Robert Frykberg, DPM, MPH discusses basic and advanced wound care for diabetic foot ulcers. Dr Frykberg focuses on the use of mesenchymal stem cells to heal chronic wounds and present a randomized clinical trial to support its use.
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Robert Frykberg, , DPM, MPH,
PRESENT Editor - Diabetic Limb Salvage
Carl T Hayden VA Medical Center
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Male Speaker: I've been talking about a number of advanced biological materials and mesenchymal stem cells have been mentioned by several speakers. And I just want to bring to your attention what I think is the promise of mesenchymal stem cells for chronic wound care. Much like Dr. Wukich had mentioned. These are my disclosures. We know from multiple studies over the years that many risk factors for non-healing wounds, whether they’d be DFUs, VLUs, pressure ulcers, or dehiscences. I'm going to combine my thoughts primarily to DFUs, but what we're talking about can relate to any one of these types of these chronic wounds. We know that non-healing wounds are often characterized by infections or even low levels of infection like bioburdens resulting in high-levels of proinflammatory cytokines, high levels of MNPs and tissue proteases. And cells which are characterized by having a low mitogenic activity and synesis, so all of these are important factors leading to that chronic non-healing wound. As well as other systemic factors and local factors like inadequate offloading diabetes, peripheral ischemia, et cetera. Hence, they need to properly evaluate our patients. As what’s said many times, the diabetic foot ulcers are really the pathognomonic lesion of the lower extremity in diabetes with about a 15% lifetime risk. That's an old factor and I don’t know if anybody really has updated that since [Polombo] [01:40] presented that nearly 80s. But we do recognize that diabetic foot ulcers are multi-factorial ideology namely neuropathy, ischemia, or more commonly neuro-ischemic ulcers which are becoming far more prevalent than they were 30 years ago. And has already been mentioned, that ulcerations are precursor to amputation and about 85% of diabetes-related lower limb amputations. Meaning that these limb amputations in diabetes have a non-healing diabetic foot ulcer in the pathway – across the pathway leading to that amputation. A number of consensus panels on wound care and diabetic foot ulcer treatment really dictate pretty much the same basic tenets of wound care which was mentioned earlier must be followed in all cases. That is management – first of all medical probabilities, diabetes, congestive heart failure, renal disease, et cetera. Management of arterial disease, addressing the wound environment, and diagnosing and managing infection and bioburden. Proper offloading is probably absolutely the most important thing. And then we also considered the use of advanced therapies as well as surgeries. Sometimes surgery is absolutely necessary to get these wounds healed when more conservative measures don't find success. So when we use advanced technologies, we're using it tradiciously and this has been referred by several previous lecturers. Good wound care relies upon offloading debridement, management of ischemia, management of infections, as we said, proper wound bed preparation. And we give it a period of observation of 4-weeks. As Dr. Wukich has mentioned that Peter Shen study brought forth that premise that if wounds are not heal by 50% in 4-weeks, it's a good indicator that's something is going to rise. So if we see 50% closure at 4-weeks, we continue on with our good standard basic wound care. However, when we do not see proper progression, now we need to step back and re-evaluate the wound. Are we missing underlying ischemia? Are we missing some indolent infection or osteomyelitis? Have we not been successful with our offloading? Have we not been successful with managing the patient's nutrition and other metabolic factors? And if all of those things are negative, then we have to start thinking about the applicability of advanced therapies. Now, I think this is the rational way to approach these where expensive therapies which can become quite confusing nowadays. 10 or 15 years ago wasn't very difficult. But now we have all these different therapies that we've seen before. These are just a listing of several of the ones. I don't use all of these, but I've used most of these different products. Recombinant growth factors, extracellular matrices, amniotic products, cell-based tissue products – we know negative pressure therapy. There’s other bio-physical modalities, as well, pulsed electromagnetic therapy, electrical stimulation therapy. There's many, many, many different things. So it becomes confusing and we have to know when do we use, what modality, what might be indicated for which wound at which time.
