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Male Speaker: Thank you for hanging in there, everybody. This will be a little bit less technical. And I guess I can say many of my personal slides, I’ve seen, produced, displayed earlier today. So it didn’t make me very happy. But it might offer some type of ingemination so that you can understand where I’m coming from on these applications of MSC. This is going to be talking about our complex wounds trial. It was of course fully funded by Osiris Therapeutics several years ago. These are my learning objectives.

We showed this slide earlier. And again, it’s a reminder of the complex interactions in the diabetic foot that we need to be aware of if we’re going to be successful in managing these complicated patients.

Obviously, neuropathy, not just sensory but motor and autonomic and all the wonderful complications attendant with those as well as vascular disease, both macrovascular and microvascular disease. Setting up the system for a high-risk foot to which when trauma is applied with the concurrent impaired response to infection due to immunopathy. We get the diabetic foot ulcer that can become infected when untreated and this cycle continues. And when enough tissue damage or infection occurs, it often leads to amputations. So it’s always good to step back and refresh your memory on what’s really going on in these complicated diabetic patients. They don’t need to be complicated. It can just be a simple patient – diabetic patient with a neuropathic ulcer. They can have always concurrent abnormalities going on at the same time.

We showed this also yesterday just showing that you need a thorough assessment on these patients. Because things can be missed very, very easily. Underlying infection, underlying osteomyelitis, underlying neuroischemia. It does not have to be significant. But there are certain levels that will preclude proper healing. So I think the most important thing is the thorough systematic assessment. When you systematically evaluate a patient, you look for all the underlying problems, comorbidities and you address each one of them obviously.

That allows us to classify the wound, is it a grade 1, grade 2, grade 3, ischemic, infected or not. And then of course, we provide for adequate treatment and always including a debridement, offloading, wound bed preparation, surgery very often and of course concurrent medical management.

And then as we stated, if we see after four weeks of good standard care that we achieved that magical 50% reduction in the wound area, we continue on with the same therapy. However, as is more frequently the case, patients get stalled along that trajectory to healing. And we don’t see that complete pathway towards healing. And then we have to start thinking about additional advanced therapies.

We mentioned this. You also saw this this morning as well as yesterday, a number of products that we have to use. So this is more of the real clinical side, not just the science side. But it’s the translation of your basic sciences into clinical applications. And if we look at the advanced types of therapies, we think of one isolated very recombinant growth factors, not terribly successful because it’s isolated. It’s an isolated singular growth factor. And I don’t think REGRANEX or PDGF, recombinant human PDGF alone is used as frequently as it was. But I was the granddaddy for all advanced therapies.

Then we have our acellular extracellular matrices or dermal products. As we can see, there are a number of them. And I’m sure Dr. Letobarone [phonetic] probably used one of those when he was talking about his dermal matrix.

Then we deal with our cell-based tissue technologies which actively produce exogenous growth factors. And to me, that’s the real meat in this application because we can produce not just angiogenesis, but we can also lay down extracellular matrix within that deficient wound. And then of course, negative pressure therapy.

As I said yesterday, many of these are often combined at the appropriate time to affect final wound closure. But we have all of these options available to us certainly in the US. Not in many places overseas has this number of opportunities for using advanced products. But these are commonly available. Very few of these are brand new for us.

So why cellular tissue therapy? Well, we know the benefits of MSCs. I certainly don’t have to speak about those benefits to this audience. But we recognize that placental membranes are rich sources of MSCs. They’re readily available to us.

[04:57]

I just go to my shelf or my cabinet or to our freezer and we can pull out cryopreserved amniotic tissue with active living stem cells. They’re full of potential of course. Although, as has been mentioned yesterday and today, we’re looking for that paracrine effect of these mesenchymal tissues to form dermis and gradually heal these wounds.

So we’re not looking for direct differentiation. We’re looking for an indirect differentiation. But that active secretion of those medicines that had been spoken about by Ronald Kaplan, whereby these are powerhouses – secretory powerhouses for producing the proper growth factors that can properly orchestrate wound healing.

