So, yeah, thanks to Marlies for going along with our ideas here to share some things that we've been studying here at the University of Utah in our amputee program these last few years. And so the first half of our session will be focused around the topic of hyperhidrosis. I'm going to spend a few minutes just kind of reviewing the scope of the problem, some highlights on evaluation, and some treatment options. But then I'll turn the time over to my colleague, Dr. Godfrey, who will share some data and some insights about using aluminum chloride as a treatment for hyperhidrosis. And then Dr. Duncan will spend a few minutes talking about the use of botulinum toxin to manage hyperhidrosis and where that kind of fits into a treatment algorithm. And then I'll finish up our half of the session talking about what starch iodine testing is and how it could be useful, particularly in the context of looking at botulinum toxin to manage this problem. So before I get started, I do just need to throw out this disclosure. Some of the work that will be presented here was supported by an award from the Department of Defense. And so anyway, so there's that language. Hold on a sec. I pulled up the wrong presentation. We'll do a quick adjustment there. So after we talk about hyperhidrosis, we'll have a little break. And then we have some guests that will be talking about some surgical strategies to manage pain and the evolution of osseointegration to finish off our session. So it should be a good session for everybody. OK, so I would be curious to have you guys join this poll. You can go to that URL or you can use the text function. And the question here, pretty straightforward, do you routinely ask your patients about skin problems? Yes or no? I'll give you a moment to find that URL. Now, how about this? Do you routinely ask your patients about sweating? Okay, so that's good. I was maybe expecting a greater percentage of folks that this isn't really a routine part of your questioning when you take histories, but that's great. Hyperhidrosis really is a significant problem for amputees. There've been a few surveys that have highlighted this. First of all, I would argue, and we'll show some examples of how hyperhidrosis can be a primary irritant for many other skin problems. But again, a couple of surveys have noted sweating to be one of the most common, if not the most common skin problem that people deal with and a significant impactor of quality of life. Even in some studies, more than wounds or pain. So we did a survey study in our cohort of patients here at the University of Utah and our neighboring VA hospital some years ago. And so this is more of an Intermountain West, kind of an arid climate. And even in that climate, up to two thirds of patients reported that sweating was problematic for them and tended to be more common in slightly younger folks and those with baloney amputations and not surprisingly a bigger issue in warm weather. Although somewhat surprisingly, it was still pretty frequent of a problem even during the winter months for many of our patients. And it kind of gets to this issue of, if you don't ask, you're not gonna know. And I think it's an easy thing to overlook when you're seeing patients and addressing potentially other issues. This was an interesting survey done kind of exploring the prevalence of observed skin problems as reported by the clinician and reported skin problems as reported by the patient. And there were significantly more problems reported by patients than observed by clinicians. And excessive sweating was right at the top of that list. And so, it is important to be asking. So what are some of the problems that can be caused by excessive moisture? First of all, you can create excessive movement of their limb inside their prosthesis, which has consequences of pain or sometimes skin breakdown or even a malfunction of the prosthesis, the slipping out of their socket, things of that nature, which can have obvious fall potential consequences. There's some hygiene challenges with odor and things of that nature. The simple interruption to desired activities. I can't tell you how many patients, when you ask them, particularly those who are quite active, how often they have to stop or alter their workouts to take off their prosthetic, dump out the sweat, dry it off, et cetera, before they can resume their activity. And not to be overlooked, it certainly can cause a variety of skin problems, which we'll highlight here. So there's a few different potential challenges with excessive moisture. Malaria is a condition where the sweat gland itself is essentially plugged up and leads to this sort of rash that you see on these pictures. Looks very much like a folliculitis, except if the lesions are centered around sweat glands, not hair follicles. You certainly can get some folliculitis as well. Irritate further things like eczema and contact dermatitis and yeast infections, I think are rather common consequences both candida and tinea type of skin infections are just breed in these moist enclosed environments. So how do we approach evaluating this problem? There are a few techniques that again, mostly used in a research type of setting to try to quantify the amount of sweat being produced. So a couple of these are gravimetry and vapometry. They're not really practical for clinician use, but there are suffice it to say some methods that have been proposed to try to quantify the amount of sweat production. The most practical, frankly, way to approach this is through a simple scale like the hyperhidrosis disease severity scale or the HDSS. This has been well validated against other larger and longer questionnaires and quality of life indexes like a hyperhidrosis impact questionnaire and the dermatology life quality index. But the HDSS is one simple question and it's really kind of become a standard in terms of measuring outcomes in clinical trials by tracking HDSS scores before and after treatment. So the HDSS is a four point scale and the patient simply asks or answers, chooses a response based on these statements ranging from my sweating is never noticeable and never interferes with my activities to the severe end of the spectrum. My sweating is intolerable and always interferes with my activities. We did some validation work in our amputee population a few years ago. We correlated this score, the HDSS score against a Likert scale question, asking patients to note how much sweating interfered with either their prosthesis fit or their prosthesis function. And to those questions, the HDSS correlated quite strongly. And so we feel like it's an appropriate scale to use in our patients and a simple way to track and help kind of direct a treatment approach based on what their HDSS score is. We further surveyed our subjects and asked them what types of treatments that they had used to try to deal with this problem. And a couple of interesting things to note from this table. First of all, nearly 40% hadn't tried any strategy, which is interesting given how common of a problem it is for these folks. Secondly, along this self-reported effectiveness score, where zero is not effective at all and five was completely effective, you can see that most of these strategies are marginally effective at best. And the most common strategies used are over-the-counter antiperspirants or prescription strength antiperspirants and using a sheath or sock underneath their liner. So to talk a little bit more about some management strategies, just to highlight a few things. In terms of prosthetic considerations, it's pretty common practice to use a sheath or sock underneath the liner to try to absorb and potentially wick away some of that moisture. And that can be reasonably helpful for some people, but certainly not everybody. In terms of prosthetic liners, liners in general are water impermeable. Water's not gonna escape through them. And there have been some work done looking at different materials and the relative thermal conductivity between them, but there is theoretically then some benefit in a silicone liner, for example, that has higher thermal conductivity than a thermoplastic elastomer liner and a TPE gel liners. If we shift from sort of prosthetic considerations to pharmacologic considerations, which will be the bulk of what we'll talk about today, there is a potential role for antiperspirants and particularly prescription strength ones. And Dr. Godfrey will talk more about aluminum chloride and our experience with that. Some other agents include this product called Hubrexa. This is a glycopyrrolate impregnated cloth that is administered by wiping the skin that you're trying to treat. It is currently indicated to treat axillary hyperhidrosis, but it hasn't been studied in patients with amputation. Theoretically, there could be some current concerns with having to treat a larger surface area than just an axilla. And then the occlusive environment, could you potentially cause some systemic effects from that treatment on an amputee? For folks with generalized hyperhidrosis, this is more just an FYI. There are some medications that are used to treat people with more generalized hyperhidrosis, but they often have side effects that aren't very desirable. Botulinum toxin has kind of become the second line treatment after aluminum chloride for hyperhidrosis. Of the different botulinum toxins, Botox is the only FDA approved medication for a hyperhidrosis indication. There is some anecdotal evidence that laser hair removal could be helpful. This study of wounded warriors was not primarily looking at the impact of laser hair removal on sweat, but it was sort of a side note. So there could be something to that. Some other strategies include microwave thermolysis with this product called MiraDry that essentially destroys the sweat glands for several months. Actually, I'm not entirely sure how long the efficacy lasts after a treatment with this product. This is not something that we had experience using. And then there is this old strategy of iontophoresis can be helpful, particularly in Palmer and plantar hyperhidrosis. And so it's not as practical to think about how would you put a whole residual limb into a water bath like that? Although folks who are used or developing other strategies for iontophoresis have even gone so far as to create this sort of like face mask. So maybe there is a way to try to adapt this by industry to help a residual limb. So this is essentially our treatment armamentarium. You can address things from a prosthetic standpoint with a sheath, look at antiperspirants. Next beyond that would be botulinum toxin and then potentially some of these other strategies which frankly could use more study in this population. So with that said, I'm gonna turn the time over to my colleague, Dr. Godfrey, and we will kind of ask to reserve questions regarding this general topic after I talk about starch testing and we will kind of go to that point. So. Take it away. Thank you, Dr. Hansen for that introduction. Let me pull mine up. All right. Everyone good on that view? Okay. So I'm gonna take over and talk in more detail about the use of topical aluminum chloride antiperspirants for hyperhidrosis in our patients with amputations and who use prostheses. And there we go. Same acknowledgments as Dr. Hansen said for our work. All right, some background and I'll go through this sort of briefly. So aluminum chloride salts, as Dr. Hansen said, are an often prescription strength. Ones are considered first-line treatment for hyperhidrosis in dermatology literature. and this is very well studied in palmar, plantar, facial, axillary, all those areas. Efficacy for axillary hyperhidrosis is somewhere between 33 and 78 percent and adverse effects are primarily skin irritation and it leads to discontinuation in somewhere between 2 and 21 percent. There are a bunch of different brand names, Drysol, Hypercare, Xerac, they are prescription strength, CertainDry you can get over the counter and they're relatively low cost. They're usually somewhere between 12 and 30 percent aluminum chloride in some kind of alcohol base, ethyl or isopropyl. So as Dr. Hansen mentioned, despite the fact that many clinicians who work with amputees are recommending and prescribing these topical antiperspirants frequently, we don't have any published literature on their efficacy, tolerability and safety in this population. And it is a concern given that they are using this and then putting on a prosthesis, there could be a potential risk for skin irritation greater than that seen in someone with axillary hyperhidrosis. There's really just this one study recently that I found that in which this team looked at coating the prosthesis liner with a 5 percent aluminum solution to see if it reduced sweating on the thigh and it was found to be ineffective. So our question clinically as, you know, physicians treating this is we wanted to know is aluminum chloride effective in, you know, treating hyperhidrosis in our patients? And then what is the incidence of skin irritation and other problems? And as Dr. Hansen mentioned, we did a study a few years ago looking at the reported incidence, severity and impact of hyperhidrosis in this population. And this was a survey based study. There's obvious limitations, but I'll just summarize what's important to this part of our talk, that 50 percent of respondents had tried either an over-the-counter or prescription antiperspirant, but about half of those reported no efficacy and 20 percent, only 20 percent reported that it was mostly or completely effective. Dr. Hansen reviewed some of this, but I'll go into just a little bit more detail. You apply the antiperspirant to the skin and the thought is that it mixes with the perspiration on the skin and in the sweat duct and that the salt forms a plug. And that lasts, you know, at least a day, maybe a few days. And application is really important to minimize effect or to maximize the effect and then minimize any skin irritation. Because if there's any water on the skin that's mixed with leftover aluminum chloride, you have the risk of creating hydrochloric acid that's thought to be the mechanism for skin irritation. So we want to minimize that. All right. So this was the second aim in a larger study. And the objective of this part of our study was to investigate the effectiveness of a prescription-strength topical antiperspirant, which is aluminum chloride, drysol, which is 20 percent, on hyperhidrosis of the residual limb. And we chose to use drysol because it's FDA approved for several different indications and it's available by prescription in the U.S. It's frequently used and common knowledge for a lot of people working in this field. Our study design, this was a double-blinded placebo-controlled four-period crossover trial. Our placebo was ethyl alcohol, which is the base for drysol. And the study drug was prepared by our research pharmacists and then they labeled it by period one, two, three, and four. So the subjects and the investigators did not know what they were taking in the different periods. The study was designed this way because it allows each subject to serve as their own control and gives us improved power in a relatively small trial. And then also better control for variability in season, because we know depending on the weather, if it's warm or cold outside and this, their hyperhidrosis may vary. And this study was going to be across 12 weeks total. And then a four-period design would have improved power over a two-period design. Alrighty. So subjects who reported an HDSS of two or more were invited to participate and they needed to be older than 18. They needed to be at least six months post their amputation surgery and use a prosthesis. They were excluded if they had any open sores or wounds, if they had any known sensitivity to iodine or aluminum chloride. He's calling me. All right. So if they were using an antiperspirant on that area, then we asked them to stop it for a week prior. And that's pretty standard for many of these studies in dermatology literature. So subjects were randomized to either start with active or placebo for their first three-week period. And they kept a log of the days that they use the treatment. And they returned three weeks later to clinic for reevaluation and to receive the next batch of study drug. And these were outcome measures, HDSS. And then we'll go over some of these other ones in a moment in more detail. Okay. Application instructions is really important. And I want to go over this because if anyone has been prescribing these medications or recommending them to your patients, it's really important that we talk them through how to use this to minimize all the adverse reactions. So this is what we told them for the study. This is what we tell them when we see these people in clinic as well. So at the end of the day, remove your prosthesis and ensure the skin is clean and dry, and then apply the medication in a thin layer to the portion of the residual limb, every part of it that's contacted by any part of your prosthesis, liner, sleeve, everything. Do not apply to areas of broken or irritated skin, then allow it to completely dry on your skin. And then before you go to bed, you can cover your residual limb in a sock or a sheath or some sort of covering if you want to. Then in the morning, remove the sock or sheath if you used one and wash it thoroughly with soap and water. So remember we want to get off any of the aluminum chloride that's on the surface of the skin. We want it gone. We just want to leave the stuff that is inside those sweat ducts. And then we tell them to dry their residual limb and don their prosthesis as usual. It's pretty standard to have patients apply the medication nightly for about a week, and then they can decrease the use to maybe every other night, every three nights or so for the next couple of weeks. And we ask them not to use any other antiperspirants on that area during the study period. Okay. So just a little flow chart of how this happened. So 39 people were consented and then two of them dropped out and never returned for us to collect any measures. 