So this is where we have to rely more on evidence and the qualities and characterizations of the wounds. But it's all predicated, of course, upon good basic standard wound care and proper, and thorough assessment of our patients. So this is where we come in to what I believe is the future of wound care, if you will – mesenchymal stem cells. So when we talk about stem cells, it's a misunderstood or mischaracterized type of therapy. But we know that stem cells, MSCs, are widespread throughout multiple tissues in the body. We most often think of these as being derived from the bone marrow but we also find them in the peripheral blood and fat, and of course placenta amnion-corion tissues. And surprisingly, the placental membranes are probably the highest or richest source of MSCs within the body. And if we think about the placenta, the newborn, the quality of those MSCs much greater, much higher then autogenous source like a bone marrow of a 66-year-old diabetic patient with renal failure. So it becomes a very intriguing source for mesenchymal stem cells. They're pluripotential. Mesenchymal tissues are the final differentiation of MSCs, bone cartilage, and tendon, et cetera. But the interesting fact is that these cells secrete multiple growth factors and cytokines and I think this is really the key. By doing that, they can orchestrate wound repair and tissue regeneration which is our goal here. I've also find intriguing is that they are responsive to the local environment as we all see. So we characterized stem cells, we have to clarify embryonic versus adult. And we're not talking about embryonic stem-cells. Neonatal derive or placental derive stem cells are considered adult stem cells. The mesenchymal and they result in the final differentiation is in connective tissues as we can see here and as we've said. So how does this affect wound repair in cutaneous tissues? Mesenchymal stem cells are immune-privileged. They are – they're from neonatal. They're anti-inflammatory. They're tissue protective and they're tissue regenerative – very, very intriguing cells. There's been a lot of research on these. They are definitely immune-privileged cells which makes them very attractive for current uses. So I post the question, "How do MSCs, if they are mesodermal origin, how do they affect wound repair which is ectodermal?" If we have the direct terminal differentiation in the mesodermal tissues as you can see to the left on this slide here. So we'd be talking about direct differentiation into cartilage or bone. But look on the right, we have the paracrine function or the indirect function of stem cells which is really what we're talking about. And these stem-cells are growth factors cytokine factors. We saw that when they are implanted into a host, they upregulate the production of multitude of growth factors – endogenic growth factors, tissue growth factors, as well as being a osteoinductive. So that's the paracrine function which influences the host cells to upregulate the local levels of cytokines and growth factors. As well as their own synthesis of growth factors to cytokines that makes these very attractive cells when implanted into tissues. And again, they do not stimulate any type of immuno-response. I’ve mentioned also earlier that they are responsive to the local environment. These are just some tissue – these are just some in vitro studies where you see on the left where you have upregulation of TNF alpha in an inflammatory state. In the presence of MSC, you see an upregulation of anti-inflammatory PGE2. And as the levels of TNF alpha recede because of the upregulation of the PGE2, the level of PGE2 goes down. This is because the MSCs responsive to their local environment. On the right, you can see how MSCs can upregulate the production of the VEGF in a hypoxic environment where you have the control on the left, on the green panel there and then stimulated. You see the increase in upregulation of the VEGF in response to mesenchymal stem cells in vitro. I think this makes these very, very attractive. So we have chronic wounds such as this and we know Ralph Woodman in 2005, in a very nice paper in diabetes care on the milieu – if you would – of chronic wounds.
Again, characterized by cells with low mitogenic activity, pro-inflammatory cytokines, high-levels of proteases, as we know, and so as in cells. It's our job to convert these chronic wounds to one of a more of an acute nature. And MSCs can actually shift the cytokine balance towards this favorable environment along with standard wound care. So shift to a healing environment which is characterized by cells with high mitogenic activity, high levels of anti-inflammatory cytokines, low levels of proteases, and actively synthesizing and proliferating cells. So that's really what we're trying to do. And because of the reactivity of the MSCs and their ability to respond to the environment, they can help drive the whole process towards the healing of these chronic wounds. So let's talk about some clinical data here because we all have growing reports about a number of products. It's been very little data when you think about it over the years for all the multitude of products that we have. So let's just talk briefly about one of the recent papers. This is a pivotal trial published in the International Wound Journal last fall. I think it was September or October; Larry Lavery was the lead author. This was a multi-centered RCT20 sites in the United States. It was single blinded and the patients were blinded and it was an open label cross over trial, where unhealed patients were allowed to cross over to the active therapy which was a living amniotic matrix tissue containing active living mesenchymal stem cells. Most interestingly about this, it was an adaptive design which more and more trials are taking on now to save cost and to save time. What an adaptive design means is that a priority in the protocol, they specify an interim analysis. And if at that interim analysis point, they have reached their outcome goals, they can stop the trial. If they have not met the goal, they will continue on to the next interim point. Now the important thing is this is all a priority which means it has to be specified in a protocol before the study even start, so very interesting here. And the endpoints of this study were, of course, complete wound closure of the index 1-12 weeks which is really the comparer that we use now because it's very difficult to compare a 12-week healing, to a 20-week healing, to 12-month healing, or 9-month healing which was seen in the long hyperbaric oxygen trials. So secondary events were timed to initial wound closure, the number of treatments required, the wound closure of patients in the crossover phase, and the proportion of patients achieving their 50% reduction going back to the magical 4-week, 50% reduction. And I tried to narrow things down here for the sake of time. So we'll go over some of the data. So this was at 12-weeks, we saw that in the active group, 62% of the patients achieved complete healing compared to 21% of the controls. And obviously, this was significant with a relative improvement of 191% margin of the effect between the healing in the active group versus the controlled group. This was the highest relative effect ever published yet on any wound care product. One criticism you can say is "Why did the controlled group heals so poorly at only 21%?" Well this was randomized. They all had standard offloading. It was fully randomized prospectively. So I haven't really been able to look into that exactly and find why that control group was so low. Because Dr. Wukich just mentioned earlier that my goal listed the study years ago which look at all control groups and found that usually about 24% of patient, 30% of control group should be healed by about 12-weeks. But it's not that far away if it is, indeed, 24%, but it seems a little bit low similar to the dermagraft trial. But nonetheless, this was randomized prospective. It was 191% relative improvement. As I said earlier, the best results, thus far, in any clinical trial. And here we can see the separation in the Kaplan Meier curve, where you see the rapid healing on the top curve compared to the standard of care. The standard of care was moist wound therapy with aggressive offloading as we said. So there's a rapid improvement here in healing in a rapid difference and effect between the two groups. Now I find most intriguing in this study is also the crossover results.
