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Male Speaker: To introduce our first speaker who is a friend and colleague, Matt Rogalski [phonetics], is a local boy from Toms River, New Jersey. He happens to have a lot of experience in wound care, works in a wound care center down in Ocean County, New Jersey, and has done a lot of early work with one of the more popular mesenchymal stem cell amnion products that we currently use now. And Matt has a good deal of experience. He has published several papers in this regard. So with that said, let’s welcome Matt Rogalski. You know how it works, right?

Matt Rogalski: Yes, sir.

Thank you Dr. Friedberg and Dr. Anneson [phonetic] and Dr. Sentrilu [phonetic] for inviting me. I'm very privileged to be here. It's wonderful having all these great conferences on regenerative medicine as the future is going that way, especially for a lot of chronic wound patients and chronic musculoskeletal conditions that we treat to. I'm very humble to be here and thank you very much.

So we'll talk about some of the good signs behind why a mesenchymal stem cells work in the chronic wound environment and their functions. So as I start giving some of these talks, I put this slide in there because in my facilities, we have five offices and four wound centers and our own vascular center, so we do quite a bit of clinical trial work, which I'm very fortunate to be involved in. It is very exciting to be on the cusp of technology and we just got done doing a large mesenchymal stem cell trail where we were injecting the calves of diabetic patients with platelet derived adherent cells to enhance angiogenic flow in the critical limb ischemic patient, which was very exciting to do but when you're sitting down with these patients that you're recruiting them and you give them consent forms and you go over and you still want to go back and talk to their family doctor about it. And you would be quite surprised as how some of the family docs that are out there tell these patients, "well, you got to be careful about this. This is still not proven science. It's still can be kind of voodooish." So it's amazing that that still comes across and so we do lose some recruitment patients because of that. But I put this slide back in because back then when we talked about new technologies, which that stethoscope was, how people were recalcitrant to that. I think sometimes physicians do think just because they are trained to do them. They don't examine why they do that. Is there something better? Is there something that works quicker? Is there something safer that we can do that can help our patients to get better. So I always like to put that slide in and this slide is kind of my salient feature of why I get into this realm of diabetic limb salvage in chronic wound patients, especially in the diabetic patient, how the mortality rates are really climbing on them and I come from a long line of diabetic people. I had to do lot of surgery on my own father because of people not following the evidence-based medicine. So I get into this field with the maybe naïve attitude of trying to help everybody that comes along and trying to heal everybody. I know that sometimes can be a little naïve as I said, but when we start to look at the morbidity and mortality that comes long in diabetic and neuropathic ulcers and then diabetes and perivascular disease go hand in hand. Diabetes engenders this, this nightmare of peripheral vascular disease and so when we look at the mortality rates for diabetic neuropathic ulcerations with 5200 people a day diagnosed with diabetes. If 230 amputations a day, 133 people go on dialysis every day and 55 people go blind everyday just in the United States, we need to find better therapies to really help these patients. So when I started doing a lot of work with stem cell, I started to look upon the trials that were going on and how many studies we are doing from them and back just in 2015 when we looked at, there was 500 clinical trials of MSCs that were going on. And now when you start to see it's even more, 720. All the different studies because now we know the power behind what mesenchymal stem cells can do and especially with so many clinical trials going on in every age-related chronic disease from head to toe. Alzheimer’s, ALS, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, cirrhosis, inflammatory bowel disease, ulcerative colitis, Crohn’s, and then of course atherosclerosis and non-healing wounds. It really is amazing the power that’s behind the cells and if all specialties are utilizing, that’s why in the wound healing realm, we are not invoking and we are not grasping this concept. In the wound healing marker, we have 67 different skin substitutes out there with about only four of them that really have randomized control trial data behind. Vast majority of them are acellular products, so it's interesting for me to understand what the science in the clinical trial, why everybody is not utilizing these most powerful products for bringing these wound to closure because even all chronic wounds have a 30% that will cause mortality, no matter what the etiology is.