And then of course, the response to the local environment as has been shown multiple times during this course. We saw this picture also. Viable multiple multipotent cells produced effective healing here. Yesterday, I showed an image. It was a little bit different to this. But it is good for us to recognize the deficiencies in such chronic wounds. Cells with low myogenic activity, proinflammatory cytokines, high levels of proteases and characterized by cellular senescence.

But the MSCs can shift the cytokine balance towards a favorable environment once we practice our good local standard care. And again, I can’t emphasize enough without good standard wound care, none of these advanced products will work.

So we know that the MSCs can convert that to a good acute wound milieu or environment that can go on to healing. And then we characterized a healing or an acute wound with cells with high mitogenic activity, anti-inflammatory cytokines predominate, low levels of proteases or characteristic and cells are active and actively secreting and synthesizing extracellular matrix.

Dr. Rudowsky [phonetic] had talked about the pivotal trial by Larry Lavery who was the lead investigator and author in this trial published in 2014. And this is just the results at 12 weeks, which was at its time, the most effective management of all these cellular products ever reported on to date. And here we say that the Grafix arm at 12 weeks affected a 62% healing compared to just 21% in the control group.

Obviously, this was highly significant. And there was 191% marginal effect between the active group and the control group, which as I said is the greatest effect seen to that date. And here we just see the survival curves and the separation of that curve – of those two curves very early on. So this was Grafix Prime which was the cryopreserved viable human placental membrane tissue with active MSCs.

So my question has always been – I’m not really interested anyway at this point with managing healthy neuropathic ulcers with these advanced products. Because more often than not and at least 90% of the time, you can get these to heal with good effective offloading or casting as it has been done for decades.

My interest has always been what about those wounds that are typically excluded from randomized trials. Those are the wounds that get me the most problem. Those are the ones I want to see new studies on because there’s very, very few studies on such complex wounds that we define as those wounds with bone, tendon or fascia exposed. Those are always excluded even in those patients with modest degrees of renal insufficiency or modest degrees of ischemia.

So we engendered in this trial. This was a phase IV post-marketing study to establish the safety and efficacy of GrafixCORE, not the Prime. Now, the GrafixCORE is just a chorionic membrane without the epithelium. So this was used on wounds as I said typically excluded from DFU studies. And these were the bad ones after debridement, after partial foot amputation, after necrotizing soft tissue infection has been debrided.

Our primary efficacy point for this trial was different than most trials. Because it was not for 100% primary closure. It was for 100% granulation tissue. Now this is important because if we can rapidly generate good granulation tissue, we can skin graft. So that was different than all other trials, 100% granulation tissue and wound healing was a secondary endpoint. And we had of course safety endpoints as well.

So the study design was a prospective open label post-marketing cohort study. It wasn’t a randomized trial.

[10:00]

It was not a comparative group and this was using the GrafixCORE. As I said, this is a viable chorionic membrane, placental membrane with living cells managing these complex diabetic foot ulcers that I treat on a daily basis. So this was my interest to study the more difficult ones, not the simple ones.

So bone, tendon, fascia was exposed and it was intended to be a real world type of situation. Again, these are the people who are always excluded from the trials and these are the people that need these advanced products the most. We provided for a weekly application of up to 16 weeks of the viable cryopreserved placental membrane plus offloading and of course standard of care. So offloading had to be excellent and the standard of care must go with at least weekly dressing changes and proper debridement. It was a registered trial in clinicaltrials.gov and of course approved by all IRBs. And an external company did the research analysis and managed the trial.

There were four study centers enrolled. The Phoenix VA was the lead center. We had South Shore Hospital in Massachusetts, Tucson VA, and University of Pittsburgh Medical Center participated in this open label trial.

So what were the inclusion and exclusion criteria? These again are different from your typical diabetic foot ulcer trials. Of course, patients could be between 18 and 85, confirmed diabetes of course but these were the deeper wounds. Usually it’s University of Texas one or two. We were going down to bone so these could have been grade 3 ulcers. A or C simply means neuropathic or neuroischemic, but they cannot be infected.