Now remember, in the crossover design, the patients who do not achieve healing at 12-weeks – which is really the end of the treatment phase of the trial are given the option to crossover to the active living amniotic matrix tissue. So here we can see the end of the trial in the down arrow, they were the patients are switched to them in the mesenchymal stem cell matrix tissue. And you can see at about a hundred days the rapid rise in healing. So the curve is really quite pronounced there. It's really, I think, a remarkable change. These are people who have not responded to standard therapy for 12-weeks. We add the active therapy at end of the trial and these 26 patients, there very, very rapid healing took place. So to me, that says a lot about the quality of the trial and the quality of the product. Obviously, there was also differences in number of our treatments necessary. I think it was 4 treatment or 6 treatments versus 12 treatments. The number of infections were gonna be half the number of infections or post-operative complication. And you'll see that with any trial because your heal wounds faster, you'll gonna decrease the complications, and you're going to decrease the cost. Here's just an example where we would be if we didn't have some examples of – clinical examples of patients. This was a very difficult patient who would have a TMA – vascular reconstruction and the graft went down. He was left with a neuro-ischemic dehiscence of the TMA. We’ve started using the amniotic matrix tissue on him and we started to see fairly rapid improvement. Now remember, this is the patient who also had ischemia. Not your typical patient was going to be enrolled in a trial. So we would see him every several weeks or so and apply the graft. There wasn't a trial, so we didn't do this religiously every week or two weeks. At 6 months, we made remarkable improvement but then he had exposure of his metatarsal. So this can become a [indecipherable] [17:12] event for many patients in many trials, but we just debrided the bone with the rongeur in our clinic because he wasn't clinically infected. We proceeded on with standard care and we continued on with amniotic matrix tissue once we were sure he wasn't infected. And we got this man to completely heal. It took us about 9 months or so, but this was a man who was risking losing his leg because there are no other revascularization options. And it's because of cases like this that we're doing a trial and exactly the similar type of patients. The ones who would be excluded from all DFU trials with exposed bone with modest levels of ischemia, with exposed tendons and deeps structures or so. So we've had a very, very nice look with this. And again, I attribute this to the living extracellular matrix tissue and the MSCs combine. Remember, these are living ECM – living mesenchymal stem cells and a whole host of growth factors and cytokines. So in summary, we know that standard basic wound care is always necessary. Always no matter what kind of products you want to use, if you don't pay attention to good basic wound care, you're never going to be successful. You can never forget that. You always have to go back and reassess your care if you're not making good progress. There are many advanced wound care products available on the market. We tend to choose those with evidence when available and there is not good evidence available for many products. And I'm happy to say that there's more and more evidence becoming available, but they are very few comparative trials we've heard about. Several of them are already this morning, there's very few comparative trials. There's no standard. You have to use this product. You have to use that product. A lot of it still comes down to evidence plus your own experience, plus what kind of patients are you talking about. Are you using a 1 centimeter ulcer or you have a 5 centimeter neuroischemic ulcer in patient with chronic renal insuffiency? So there's a lot of variability here. I think that the mesenchymal stem cells offer a unique mechanism of action as we said. They are neonatal. They are actively proliferating high concentrations of these cells in the amniotic matrix tissue. They respond to their environment. They're anti-inflammatory. They're immune-privileged. They're tissue regenerative. So I think, just those qualities make these really an exciting and attractive option for your chronic non-healing wounds. And as I hope I’ve illustrated the most recent RCT provides superior evidence of clinical efficacy in healing DFUs. And again, we look at trying to compare one trial with another, it's not always easy to do so.
But I think the most important thing is looking at the evidence, making sure that there are some kind of biological plausibility that makes sense to you and why you should use these kinds of products. Again, they are all expensive but the savings come in the long run. You get your patients healed faster, you have fewer complications, fewer hospitalizations, fewer surgeries, fewer amputations, and the longterm cost are always cheaper in the long term by using these products. So with that, I want to say thank you very much.