37 enrolled and had their baseline measures taken and they were randomized to start with either active or placebo. Seven dropped out before they ever came back for the first follow-up and we never got any data from them. So for the rest of them, we had in-person visits at week three and we collected the HDSS and then IST is the iodine starch test. SIVS is a visual representation of their starch test. I don't have data on that today, so we won't spend a ton of time on it, but we did take that. And then these measures of fit and function and the effect of sweating on the fit and function of their prosthesis that Dr. Hansen mentioned. And then they came back and just had phone visits or they didn't come back, but we had phone visits at six and nine. And then they came back at week 12 to repeat all the in-person tests. So they did an iodine starch test. They did the visual tests and they, we did the measures of fit and function and the HDSS. All righty. So some basic demographics is we had 24 trans-tibial amputees and six transfemoral. Average age was 48 and the range was 21 to 73. We had 23 male and seven female. And our outcome measures. So our primary outcome measure, as I mentioned, was the HDSS. So a change in HDSS from baseline for the placebo and aluminum chloride conditions. And then our secondary outcome measures, we looked at this change in the iodine starch test and a visual scale for that. And we are still analyzing that data. And then we also looked at the interference on prosthetic fit and function. That was on a Likert scale from zero to five, zero being no effect, five being the most effect. And then we also reported any adverse events. Okay. And then these are what we looked at. All right. So some baseline measures. So just remember that all of our subjects alternated between active and placebo for four different periods. So they all had exposure to both study conditions. So we can see that the baseline HDSS seems to be mostly twos and threes, just a few fours, which would be the most severe effect. And then when you look at the effect of the hyperhidrosis on the fit and function of the prosthesis, it sort of scattered, but kind of worse in those middle ones, one, maybe two, three, and four. All right. So give you some of our preliminary results. So there were 120 total observations because there's 30 subjects in four periods. So this is showing the change in HDSS from baseline between the active and placebo. So we want those numbers to go down. So minus one, minus two, minus three are going down one, two, and three points on the HDSS. Those are improvements. So these are all just raw numbers. But what we cared about, highlight those, we cared about was this, were these, these changes here. So an improvement of at least one point on the HDSS that's considered to be clinically important for the patient. So we saw that in, when patients were taking the active drug, 55% of them reported an improvement in HDSS by at least one point and 47% taking the placebo reported an improvement. So this wasn't statistically significant. It's obviously a fairly modest difference between the two conditions. All right. Let's look at the effect of hyperhidrosis on the fit and function of the prosthesis and the change in baseline under the different conditions. So I'm going to orient you to these box plots. So the gold is placebo and the black is, well gray, is the active drug and the X axis is change from baseline. And remember that a decrease is an improvement. So a reduced interference on fit or function, the sort of darker bolded vertical line in the middle of the box is the median. And then we have quartile one and three on the left and right. Those are the vertical lines on the left and right, and then the minimum and the maximum on the left and right again, the horizontal lines. So when we look at these two box plots, we can see that the median change is very similar between the active and placebo. Subjects improved about a point on our Likert scale with their fit and function, regardless of whether they were taking active or placebo. So let's look at the similar way of looking at this data for the change in HDSS from baseline. So we can again see that the range for both treatment conditions is very similar, but we can see that the median improvement, the darker line in the middle of the box when taking the active was very close to minus one, which means a drop in the HDSS score by one point. And then the median improvement when taking the placebo is very close to zero. So due to the complex design of our study with the crossover, our statistician had his work cut out for him, but we were able to determine with statistical significance that active treatment was superior to placebo at improving the HDSS score probably around one point. And again, that's a modest improvement, but it is important clinically to reduce the HDSS score by one point. All right. Adverse reactions. We have no serious adverse events related to the study drug. There were five events that were reviewed by our independent safety monitor and felt to be unrelated to the study drug. They were all felt to be related to a poor fitting prosthesis and, or changes in the residual limb volume, but they included things like cellulitis, some pressure ulcers, abscess, even one abscess that required an IND. So there were some severe things, but again, they were reviewed by an independent monitor and they were felt not to be related. And then we had just two events that were felt to be related to our study drug. But both of those subjects elected to continue with the study. So one was just a rash on the residual limb with some mild blistering. And when we later broke the blind, we found that that subject was on placebo. But before that, we reviewed the application instructions with the subject and this person elected to continue and continue the rest of the study without any more issues. And then another one, interestingly reported that his beard area was itchy during the periods one and three, which is when he was taking the active drug. But again, he didn't want to stop the study, but we think that perhaps he maybe transferred it to his beard area during, after applying and maybe didn't wash his hands. All right. So just to sort of summarize this part, we feel as a team that aluminum chloride is a reasonable first-line treatment for hyperhidrosis in our patients who have amputations who use a prosthesis. It may decrease the HDSS by around one point, and that might be enough to provide some of these individuals with enough relief. It's also inexpensive. It's well tolerated. It is easy to apply. You don't have to get a bunch of injections into your skin. And very few people had adverse reactions, at least in our study. And I think we minimize many of those by doing a very good job of explaining to our patients how they needed to apply this solution. But for those of our subjects who do not have adequate response, we would consider the next step for treatment. And so at this point, I will turn it over to Dr. Chris Duncan. Hello, and thank you. Let me get my video going here. Hmm I'm just going to have to restart zoom, it won't let me share according to privacy, but I will just read over join in 30 seconds to shortly. So while we wait for Chris, I just want to do a big thank you to Colby and his team for putting this presentations together. We've, you know, we were very excited to hear new topics and for all of those that are part of the clinical community, we want to welcome you and make and make sure that you let us know what you think of these topics and what we can do on next events to make sure that the topics are relevant to your everyday clinical practice. I also want to remind you that there will be an in-person event next week at the AAPNR meeting. Please look at the program is scheduled on Thursday morning. So we'll be very glad to meet with you and say hello and again, have some conversations about the topics that are important for physiatrists who practice with patients with limb loss. It looks like Chris is back. So I'll let him proceed. Chris, whenever you're ready. Presentation looks good, but you're muted. There we go. Thank you so much for your patience, new computer, new operating system. So I wanted, thank you so much for letting me join in this conversation about the importance of treating hyperhidrosis in the amputees. I particularly appreciate Colby, Dr. Hansen's recommendation about asking the patient for what's important to them and how they rank order their priorities. Just as a quick overview, botulinum toxin works by inhibiting the SNAP25 protein at the presynaptic cholinergic nerve that then prevents the eccrine glands from releasing sweat. It's not as effective for apocrine glands, which have a sebaceous content. The schema is over on the left. And a reminder is that we are host to a broad microflora of bacteria and other fungus, which is only exacerbated and changed when incorporating a prosthetic. So the benefits of intradermal Botox is a second line treatment for amputee associated hyperhidrosis is to hopefully reduce the quantity of sweat improving the activity and thermal envelope in which a person feels comfortable, improve prosthetic fit and function. I'll share that data with you at the end and improve residual limb ulcer healing. We published a case series this year focusing on two Paralympians looking at chronic non-healing ulcers that were recurrent over years that we ultimately decided to apply botulinum toxin for in the surrounding areas, not in the wound bed itself. And we saw subsequent healing over the next few weeks, which was fairly dramatic and felt that it was worth sharing. Malodor reduction, this I believe helps improve embodiment of prosthetics. And then a consideration that I hadn't thought of until I talked to my prosthetic colleagues was that especially in expensive upper limb prosthetics, there's a fair amount of salt intolerant electronics and many upper limb prosthetics have been invalidated just because of saltwater incursion into the electronics costing 50, 60 to 100 K in damages, which is sometimes unredeemable. And so I referenced that we had a case series earlier. This is the same kind of clinical presentation that started this pathway down investigating whether or not botulinum toxin could help heal wounds with Dr. Gonzalez-Morales and Dr. Pasquina and others starting in the early 2000s. And the wound closed quite dramatically. You'll notice it's over the incisional area where friction and shear forces accumulate. By reducing the hydration of sweat alone in a water impermeable membrane, we reduce the chance of maceration. We know maceration is an inhospitable environment for the stratum corneum and for the dermis below that. And we know from mechanical studies that the stratum corneum is thin and disassociated from the underlying epidermis. Temperatures influence it and make it worse and likely contribute to dysbiosis, the perturbation of microflora that is usual for skin bacteria. And then of course, with micro-injury, there's a risk of bacterial incursion leading to cellulitis or flares and other chronic skin conditions like eczema, psoriasis. And there is no strong rigorous data for a prospective cohort of healing prosthetic residual limb ulcers. So the closest analog in my mind was to look at diabetic foot healers, foot healing, and as it relates to maceration. And so this is a plot that looks at degree of maceration and change in surface area over time. You can see that maceration was negatively correlated with healing. This is just a further evidence to support the clinical suspicion that pressure accumulates in known areas at the distal end of the tibia and inadvertently across the distal anterior margin of the incision. Notably, that scar tissue is often devoid of elastin and therefore is at higher risk for tension-related injuries due to a lower Young's modulus. It's always reaffirming, of course, to find convergent treatment. This is a perforated liner. This is the effect of a perforated liner on the left before application, and then later, three months of use. You can see the skin. We're deeply attuned, of course, to looking at skin and noting its quality. And on the right, there's a pretty dramatic evolution over time. And so this is another approach. As far as I know, there are no commercially available perforated sockets other than at least in the gel format. I had mentioned earlier about dysbiosis. This is kind of a seminal article about the skin microbiome from 10 years ago, but it shows that in these microclimates, much like Dr. Hansen alluded to, being an arid environment in Utah, there are other environments on a micro scale that harbor different bacteria. And the populations are quite different, as seen here by the families of bacteria, by location. And then on the right, there is a histogram of different bacterial colonies as they relate interpersonally. So different persons harbor different bacteria. We know from other applications of onobotulinum toxin for axillary hyperhidrosis that a decrease in malodor, a decrease in bromohydrosis is achieved after weeks generally. And in studies looking at dysbiosis and the types of microbiomes present in below-knee amputees out of Africa, we see that there is a reduction. This is the graph on the right. We see that there's a reduction in diversity of bacterial colonies. This is a weak study, certainly, but it's the only one of its kind that looks at, as far as I know, other than in osseointegrated populations, that looks at number of bacteria in an area. It could be, the design could be much broader, of course. But what we see is we see in a lot of dysbiotic states is a reduction in bacteria so that we have a less fit microbiome by diversity. I had mentioned prosthetic corrosion. These are just some of the pictures I was able to glean from practice. On the left, it's a pyramid that's corroded. And in some of the center pictures, those are transradial prostheses that have terminal wear due to saltwater incursion from hyperhidrosis. Pivoting to the tangible aims of this discussion and an extension of the DOD grant with Drs. Hansen and Godfrey, the last arm was to look at injection of botulinum toxin in the residual limb at 100 units per four milliliter dilution. Usually, it required a fair amount of injections, 50 to 100 at 1.5, one to two centimeters apart. And each injection, of course, was a small fraction of the total volume. Mostly to try to, of course, engender repeat customers, repeat patients, we avoid the sensitive or inflamed areas. This is a makeup of the demographics for those that failed the application of aluminum chlorine. Firstly, we see that patients are approximately 50 years old as a preponderance of males, kind of matching some demographics, especially if favoring trauma, as we'll see a little bit later. Baseline HDSS score is moderate with fair amount of severe and level of amputation favored the epidemiology of amputation below knee. Baseline sweating interference with prosthetic function, this is quite telling. It's a four and very high baseline sweating interference with prosthetic fit was not included here, but it mirrors sweating interference with functioning in many ways. Cause of amputation, this is a little abnormal, I think, from epidemiology and may limit the generalizability of these results, but acute traumatic amputation was the largest group followed by infection and then microvascular disease, presumably with lastly macrovascular disease at 20%. So it's a representative sample that we looked at over the course of two and a half years. These findings are fairly dramatic, right? The HDSS score at baseline at time zero was 3.08. And then you can see that the HDSS scores at eight weeks had an ADIR at 1.65, a statistically significant change relative to baseline with a P value of 0.001. And that's true of all time points. I'll flash a table here at the end to just confirm that. But what we see is a very strong statistically significant trend towards improvement in the HDSS perception. It's looking more between interpersonal variation and duration of effect is a potential aim for future study. But as we see, there is effect lasting out 20 weeks. Prosthetic fit after