Barry Rosenblum, DPM, FACFAS reviews the specific cell components of normal wound healing and delayed healing of chronic ulcers, emphasizing the biochemical differences and cellular abnormalities. Dr Rosenblum relates the role of cell therapy for chronic wounds and describes the benefits and indications of Apligraf and Dermagraft as an advanced therapy for non-healing ulcers.
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Barry Rosenblum, DPM
Assistant Clinical Professor of Surgery
Harvard Medical School
Associate Chief of Podiatry
Beth Israel Deaconess Medical Center
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Robert Frykberg: Dr. Rosenblum to come back to talk about another very important advanced technology and that is on Living Skin Equivalents. And I really want him to focus on the science behind the cell. Once you understand the science and the biological plausibility, it makes sense as to why these products are helpful for us. So let’s welcome back, Dr. Rosenblum. Hopefully, everything is going to work better. Okay. Thank you.
Dr. Rosenblum: Thank you. Thanks Bob. While they’re setting up the presentation, couple of things I think that everybody should be aware of and you’ve heard it all throughout this morning’s talks. Most important things to keep in mind are the basics. You’re going to hear and see a lot of advanced skin products. You’ve heard amniotic membrane. You’ve heard about mesenchymal stem cells. The key thing is get your own protocols. Use what works in your hands. Then if you need to, go to an advanced product. One of the other things too, and I’ll take just about 30 seconds on my soapbox here, is you’ll hear a lot of products talk about wound healing and you’ll hear a lot of products and representatives talk about limb salvage. I think one of the things that Marie talked about that’s very important is that in my opinion, limb salvage isn’t truly defined until you have a patient who scheduled for a baloney amputation in one room and you heal them and prevent that amputation. Healing a wound does not necessarily equal limb salvage. Because you have a lot of wounds that are small, superficial. You might be able to get them healed. You might not be able to get them healed. They might go on to another modality. They might go on to an operation. But true limb salvage is the ability to avoid an amputation. I think that’s an important take home message, at least in my opinion in what I practice. Now, I have nothing to disclose. As Bob said, I’m going to talk a little bit about the science behind the cells, specifically with living skin equivalents. You’ve heard this morning, primarily from Larry and Marie, a little bit about the science behind the cells and how the skin healing, wound healing takes place in the normal patient. What I’m going to talk about a little bit is what happens in the diabetic patient, and then wrap up with what happens with the living skin equivalents when it comes to the science behind the cells. I think what we’ve heard and what we’ve seen is that some of these products, many of these products are very expensive. But George Bernard Shaw in the Doctor’s Dilemma, can you start the timer, please? George Bernard Shaw had this quote, “I marvel that society would pay a surgeon a large sum of money to remove a patient’s leg but nothing to save it.” I think what’s important here is that this is when the expense I believe in the short run is important to prevent that expense in the long run as we see with an amputation. This is a patient who presented to the office, had had numerous trips to the hyperbaric chamber, had had numerous advanced skin products, was never adequately offloaded. I think it’s important that we understand where advanced therapies need to be utilized when it comes to trying to heal our diabetic patients. When it comes to the components of wound healing and you’ve seen this and heard this, this morning from some of the other speakers, I think it’s important to understand the phases, the components of wound healing that takes place in the normal wound healing cascade. We know that there’s the coagulation process that starts right at the time of injury that last a few hours primarily taken up by the platelets. The inflammatory process and we’ve heard this over and over again about how these patients whether it’s due to weightbearing, whether it’s due to faulty cellular mechanisms maintained, they're “stuck” in that inflammatory phase. I think that’s important to understand. Then what we need to do when we look at all of these advanced products is understand how they get us to the next phase of the inflammatory process. When we look at normal wound healing, we know that there’s an acute tissue injury that takes place. Sometimes it’s difficult to try and project what happens in the lab to what happens in the office, in the clinical setting. I think Dr. Lavery discussed that. When it comes to patients, still in the inflammatory process due to weightbearing, due to ineffective offloading. We know that platelets degranulate. There's a release of growth factors. We see inflammation with an influx of neutrophils and macrophages. There’s a release of proteases from those neutrophils. We’re going to talk a little bit more about proteases and how those work effectively and then more importantly how they work ineffectively. There’s activation of macrophages with release of both cytokines and growth factors. There’s an influx of fibroblast. Some of the things that we’ll see when it comes to fibroblasts and endothelial cells is how there’s a little bit of a dysfunction in the patients with diabetes. There’s collagen synthesis that takes place as well as angiogenesis. Then ultimately the last final phase in the wound healing cascade, scar formation epithelization and remodeling. However, when we have patients with chronic wounds, when patients have delayed healing, we note that there’s a prolonged inflammation. We see that there’s normal stimulation of macrophage and neutrophils to the wound bed, but there’s prolonged inflammation in those patients.
