Okay, let's get going. Thanks. This is me. I'm done. Thanks. Well, again, good evening, everyone. I'd like to additionally add a warm welcome for you guys here today. This could be a great course. I also wanted, just as a prelude for tomorrow, just to also let you know the unique part of the laboratory tomorrow is we have whole cadavers. And that's a very unique thing for anyone who has gone to a previous ultrasound course. And so then the reason for that is for really all of you to take the time and go rogue to explore all the different regions. Instead of having a knee available or a lower extremity, we really have everything. And that, of course, is because of the tissue harvest portion, which could be, of course, from the abdomen that we'll talk about, but also from alternate sites. And so then it gives that real good opportunity to really explore ultrasound in different areas. The next is the opportunity for multiple different ultrasound systems. So again, oftentimes, it's just one. Get your hands on both. Each station will have two different companies' ultrasound units. So that would really be good to flip back and forth on both sides of the cadaver to really get used to two different panels and different ways of looking at the ultrasound. For those of you who do not have the laboratory portion, so didactics only, we're also going to have a separate program in this room for you. And that's with our industry support are going to be giving you live demonstrations here while the rest of the lab is going on. So we want to take full advantage of the time that you guys are in here. We're going to pipe in all the demonstrations in the lab. But in between those demonstrations, there'll be additional types of industry demonstrations. That's right here in this room. So it'll be a full day for you guys tomorrow. So that's to look forward to as well. So we'll begin this, quote, rapid fire of technology. There will be some redundancy, which is built into this. I think you're also going to see some discrepancies between the different speakers. And that's also legitimate. In this current technology where we don't have full data on everything, for two speakers to come up and give a slightly different view on the best therapy is very reasonable at this time. Because of scheduling, we're going to change things a little bit. So in comparison to what's on your schedule, we're going to have Adam Antz come up first. He has a flight and another obligation later on tonight. So we're going to welcome him up first. For those of you who don't know Adam, he is an incredible researcher from the Andrews Institute in Florida. He's safe from the hurricane, I'm proud to say and happy to say. But also, he's been one of the founders in this Western world of peripheral blood stem cell therapy. He's done a lot of work on BMAC and bone marrow concentration techniques. So he's here to share a couple of different talks with us, including the use of BMAC as an adjunct and biologics for ACL management. So thank you, Adam. All right. Good afternoon. Apologies for having to talk and run. I have a talk tomorrow morning at 745 in Tampa. And so that's why I've got to hit the road and get a flight. Can you hear me? Am I talking close enough to the mic? So I'm going to talk about bone marrow aspirate. I'm going to talk a little bit about some lessons learned from our institution. We've been using bone marrow aspirate and bone marrow aspirate concentrate for about seven years now clinically. So we've learned some lessons that I would like to just share from our perspective on it. Also, I want to show you some early thoughts that we have on adjuncting the ACL. We have two clinical trials underway that I wanted to introduce and show you some of our early thoughts on that. I have some disclosures. They're in the program guide if you would like to review them. But my main disclosure is I'm an orthopedic surgeon. So take what I speak with a grain of salt. And the one thing I do know is that I do not know everything. And that may be the key in this space is for us to understand that we're trying to make clinical application decisions with only seeing the tip of the iceberg. And so in order to really develop these technologies further, we have to look underneath the surface. And we also need to understand the developmental process as well. Basing what we do with a very firm base of preclinical animal study and then developing clinical trials so that we can ultimately understand what we're providing to our patients. So that's a little bit of a soapbox. And I'll go ahead and get off of it and go from there. These cells functionally do a number of different things. In the right environmental niche, predominantly our bone marrow, they can replicate. They can self-renew. Also under the right environmental niche, they can differentiate. But they probably don't do that naturally. So under the right setting, they can differentiate. But it's probably more likely that they're going to tell other cells what to do. They're going to monitor their environment. They're going to become mobilized in a number of different settings. And then we're going to release these secretomes or these exosomes. And those exosomes are what have an effect on their environment. And so that's how we've migrated in terms of our understanding of these cells. We first became very infatuated with their ability to self-renew and their ability to differentiate. But now we're trying to understand and leverage their ability to monitor environments, mobilize, and affect change in their healing environment. So understanding bone marrow aspirate may ultimately mean we need to understand what these cells do in their own natural environments. And for that, I'm going to show you some other domains literature, some literature from the cardiology space, which says that in settings of myocardial infarction, you mobilize these cells to your peripheral circulation. And also