favorite amniotic membrane product. Because all kind of sounds the same, and then people site studies that are in published without sometimes referring to the exact source materials. And since my name is in some of those publications I happen to know what the source material was, and so I always ask the question, wait a minute, where do they come from, what parts were involved, how did you study it, how was the process, what type of assays were done? So that’s one, putting on my scientist hat to really ask that questions, so that I can be sure I know what is being presented to me. So, I intended to actually insert a picture here of sport fishing because Frankburg mentioned fishing. You know, I don’t like to take things, hook, line, and sinker. I like to really understand what the data is, and so one of the things that I started to ask myself is, what are the differences between amniotic membrane products as different in the biology of mother nature’s amniotic membrane is what’s out there? And I don’t have the full answer to this, but I will tell you that, you know, we were very interested in setting amnion and chorion, the whole shebang, because we wanted to understand what break came right out of the month. And so to have a more complete understanding, and what’s interesting is that out of a 100% of all those growth factors at 220 some growth factors that I’ve showed you earlier, 20% of it is actually found in the amnion and 80% is found in the chorion.


    And so again, the layers matter because the distribution of the growth factors also matter. Will that make sense? You know, if you’re a miner and you’re going in to look for gold and you’re taking a look for the different surfaces in the mine, you going to take a look at what’s actually in these different layers. And so, we don’t know the biological function of what this does in he mom yet, but it does lead to the question of “okay, so we’re actually selecting one or the other, removing one or the other, or using them all together, how might the package of growth factors play and make a difference as well?” So again, the data I showed you is really on with dHACM is really on the amnion and chorion, which is where the research was done. So the next question was asked, is that within the distribution of chorion and amnion, if you would take a nine different growth factors, can we quantify how much of each growth factor is in the amnion part, which would be the red versus the chorion part, which is the blue, and you kind of see like for example for basic fibroblast growth factor, 23% of the FGF in the entire product was found actually in the amnion, and 77% was found in the chorion. So that’s an interesting way to actually look within the same tissue, the same platform with the distribution of this material actually is. And then other question will be, how does it get released from the material, how quickly does it get released, and is there a point in which you spent the materials, all the bullets are out of the gun, because this is Arizona, I’m saying that. And we’ve actually delivered all the growth factors that we can. Now, this lead to this issue about comparing sort of amnion and chorion versus amnion alone, and so again, fixing it against a 100% of having the whole shebang there, if you then take a look at the single layer amnion and do head to head comparisons of some of the growth factor, you can see in the gray bar on the right that there’s markedly diminished numbers of growth factors. Does that mean that that’s less clinically effective, that’s not what I’m addressing here, what I’m addressing is that there’s just less, and so it’s different and that difference is what we actually need to be able to receive and understand, and critically kind of think through as we appreciate what we’re actually able to apply in the clinic. Now, I did show you sort of taking a little bit of a journey, I talked over regeneration, I talked about a reservoir to discover regenerative factors, I’ve shown you sort of a scientific methodology that you could take to take a look the amniotic membrane and break it down into knowing what’s in there. How does it actually distribute differently to different layers and what the differences might be if you were to compare set of different products that are out there. At the end of the day, in a meeting like this and a community like ours, does this work in a clinic? All the research in the world is great for academics meetings but we want to know if there’s any differences that this type of research leads us to have in a clinic, is there any correlation. Which is the science translated into measurable clinical benefit. And again, it’s a little bit overwhelming to even think about what 226 growth factors do, in fact it will be impossible to ever breakdown each one of this to look at their individual contribution. And the wound itself is so microenvironment that is able to receive these signals and utilize each of the signals in their own way. And then patient with diabetes or cardiovascular disease or both or venous disease or aging, or smoking, or drinking is going to then have all sorts of microenvironment. So, this is a really complicated stuff to really think through at the clinical level. But we can run clinical studies. And the types of clinical studies that are actually being done now, this modern day where the clinical evidence actually goes directly to understanding, are we getting more value out of the treatments in comparative effectiveness research. This is the study that I was involved with Chuck Cell [phonetic] and I think he showed it earlier that 60 patients, a study in which there were three arms 20 patients in each group with diabetic or extremely ulcers and were treated comparatively to receive either standard of care or standard of care plus allografts or standard care plus dHACM which is of same material, or EpiFix that I showed you earlier. And this Kaplan-Meier curve really is sort of outcomes-based analysis shows you on the vertical access to present the patients with complete wound closure. And you can see already the differences in the dHACM that has 220 some factor released versus the standard of care versus the other bioengineer products that’s been around for a long time. And that is also clinical useful. So, what we believe is that, just as in the other areas of medicine and surgery, compared to effectiveness research when combined with basic or translational research gives us a handle on what types of technologies we can apply in what circumstances.