[05:07]

That’s even more than breast. That’s even more than prostate itself. So the role of stem cells in tissue repair is that we have this stem cell that -- all cells in our body have to stem from something. So stem cells gives rise to its progenitor cell and then from the progenitor cell to terminally differentiated cell. And all tissues in our body has to turn over. When cells are being depleted and we are not growing and we are not expending, we are aging and aging itself being a mark of cellular senescence and then it can lead to dysfunctional tissue and then of course failure of those tissues, but it's interesting to understand how MSCs can function within this entire realm. The amazing capabilities that they have by the microenvironment that we put them into. So being angiogenic, being anti-inflammatory probably being their most salient feature, antioxidant, antifibrotic, anti-scarring and then the ability to wake up the endogenous repair mechanisms within our tissue. It's quite amazing that they can home to these areas and then they can fix these aberrant immunological and inflammatory conditions and then repair that tissue. So in the wound healing paradigm, they can participate in the all phases of that. Like little physicians, they can go in and they can diagnose what is going on in that microenvironment. With the secretome of over 600 different proteins that they are able to secrete depending upon how they are stimulated. So just with the aging process, the free radical theory of aging and the generation of all these different reactive oxygen species due to our inadequate transfer across the electron transport chain and we generate all these reactive oxygen species with their mitochondria just cause aging. And just as we see as we get older 70-80 years old, when you look into bone marrow, we find one MSC per 400,000 cells within that bone marrow context and in the placenta, obviously it is one out of four nucleated cells is going to be mesenchymal stem cells, but we can start to see that these are going to drop off as we get older just from the process of oxidative stress and from aging. So when we look at therapy, I used to use a lot of PRP, still has some good adequate characteristics to it, but if we start taking these from the aged individuals, the capability of those cells become very declined as they grow older because their ability to home their chemotactic and mitogenic capabilities are dramatically reduced. I think we could spend hours and hours just talking about this phenomena that happens and with cellular senescence and the aging and how that occurs because as we get older and even in just in the diabetic state, which causes premature senescence of these cells, these cells become stably growth arrested, but they are metabolically active in a very pro-inflammatory way and how they are triggered to do that is can be by several different things whether you have some type of DNA damage and of course telomere erosion that we see and replicate the senescence, the oxidative stress that occurs, strong mitogenic signaling from over expression of oncogenes. We have disruption of epigenetic regulation and ectopic expression of tumor suppressors of course, but all leads to this problem where these cells are not growing. They are not moving or advancing, but they are highly metabolic in this pro-inflammatory way and they secrete all these different substances called the senescence associated phenotype. So all of these high levels of pro-inflammatory cytokine and chemokine and growth factors and proteases not only slow their healing but it also can lead, especially in a diabetic population, to higher rates of cancer that can develop. So this is a fascinating concept and we could spend very long time talking about it, but how diabetes can affect those causing that premature aging of even our stem cells. Cellular senescence can affect every cell line from fibroblast and epithelial cells and endothelial cells, keratinocytes, glial cells, renal mesangial cells, and even our own tissue resident stem cells because diabetes in part is due to that complex of these aging-related mechanisms and underlying age-related tissue dysfunction. When we talk about chronic, sterile, low-grade non-pathogen associated inflammation, macromolecular damage, progenitor cell dysfunction and cellular senescence of diabetes causing that premature conversion until these cells or irreversibly growth arrested but quite metabolically active in a pro-inflammatory way. So we need these cells to repair in diabetes. Now, it's because of all the advanced glycation end products that are producing this oxidase stress, DNA damage, mitochondrial damage, and then hard for these cells to replicate, hard for these cells to secrete those factors. So we need to find some reparative mechanisms that we can put into these tissues to help because we know the long-term effects on diabetes and the longer that these wounds stay open, the greater risk for morbidity and mortality for these patients. When you look at the cellular complex that goes on inside with particularly fibroblast are unresponsive, chronic wound fibroblast, unresponsive to growth factors signaling.

[10:01]