literature from just physiologically space, where they say that if you are stressed as an ecosystem, be it hypoxia, you mobilize cells to your peripheral circulation. And some of those cells are filtered by your liver and your spleen as well. Also, we know that exercise is a stressing environment for your system. And that with exercise, acute bouts, you increase circulating in your peripheral system of these cells, where they'd be these hemopoietic progenitor cells or these very small embryonal-like cells. They're both increased in your peripheral circulation with exercise. And that may ultimately be things to consider when you move clinically. You need to consider your patients and whether your patient is sedentary or whether your patient is an athlete, because those are two very different birds. This was a study looking at rats, and half the rats went in their cage. And the other rats went on little rat treadmills. And you can see that the bone marrow concentration is very different between a sedentary rat and a rat who's a conditioned athlete. So those are things to consider naturally, which then will help you understand how you can leverage or consider trying to leverage bone marrow in your clinical practice. So now, talking about what do these cells do naturally, I always think about this video, which really infatuates me every time I see it. This is a healing wound in a zebrafish. As you watch the blood vessel at the bottom of the video, you'll notice we're going to circle a pericyte, which is a cell that is latched onto the outside surface of a blood vessel, migrate to the wound, and participate in wound healing. And there's also a number of other cells that are spitting out from the bloodstream as well. So thinking about this natural process of healing and how can we leverage it in a more effective way is the underlying basis for understanding bone marrow aspirate. So our bone marrow is part of our immune system. And that immune system, when I was in medical school, all I remember was that it responded to infection. That's it. Next subject, I'm going to be an orthopedic surgeon. I don't care. But I'd submit to you that it's more of an alert system, a response system, an attempt at a repair system, and then an adaptation system. So it's part of that whole response that you as an ecosystem are alluding to with these cells. So when we think about our bone marrow, there's a number of different cells within that tissue. And those cells are immature on some stance. Some of them are mature. And there's a number of different progenitor cells there too. These hemopoietic progenitor cells are there. These other cells that support the whole niche are there. And they have stem capabilities as well. There's also some myeloid progenitor cells. So you have a whole potpourri of cells within your bone marrow. And that's important, because your bone marrow is a major player in your immune system. And right now, it's what, from our stance, we are currently leveraging within this space. So I like to think of bone marrow aspirate and bone marrow aspirate concentrate as a point of care bone marrow product. Like we were just discussing, we can't really use the term stem cells. We need to think more about what this is. It's a bone marrow point of care product that we are manufacturing at the point of care. And so to understand this, just think about two simple principles. One, centrifugization creates a density gradient. If you take a fluid, you put it in a centrifuge, it will layer out based upon the density of what's in that fluid. And also selective harvest, based upon what you take from that layered out product is going to be your product that you're using in your clinic. So for instance, say we take this tube of bone marrow, we put it in a centrifuge on a long spin at a high RPM, we're going to get what some people call a hard stack. And that hard stack is going to layer out based upon what's in the bone marrow. And if we select this middle buffy coat, we're going to get our product there. And if you'll notice, how the white blood cells stack up in that buffy coat is dependent upon their density. So the neutrophils are pushed to the bottom of that buffy coat, whereas the monocytes and the immature cells are more towards the top. So if you understand those two principles, you'll understand how to make a point of care product with bone marrow aspirate. I think that may ultimately be the basis for understanding how we move forward clinically. Now we need to ask again, what is in this product that we're making and how does it compare to platelet-rich plasma? And Lisa Fortier published a study from 2016, which I think is beautiful because it's the one out there that helps me understand that. She compared bone marrow concentrate compared to platelet-rich plasma for cellular composition. And here's what she found. The bone marrow concentrate is a much cellular product. It's not just neutrophils, it's also monocytes and lymphocytes, eosinophils, basophils, and platelets as well. She also found when she looked at the chemokine concentration of these products, that the bone marrow concentrate is also a higher growth factor product as well. Platelet-derived growth factor, VEGF, as well as the inflammatory proteins as well. So it has a chemokine concentration that much higher than platelet-rich plasma as well. It also, when you compare bone marrow concentrate to platelet-rich plasma, you will find cells that will culture, that if you smear them onto a Petri dish, you will have cells that grow in that Petri dish. In her study, she compared bone marrow aspirate and found 7.1 CFUFs per milliliter, whereas the concentrated product had a higher growth as you would expect. A little bit of a side note, if you're trying to compare sites that are doing culturing of cells, you're gonna get variation. You really need to think about all these studies independently as independent silos, because what cultures in one