What ends up happening is ultimately we see an increase in the production of matrix metalloproteinases and there’s lack of or diminished inhibition of those matrix metalloproteinases. What we find is that here’s a degradation of the extracellular matrix. When that happens, we see degradation of growth factors, we see impaired connective tissue deposition and ultimately as Dr. Lavery referred to, we sometimes see heavy bacterial burden and we do see even though we can’t really see it, we see it. We presumed that it’s there, increased biofilm and bioburden. What are the biochemical differences between healing wounds and chronic ulcers? What we see in the healing wounds is there’s a decrease in the proinflammatory cytokines whereas in chronic ulcers, there’s an increase in those proinflammatory cytokines. I think what’s important to understand as well is that in a healing wound, there are cells that are capable of a rapid response or a normal response. Whereas in the chronic ulcers, there are those cells that are more senescent. What are the roles of keratinocytes in wound healing? I think one of the things, and this might be a little bit what differentiates a product say Apligraf, which is bilayered versus a Dermagraf, which is more of a dermal matrix. We do see a role of keratinocytes in wound healing. We see that they play a role in the migration and proliferation of cells. We see that they play a role in the production of the extracellular matrix. We also see them play a role in the deposition and production of growth factors in cytokines, angiogenesis as well as matrix synthesis regulation. In addition, we see a role of fibroblast. This is what we see constantly and chronically throughout the discussion of advanced care products, advanced wound healing products. Specifically those that focus on the fibroblast and the role that they play in the deposition, the migration, the calling out to growth factors when it comes to the wound healing cascade. What happens in a diabetic wound? Well, we see that there are growth factor and cytokine deficiencies. We see this both in diabetic mouse models, as well as diabetic human wounds. We know through studies that have been done at a number of different centers around the world that there is endothelial dysfunction. We know that neuropathy and arterial occlusive disease plays a role in the abnormalities we see in the diabetic wound. We see decreased angiogenesis. There are abnormalities in fibroblast function. Then lastly, one of the things that we see that we've only recently over the past number of years attributed to the matrix metalloproteinases, we see an abnormality in the extracellular matrix with the decrease in the cellular infiltrate. So why do we look for cell therapy for chronic wounds? Well, I think what we’ve heard from previous speakers, cell therapy, there’s a smartness. Cells are able to adapt to their micro environment. Cells may bypass our lack of knowledge about what’s required in specific situations. They’re commercially available whether you see living skin equivalents or any sort of other product and they could be genetically manipulated. What is Apligraf? Well, Apligraf is a living bilayered cell therapy. It’s got a cornified layer, the epidermal layer as well as a dermal layer. The epidermal layer as I stated maintains or possesses human keratinocytes and the dermal has fibroblasts. In addition, it’s got an extracellular collagen matrix. Basically they have as bovine and human collagen with additional extracellular matrix proteins. The science behind it, as I stated earlier, cell therapy provides growth factors, cytokines. There’s a barrier function as well both the physical, as well as biologic. Lastly, the dermal matrix, again which is similar to what we see with Dermagraf, has a matrix for cell migration as well the a substrate to quench the proteolytic enzymes. Those enzymes that are normally there, those proteases can be involved more with this dermal matrix and allow the other extracellular matrix to be deposited. What happens with that functional dermal matrix? Well, it provides a matrix for migration of cells into the wounds. The fibroblast may produce, and I’ve stated this earlier, new human collagen and other matrix components and lastly, there is evidence that the bovine collagen matrix can quench the proteolytic enzymes that we see. The matrix metalloproteinases that otherwise may run amok in a chronic wound. What are the potential mechanisms of action? It delivers young active fibroblasts and keratinocytes. It’s been shown to have persistence of cells in the wounds to six weeks. There’s no matrix material. There's is also recruitment of other cell types including stem cells to the nonhealing wound. Now, what’s Dermagraf? Well, Dermagraf is a fibroblast-derived dermal substitute, again, used in the treatment of full thickness diabetic ulcers. Its composition as opposed to the bilayer of Apligraf is a fibroblast extracellular matrix on a bioabsorbable scaffold. Its mechanism of action, again, very similar is reepithelialization that assist in the restoration of dermal bed allowing wounds to heal. Now, here’s a take home message. It doesn’t matter to me, I’m not here to talk about Apligraf or Dermagraf specifically. What you need to understand is that those wounds that aren’t healing, you need to, as I mentioned in my previous talk, reassess and determine when to use an advanced product.