    Now, the other interesting thing that needs to be done with studies like this is to ask what patient subpopulations respond at all to these inventions, who doesn’t respond and are there biomarkers that allow us to predict or therognostic markers to predict whose respond better. We want to know when to treat and when not treat and, I’m very intrigued by the promise of fluorescents micro and geography, because it maybe possible to do a type of imaging that was descried earlier and get a sense of what the vascular patterns are that might predict healing. So that’s another area of convergence between the last lectures you heard and mine. I’ll just close by, you know, because I’ve shown you a lot of information a bit about the future, because we’re going to be here in another five years at Desert Foot hopefully, talking about how far we’ve come with regenerated medicine. And what you’re hearing today will be really the old school stuff, and what’s interesting is to think about where we want to go into the future. So I’m going to just present to you a bit of a crystal balling of where we’re going to be in five years. In five years we’re going to have biological factors in stem cells, we’ll have biomatrixes that come from humans and non-humans. There is a technology that’s even based on fish material, and I’ve done some evolutionary comparison between the extracellular matrix that was conserved through evolution, so it doesn’t even have to be humans, the pharmaceutical companies moving into our field. We’ll continue to be mechanical, chemical ways of actually manipulating the tissue to be able to regenerate class regeneration and other modalities that we haven’t even begun to think about yet. Maybe gene therapy, nanotherapy, maybe even radiation that can actually stimulate regeneration and wounds in soft tissue. And we’re not going to be able to actually come up with all the answers ourselves, in fact what we need to be doing is following the research publications in our own field, which is hard enough to do. But I think we should be recognizing that there are many other researchers that are focusing on other organ systems that are doing exactly the same thing. And if we talk to our colleagues in cardiology, or neurology, or pulmonology, or hepatology, or orthopedics, and pediatry looking at specific ways that they’re looking at regenerative medicine, it will be really interesting to see what we can learn from within our field and outside our field and we can share insights from what we’re doing that could help the cardiologist as well. So we’re really coming into new field, a new era when it comes to regeneration and androgeneses where there’s going to be a lot of crosstalk in order to be able to improve everybody, sort of “What’s good for the goose, is good for the gander.” And what we want to do is to give a benefit from other fields are learning about regeneration, and we have something to share as well. At the end of the day, as I mentioned, every one of these tissues that regenerates requires androgeneses. So, we don’t know what’s going to happen for the future that we’re dealing. Well we probably won’t have liquid metal, but what it interesting by the way is that they’re actually developing polymers that are self-healing just like this, which is why I pulled the picture. And so will there be some ways that we can actually envision the future that’s far beyond ourselves. If you go to conferences like TEDMED, or Ted, or you take a look at thinking digital, you can see some of thought leaders that are dreaming the future and I encourage you to actually do that and think about the first principles. Number one, we have to first think about our patient, caring for chronic wounds is about caring for the patient not just closing the wound. Number two, is that science really leads the way, and scientific evidence is really how innovation occurs. Clinical evidence is what we need to actually have, so well-designed trials will actually allow us to know, is does it work, and in what circumstances. We are so far beyond anecdotes and case studies, while those are important that’s not where the present or the future is, we need good well-designed randomized control trials moving forward. And the investment now is actually going in those directions. And then we need to allow our imaginations to guide us because that’s really where we going to go with wound healing. Again androgeneses regeneration, think about better healing, better quality repair, ask yourself when you’re actually using a regenerative product, am I getting better quality? How could I actually assess that, we need that information from the trenches in the clinics, people who are practicing. So I’ll just close with this quote by the Nobel Laureate Richard Feynman, a physicist, who basically said, “The best way to predict the future is to invent it.”


    I think there’s a lot of people working on this research area right now, I’m just one of them. But we should all band together, because together we’ll be able to actually find new ways to invent our future. Thank you.