Botox has a similar shape of a different magnitude, certainly. So remember that a scale of four is moderately to severely affected. And as early as week four, we see a drastic improvement following up by week eight, again with our ADIR, most efficacious then. And then peaking out to week 20 where we're still seeing a result. Function, again, I mentioned that the curves were mirrored. Here we see a significant drop in sweating interference, which translates to more activities, more use. And then just to confirm, just so you have solid numbers to rest your confidence upon, we see a P-value of less than 0.0001 on all four of those timelines. And that is true for every single time point reviewed here. Very, very strong findings. And it lays the framework for additional research with a larger group. I wanted to address a few things because beyond clinical efficacy, I think there are limitations for adoption that kind of need systematic consideration to better apply this therapeutic treatment. Firstly, cost. The cost is for 400 units where patients usually require two to 400 units. It's $2,400, which is beyond the reach for many people, especially the lower SES of dysvascular populations, requiring prior authorizations, time, procedural discomfort I'll touch upon in a moment, but it's not trivial and dissuades patients from returning. Time cost, assuming that pre-physician or pre-provider preparation takes 10 minutes, and an additional 20 minutes to inject. The CPT code as it stands for chemo-denervation of an extremity is not as beneficial as it could be, I think. And then lastly, I wanted to touch upon this non-trivial consideration that patients remember the most. And I think that we need to mitigate strategies for perception of pain. EMLA, or as it's known generically, prilocaine, lidocaine, 2.5%. In combination with ice has proved effective, but there are no great robust data sets to support that. It's just clinically been indicated with a few case series. In the dermatologic literature, there is a new modality of delivering on a botulinum toxin through the CO2 laser, which requires 50% more drug, but it is less painful. And the magnitude of the effect is not clearly published, but approximately 50%. Reduce the number of injections, considering that a below knee surface area is about 1,200 squared centimeters. It's challenging, but the starch iodine test will help focus where one needs to go. Small, the smallest needle possible, I think that's self-evident. And then avoiding sensitive or inflamed areas, such as the tibial crest, the patella, and the distal, very distal margin of the residual limb. And of course, anxiolytics could be helpful to tolerate the procedure. I have some, just some acknowledgments, and I'm grateful for the Department of Defense for pursuing these lines of research, because it contributes to the greater body of knowledge and to getting warfighters back on their feet in a meaningful way. That is my presentation. Thank you very much. Thanks, Chris. That was great. Sorry you had the technical difficulties there. I switched to a Mac all day and nothing got me. Let's see. Let me have you stop sharing. Great. And then I will re-share my screen. Okay, so I'm gonna finish things up here with a brief discussion on starch iodine testing, what it is, why it should be thought of in this context, and just some logistics. So again, the same disclosure, as Chris just finished talking about botulinum toxin can be quite an effective treatment for this problem. But when we think about amputees, as opposed to treating an axilla or a palm, the obvious challenge is surface area. So botulinum toxin is generally recommended to be injected every two centimeters in the area of interest. And if you take a standard kind of acid, average residual limb, like I've shown here on the left, you can take some simple measurements and you can estimate a surface area. And we did this with all of our subjects in our studies. And the average surface area was around 1,400 square centimeters, which if you followed the dosing guidelines for botulinum toxin, you would need over 1,000 units of drug to cover that entire surface area that would be covered by a liner or a sleeve or something along those lines where the hyperhidrosis might accumulate. So that's not really tenable in a lot of situations. A, it's many, many injections. Every hundred units is, 40 pokes. And so there's a discomfort cost there. There's a time cost there, et cetera. So some questions that that poses, do you really need to place botulinum toxin everywhere and do patients sweat in a diffusely all over or more so in focal areas? And so this was the subject of some of our study. There is this procedure called a minor starch iodine test. It was described by presumably a dermatologist, Victor Miner back in 1928, the original publications in German. So I don't speak or read German, but it's really a rather simple test, which is great. Basically you apply iodine over the region of interest and then let it dry. And then you place a corn starch on top of that with something as simple as a makeup brush. And then you allow the area to sweat. And what happens is the sweat will react with the starch and the iodine. And it'll turn dark. It'll turn an obvious dark color. And so then you can have a higher degree of confidence that you're targeting the area that needs it the most. Could the starch test be used as a semi-quantifiable indicator of severity? So this was proposed by some clinicians in 2010 in this publication. And in this picture, I think is a nice illustration of this idea that you can have a spectrum of a reaction on your starch testing from obviously no reaction to starting to get just a very, very light discoloration, discrete sweating occurring to then it starts to become a little bit more prominent and so on and so forth on down the scale. And so they proposed this sweating intensity visual scale or SIVS as a potential tool to indicate the severity of sweating. So how do you do the starch test on an amputee? Was one of our early questions. Because simply putting the iodine, the starch on their residual limb and letting them just kind of hang out there in the room, that is not gonna generate a sweat reaction, at least in most people, unless they're super heavy sweaters. So you need to kind of get them moving, get them walking, get them doing some activity that will generate some sweat. And so we played around with some different approaches as to how to do this without making a mess of sweat and starch and iodine getting all inside their liner. We thought, well, maybe we can cover the limb with something as simple as a plastic wrap, saran wrap. And which is a little cumbersome and a little too effective, as you can see here. Within minutes, the entire limb turned completely black. And we did a handful of subjects kind of playing around with different methods. And this was a very common scenario that played out for us. And so we switched gears and we thought, well, that's probably not the best way. So what we did is we started to apply a prosthetic sheath. It's thin enough that it isn't too bulky and still allows them to kind of wear their prosthesis in the same manner as they're accustomed to without having to do too many adjustments. And as we'll show here, it turned out to be fairly effective. So here you see, you know, painting the limb in iodine there on the left and then in the middle, sort of what it appears when you try to dust it evenly with cornstarch, which making an even dusting is actually a little trickier than you might think. But then placing the sheath on top of that. And so then what did we observe? Well, we observed that in many patients, as you can see here in these pictures, first of all, you can see these areas that start to turn black that are reflected on both the skin and on the sheath. So in some ways that sheath sort of created a little bit of a map for us of where the sweat was occurring. And we observed that it didn't in fact sweat everywhere. So this is the back of the limb of that same individual and it's pretty clean. There's not hardly any reaction there as opposed to the front of the limb, around the knee and down below the knee, you definitely see sizable patches of reaction. So our approach then, take off the prosthetic, dry the limb if needed, apply the iodine to the entire limb or the area of interest and let it dry. Then you dust it with cornstarch using a makeup brush. That all takes just a few minutes to do. Put on their sheath and then put on their prosthetic and then ask them to walk or do some other activity. For some individuals, they prefer to sit at like a recumbent bike type of a setup in our gym or some other activity for about 15 minutes. When we did this initial pilot work, we sort of settled on 15 minutes as kind of the ideal time that wasn't too long, but it was long enough to kind of get that initial sweat reaction going. Then take off the prosthesis and inspect and mark out your areas. So let me show you some examples. Here we have an individual, one of our control subjects, HDSS1, meaning they had reported no interference with sweating. This is after we had them walk. There's no reaction at all, either on their limb or on the sheath. And so that's a good example of a no reaction to the starch test. This individual reported an HDSS of two. So mild sweating, some interference, some occasional interference with their daily activities. And you see here, it's a little hard to appreciate. It's not super dark, but it again illustrates that just the initial kind of bit of reaction with some sweat production there around the knee and a little bit reflected on the sheath. Not super dramatic, but there's a little bit of something there. This is an example of one of our subjects who reported an HDSS of three. So more moderate sweating. And you can see here, definitely pretty obvious large patches. This is around the front of this person's limb. Pretty large patches, a little darker, certainly more noticeable than the previous example. Again, reflected on both the skin and on the sheath. And then you do have some individuals who are HDSS4 who sweat everywhere. And it's good to know that. So this is one of our patients who quite dramatic on both the limb and the sheath. Basically everything's black. And so there are some of those. They're not the majority by any stretch, but there are some that sweat diffusely. And so you're sort of left back to that original challenge of like, well, either we use a very large amount of drug or we can only use it in certain areas. But this, the whole point of this is to help facilitate the subsequent botulinum toxin procedure. So here's an example of one of our subjects. We'd kind of mapped out these areas and the reason the map looks so funky here is because we were trying to incorporate both those areas that were reflected on the skin and on the sheath. And it looks more complicated here than it needs to be in clinical practice, but for what we were trying to keep track of in our study. But you can see here on the picture on the right, I'm simply starting to map out every couple of centimeters, a little dot, where we're gonna put a little injection of botulinum toxin. And so when we're all done, we kind of have that mapped out. We have some areas that we've X'd out that we think didn't have much of a reaction at all and we're gonna leave those areas alone and then we're gonna inject into the other areas. Here's another example of that process kind of from start to finish. So this is one of our first subjects. I don't remember what his HDSS score was, but to his starch test, you can see there in panel A, some definite areas of reaction, which we started to mark out in panel B and then with that area marked out, mapped out our botulinum toxin procedure. Panels D and E show the same subject coming back four weeks later and doing the same starch test and showing a pretty clear reduction of the starch reaction. There essentially is no reaction indicative of producing an anhydrotic effect from the procedure that we did. So that's, I like that example, kind of showing that process from start to finish and also the beneficial reduction in sweating. So some things that we've learned along the way. Individuals usually don't sweat diffusely inside their prosthesis. Some do, but most don't. Starch testing with the method we described is well-tolerated. There are very few reports of causing discomfort. If they were, they were just very temporary, minor kinds of issues, but nothing that led to a significant problem. Some of our validation analyses comparing starch test results between subjects with HDSS 2, 3, and 4 compared to group of control subjects are ongoing, but early results look promising that there is the ability to, there is some correlation rather with our starch test to HDSS score. From start to finish, I would estimate that starch testing takes about a half hour to get everything set up, have them walk, come back, take things off, mark it up, and then identify those areas. And so if you combine that with the chemodenervation procedure all in one visit, it is rather lengthy. And as Chris alluded to, that is one potential limitation, I think, for clinicians to be eager to adopt this. But I would, this procedure works and it helps people tremendously. So I would encourage folks to kind of overcome some of those maybe hesitancies and try to adopt this in their practice. I'd encourage you to take good pictures so that you can use them for future reference. Oftentimes, we get a good result with our initial starch tests and kind of create that map with some good photography. You can use that as a reference for subsequent injections and spare you that time. There is a CPT code for kind of a procedures, well, in quotes, procedures in the integumentary system that do not have a specific code. We have tried to use, well, we have used that code for our starch test procedure. Then there's a separate code for the botulinum toxin. Because most of our experience with this was under the oversight of our grant, I don't actually have much knowledge to give as far as does this procedure get paid or not when you bill it. But anyways, that is what we have to share about hyperhidrosis. This is a picture of the Craig Nielsen Rehab Hospital, newly constructed a couple of years ago. It's a beautiful facility here on our health sciences campus with the Wasatch Mountains there in the background. We'd be happy to take questions for myself or Dr. Godfrey or Dr. Duncan for a few minutes. And then I think we're segwaying into a break. Thank you. Thank you to our presenters from today. Feel free to unmute your microphone if you have questions or type them in the chat. I'm really interested since I don't see any questions of Colby to understand what has happened after the conclusion of the study, like have patients returned for botulinum toxin injections and how much is your dropout rate because of the number of injections? Yeah, it's a good question. There were certainly a few candidates that we had that were a little reluctant to try the procedure because of that kind of fear of the discomfort. In terms of once they get it done, I can think of one individual who, in spite of it being helpful, didn't wanna do it again. That's my personal experience. I don't know about Dr. Duncan or Dr. Godfrey, but many of them are coming back now. In regards to coming back, you do have some really heavy sweaters who are lining up to do this every three months, but there are many who get a good six-month benefit from this or longer. And so they will often try to come in around June, kind of when it's starting to heat up, get treated once, and then they're good for the hot part of the year. And then the problem isn't so bad for them during the cooler part of the year that they only do this once a year and they're very happy with that. Great, we have some. I'll just add really quickly, because I probably do the most of this clinically. I'm at the VA, so I can get our pharmacy to approve Botox in people who have failed dry saw. And so I have probably 15 veterans that I do this on regularly. And I have a few that do it every three months because they sweat quite a bit. I have a few that come in in like May and June when things are starting to get hot. I have one on my schedule this morning, bilateral trans-tibial Paralympian. There was a question here in the comments, are we using 30-inch, half-inch needle tips on angle? I think 45 degrees is appropriate. I think that the risk is back pressure usually if you're not deep enough below the stratum corneum and you'll have it just escape out your injection site. And then I think just to echo Dr. Godfrey's thoughts is that patients are believers in this treatment more than anybody else. And so them incurring the cost of pain, travel, time, and become repeat customers is really indicative of the treatment's efficacy. Thank you. Dr. Carr? Have you guys noticed that patients that you do it more than one time on, it seems to last a little bit longer, kind of like a compounding effect or maybe some residual from the last time they were injected? I have noticed that. I have a particular patient who has