It's quite a complex that prolonged inflammation and then you have imbalance of regulatory growth factors and cytokines. We have a defective ECM that is degraded. It's corrosive. It fails to support keratinocyte migration in the modified fibroblast function that we have and then defective capillary formation and all engendering into the chronic wound. So these fibroblasts are quite unresponsive because of the high proteolytic environment that we put them into and target receptors are dysfunctional then though we have some that are inopportune, that are upregulated at that time that fail to support that, that fail to support proper cellular spreading and cellular behavior and the cellular fate. So one of the reasons in chronic wounds that we debride is we want to be able to remove that bio-film that is in there, that nidus for inflammation. All of these have senescence cells. When we look at the complex that goes on within this chronic wound environment, it is quite fascinating to see how this chronic inflammatory cascade affects all cell types that are involved in there and we talk about these wounds being stuck in this inflammatory phase of wound healing. Now, we have the capability to put cells into that, their salient feature being of anti-inflammatory capability and atrophic capabilities to regulate that chronic environment because we know that stem cells have the capability to regulate both the innate and the adaptive immune system through various mechanisms and downregulate that over the aggressive immune migration as dictated by the chronic inflammation that occurs a lot from the bio-film. So when we have all these inflammation going onto the wound, we have disruption of the extracellular matrix and then we can't have proper cellular attachment, cellular migration for that to help with proper epithelization, but even more importantly, that all these cell lines that are involved in that become senesced because of this pro-oxidant and pro-inflammatory environment. So we have these capabilities now to put things into it that can help to wake up these endogenous repair mechanisms and help to regenerate that tissue. So this slide here I think we could spend hours breaking this down and talking about which is quite fascinating. So MSCs are going to be able to support healing throughout all of these phases. As we put them into that, they are able to be drawn to those areas. That’s what a great thing about their bio-distribution is very targeted. It's very focal into that area and then they can be able to prevent tissue damage and then they can stimulate the repair of the damage that has been happening and then have to reverse the inflammatory in these aberrant immunological reactions that go on because of the power that they have in their secretory capability regulating all phases of that. And then of course chronic wounds that we see are characterized by tremendous scarring and tremendous fibrosis. So they have that capability to downregulate fibrosis and scarring, that’s why they have tremendous pleiotropic capability not only in chronic wounds but all kinds of chronic or immune diseases where we see quite aberrant and excessive ECM deposition. So it's nice to have all this wonderful science and it's tremendous to have all the science, but can we actually -- does it work in the clinical setting and so when we look at native placental tissue and cryopreserved placental tissues, looking at all the various factors that are found within, I always wondered as when we look at these heading of skin substitutes and you try to go down the list and you look at all these different skin substitutes that are on the market, some have these factors and some are missing these and some have those. I have always wondered why we call it a skin substitute if it doesn't contain all of those factors that are involved in that. Certainly when we look at placental tissues, living cryopreserved placental tissues containing all of these different factors and there is vastly much more. There is over 300 different ECM proteins and 82 different types of growth factors, but I think that we need to have an entire functionality because when we have chronic wounds that each one of these factors are going to be downregulated or dysfunctional. Placental tissues, living cryopreserved placental tissues maintain that integrity, maintain that function and that are able to foster that regenerative reparative capability because it's fully intact and integrated. When we look at the structures of amniotic tissue that is going on and it's amazing robust tissue, how we can protect the fetus from injury and repair that in scar-less fashion, how it can downregulate our immune system because obviously the fetus is recognized by the mother as being a parasite, will want to attack it. So these tissues are incredibly regenerative and it's amazing that we used to be able to discard this tissue instead of utilizing that capability, not too long ago. So when we look at these different layers of that and what they contain the amnion and chorion, the decidual and trophoblastic tissue and the functionality that they have, we get more into these talks in the afternoon about living cryopreserved tissue of both amnion and chorion and the functionality that they are able to do.

[15:10]