We had larger wounds in this but they could only be large enough to accommodate three pieces of this 5 by 5 cm tissue graft. And we did allow for wounds with modest degrees of ischemia. Normally, an ABI, ankle brachial index of below 9 is excluded from trials. We allowed ourselves to go down to just above 0.5, total blood pressure had to be over 40 which is on the margin of what would be acceptable and transcutaneous oxygen tension just had to be above 30. So these wounds could have been ischemic and the patients were allowed to have modest degrees of renal failure as well. We actually allowed patients with an A1c of less than 14% into our trial which is very much different than other trials where they usually have to be below 8%, 10% or 12% depending on the exact trial and they cannot be infected of course.

So now let’s go to our results. On the intent to treat population, we had 31 patients entered into the study. That was the entire study. This was not meant to be a huge multicenter trial. Four patients dropped out, so we were left with a per protocol population of 27 patients who actually completed the study and completed all study requirements. Four withdrew due to noncompliance or necessary surgery. Remember, when we’re talking about diabetic foot wounds with modest degrees of ischemia and exposed bone and tendon, these people can get infected very, very easily. And we did have two patients who had to leave the trial early – at an early point to have transmetatarsal amputations.

So our demographics of our population, this was the intent to treat demographics, we can see the typical age of about 63 years old. Mostly males because most of the patients were in the VA, veterans population, but these were older wounds, seven-and-a-half months old. And as you can see, the baseline area was almost 15 cm2, which is larger than most diabetic foot ulcer trials, 45% of the people had prior amputations and many of these people had prior advanced wound therapy recorded usually in their past, but not for these particular wounds. So the wounds were older and they were larger than typical ulcer trials would allow patients into.

So remember our primary endpoint for our trial was 100% granulation by 16 weeks or earlier for these very difficult wounds. The per protocol population achieved this endpoint a rate of 96% by 16 weeks and the ITT population was about 84%, achieved 100 percent granulation tissue by 6 weeks. This was the primary outcome as you could see, a nicely granulating wound. Anybody in this room especially plastic surgeon would jump to do a skin graft at this point. Now, what’s the whole rationale here? To get these complex wounds that had previously had exposed bone or tendon to granulate so that they could be moved on towards a skin graft.

[15:01]

Obviously, we didn’t include skin grafting in our trial for obvious reasons. This is the Kaplan-Meier curve, just showing the rapid trajectory of achieving 100% granulation tissue in our population here.

So secondary endpoints. Remember, this is different because our secondary endpoint or primary secondary endpoint was complete wound closure. And in our per protocol population of 27 people, we can see that nearly 60% of patients were able to achieve complete closure and the ITT population of 31, just about 52% achieved closure. Mean percentage wound area reduction also a secondary endpoint, 92% in both groups. Mean number of Grafix applications to achieve 100% granulation in 16 weeks, 6.8 in both sides. And mean time to achieve 100% granulation was 6.8 weeks. Remember, we applied these up to 8 – we can apply them up to every single week during the trial but we achieved our endpoints at 6.8 weeks for the 100% granulation tissue. So we had rapid improvement, rapid trajectory towards achieving that 100% granulation point as we saw from the previous survival curve.

Now, on a post-hoc analysis, we looked at the mean number of Grafix application to achieve 100% epithelialization, complete wound closure and we had 9. And of course, it was about 9 weeks to achieve that. Now, if you look at most diabetic foot ulcer studies, for typical neuropathic ulcers, approximately 50% of patients will heal at 12 weeks or so for simple neuropathic ulcers. Here, we were seeing the mean number of weeks to achieve a complete healing in these more larger and these more complex wounds was just 9.1 weeks. This was another picture of a patient, a typical example. Post amputation, obviously that’s a chronic wound. If f it doesn’t heal within four weeks, it was considered chronic and we would allow this into the trial.

And here we can see a complete closure only by using this cryopreserved membrane. No other adjunctive therapies were allowed of course, no negative pressure was allowed once the patient was enrolled into the trial. We would usually just apply the GrafixCORE to the wound, put an absorptive dressing on, usually a foam dressing and leave it for a week with proper offloading. I tend to use products that we can apply weekly because I don’t want my patients touching their wounds because I don’t know what’s going to happen. So I control it by doing it this way and it was very effective in that regard.