So, there are plenty of practitioners out there who don’t “believe” in the advanced product. There are a subset of patients that need it. It’s knowing that it’s available and when to use it and which patient to use it on. The possible role of Dermagraf in wound repair, again, going into that wound healing cascade, Dermagraf provides growth factors, matrix proteins and glycosaminoglycans early on in the initial wounding just after wounding, after platelet aggregation in order to reduce inflammation and then ultimately it forms substrate for epithelial migration. Again, very similar to Apligraf, not the bilateral. There’s no keratinocytes present but the important take home message are the fibroblasts and the extracellular collagen matrix. What’s the efficacy of Apligraf in wound closure. We saw this data from Dr. Lavery. You can see at 12 weeks with wound closure overtime, 56% compared to 39% in the control group. It’s also shown to promote rapid closure of diabetic foot ulcers, the median time to 100% wound closure, 65 days in the Apligraf group, whereas 90 days in the conventional group. What’s important there is that in those 25 days, this patient may be back to work, may be back into their activity of daily living, that’s 25 extra days of wound healing, 25 extra days of being in bed, off their foot and not working that you see in the conventional therapy group. Here are a couple of examples. Again as Marie mentioned, the pictures tend to speak a thousand words. Charcot with an ulcer in the plantar aspect had Apligraf applied and here you can see a complete epithelialization. Here’s another case of patient that presented with a wound on the dorsal aspect of the foot, significant fibrin deposition, was taken to the operating room for a debridement. We instituted negative pressure wound therapy as Dr. Bevelock would discuss. Here you could see pretty much complete resolution of this dorsal ulcer. Patient with an ulcer had a bypass. As you can see from this incision here, in the medial side of the heel, had Apligraf applied shortly after the bypass and debridement. There you could see about six weeks later complete epithelialization. Another example of a patient again with a wound that started on the heel and extended all the way over to the medial side of the foot. Here you could see the debridement. You could see all of the stuff. This is was a patient who clearly had a below the knee amputation recommended and then after complete healing with negative pressure wound therapy and application of living skin equivalent. When it comes to Dermagraf and I want to talk just briefly about the data with Dermagraf. Again, you could see at Week 12, significant improvement when comparing Dermagraf to control group with ulcers that were present for greater than six weeks. So, it’s important to understand that in those patients who aren’t healing and as Dr. Lavery pointed out with the Margolis data, it’s significant when you look at those controlled groups whether it’s 24% at 12 weeks and about 30 something percent at 20 weeks, there’s a significant number of patients that despite your best efforts are not going to heal. These are the patients to consider a next step such as a living skin equivalent. When it comes to angiogenesis in Dermagraf, looking just of Newton looked at seven full thickness ulcers in five patient, what they found with weekly applications of Dermagraf, they looked at the blood flow and assessed that using laser Doppler imaging and they found that blood flow was increased by an average of 72% at the base of five out of those seven ulcerations. Here you could just see schematically, immediately before treatment with Dermagraf after two weeks and after five weeks, you can see using laser Doppler significant angiogenesis. In summary, the use of advanced technology in wound healing, understanding what takes place at the level of the cells, understanding the science behind it, understanding that it’s available, use good wound care and again, this wraps up both of my talks, as well as the other talks from this morning. Have a period of observation where you understand changing gears or using something a little bit more advanced. If the patient is showing healing, continue offloading, continue your regular followup. But if the patient is not showing healing, you can use whatever parameter you want, three weeks, four weeks, consider adjuvant care, reassess for infection, check their circulation. If everything else is going well, then consider advanced therapies. I thank you for your attention.