a trans-radial amputee, has a myoelectric prosthesis, and we were doing him every three months for quite a while. And then we were able to space it out to like five to six months. He's really active and exercises a lot. So he would notice when it wears off. So yes, I think that can be possible. The axillary literature says four to nine months for efficacy. So I wonder which time point that was with initial introduction or later on and whether or not those time periods were averaged. But good question. Yeah, there is also a question about dilution. You mentioned 100 units per 4ml. Is that what you all typically are using? Yes. Yes. That was a quick answer. Yeah. And then you inject like a, you put them in 1ml syringes. And so you can easily inject a 0.1ml kind of aliquot into each spot, which is two and a half units per site. Any other questions? I've got another question. I've done a good bit of Botox down here at UAB. How do you guys prevent leakage? I've noticed a lot of leakage when I'm injecting, you know, multiple spots, and I'm not sure if I'm going... I mean, I get good results, but I'm wondering if maybe I'm not deep enough. You know, I try to do it pretty superficial when I go in. But have you guys noticed a lot of the leakage just when you're coming out? I have, yeah. And I think that's unavoidable, especially if you're going to try to make this commercially successful, if you don't have the luxury of practicing at the VA or under a grant, I think you have to move quickly and you're going to have some leakage. But I think that you can't contest good results though, knowing how much you... There's potential there for sparing some costs and drug costs if you had less leakage, but I don't know. I don't know if it's clinically meaningful. What do you think? Yeah, I think it does happen. I think most of the time it's like a drop at every site. I feel like I'm losing just on the surface of the skin, but sometimes there is more like a little squirt that sort of comes back out of the track. Yeah. Some areas of the skin are just, I don't know. I don't know how it... When you do this on different areas of the skin, you just notice that some areas seem to take in... You take it, you know, the solution in easily and some do not. So, yeah. I'm done. I'm sorry, go ahead. I was just going to say, I typically do a one to two dilution and just spread it out. I haven't done the starch iodine test just because I'm so busy. I wouldn't have time to do it. Yeah. If there was a faster method, I could do it, but I have really, really good results. I mean, I think... I don't know how many I've done, you know, probably over a hundred and probably only one or two have said that they had poor results. Yeah, it does work. And, you know, patients may have enough kind of insight to, and I think if you direct them to kind of reflect on this, they could probably help you self-identify where they feel maybe they need it the most. And if you look at the hyperhidrosis treatment algorithms produced for like axillary hyperhidrosis, they will recommend, you know, first line aluminum chloride or the glycopyrrolate clots, second line botulinum toxin. And then if that's not effective, then kind of reconsider, re-evaluate with starch testing or, you know, something along those lines to try to hone in on where maybe it's most beneficial. I'm not sure if you guys touched on this. I may have missed it. Sorry for all these questions, but it's a great topic. I enjoy it. I actually have hyperhidrosis myself and during the interview trail early on, mostly Palmer. And so I've actually had Botox years ago when I was in the application process. So it's kind of near and dear. I understand what they're going through, at least from a Palmer perspective, shaking people's hands and it's sweaty and that kind of stuff. But anyways, I was wondering, do you guys recommend that the patients continue to use their DrySol or other formulas, I guess, after they've done it? For mine, I just kind of say, you know, if you start to notice it's wearing off, you know, start, you know, you can use the DrySol or some just prefer just to kind of continue to do it to get back some more relief. Yeah, I think that's a reasonable approach and I don't, I neither discourage nor encourage that, but yeah, I think that's, to me, that's, I don't know if Brady or Chris feel otherwise. Yeah, I don't know. I don't think comboing them because their mechanisms of action are so different as any real risks, although, you know, it's not speaking empirically. Thank you. Thank you very much to all the presenters and for the very thoughtful questions. I think we are due for a short break and we have Dr. Garza here, our next group of presentations address different topics, starting at 2.30 with a presentation on new techniques to try to transform skin. Very interesting, stem cells type of work and Dr. Garza will start us and then we will be talking about phantom limb pain surgical management and also integration. So take a quick break and be back at 2.30. Thank you, Luis, for joining. Thank you. Well, welcome, everyone, again. It is my pleasure to continue the session with the next presentation by Dr. Luis Garza, Dr. Garza's Professor of Dermatology, Cell Biology, and Oncology at the Johns Hopkins University School of Medicine, where he consists areas of clinical expertise, concentrate on hydrodinitis separativa, alopecia areata, and general dermatology. But in addition to treating patients, Dr. Garza runs a molecular biology lab that studies skin, stem cells, and wound healing, with an emphasis on identifying the next generation of wound therapeutics and diagnostics. And thus, he's bringing some of his findings to us today. So thank you, Dr. Garza, for joining us, and the floor is yours. Thanks, Dr. Gonzalez-Fernandez. Thank you for the introduction. I'll share my screen here. Let's see. Okay. Great. Does that look correct to you guys? Is it the normal view? Yeah. Just changed. Thank you. Super. Okay, guys. So in the next 20 minutes, I'll tell you about some cool research. Hopefully, you'll agree on work we're doing to try to help out amputees. It's a real pleasure and honor to be here. And please interrupt me if you guys have any burning questions, and I'll answer questions at the end. So what really excites my lab is this idea of, can the body go under morphogenesis again as an adult? Our inspiration is a salamander, of course. And today, I'll talk to you about what we can try to do if we can't regrow a whole limb, at least today. So the problem, of course, is amputations are increasing. We certainly know that diabetes is increasing, and that's a major cause for amputations. And you guys know better than I do that, even at its best, if you just ask for a routine upper limb amputee how often they use their prosthetic, it's not as often as one would want. I feel somewhat sad if somebody told me I could only use my arm for 24 days out of a month. And this is even outside of normal problems. And we do know that there can be like skin breakdown and a whole bunch of other associated difficulties at the stump site. And we want to try to address that problem with this question. Can we create new palmar plantar skin at the stump site? Because you didn't think twice when you put on your shoes this morning, because your skin is adapted to bear weight at the palms and soles. I joke that 500 million years ago, we crawled out of the ocean. And the moment our fins touched the land that started 500 million years of evolution of like adapting to pressure at these palmar plantar sites. So the question is, can we imbue some of those properties at the stump site of an amputee or at the distal end of the residual limb? So the goal then is to kind of convert from this normal fin epidermis we have on our skin to this thick type that we see on the volar sites or that is palmar plantar skin. And there's unique keratins like this keratin 9, for example, that is like a steel cable that helps impart some of this friction resistance and irritant resistance. And how to accomplish this? Basically, there's a super cool paper that was published a long time ago. It was in 1966. This is from the New England Journal of Medicine. And this really smart guy, Dr. Silvers, was doing these combinations where he put epidermis together with different dermis. So there's two parts of the skin. There's the epidermis and the dermis. And he would mismatch them and ask what would win. So interestingly, even if you used different types of epidermis, like from the sole or the ear or the trunk, as long as he used sole dermis, the final phenotype of that epidermis, the final appearance of the epidermis was like the dermal component, like the sole. So he basically showed that when you mismatch epidermis and dermis, the dermis wins and sole dermis can reprogram the epidermis to make it thick. And so that was an inspiration for what we're working on. The question is going to be, can we inject like molar fibroblast stem cells into the dermis, just like kind of Silver did, Silvers did, and then have those cells reprogram the epidermis to make it thicker and more friction resistant and irritant resistant. And so the general idea is, can we have a fibroblast cell transplant at the distal end of the amputee to try to improve their weight bearing. And so the first question is like, well, are these cells special? And I'll show you guys some cool like videos where you look at that, they even like move differently. So these are volar cells and you can see they've got this like cool, like slingshot movement where they move. And then the, I'm sorry, then the non-volar control is on this side. This is how normal cells move is like, they're kind of a steady kind of movement, but you can tell it's clearly different. The volar cells, they're smaller and they have this cool like slingshot movement, like where they'll jump. So they definitely look different, even just the way they move in a dish. Here's like a neat time-lapse video where we took pictures with our collaborators, Dr. Chen at bioengineering. We took pictures like, you know, every second or so, every several minutes. And for normal cells, you can see you capture kind of a normal kind of progression of those cells, but you could tell with the volar cells, there's this gaps where they go through this really rapid movement. And so that's the kind of that slingshot migration. Another cool thing is like in some work we're replicating now is if you put cells under pressure, these volar fibroblasts look like they divide more. So when, you know, in control situations, they're more or less similar. The volar cells do divide a little bit more, but when they're under pressure, they seem like they were dividing a lot more. So like I mentioned, then the goal is the question is, can we inject volar fibroblasts in the dermis and have them re-change that top layer of the epidermis? So, you know, what's a good marker for changing this? This is just looking at normal pomoplantar skin and trying to say, like, what's a good metric for improvement? And this is asking how much increased is a particular gene in the pomoplantar skin. And this is the inverse of its P-value, so how significant it is. And this one lone red dot is this one gene called keratin 9. And it just basically means that keratin 9 is a protein that's significantly upregulated in your pomoplantar skin. It might be how it has that resilience and strength. So the first question is, well, can we induce keratin 9 with these special cells, with these volar fibroblasts? So this is looking at mRNA and protein, and we're taking keratinocytes from foreskin that we collect from the newborn nursery. And then we're adding different fibroblasts to them in vitro in a dish. And we can see that if we add sole fibroblasts, we can induce this keratin 9, which is really exciting. And then we started looking at a mouse model. So here we take paw fibroblasts from a bioluminescent mouse, and then we transplant them into immunosuppressed host to see if the fibroblasts can change the skin. So here we took these volar fibroblasts, and then we injected them back into the paw or into the ear to see, can we partially turn the ear into a palm-like skin? And this first just shows you the engraftment was good. We could see that nice bioluminescence continuing over time. And then we stained for that keratin 9, we could see some good, nice keratin 9, and the skin does look thicker in the ear after injecting these fibroblasts. So it looks like it works in vitro. It looks like it works in mice. So then we next said, well, can we bioprint skin with this different type of cells? Does that work? So if we bioprint like a skin using a machine, does the skin look more like volar skin if it has those special fibroblasts at the bottom? So we use these neat bioprinters that kind of layers one layer of skin at a time. And we had it layer either volar or nonvolar fibroblasts first. And then at the top, we put in every condition, we put the four skin keratinocytes. And then we let that bioprinted skin mature in a dish. And then we took sections of it to see what it looked like. And so this is the bioprinted skin. If you use nonvolar fibroblasts, you can see the epidermis is more thin, but if you use the volar fibroblasts, you get a thicker bioprinted skin with a thicker epidermis. So again, this is nice proof that those volar fibroblasts can make the skin more thick like in a human bioprint. So this is just the quantification of those results. So showing that like if you look at epidermal thickness across three people, it's significant. If you look at the keratin 9 staining, that went up too. And if you look also something I'll mention later, the cytoplasmic size, like the cells look bigger too after you inject those cells at the bottom. So the cells from the bottom, those fibroblasts are chaining the cells at the top to be a thicker layer of tissue and also bigger cells, like bigger cytoplasm, which is pretty cool. So summary so far is that the volar fibroblasts are unique in vitro, and they can induce keratin 9 in vitro in mice and in bioprint. And so the next question we said is, well, what will they do in humans? So luckily we've had some grants from the Department of Defense because they're very interested in trying to help some of our wounded warriors from Iraq and Afghanistan who have had limb loss. And so they've allowed us, them and the National Institutes of Health, NIH, and also the University of Maryland, I'm sorry, State of Maryland Stem Cell Fund have also funded us to try to do this in people. And of course it took a lot of regulatory approval. But the question was, if we inject these volar fibroblast stem cells in a people, can we induce keratin 9 and can we see any tissue changes? So this is what the protocol we did was, is we biopsied the sole, first on healthy subjects, I'll show you that first. We biopsied the sole and the scalp of healthy subjects. We grew up their fibroblasts for about two months in the lab to expand their numbers. And then we injected just into an ectopic, a new location into the buttocks or thigh, vehicle only, sole fibroblasts or scalp fibroblasts. And we waited five months. And then we injected, we excised the skin, we injected, and then we did a bunch of tests on it, like histology, immunofluorescence. And then we also did bulk and single cell RNA sequencing that I'll tell you about. So this is what it looks like. The cells are just kind of a slurry, like a gray kind of slurry. So it looks just like a cloudy solution. And the first, one of the first things we did is look at skin firmness. And here's looking at vehicle and vehicle doesn't really change skin firmness too much. But if we look at either the ones that were injected with the scalp fibroblasts or the sole fibroblasts, we can see a nice increase of skin firmness. So that was a good indication that we're changing the tissue architecture by making the skin more firm by adding these cells. And next, we said, well, what about that keratin 9 marker? And this was one of our best responders. We found normally there's not much keratin 9 in humans outside of the palm and sole. But when we inject these fibroblasts, we can see really nice increase. It's in the right location too. It's normally, it should be super basilar, like above the basement member, above the basement memory in this, above that, above that first layer of cells. And we can also see the cell size is bigger. That cytoplasm is bigger, just like we saw in the bioprint, for example. So that was a good indication that you're working. This is averaged over a lot of subjects now. We've done more than the 30 subjects. And we can find that the, this on the left is just your normal native skin showing that keratin 9 is normally higher in your volar surface than your non-volar surface. And then after injecting in these humans, of course, there's a lot of spread because people are, you know, everybody's different. And so it's not as tight as a bioprint. And, but we can see a statistically significant increase in keratin 9 protein and mRNA in our subjects after injecting the cells. So then we took a look at epidermal thickness in our human subjects. And