Just a cross-section of the different types of the tissue that we do find within the placenta is a really interesting stuff not only of the amnion and chorion but also living umbilical cord, Wharton's jelly stem cells that we are going to be doing some really significant work on and reparative mechanisms for that. It's just amazing and I, as a clinician, want to thank all of these companies that have these capabilities that do all these research because without them, I couldn't do the things that I do. So I'm very thankful that we have people that are committed and passionate and really enjoy what they do in trying to help people. So we look at all the different types of placental products and I'm not posturing one over the other, but I'm just trying to show you what some of these products have out there that they have and what they don't have. So we know tremendous capabilities of placental tissues and that we have a cryopreserved placental tissue that’s almost identical and intact, too fresh placental tissues. We think about all of these different factors that are in there and you can see some of these other products that everybody is quite familiar with. That’s why I'm always confused when we talk about skin substitutes. If some of these factors are lacking, particularly the cellular component and that functionality that cells put into that because I have always wondered I have heard people say, "well, do you need cells to heal, do you need cells to repair bone, do you need cells to regenerate adipose tissue." I have always wondered that question. I always thought that when we cut ourselves, it's a cellular process to heal. When we regenerate tissues, when organs are growing, when the placenta is developing, that’s a cellular base process. So I can't figure that out why people think that we don't need cells to heal. It's interesting I'm not as smart as the people are in this room that do all of these research, but I have always wondered why I always thought that it was cells -- when I do plastic surgery -- when I was trained to that, that they always -- my attendings always used to say we take skin grafts because we have cells, they never mentioned because you have decorin or elastin or fibronectin and they never mentioned they had platelet-derived growth factor, hepatocyte growth factor, epidermal growth factor. They always used to beat it into us that we have cells, we have cells, we have cells that regenerate that tissue. So I have always wondered that why people talk about acellular products as skin substitutes. So when we look at the viabilities now how these cells that are going to be living within these tissues and we have to be able to maintain and some people think cryopreservation can affect the cellular outcomes and once we are looking at post thaw and we try to show that these incredible processes that people that are far smarter than I could ever dream to be being able to develop this and maintain that functionality of these cells because it's critical that we have their integrity that maintain their capabilities to proliferate and to secrete and all the different processes that go out there to process tissue whether you're talking gamma radiation and dehydration, which can lead to. It's a very cruel process that can lead to a decrease in protein function and then obviously with a loss of proteins and denatures those proteins, it has a great capability upon the structural and functional integrity of those extracellular matrix protein. So in cryopreserved tissues that we can see that the recovery of these post thaw is substantial to what we see in fresh tissue. The environment that we put these cells into is very nasty. It's very corrosive. It's destructive. The different types of proteases that are found within the wound bed, these different types of bacterial species producing hundreds of different types of proteases are incredibly corrosive. Are these cells being able to withstand in there? Lot of people always question how long do these cells persist, are they are able to secrete, do they do those things and it's amazing to see the placental tissues now that we have in published reports to show that MSCs can survive within these tissue for up to eight days rapidly secreting all these different factors hundreds and thousands of times of greater than we could see with any type of acellular tissue. So they are able to maintain within that status, within that chronic wound pumping out all of these different factors that we need. Different chemotactic signals, different mitogenic signals, drawing other cells into that area and up regulating the endogenous reparative mechanisms. It's fascinating. It's amazing that we're going to be going that way here in regenerative medicine, I hope, someday because I come from a long line of diabetic people and I'm definitely afraid of developing diabetes. So hopefully, we will be able to have IV infusions that we can just take for that and then be able to reduce the inflammation and help to repair our beta cell mass because I see it every day these poor people that come in and just being rotted away and eaten to the bone and I'm so thankful that we have companies that are able to develop these products.

[20:02]