Now, this graph shows the primary endpoint, which was time to achieve 100% granulation. The blue curve up above is the per protocol group and then we can see the ITT group which, you know, 27 versus the full group of 31, we can see lagged a little bit behind obviously. And the bottom curve is our secondary endpoint for complete closure. Again, blue is the per protocol and the yellow is the ITT group.

But we can see a steady climb towards achieving those two endpoints in both the ITT as well as the per protocol group. So we are fairly happy with the results here. Safety analysis, 59% versus 61%, there was no difference and the mean adverse event would have been maceration for the most part because we left these dressings on for a week, but there were no serious adverse events related to the study product alone.

But remember, several people had to drop out. Hospitalization for an unrelated pleural effusion and hospitalization for cellulitis and non-indexed wound, and we had four early terminations as well which were patients unwilling to comply with the protocol as you always have, plus two patients who as I said required the TMA surgery. Very, very, very common complications in this cohort but none related to the product itself.

This is just a picture of – I think this was the very first patient we enrolled into the trial. This was uncontrolled diabetic patient who came from a long distance. He had a severe necrotizing infection which we see very, very frequently and he was post amputation and debridement. You can see that this was a type of wound that we initially started with.

[20:03]

Once we achieve a decent granular bed, we enrolled him into the trial. These are the ones would be while the patient was in the hospital. We chose not to start the patients to enroll them into the trial until they left the hospital and we can treat them as an outpatient. Here’s where we would have started with this patient. That’s still a fairly significant wound with some fascia exposed and bone exposed at the underlying portions of that wound.

And here, we see a gradual progression. And with this patient, the first patient never quite achieved 100% healing. We later found out he had underlying osteomyelitis on here which would certainly preclude them from healing, but we saw a great progress by using the placental membrane.

Here’s another patient. This is our second patient enrolled. This is fairly typical but this patient would be excluded from any diabetic foot ulcer trial because capsule was exposed here, okay, and joint capsule would be a level, a grade two or a three, and this patient was post incision and drainage for an infected wound, but we started him on the trial and he healed very nicely as you might expect. This was not the most severe wound, but it still met all of our inclusion criteria.

This patient was a little more complicated. I was a little more nervous about him because as you could see, he had already lost his other leg. He came into the emergency room with a very high white count and we can see this abscess that he had in his heel. This was from stepping on a nail that was in his shoe, also a very typical problem. So, we obviously took him to the operating room. Here, you can see fascia exposed. He luckily did not have any osteomyelitis and he was not really ischemic. He was just a very high-risk patient with very bad neuropathy obviously and a great risk for further amputation.

Here, we can see post I&D. He was still in the hospital here. So, once we got him out of the hospital, he did have a fairly healthy wound, but once again, it was a deep wound with the fascia exposed. You could see the fascia exposed underlying that wound. So, these patients would be excluded from your typical ulcer trials. And due to their complexity, they just generally wouldn’t meet the criteria. We saw a very rapid improvement in this person’s wound as well and he affected final closure. Obviously, this patient was very worried, but we had really great success with this patient in using this protocol.

So, in concluding remarks on this study, when we applied this product weekly to chronic complex wounds with exposed tendon, muscle, bone, or joint capsule with a standard of care, we found that 96% of patients completing the protocol achieved 100% granulation tissue by 16 weeks with a mean of 6.8 applications at a mean time of 6.8 weeks. So, we were allowed to apply the product every week if we needed to. But we found we just didn’t have to, to achieve our 100% granulation because we achieved it fairly early. 59% achieved complete wound closure, defined as a 100% reepithelialization.

Now, remember also that most diabetic foot ulcer trials, simple neuropathic diabetic foot ulcer trials, many of them don’t even achieve 50% complete healing for a simple superficial neuropathic ulcer. We were able to achieve about 60% healing in about nine weeks with these deep complex wounds. So, it was very remarkable for us when we realized what we were doing here because that can compete against any other trials that’s really on the products that are on the market. So, we had 92% mean percentage wound area reduction at 16 weeks for the entire cohort as well. Remember, 16 of our 31 patients achieved complete closure in this trial. Two patients required TMA, which compares favorably to an 18% reported rate for patients with chronic wounds.