again, comparing our normal volar skin has much thicker epidermis. But we can see an increase of that epidermal thickness when we inject the sole fibroblasts in our subjects. So that was nice to see too. And then we looked at that cytoplasmic size in our human subjects to see if the cells get bigger. And again, we know the cells are bigger in our volar skin at the epidermis. And then after injecting these fibroblasts in the bottom layer of skin, we could see that cytoplasmic size also increasing. And then we looked at collagen length, like using second harmonic generation and collagen is just like a steel cable that lives in like your dermis to provide strength. I mean, it's normally those collagen bundles that are longer in our palms and soles. And then we can also see after injecting those fibroblasts, we could see a nice increase of that collagen length as well. So basically to summarize so far is I've shown you that the, after injecting these volar fibroblasts down here, we can change the, change it oops, so the collagen gets longer. And then we can also, we make the cytoplasmic bigger in the top layer and there's more keratin 9 and the skin is thicker. So what about, what about gene studies? Like, so next we did a single cell, we did RNA sequencing rather. So we just took all that skin and we assayed like every single gene that was upregulated. I mean, every, sorry, every single gene that's present. And we asked the computer and we said, oh, you know, if we, these are the results, these are the genes that were up after our cellular therapy. Like what does it look like to you computer? And these are some of the structures it's called a gene ontology analysis. So the computer spit back that what we're doing is we're doing anatopical structure morphogenesis, a regulation of cell differentiation, tissue development, animal organ development, which is really cool. So a computer looked at the genes that we turned on and says, yes, this is morphogenesis, which is great. Cause that's how I, the computer didn't know that I start off my talks, like pictures of a salamander. And I like, we're trying to do morphogenesis in people. Cause that's, that's great. Cause that's what the RNA sequencing is telling us we're doing. So that this is what we call bulk RNA seq where we're sequencing like every single, all the genes together as a group. I mean, all the cells together as a group. And then we did single cell RNA sequencing where we take the RNA sequence from each individual cells. And this just goes to show you that what we think the fibroblast is doing is it's acting on cells as they leave this top layer, this bottom layer rather, and they go towards the top layer. So that's where we think the fibroblast is acting on. Cause if you look at, this is just like what we call principal component analysis. So just saying how it's like a mathematical test of how similar or different cells are. And if you look at those basal layer keratinocytes, whether we inject the scalp fibroblast or the sole fibroblast or vehicle, they're all kind of clustered together. But if you look at the super basal keratinocytes, they really start to spread out. And so that's where we think these cells are changing is at this kind of event where, where the bottom layer keratinocytes are maturing upwards and that's where they're acting. So based on these positive results, we're starting to test in amputees. And so if you guys have any referrals or any amputees you think might need help, like send them our way here at Hopkins, we're happy to enroll them in the study. And I can just close off by telling you who contributed to the work. This was done work here at Hopkins. And this is the Hopkins team. It's been really grateful for Mark and Marliss and Ed's help for recruiting patients. Yuan and Junjie are the bioengineers who helped us with some of the cell characterization. And Donghuan did a lot of the work on the keratinine. And then these are some of our DoD collaborators that we're really honored to work with. And yeah, so I can close for any questions. Thanks so much for your attention. Thank you very much, Dr. Garza for that presentation. I think we'll have, we'll have a few minutes for questions before we move to Dr. Shores. Any questions? It's nice to hear that we're not talking about regeneration in sci-fi movies anymore, that we are trying to move the needle to better understand how we can regenerate. And again, starting with the skin and perhaps we'll move back all the way to the salamander. So thank you. Thank you, Dr. Garza. Thanks for the invitation. Thanks guys. Thank you. Well, and our next presenter, which I'm glad to see was able to make it in person because he's on call for the surgery services, Dr. Jamie Shores. He is associate professor of orthopedic surgery and plastic and reconstructive surgery at the Johns Hopkins University of Columbus School of Medicine, where he's part of our limb loss rehabilitation team as the main self-tissue surgeon. He is also the co-founder and clinical director of the hand-arm transplant program here at Hopkins. So without much further ado, Dr. Shores. Your mic is muted. Your presentation looks great. Sorry about that. Thank you. Can you hear me now? Yes. Thank you very much. Go ahead. All right. Thanks, Marlis. So I'm talking about surgical techniques for the treatment of refractory neuroma and phantom limb pain, and I have nothing to disclose here. So I'll just start with a quick discussion of the current state of amputation. Dr. Garza is already kind of talking about this and some of the after effects, but when we do the surgery itself, it can be done by a lot of different surgeons, orthopedists, general surgeons, plastic surgeons, and a lot of different venues, trauma, tumor, infection, vascular insufficiency, even chronic pain we've done amputations for. And in general, when this is done, depending upon the level, there are certain fundamentals to that operation that stay the same no matter who it's done by. This is an excerpt from Cameron's seventh edition of Current Surgery, and I just wrote not an amputation chapter, but a hand infection chapter for their next one coming out. But most of the steps in performing amputations are the same as they've been for decades and decades and decades, if not centuries. Some of these things, keep an adequate muscle and fascia cutaneous flap to pull over the bone. Lots of options for this. Perform a myodesis with tendons or muscle flap to the bone. So you have limb control and stability in the prosthetic socket. And then what these books still say to do with the nerves is perform this thing called a traction neurectomy with or without ligature. And a traction neurectomy is where you pull the nerve, put it on tension and stretch, cut it at the end of the stump flush with the other tissues. And then it just kind of pops back like a rubber band up inside the muscles and the rest of the amputation stuff. And hopefully it's hidden and it doesn't cause any trouble. And some people will put a ligature on that because big nerves like say the sciatic nerve and the median nerve, these nerves are the embryogenic blood supply to the limb while it's forming. And so even after the brachial artery and the femoral artery and SFA and everything formed, there's still a pretty good sized vessel in those nerves. And so they'll bleed. And other nerves who aren't the embryogenic blood supply will also bleed. They can have pretty good sized nerves so, or blood vessels. So many people will ligate these, which also just kind of hurts the nerve further. And that's pretty much standard practice. We're making inroads in education and new textbooks and stuff saying we should do other things up front with the nerves primarily, but this is still considered standard practice. So if you think about, if you think about the afferent and efferent circuits in our bodies, going back between our sensory receptors and to our muscles, and you think of the, just the, this kind of motor unit, motor neuron graphic that I have here, I think of them more as like these little booby traps that are easy for us to set in our patients, where if you give a little stimulation, it's just going to explode on. And sometimes they don't even need any stimulation because these neurons will sometimes self-stimulate. So we know that when we do these things and we treat the nerves this way, this gives us a certain incidence of chronic pain. And, you know, it's estimated between 50, 85% in this study and another study, 10 to 76%. I will say that there are studies that have pretty low pain incidents, but I don't know if those studies really focused on trying to discern pain as their primary outcome in these studies. And I think it's just, you know, it's just a secondary thing that they're looking for. And I think they also don't follow the patients very closely when they say that. When I asked my own vascular surgeons who do amputations for ischemia and infections, why don't you let me come and do something with those nerves for you when you're doing these? Because, and they say, why? I said, so the patients don't have chronic pain problems, or they at least have reduced chronic pain problems. They said, Jamie, my patients don't have chronic pain problems. And I say, well, how long do you follow them for? And they say six weeks. And then I turn them over to the physiatrist. And then, you know, then they end up in your clinics and you guys see the chronic pain problems they have, but the surgeons doing these primarily don't because they don't watch the patient long enough. So we know going back all the way into the 1980s that people were looking for ways to improve nerve pain in people. Now these aren't necessarily major limb amputations. These are more sensory nerve injuries and digit amputations, but Lee Dallin, who's from Baltimore, Susan McKinnon, who's Canadian, but is at Wash U and was chief of plastics there for a long, long time and is a world-famous nerve surgeon. They started looking for ways to try to make these smaller nerves that had painful neuromas more manageable. And they did it by just burying the nerve in muscle. And because of this paper that showed profound improvement in pain, burying the nerve in muscle became the standard of care for us. And it didn't help everybody, you know, patients who had workers comp didn't do as well, patients who had three or more prior procedures for pain, which means people, they had pain for a long time already, uh, didn't do as well, but it even worked well as superficial radial nerve neuromas, which I consider the pancreas of hand surgery. And those of you who are physicians will recall back to your general surgery rotations in medical school. Remember the three rules of surgery, eat when you can, sleep when you can and don't mess around with the pancreas, right? Uh, however, burying those nerves into muscle is just kind of finding a cushion for them. They're going to form their little neuroma. Hopefully it's in a little bit of padding and it's away from direct stimulation, but it it's, it's not reasonable to think that that muscle, which is already innervated is suddenly going to allow itself to be taken over by new innervation from this nerve that's been put in there, which means that nerve still doesn't have anything to do except grow an aroma. So my philosophy of neuroma management is that nerves are like teenagers. We need to give them something to do, or they're going to cause trouble. And that trouble for our patients is pain. And maybe you guys are better teenagers than I was. And I'm just projecting my adolescence and youth onto nerves, but I stand by this statement. I think it holds true. So my goal is to give nerves something to do with, uh, with these surgical techniques that we have. So they can include giving them back to original function. So let's say they've had an amputation and we have the amputated part, can we reattach it and just try to repair the nerve and give it back its function? If they just have a peripheral nerve injury that could just be repairing it or reconstructing it, but it can be replanting an amputated part, or it can be taking somebody who has a remote amputation and performing a transplant on them. So we're giving them a new limb. Could it be giving that nerve a new function? So we're not, we're not replacing it with what was lost. Like we were when we were replanting things or transplanting things, but we're just giving it a new function. And, uh, and that's going to be through nerve transfers or putting some kind of muscle that can accept innervation around it. Like a nerve graft is in the regenerative peripheral nerve interface, the RPNI or a vascularized piece of muscle called the, which we call vascularized direct muscle transfer, direct muscle target, medium T. There are some other strategies that people are starting to use, which we don't have great data for. And I'm not sure how, you know, in theory, I just don't see how they can work very well. One is putting these surgical caps on the nerves, usually made out of either, uh, polycaprolactone or, um, SIS, which are supposed to somehow inhibit the axons desire to, uh, regenerate and send out axonal sprouts to form these neuromas, which is hard for me to believe. And then the allograft, acellular allograft bridge to nowhere, which I did many times, uh, with mixed results. And this is putting a long allograft on. And when you put that on the end of a cut nerve, after you've resected the neuroma, the nerve will try to regenerate into it. But if it's long enough and there are no growth factors and support cells to help guide it along that regeneration just fizzles out inside of it, at least that's the theory. So we're going to talk about these other more active things. So, you know, getting back to the acute injury, the best solution may be to immediately fix the problem. So this replanting the arm and with a lot of work and a lot of operations like this girl had, we were able to get this to live, took six weeks in the hospital, multiple operations. Uh, her family got a $2 million hospital bill that they had to declare bankruptcy for because her insurance wouldn't cover it, but she doesn't have chronic pain. She has a hand that sort of works not as good as a prosthesis, like a regular prosthesis, not a super advanced one, and she's got some elbow motion. She says she still thinks we did the right thing, but some could argue we should have just performed an amputation revision. On the other hand, we don't always have that opportunity like this gentleman who presents 40 years after his shoulder disarticulation and transradial amputations. And so we need to have other strategies available. So what about transplantation? The biological prosthetic, the prosthetic that can heal itself, that doesn't have batteries that run out, that doesn't have to be donned and doffed, that, uh, if it does need repairs, we do in situ, we don't send it back to the company for six weeks hoping and, you know, asking for a loaner. Um, and then it can't be forgotten in the bathroom stall, uh, in the building that my clinic is, which I have seen before where somebody forgot their prosthesis, uh, in the bathroom. Um, so here's an example, somebody, uh, traumatic amputation from an avulsion injury, which is a very bad type of injury, eight out of 10 to 10 out of 10 pain already on enormous doses of opioids, failed prosthetic trials. Um, this is them after their arm transplant pain, not eliminated, but substantially reduced on less pain medication than they're on before here, knocking out more pushups than I can do, but not more pushups than Dr. Forsberg can do. He's recently retired from the United States Navy. Thank you for your service, sir. But that's pretty good. However, anybody who is interested in transplant, I always refer to this article by David Dobbs who, um, you know, profiled some patients from our own, my own transplant program and some other transplant programs showing that some of them do very well, but some of them don't do well at all. They can have functional and medical problems, uh, medical complications from the immunotherapy and things. And so this is not to be taken lightly, but for the right candidate, it's life-changing like this, lost both arms in a corn picker, helping his farmer friend neighbor harvest his crop, uh, trans humor on one side, trans regular on the other side. Uh, this soldier, who was the first known survivor of, uh, losing all four limbs in a combat theater, uh, you know, before he had been evacuated back to any, you know, more further echelon of care, uh, for him, this turned out to be a life-changing, uh, uh, uh, operation so that he could gain back his independence, get his life back, transhuman on one side, proximal flow on the other side. And when your surgeon will let you drive them around Baltimore in their vehicle, I think, you know, that the surgeon actually believes in the therapy that they're trying to promote, uh, you know, as opposed to kind of hiding behind things. And so I let these guys drive me around and, uh, this, this really is life-changing for the right person, but for most people, they're not going to be candidates for this. So what can we do for people