I'm so thankful for that. But when we look at how the tissue is functional and when these cells that they are able to diagnose, when we put them into the microenvironment, they are upregulated. They are stimulated by various pro-inflammatory molecules to upregulate their production of specific factors that they are like little physicians and then they can go in and diagnose what is going on within that micro environment and being able to upregulate the capabilities that we need to repair those mechanisms and it is no wonder that when you have a living cell that is in there and you put it up against more of dehydrated-type tissues that don't have, they have a finite amount of those capabilities, if they have them at all that we can see the tremendous upregulation of these specific types of growth factors that we need to establish the mitogenic signal and draw other factors and cells to that area. Again, we talked about salient feature being the anti-inflammatory capabilities that they have. When you look into these chronic wounds, the tremendous amount of TNF alpha and gamma interferon that are flying around in there that can upregulate stem cells to secrete a tremendous of anti-inflammatory type peptides. When you have high amounts of TNF alpha, they are going to pump out PGA2 that has a capability of up regulating interleukin-10 from M2 macrophages which is tremendously and potently anti-inflammatory. With gamma interferon that’s in the wound, it upregulates their capability to produce hepatocyte growth factor, indolamine and TGF-beta. So in those pleiotropic capabilities are tremendously anti-inflammatory capabilities. So they are anti-inflammatory and their trophic capabilities are just outstanding and in the hypoxic environment, they can upregulate their tremendous production of vascular endothelial growth factor to enhance endothelial cell migration and then obviously endothelial tube formation and we can start capillary formation bringing more blood because all chronic wounds are hypoxic. So when we look at the comparison between these capabilities, between living and dead tissue, I think it's amazing that we find how much upregulation in this tissue that we can see mesenchymal stem cells and even amnion epithelial cells, which are incredibly robust, incredibly immunomodulatory and immunoprivileged as well that have tremendous stem markers and differentiating capability. The upregulation of these factors that we need because all of these chronic wound no matter what the etiology of these are going to be is that they are incredibly inflamed. There is some tremendous amount of oxygen that are flying around, that there are tremendous amount of fibrosis that is going on with tremendous cellular senescence of all cell lines. So we have that capability now to resurrect into repair and to regenerate those endogenous mechanisms that are dysfunctional caused by the chronic inflammation. Fascinating to see how much it is. And then we talk about the bio-film being huge within the chronic wounds. Do stem cells have that capability? Biofilm is very difficult to get through. Our own immune system can't penetrate that sugar coating that they do put around themselves. It's one of the reasons why we are trying to find strategies to defeat the bio-film because the bio-film is one of the great niduses for the chronic inflammatory signal. And so I have often wondered, can stem cell secrete because they secrete a tremendous amount of an antibacterial peptide repertoire, can they have a capability to downregulate the bio-film and be able to have a capability to destroy some of these bacteria and you can see the log reductions on these bacteria which are probably the most and some of the most resistant types of bacteria that we find through DNA testing within the chronic wound environments. So stem cells do have that capability in these both reports to show their downregulation of and be able to attack some of the bio-films that help us to reduce that nidus because it's certainly very difficult to get through the bio-film and certainly NIH is talking about 80% to 90% of all infections that we have in the body are due to bio-film. So when we look at these constructs between acellular and cellular matrices and all the aberrancies that are going on their eye. I still find it interesting that some physicians are still not grasping that concept because in the chronic wound as we talked about them being tremendously hypoxic, because with the low O2 grade and we don't have collagens synthesis, you don't have angiogenesis, you don't have resistance to infection, you don't have epithelization without proper O2. And then with the chronic inflammation that is destroying the extracellular matrix, thereby inhibiting keratinocyte migration and then chronic inflammation also downregulating fibroblast function making them blunted in response to those signals. We look at acellular versus cellular matrices. I find it interesting that people still are not utilizing more of living cell products because of all the tremendous cellular information that we have out there and also all of the information understanding what is going on within that chronic wound environment.

[25:00]

So it's nice to have all that good science but can actually translate into clinical outcomes and clinical success. You have to spend money, millions of dollars to do this, but you have to be able to show that you do better than the standard of care and I know people always say, "well, the standard of care is sharp debridement, moist wound balancing and offloading in diabetic foot ulcers," but don't you think it's going to do much better and can't you put it up against another product and let’s actually have a good head-to-head comparison, but the FDA doesn't require that. FDA simply requires what you're doing against standard of care. So when we look at -- you got to perform multicenter national clinical trials overseen by third party and you have to be able to show that you do what you are claiming to do. We all know about the science and the stem cell were isolated 150 years ago. So we know their capability, but we actually have to prove that in the controlled environment. And so as we can see the tremendous outcomes of utilizing cryopreserved placental membranes with its fully functioning capability of the cellular repertoire, the ECMs and the tremendous amount of hundreds of different proteins, it did substantially better in the randomized control trials than we see with those products that are already out there, which are some good products. But we can see that this type of tissue was far and above that because of the tremendous pleiotropic capabilities that we have with cellular mechanism.

So I thank you for this opportunity. It was wonderful to be here and I look forward to giving more lectures during this stay, but I just wanted us to give us a good scientific background. We are preaching to [Indecipherable] [0:26:34] a lot of people understand the amazing cellular capabilities, but hopefully in the future that we'll be all more embracing and advancing these technologies. Thank you very much for your time. I appreciate it.

[Applause]

Anybody have any questions at all and any of that or?

Male Speaker 2: Could you elaborate on the graft host problem with using placental cells for therapeutic treatment?

Matt Rogalski: Can I elaborate on what?

Male Speaker 2: The graft host interface with placental stem cells.

Matt Rogalski: Well, graft versus host disease that type you're talking about?

Male Speaker 2: Yes.

Matt Rogalski: Well, I know that living cryopreserved placental products here and company that’s out, Osiris had one of the first products in the treatment of acute graft versus host disease which was able to downregulate, not in the chronic realm, but I know in the acute realm that they did have that. I being obviously a foot and ankle guy, I do not use that product. I don't treat graft versus host disease. I'm simply relating my science to the chronic wound, but there are scientists that are here that help to develop that if you would like to discuss that with them.


TAPE ENDS - [27:53]