And again, they did not have complications as a result of the study product. They just had intercurrent disease and became infected as is so typical with these types of complex patients. So, in summary, I know you saw this already today, too, but this is a more simplified version of our algorithm. And the reason I do this is to stress the importance of a good thorough systematic evaluation of these patients.

[25:01]

And the more systematic you are, the less likely you are to miss something. It’s very easy to miss very critical points on these patients. So, we always look for infection. We use the IDSA, the Infectious Disease Society of America guidelines for diabetic foot evaluation. We always probe our wounds and if we probe to bone at the base of the wound, we know there’s a high likelihood for osteomyelitis. So, we always do that.

Is it ischemic? We always take our pulses, but pulses aren’t good enough in most cases. So, we always get our ankle-brachial indices or other non-invasive tests and if those are very much abnormal, we’ll do a vascular surgery referral for angiography. So, with one hand, I’m taking a pulse. With the other hand, I’m probing that wound to see how deep it is. So, within the first 10 seconds, I’ve already made an assessment as well as a visual assessment of what we’re dealing with.

Then, we obviously need to provide for a proper offloading usually with a cast, total contact cast or removable cast walker and of course debridement. Debridement is critical as we all know. Any surgeon knows that bad, nasty wounds need sharp surgical debridement first and foremost and those are just the basic standards of care that everybody needs to follow. Then, once again, if the patient is improved by standard care, then we continue the standard care and standard offloading until the patient heals. And then, once they’re healed, we put them into a prevention program, surveillance program, and proper footwear. This is standard based on any practice guideline anywhere in the world whether it’s international or US.

However, many patients have difficult, complex wounds, or they do not go on to normal healing. Then, we need to step back, reassess and offer them advanced therapies as necessary. And the advanced therapy that we use is usually based on some type of evidence or some type of science to give us enough confidence that this would be the appropriate type of therapy to go to.

Now, normally, I use multimodal therapies. During a trial, obviously, you cannot do that, which means that most diabetic fool ulcer trials are not real-world circumstances because very rarely do we use one product from start to finish. We usually change. As I said yesterday, wounds change with time from week-to-week. So, you need to step back and change your therapies. But that can’t be the case obviously in a controlled trial or any clinical trial such as this.

But good thorough assessment, good understanding of the magnitude of the disease and all the possible underlying metabolic perturbations will make you more successful in your attempts to manage these very difficult patients.

So, thank you very much for your attention. Congratulations also for sticking it out all day. I don’t imagine there’s any questions, right? We’re all tired. We all want to get out of here, but I’ll –

Male Speaker 2: I have.

Male Speaker: Yes, sir. Oh, we do have. Go ahead.

Male Speaker 2: My question is if it’s not per protocol, how do you know when to reapply Grafix?

Male Speaker: The ITT population is the population by definition that signs a consent and at least gets one application of a studied product. A per protocol population which is not as good, per protocol means it’s the population of people who completed the protocol as it’s supposed to be you want to follow through the end. So, in this regard, we were by protocol allowed to apply the study agent, the placental membrane product every week until 16 weeks.

Male Speaker 2: Okay.

Male Speaker: So, that’s the difference and that’s pretty typical for these types of things.

Male Speaker: Got you.

Male Speaker: Okay.

Male Speaker 3: I really learned a lot from – about wound from you in this meeting. You should be congratulated particularly –

Male Speaker: You’re a plastic surgeon for crying out loud.

Male Speaker 3: Well, I mean, you should call us more. That’s what I learned. No. But, it’s really a testament to your expertise in caring for these difficult wounds and having that good sense to not try to get them all closed without skin grafting them and I think you should be congratulated for that.

Male Speaker: Yeah.

Male Speaker 3: Thank you.

Male Speaker: Yeah. I think we really should probably skin graft faster than we do, but many of these patients, like all these patients were treated completely in the outpatient setting and even with that, we achieved good wound closure. But the reason I have any expertise in wound care is because I trained in Boston – Boston Clinic, okay.


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