who don't call, who aren't these select few individuals who fall into this category because of all the other things it takes to become a transplant patient? Well, we've got this cool thing called targeted muscle renovation. And so TMR is simply performing a nerve transfer from a nerve that used to go to something that has been amputated. And it's no longer present say the media nerve, the ulnar nerve, and the distally destined fibers of the radial nerve that would become PIN and superficial radial nerve that say in an arm that you don't have anymore and transferring those into some of the more approximately, uh, uh, approximately must, you know, innervated muscles from musculocutaneous and more proximal radial or any other muscles. So that instead of those things doing the function that they did, when you would think straighten your elbow, flex your elbow, uh, maybe one head of your biceps contracts when you think make a tight fist. Uh, and the other head of your biceps is left with its regular musculocutaneous innervation. And it still contracts when you think flex my elbow. So we can do this to all these different muscles. Todd Kiken is a physiatrist who is affiliated with Northwestern did his PhD dissertation on trying to do these nerve transfers in rats to see if they could power, uh, my electric prosthetics. He then recruited Greg Dominion, who's a plastic surgeon there to helping translate this in humans. And so they've come up with multiple, multiple nerve transfers that, uh, people can do in a standard fashion, as long as the anatomy is normal, proximal level of amputation for, you know, upper and lower extremity amputations. And the most, you know, the prototypical one is the transhumeral where we've got two heads of biceps. We'll keep one to musculocutaneous. We'll donate one to median. If they've got brachialis, we can donate that to ulnar. We'll take part of the triceps and we'll loop the more distal radial nerve that hopefully has PIN fibers back to that branch of radial into the triceps, uh, to try to get good, you know, uh, intuitive motor control with five inputs, instead of having people have to switch back and forth manually. Here's our first TMR patient for Johns Hopkins, Rick and I did this surgery, Albert Cheed enlisted us to do this. And he's got the MPL, uh, which is the modular prosthetic limb from the applied physics lab at Johns Hopkins, which in my opinion, is the most advanced prosthetic that I have ever seen. He's had osteointegration from the university of Pittsburgh. This is before Dr. Forsberg joined us and started our osteointegration program. You can do this also for shoulder disarticulation patients going to the chest wall. This was our first shoulder disarticulation patient that Rick and I had done together. And I've either I've lost computing skill or the security features have gotten much better. So I was unable to steal the video clip of this to embed into the thing, but here's the link. If anybody wants to watch this guy with bilateral MPL limbs, I think it's just incredible technology. So interesting observation that Greg and Todd made while they were doing these patients. These are, again, we're just done for motor control, but they found that 14 out of 15 patients said, Hey, that pretty severe, profound neuroma pain I had is like gone, or it's nearly completely gone, if not completely gone. And so that led people to believe, well, even if we're not doing this for motor control, I wonder if we should be doing this just for pain control and amputees as a standard, as opposed to just doing a traction erectometer, just burying the nerve and muscle, which is the previous standard that we've been taught. So to try to answer this question, investigators like Dr. Kamanian, Dr. Kiken, and people from Walter Reed National Military Medical Center in Ohio State tried to perform what we consider in medicine, right? The gold standard, a randomized prospective trial, wasn't going to be blinded, unfortunately, but nonetheless doing the best study they can design to try to discern this. One group of amputees is getting TMR. The other one's getting just nerve buried in muscle. They hope to recruit 200 patients. They had to stop after 28. It turns out most of the people who are being recruited were from the military. And when folks from the military come back with these amputations to the major military treatment facilities, like Walter Reed, they have an incredible medical support network there of professionals that we should all be envious of. But at the same time, they've got this incredible peer support, all their battle buddies who've come before them come and support them. And these guys started telling each other, hey, they're going to try to recruit you into the study. And if they assign you to TMR, that's great. You should always ask for TMR. But if they say the regular nerve stuff, you should decline that because what you want is TMR. So you suddenly couldn't recruit anymore. And the good thing though, was after 28 patients, they still had statistical significance. The power of the study was enough because the difference was enough. The 28 patients were able to show. And so when they looked at all comers for primary outcomes, the TMR group had a substantial reduction in their phantom limb pain and residual limb pain compared to their baseline at one year, much more profound than the standard of care. And on phantom limb pain, standard care didn't even have much of a change. They then went to intention to, you know, they divided all comers back out to the intention to treat without any crossover. The difference is even better. And then they let those folks who had standard of care, who didn't have great relief, then cross back over to the TMR side. They still saw a significant improvement, although not necessarily as big of an improvement as people who got it as their first primary procedure. So interesting observations, the longer patients had chronic pain, the less chance they had being completely pain-free and the lower the benefit, though they all still had a significant benefit, even if they were in the group that had lower benefit, it was still profound and well worth it. There was also no comparison to RPNI, which has also demonstrated improved pain relief from neuromas and phantom limb pain. The RPNI is illustrated here is created by Paul Sodern at the University of Michigan is taking a thin graft of muscle, wrapping it around the nerve and sewing it to itself, creating this little burrito that the nerve is, is intubated in. This doesn't have a blood supply. So part of that graft may die. And the bigger you make it, the more likely it is that you're going to have ischemic cells that just form scar. But the hope is that it's the muscle piece is small enough that it can survive by diffusion. And then a blood supply can grow into it from the wound bed and keep it alive. And what he has found is what he thinks is a three by one by 0.5 centimeters, kind of a sweet spot for the graft size. And the nice thing about these is because it's not vascularized and it doesn't require a nerve branch to sew into, as long as they've got a source of muscle, you can keep taking grafts. And so you can put a zillion of these things on assuming that the patient has good enough supply. He published his initial clinical series of these in 2016 in PRS Global Open. And what, and these patients had to have at least three months follow up. And I don't think the average was a year. I think the average is more like nine months, but they found a profound improvement in neuroma pain. They had a significant reduction in phantom limb pain, a significant reduction in pain interference scores, and their pain medication went down some, not as much as you would guess from what the data show, but maybe they had pain from other sources. So he then followed that up about two years later with more patients as still a retrospective study, but 47 RPI patients compared to 50 controls and almost a year's worth of follow-up. And the patients who didn't get RPIs, you can see this lower right-hand table, what they had traction neurectomy, suture ligature, which is the most common or an unknown second most common. But essentially none of the patients who had RPIs had symptomatic neuromas. Only six in the controls had symptomatic neuromas, which I find kind of surprising actually that they didn't have more, but 50% of the RPIs still had phantom limb pain versus 90% in the control. And so I think we are good at finding ways to reduce these things, but we have yet to find a way to completely eliminate these things. Because RPI is being performed worldwide now, because of Dr. Soderma's early work, there are many places that do it. We do it at Hopkins. Lots of other people do it. This is a study of patients from Macedonia who had semi-elective amputations. So these are all diabetic patients who are undergoing AK or BKA due to lower extremity infections. And so they were able to plan their surgeries up, 14 had RPI versus 14 had it being standard, mean follow-up almost a year, pretty significant VAS, you know, visual analog scale scores and their promise pain interference score was also lower. So we've seen what we can do with nerve transfers into living muscle. We've seen what you can do with dead little muscle grafts. What if there were some happy medium and that's the BDMT, the vascularized denervated muscle target. And so this is denervated muscle, so it can accept new innervation, but it's got a blood supply and there's lots of it available based on perforator anatomy. And so surgeons such as myself have been doing this on a large scale for years and years without having a name for it. We would just denervate a total, a big muscle, do a flap, maybe even a free flap, and we'd start plugging nerves into this thing. Sammy Tufaha, who's one of my partners and people in his lab really went further, drilled down on this, became very innovative and came up with this name, but they found that if you could take tiny little perforators coming into muscle. You dissect the nerve fibers off, but you keep the vascular fibers intact. You can implant nerve into that just like you can have EDMT, but this is a lie. And it's not going to undergo the level of fibrosis that those RP and I's do. And so, plastic surgeons are great at finding perforators with Doppler's in the operating room. That's what we do for a living half the time. And so, I think we are well-equipped to do this. And so, Sammy did this in an animal model, transitioned it to clinical. We're doing this in a clinical now, and we hope to get funding to do a randomized prospective trial. Does it have to be one or the other? The answer is no. Is this a patient who's missing their wrist and bone? And it's not because it got chewed out by the Rottweiler. It's because of infection from injecting drugs. So, this is a person who's at high risk for chronic pain problems already because of their opioid exposure and addiction. So, I'm going to do a trans-cumeral amputation, actually through the elbow amputation. And I'm going to make sure that I give them what I think is possibly great motor control capability in case someday, if they have health insurance and a desire to have a myoelectric advanced prosthesis and get some rehab, they can do that. But at the same time, I want all their other nerves taken care of too. And so, I do a combination of TMR and RP and I and BDMT in this to make sure that all of our sensory nerves and our motor nerves are all good. Here's another example of how we can mix these. A patient who wants osseointegration and a myoelectric prosthesis, but they don't have enough upper arm muscle to control the prosthesis. So, I'm going to bring a new muscle in, a gracilis preflap. I'm going to do a TMR with the median nerve to that. I'm going to do a BDMT directly to that gracilis nerve with the ulnar nerve. And I might even do that with some of the sensory nerves. I think I did that with at least one sensory nerve, then RP and I, so the other sensory nerves. And so, here's the gracilis flap going in. There's a skin paddle with it there, so we can monitor it, make sure it stays alive. And then, you know, three to six months later, we can come back. Once the swelling's all gone, cut that skin paddle out. Dr. Forsberg jumps in. He can slam an implant in there. I say that facetiously. He's not, you know, it's actually a very nuanced technique. And this is his osteointegration compressed device. And this is him using his prosthesis for the first time after his rig has been totally set up by his prosthetist. He lives in New York. This is how he says, hello and thank you. Pretty cool. So, of course, there are way more lower extremities than upper extremities. I do the lower extremities because I also want the upper extremities. And if you want to be good at this, you need to just do all of them, so you get as much practice as you want. And if you want to capture the more rare ones, you got to do all of them, so you cast a wide net. But when I started getting involved in this, I found out, as Marla and some of her partners taught me, that a lot of these lower extremity amputees are really kind of surgically abandoned patients, and they need somebody to help them. And so I'm happy to do so with all kinds of things like contouring their stumps and doing these nerve surgeries for pain and then helping Dr. Forsberg with osteointegration, which he'll talk about. Which brings me to our OI program and our Center for Amputee and Limb Loss at Hopkins. And so I have these great partners here, Mark Hopkins and Ed Lyons, Marlise and Jonathan, who you'll hear from soon. We do OI of just about everything that Dr. Forsberg thinks he can design an implant or find an implant for. And here's the link to that if anybody's interested. And we got lots of future developments and advancements in that, and coupling the nerve part with the implant part through direct wires. And I think Dr. Forsberg is going to talk a lot more about osteointegration and maybe he'll get into a little bit of this stuff. So I'll stop by saying, what are the future developments? What do we need to be looking at? Well, we have to recognize that the peripheral nerve doesn't explain all the peripheral nerve pain that people have. TMR doesn't take all the pain away. I also don't know why TMR even works. The nerve is so big compared to the nerve that we transfer it into. There's probably 10 times the nerve fibers in the nerve that we're trying to get rid of pain from compared to the amount of fibers that are in the little branch we're putting it into. I don't know what happens to all the other fibers. The fact that most of those fibers are motor, but most of the nerve fibers are sensory. And, you know, so there's a lot of mysteries around this. I also don't know why mirror box therapy works because that's not peripheral nerve intervention as much as a CNS, maybe a plasticity intervention. Why do some of these patients with chronic peripheral nerve-based pain, we think, not get in, not get all of their pain relief when they get a really dense peripheral nerve block? It doesn't make sense to me. And so how do we help these people have centralization for lack of a better word or a better explanation of their pain? And will giving a fair input help them such as with a peripheral nerve stimulator, can we do something better by putting directly implantable cuffs on these nerves and things? So a lot of questions here. And then I don't know what to do with these people who have nerve damage proximal to their nerve stumps. They have more than one injury. They've got the amputation injury, but they may have injury that, that propagates the entire length of their nerve up from a traction injury, such as a motorcycle collision, which usually results in an avulsion amputation injury. Or patients who have high energy, high kinetic energy injuries, say to a bomb blast, maybe the leg was removed or the arm was removed. And to get, you know, all the soft tissues and hard tissues injuries results. But if you look at their peripheral nerves under MR neurography, they've got scarring and thickening that go all the way up to their neck or their lumbar plexus. And in some of these patients, like the motorcycle patients will even see partial nerve root avulsions, or maybe a complete nerve root avulsion, say T1 is avulsed, but C8 and all the others are intact. And so we can get, maybe help some of their pain from all those others, but not the T1, or maybe their C8 is also involved. You know, so you get these degrees of things where we're treating the amputation nerve stump and nerve neuroma pain, but then we have more problems with that nerve going back approximately that we don't have a great solution for yet, maybe DREZ, but that doesn't really work all the time either. So we still have plenty of questions to ask. And we're in the infancy of, I think, surgery on these peripheral nerves to help people in their pain. And we have a lot of runway that we still need to get down before I think we're really good at our jobs. And I'll stop there. Thank you very much. Thank you. Thank you, Dr. Shores for that presentation. And maybe we'll take a couple minutes if there are questions. If not, we can move to Dr. Forsberg's presentation. Well, I'll just, then if I don't, if there are no questions, I'll just finish out by saying it's great to have awesome partners. Marlies, you're one of them. Jonathan, you're another one of them. And Ed and Mark, I don't know if they're on, but they're there as well. Princess, if she's on, she is also for our upper extremity. She's our therapist for transplant and our upper extremity team are. So it's great working with a fantastic team like this. So thank you guys very much. Well, thank you, Jamie. And on that, I will move to Dr. Shores, sorry, Dr. Forsberg's lecture. Dr. Forsberg is an orthopedic oncologist in our area. As Dr. Shores said, he just retired from the military and he specializes in treating people with bone and soft tissue sarcomas. And as part of his practice in the military and now in the civilian world is performing osteointegration surgery for our patients. And we'll be talking about that topic. Thank you, Dr. Forsberg. Thank you, Marlies. Jamie, that was a tough act to follow. What a wonderful lecture and review for everyone. Can you hear me okay, Marlies? Great. We're going to talk about osteointegration today, which is an up and coming surgical technique. And we have a few years under our belt, but we're still learning. Just like Dr. Shores alluded to, we're still learning about the nerve work and we're still learning about the role that bone and soft tissue have to play in a transdermal orthopedic device. I have a few disclosures on the PI, was the PI of the study that I'll show you. I'll give you a sneak peek into the outcome there. And then the Zimmer Biomed has been supportive of our research in this space over the years. I do some consulting work and I also own a patent that may or may not be used in this space. And we also do a very important job advising the FDA on how to evaluate transdermal implants, because this is not a typical orthopedic implant. I don't need to belabor this point, but we have a preponderance of lower extremity amputations or major extremity amputations in the military. And we don't quite have the world's greatest solution. And this audience truly understands that problem more than most. But Herr Ingvar Brønemark investigated, he was investigating something that came across a topic that is now the subject of this lecture, and that is osseointegration. And he was looking at how rabbit tibiae responded to trauma. He was looking at neovascularization and other things. So he had these specialty components made and they're made out of a brand new metal at that time called titanium. And there was a quartz lens. So he could look in from the top with a microscope and see the bone healing and see new blood vessel formation, things like that. And these were so expensive that they had to be reused. And he noticed that when he tried to remove these things, he couldn't do it without taking out a chunk of bone with it. So he spent countless hours with a dental pick, picking out the bone from the thread so these things could be reused. And what he coined, the term anyway, was osseointegration, in which bone will grow onto a titanium or a metallic implant without any intervening tissue. And orthopedic, and this formed the basis for the first dental implant, an orthopedic surgeon, not to be outdone, said, you know, we can do this in hips and knees. And there were high fives all around. But later on, an amputee who was injured in Yotobori asked the inventors of this technology if the same thing could be done for her residual limb. The first answer was no. And the second answer was no. Third answer was no. And finally, she was implanted with the first generation Oprah device. So now when we say osseointegration, this is what we're talking about. We're talking about patients who have no other option for a socket prosthesis or documented difficulty or frustration with sockets, or patients who we know are going to have difficulty because as an oncologic procedure, I may take out everything under the sun. I may take out the abductors or the adductors and not leave any soft tissue to support a socket. So what is this transdermal bone anchored implants? This has got bad written all over it, right? There must be some mistake because we can't have a piece of bone sticking out. It's going to get infected and patients are all going to do terribly. Well, let's just see if that bears out. So in this country right now, we can put in one of three devices. We can put in the Oprah implant system on the left. We can use a long stem device. And there are many other than the OPL. There are three different types. And then the compress is going to come online once again early next year for a clinical trial. But the only FDA approved implant is the Oprah implant system. It's done in two stages. And it's done in two stages because we rely on bone healing. I'll explain why that's important in a minute. And then over that bone that's healed, we do a full thickness skin graft. And the idea is to minimize motion at the skin penetration site. And if you want to go on YouTube to search for osteointegration stage one and two at Hopkins, and you'll see this, but not everybody likes it because this is real surgery and it shows all the steps necessary to do this. I originally made it as a teaching video for the residents, but the patients like to watch it as well. And what we quickly realized is a lot of patients have redundant soft tissues. So having a plastic surgeon involved in a program like Dr. Schor's is absolutely critical because they, Dr. Schor's and others taught me the value of maintaining or tightening up the soft tissues and borrowing a technique from the aesthetics field to do what's called a thigh plastic. But once we get the soft tissues taken care of, we go through a series of steps. We make an osteotomy in the upper left-hand corner. We then ream by hand to prevent thermal necrosis. We want happy osteoblasts at the end of this. And then we do something that orthopedic surgeons don't do very often. We're plastic surgeons and we tap the endosteal canal. And here you can see the threads in the endosteal canal. And that will allow us to thread this titanium implant into the bone. Now the whole thing's titanium, but the proximal two-thirds have been roughened with the laser, which changes the mechanical properties a bit. But when you insert this thing, it's very clear that what you're achieving is a circumferential press fit. And a threaded implant has three times the surface area of a similarly sized porous coated stem. So there is a ton of healing potential here. And if we've done our job well, we end up with a nice residual limb that's going to be suitable for stage two. And this is what we strive for after every stage one procedure. Eliminate the redundant soft tissue as much as possible. So here's what I was talking about. The full thickness skin graft over the distal femur is very, very important. And we do this through a series of steps to achieve what looks like this. Now this is the skin penetration site. This is where the A-plus in skin penetration site. Notice that as a patient moves around, there's not going to be any motion at the skin implant interface, no tugging, and there's also no drainage. So more motion equals more inflammation. More inflammation means a higher risk of infection. So patients can actually get out there and do the things that they like to do. Early in the rehab program, we have them do supervised weight training. The skin penetration site in this type of implant system is so robust, we let patients swim. This is patient number one in our transfemoral trial. I'm sorry, patient number one in our transhumeral trial and patient number, I don't know what he was, 10 or so in our transfemoral trial. But he puts a little Neosporin around the aperture, also known as the skin penetration site, and wipes it off when he's finished swimming. And that's a saltwater pool, by the way. In the rehab protocol, we have to teach the osteoporotic bone what it's like to bear weight again. So it's a graduated loading protocol. Sometimes this might be modified and head lions might consult with us and say, look, this patient's having a bit more pain with loading, so we need to back off, give him a break for a week or two, or go to a half speed protocol, which takes a bit longer. But once the prosthetics fit, most of these patients are experienced socket users. They take to walking very well. And even at six months post-op from stage two, I've had some walk with a barely perceptible limp. And this is what it's all about right here. So what's going on in the bone? The bone will remodel. It's slow. And these bones remodel molecule by molecule. And you can see the bony hypertrophy up toward the proximal aspect of the implant. Here's another example. And the bone continues to remodel more than two years out. So I'll show you a sneak peek of the results from a clinical trial. These are all femurs. And we had patients, mostly military, at age greater than 22, all with documented difficulty with socket wear. They were all referred from the process. They all had a trial of sockets and were unsuccessful. There were one or two that had expected difficulty after primary amputation. These are oncologic patients, but the vast majority had been socket users. They all had to have sufficient bone stock and they all had to have mental health clearance because these patients are all traumatized. And we rely on them to complete their 15 months of rehab after surgery. This is very, very important. So it's negative screening for nicotine use. In terms of outcomes, we measured everything under the sun because this was the first clinical trial in transfemoral patients done in the United States. So we didn't quite know what was important. So we measured it all. And we're now in a position to advise the FDA on what is actually important to patients and what's not. So future clinical trials can be streamlined to include only the outcome measures that are appropriate. So we had one patient who was lost to follow-up. Unfortunately, he had a motorcycle, crashed his motorcycle after stage one surgery, and he's no longer a candidate for osseointegration. So we had 49 patients for analysis and median survival was two years. And this was an interim analysis of an observational study. So here's our mean age. And if we overlay this onto the world's international osseointegration registry, we see that our patients are a little bit younger than the patients treated abroad. Most are male and most were injured due to a blast injury, but we had a smattering of other diagnoses. Most had unilateral amputations, but we then started enrolling bilaterals and even triple amputees. Obviously they were bilateral lower extremity amputees with an upper extremity. And most were less than 20 years from amputation. And this is important when one considers the bone stock you're working with. And this is about the same distribution of what we see on the global scale. So in terms of patient reported outcome measures, I'll just boil this down in a series of graphs for you. Mean prosthetic use per day. This is very important to the FDA and they consider it to be the end-all be-all in terms of prosthetic use. But we did see a little bit of dip during COVID for most patients because it was so convenient to don it off. They would take it off when they were on the airplane or take it, they took it off when they're in the car or when they're sitting around, you know, doing office work. It's just so easy for them to take it off. So, so we had to come up with a surrogate for a prosthetic use in hours per day and convert it to a denominator of every, whenever you wanted to use the socket, were you able to do that? And, and by far it was near a 100% of the time. We have mean prosthetic use in days for the week. And then the patient reported outcomes were few and far between. We had problem scores, we could do the pain score, but the questionnaire for transfemoral amputee happened to be the only validated PRO in patients with osteointegrated limbs. And what we did see here is improvement at all domains and fewer problems. Problem is with the QTFA is very cumbersome. The research coordinators hate it. Patients hate it. It takes forever. And there are better ways out there like the computer adapted promise scores. Now take a look at how similar these curves are from pre-op to 24 months. We did see a statistical significance or statistical difference in all of these domains, but for the FDA, we need a little bit, we need a little bit more. And we came up with a composite income per end point based on what I want to see as a surgeon. I want to see more function and less pain if I'm, if I'm intervening. And that's exactly what the curve on the left shows. And then physical function per hours of use per day. This gets past the COVID convenience factor, you know, Don and Doffing simply due to convenience. So these composite end points are what we're going to report when the publication comes out next year. You're all probably wondering about infection. Yeah. Infection does occur, but in my opinion, the infection is absolutely due to the degree of motion at the aperture. If you have a lot of motion, you have a lot of, a lot of inflammation, you will get an infection. And here's the, here's a patient with even a perfect looking aperture and he developed an infection, which resulted the oral antibiotics. Here's the waterfall plot plot of all the patients and the all the limbs rather of all the patients in our femoral study. And what we can see is about one, one in three will develop an infection. 10 of those had multiple infections and three of those patients had deep infections. But the most striking outcome on this is 68% of limbs had no infection. I mean, we had, I didn't believe it when I first saw these data, I thought it was absolutely wrong, but that's, this is real. This is a, this is what's happening. Implant removal. We have one implant removed in this particular study in a patient with multiple infections, and he did have osteomyelitis. I can tell you anecdotally that we've, we've removed two implants. I personally have removed two implants in the civilian population due to aseptic loosening in both cases. And we can talk about the reasons for that if you have any questions later. But the probability of poor outcome is directly related to BMI. And this is hard data from the transfemoral study in the military. We did see stress fractures in three patients, all were less than eight months post-op, excuse me, and all healed with protected weight bearing. This one I also thought was due to infection, but we ruled that out with some blood tests and a tagged white cell scan. So even with multiple infections, even with complications like stress fracture, we asked all of our patients, would you have this procedure again or go back to a socket? And they all said, yes, I'd have it again. So that's high praise. So in conclusion for that portion of the presentation, we showed statistical improvement at 24 months in everything except pain behavior and walking aid in the QTFA. But most patients had higher function and less pain on the composite income or composite outcomes. Infection rates of 32% are comparable to the historical literature. Deep infections are relatively rare. And QTFA and PROMIS domains don't correlate at baseline, but are strongly correlated, moderately or strongly correlated to your follow-up, which means we can ditch the QTFA for our long-term follow-up patients. Now I want you to think about this very new surgical technique in the context of orthopedic surgery. The first hip replacement and knee replacement were done in this country in 1940. That was 82 years ago. At that time, they didn't have access to longitudinal studies. These surgeries were only done in a handful of centers. The first hip replacement was done in Charleston of all places. Some orthopedic surgeons said it was crazy, shouldn't be playing God. And we've heard the same thing with osseonegration. It's a little bit out there. So we designed early on an international quality registry to look at radiographs, photographs of the aperture, rehab assessments and complications over time. The rehab has a mobile interface, so we can push out OPUS scores. We can push out PROMIS scores or pain scales or ad hoc questions to the patient's cell phones. And in our experience, the patients don't want to come in for a follow-up. They're out. They're living their lives. They don't want to come back to the hospital where they spent a good portion of their lives. So our research assistants do a good job of tracking them down, speaking their language. The FDA loves the registry because they use it for post-op or post-marketing surveillance, rather. And we can ask some very relevant questioning. Does a refashioning procedure revision thioplasty, does that represent a failure of the implant system? Or does it represent the fact that now this patient can get out and walk and exercise and they need a little nip-tuck to protect that skin penetration site? Is a superficial infection considered a failure of the implant system, even though it doesn't limit any mobility and they're still allowed to don their prosthetic and walk as much as they want? And is stress shielding a problem down the line? So I want you to think about that. I'm going to close with a couple of considerations here and then show you some next steps. Our soft tissue considerations are very, very important, which is why a plastic surgeon must be involved. Here's an example of one of our first patients at Walter Reed. We put him, we put this osteointegrated implant in and we put the hole in the wrong place. And the first thing he did is he sat in his wheelchair and he dug a trough through the soft tissue. So I zipped him up once, I zipped him up twice. And this is here, this is him at three years and he refuses to have a soft tissue procedure because he's active. He's got small children and he likes to swim with them in the pool. And he also does like all kinds of things that I told him not to do. So he's, he's just out there living his life. He doesn't want to come in for a soft tissue procedure. And this is, you can see on the left, the compress style transdermal implant system. Here's one with an Oprah patient's dissatisfied because the medial skin touched the the fail safe mechanism, the XOR2. And remember these patients did everything they could to get out of a socket. And now you've got something on their residual limb touching a piece of metal or a plastic. They don't like that. So this is a thighplasty procedure done by Jason Sousa at Walter Reed, which helped do a nip tuck on that medial thigh, which is very difficult to get a handle on. By the way, it always sags a little bit. This is a patient at Johns Hopkins who had failed the compress. And I gave him another chance. As you can see, he's got a generous soft tissue envelope and he, the split thickness skin graft eroded to the point where all we're seeing is periosteum and a little bit of granulation tissue over the bone. And this is a horrible situation. And thank goodness Dr. Shores became involved and ultimately did a split thickness skin graft and vac'd it to the point where it is, it's probably now one of the best apertures we have because that skin graft healed right to the end of the bone. And there is zero motion at that skin implant interface. Here are a couple of cases where we're pushing the envelope. I mentioned some of these are oncologic patients. This was someone who was on second line chemotherapy for osteogenic sarcoma. She needed a lot of extra time for bone graft healing. Ended up doing a, doing a first look and then a second look at the bone graft, but the soft tissue will heal despite chemotherapy. Here's a patient with, who had a vascularized composite tissue allograft and he was on tacrolimus. So we, we weren't sure whether, whether he was a candidate, but we tried it with the transplant team. Very well involved. They're very, very heavily involved. We noticed that the bone graft healed on time. The soft tissue also heals, but his lifelong risk of infection is to be determined. But as of this moment, he's, he's still doing, he's still doing well. This is a 50 year old female with what can only be called a catastrophic antiphospholipid syndrome or a vasculopathy, not otherwise specified. She, she does not have a diagnosis despite being at multiple centers around the world, but she, we did osseointegration that she had some exposed bone and we had noted this at her stage two procedure, but elected to proceed. And she ultimately healed six months later, but you know, this, this cosmetic problem at the end of the bone at the aperture didn't affect her weight bearing. We ruled out deep infection every chance we got. And with a little patience, she healed it up. Dr. Shor showed you this slide. Here's a, here's an example of a patient who has implanted electrodes and notice he, he doesn't choose to use a whiz bang hand. He chooses a rotational risk rotational risk grasp mechanism because he's much more functional and he's trimming the heart of some wild animal that he shot not too long ago. And then you saw this x-ray, but I can go into detail a little bit more. This was a patient with a shoulder disarticulation who's got no options, you know, very, very few options. And he came to us with a phantom limb pain that Dr. Shor has treated with, with TMR and RPNI. And then we decided, he asked if we could create an arm for him again. And the first answer was no. The second answer was no. And the third answer was, gosh, what if we could? So we brought three orthopedic equipment manufacturers together with the Johns Hopkins Applied Physics Laboratory to, to bring this technology to him. And I he's coming back next week to be, you know, for an assessment to be to work with the APL in terms of pattern recognition and all that. So more to follow on that, that is, that is bleeding edge stuff. And if it works great, if it doesn't work, don't, don't ask me about it because it would have left a permanent mark on my soul. But that's all I have for you today. Thank you very much. Thank you, Dr. Forsberg. And we'll open the floor for questions. We already have a question in the chat. And people are asking about the press that osteointegration has received, especially with a 60 minutes presentation about the Australian program. So can you give us some comments about, and, and kind of put this in context? I will definitely put it in context. Yes. So what you're referring to is a long-stemmed implant made popular by Munjid Al-Mudairis. And Munjid, he's a, he's a collaborator and he was kind enough to host us in Sydney twice for us to learn the surgical technique and also to, to meet his team. This is a challenging operation in a challenging patient population. And just like we do in any other, any other setting like this and transplant or oncology, it's critical to have a team approach and you need depth on the bench to take care of these patients. They are very needy. We're dealing with a skin penetration site that's never been, that's never been seen before in orthopedic surgery. So, so we are figuring it out, figuring out how these wounds evolve over time as we go. So if you ask me what the 20 year complications are going to be on these things, I won't be able to answer you until we're there. Now, what happened in what happened in Australia is that certain patients felt marginalized, felt marginalized and abandoned. And that is never the right answer, no matter what you're doing. And once that, once that happens, you're sunk. So it may, it may be a case where Munjid Al-Mudairis got a little bit, operated a little bit too quickly and didn't prioritize the follow-up and the patient relationships. I mean, he's got huge numbers. He's, he's done, he's done thousands of these. So it's hard to be in a place like Sydney, especially during a pandemic and to have a follow-up with all your patients on a regular basis, especially ones that come from far and wide across the globe. Yeah, it makes sense when, you know, he, he has a very successful program from people coming and their typical stay, correct me if I'm wrong, Jonathan, is about a month after the procedure. I think you're right. I've heard patients who were there for under two weeks and come back. And that's, you know, that's certainly not the way we do business. You know, if I have a patient that comes from afar, we, we see them at regular intervals, but over time they start to complain about that, but we remind them that, Hey, this is what you signed up for. This is not, this is not something that you can rehab, you know, with your local rehab physician, unless they're experienced and trained to, to deal with osseointegrated devices. And I will say our, our, we've had two recent failures, and I promise you, I'd give you a little bit more detail on those. Both of those were aseptic loosening, which means there's some force that was put on the implant at some time during the rehab that overwhelmed the osseointegration that was trying to occur. So too much force, maybe in the wrong direction, maybe it was torque, maybe it was too much of a lever arm too soon. I can't be sure. But one of the reasons I believe that, you know, that, that our, our group and the DOD group is so successful is because we do our best to standardize the rehab across all patients on all sites. Thank you. Any other questions? Please feel free to unmute your phone or use the hand up sign and we will, we will listen. Jonathan, how do you, you know, you've got these three implants that are available in the United States, and they're all, they're all pretty different from one another. How do you decide what is going to work the best in the patient? It's a good question. And, and we've, we've debated this many times. So we've had discussions with patients many times. And as an orthopedic surgeon, I, I need to choose the right implant for the right application for the right patient. And, and that's a, that's a really deep discussion. So, you know, if you have a bone that's long enough for a long stem implant, it doesn't mean you have to use a long stem. It just means you could, if you needed to, I prioritize the availability of modular components down the line. And think about it, if you're, if you're buying a car, you wouldn't buy a car that has no service center near your house. That's just asking for trouble. You wouldn't buy a car that if, if something broke, you weren't able to get parts for it, because that would mean the car would sit in your garage or on the street and not be, not be able to be used. Same thing happens with some of these implant systems. You buy, you put in an implant system without good representation in the United States, you're going to be waiting for parts. And I have a very angry patients who had the compress early on that we're waiting for fail safe for months, which meant they're not in a socket, they're back in a wheelchair for months. So, you know, it's a, it's a, it's a, it's a, it's a, for months. So Integram has done a good job making sure that we're able to get replacement parts whenever we need them with rare exception. Also the, the Australian group has done a, done a good job with that as well. But if you fit the right implant to the right bone and the right person, you, you won't go wrong. And I say that, let me just tack that on. There are surgeons in the world that will modify a bone to fit their particular implant system. And that's, that's not the right answer. Yeah. I've always thought our program had an advantage in that we have an orthopedic surgeon and Dr. Forsberg who understands the benefits of all three of these devices and is capable of using all three. Whereas if you go, you know, to most other places in the world that are doing this, you get a surgeon who just does one kind of implant. That's it. And so we're, I think we're fortunate here to have you and to be able to let you use your judgment and your understanding of how all three of these work with your experience to pick, pick what's right for the patient. Thank you. Any other questions? Well, if I was going to say, if Colby can come back to, I want to just say thank you again to Colby and his team out in the West coast for teaming up with us to put this session together. We're happy that you've joined us and if you have any parting words, Colby. No, that was great. I, I was actually going to ask Dr. Shores a quick question, you know, when, when he would use the RPNI versus a TMR, maybe I missed that in his, in his discussion about it, like choosing one versus the other. Yeah. So I, I, uh, I frequently will mix them together, but in my order of operations, I think that the TMR right now is, uh, better if I want motor control for something, Dr. Soderna has demonstrated the ability to obtain motor control from RPNIs, but it's, it's less straightforward than, um, our standard transcutaneous EMG pads that we can fit into sockets or, uh, you know, uh, integrate with a OI implant. Um, and so if I'm looking for motor control, then, uh, then I think TMR is better. And so I'll prioritize nerves that I want motor control from, for the TMRs, and then I'll use RPNIs for the others and BDMTs for the others. But if I'm not, if I'm not looking for motor control, I may still prioritize TMR because you can get a bit of both. When I do that technique, say, um, you know, I can take a little bit of a length of that, that nerve. So I've got a little bit of a, on the, on the recipient nerve that is the nerve going into a muscle that's feeding it. And when I clip that and I take that other nerve, I can create a trough in that muscle where I can put those two nerves together, sink it down in the trough, and then so the rest of that muscle belly over it so that you're getting kind of a combination of both TMR and this BDMT sort of thing at the same time. And so I think you get the best of both worlds because I think the most efficient reinnervation comes from, um, direct, uh, nerve regeneration through motor end plates with the way that our physiology is set to capitalize on, on motor inputs. RPNI and BDMT skipped the classic pathway of, uh, going through the regular nerve, then it's branch pattern going throughout the muscle and then hitting these motor end plates. And it's doing some sort of local, uh, reinnervation that probably does involve the motor end plates somehow. Maybe it's just through random axons, finding other random axons and, and trying to regenerate with them. But I think the process is probably more efficient when you've got a nerve branch, you can put it into. So if I've got that capability, that's my priority. That being said, there are way fewer of those opportunities in these, uh, legs and their, uh, and arms than there are for, uh, RPNI and BDMT, just because you don't have to worry about finding a nerve branch and finding these nerve branches can sometimes be difficult because you don't have them just everywhere. They happen at certain regions and certain lengths and certain, you know, along each limb, and they're pretty specific. So, uh, if they need motor input, then that's when I prioritize TMRs. And then, uh, for everything else, if they don't have those, uh, recipient branches, then I think RPNI and BDMT, I use those like crazy all the time. How often are you guys doing these, uh, prophylactically at the time of initial amputation, as opposed to, uh, coming back later to address a complaint, you know, of pain or whatever? I'd say 95% of them are secondary procedures. A few, the few that I get to do are because people like Dr. Forsberg or somebody that he's doing a primary amputation on them for, for either a tumor or, uh, or horrible chronic pain that is just, you know, uh, overcome every other attempt, uh, at keeping the leg and getting rid of the pain. Um, or, you know, maybe a few of my traumatologists that I have, uh, been able to convince that this is a good idea for them. But, uh, most of them, the vast majority are all secondary procedures. Got it. It's coming into the mainstream to be, uh, to be done with every index amputation, at least in the oncologic field, or, uh, we're, we're, uh, advocating TMR to be done at the index. And then, uh, within the military, all the patients get, get TMR at their, uh, at their definitive amputation, but that's, uh, that hasn't trickled out to the civilian traumatologists yet. And most of that TMR data, like from that randomized trial, that all came from that military population. They, uh, the peripheral nerve surgeons and the plastic surgeons in the military and the orthopedic surgeons in the military are really, uh, cutting edge surgeons. And so the rest of, you know, our civilian population is learning from the advances that have come out of, you know, uh, the last two decades of conflict, uh, with, uh, what our military surgeons had to have to come up with or take principles that Greg Domanian and Todd Hyken had, and then incorporate those. And, uh, one of, uh, Dr. Forsberg's partners, uh, Jason Souza, who helped him for years and years with all these things was a trainee under Greg, uh, Domanian. And so it was able to take that excellent training and help unleash that in these other, you know, incredibly deserving patients who need it more than anybody. But when you do it upfront, the way that Dr. Forsberg is saying that most of the patients in the military get, they're, they're going to have better outcomes. We know that the more procedures you have and the longer it takes to get that procedure, the less good the outcome, the outcome is still better, but it's not nearly as good as it could have been if you did it as an index procedure. Yeah. Great. Thank you. Any other questions? If not, we're, we're finishing a few minutes early. So again, thank you everyone for spending the last, the past three hours with us and to our presenters for sharing your data and your experience. Thank you very much. And we'll see you next week in person in Baltimore. So everyone, uh, welcome to Baltimore next week.

hyperhidrosis

amputees

evaluation

treatment options

aluminum chloride

botulinum toxin

starch iodine testing

TMR

phantom limb pain

residual limb pain

osteointegration

prosthetic control

clinical trial

pain improvement

soft tissue considerations