person's site is gonna be different than what cultures in another person's site because of their techniques. Something to consider in food for thought. Also, a surprise was found in Lisa Forte's study, and that was IRAP. She found a pretty significant concentration of IRAP in bone marrow concentrate, and Dr. Cole already discussed that, so I won't belabor that point. So now I wanna talk about some principles of harvest. So if we're gonna do it, I would submit to you it matters how many cells we harvest, and that's the thoughts of Philippe Harnigot out of France, and some of these thoughts are here. You need a sufficient local force to mobilize these cells that are adherent to the spicules. If your vacuum is too low, you're likely gonna get peripheral blood. Also, pressure is dependent upon us as the operators, but then also upon your syringe geometry as well. The smaller, narrow syringes have a higher pressure. Also, we recommend that you do several small portions and small apparition volumes, and then move your needle, so five mLs, and then rotate or withdraw, and create a new vacuum, and that's how you can augment or get the most cells that you would like from a number of space. Also, after you aspirate in one location more than three times, you've likely aspirated all that you will from that location, and you'll have authors argue about whether a parallel insertion or a divergent technique is superior. Philippe Harnigot says that parallel aspiration is superior, and Kristen Oliver will tell you that the divergent tech is superior, so at this point, we don't really know. Those are some of the principles of harvest. Now, let's talk practically some lessons learned. Posterior iliac crest is where we started with our harvest, then we moved to anterior iliac crest, exactly like you were discussing, logistics. With our surgical setups, it's easier to get anterior iliac crest. We then also moved to distal femur and proximal tibial because of ease of harvest. But I'll show you our story about why we now predominantly do posterior iliac crest. And that story is a little bit of literature, but it's also some personal experience that I'll share with you now. There's some pros and cons to both. With posterior iliac crest, it's relaxed position for the patient. It's also relaxed and ergonomic for me. It's easier for me to put a needle in like this and to put a needle in like this. So for me, that was one reason that I found it easier to do posterior superior iliac crest. Gravity is also your friend when you're moving a viscous fluid. Just think about a milkshake and pulling it through a straw. With posterior and the lateral cubus position, I basically just create a flow and then gravity helps me out in terms of moving that viscous fluid. Some anterior iliac crest has some pros and cons as well. It's definitely very awkward if the patient is awake. It's not ergonomic from my experience with it. And gravity is no longer your friend. It's actually now your enemy. And you're pulling a viscous fluid up a very narrow straw. Things to think about. However, we initially thought that this would be the best way to harvest because it's going to be quicker. So we moved into our surgical space with anterior iliac crest harvest. But what we found is we were spending more time with our harvest when we did it this way as opposed to lateral cubitus. And we're getting a lot better harvest with the lateral cubus as well. And I'll show you that data. Some pros for anterior iliac crest is you can set it up for your surgical procedures and you can make it nice and cute with Ioban. But I'll show you that we've learned that even these other sites, although they're accessible and you can improvise with them, our experience has been lower cell counts. So before I show you our data, here's a harvest lit review on this subject. When we first got excited about harvesting them peripherally from the proximal femur and the distal femur, it was from Mazaca's group in Connecticut. Arthroscopic aspiration was found to harvest and yield cells. However, the trauma literature started comparing the central locations from the peripheral and found higher cell counts from the iliac crest. Then you had a study looking at iliac crest compared to distal femur and proximal tibia. And again, iliac crest performed superiorly. And then another study looking at posterior crest compared to anterior crest and found that posterior crest was again superior. So all this literature review, I didn't really believe at first. I didn't think that practically it should overcome what seemed most logical in our OR. So here's some of our data to show you what we found. This is a study where we were looking at donors and we were running the numbers on them. And we started with distal femoral harvest. Now granted, we were sending these samples all the way down to South Florida from Northwest Florida. And you'll saw with distal femoral harvest, we had very poor growth. We then moved to anterior crest harvest. You can see we had better growth, but it was still very variable growth in what we were producing. Then we moved to posterior crest harvest. We found that our times were quicker in the OR. And we also found that we were having better growth. So that's our practical experience applying the literature, but having to figure it out for our own as well. Now, two things to consider before we move on is maybe we need to mobilize these cells to the peripheral circulation and then harvest them from the peripheral circulation. This is one thing that Jason was alluding to. So we had a thought that we could use neupogen, which is a synthetic form of a hormone, to mobilize these cells to the peripheral circulation. And then we could collect them from the peripheral blood. So we took 10 subjects. We did bone marrow aspirate on these subjects. Then 30 days later, we mobilized them with neupogen and just did a simple blood draw. And here's what we found. We found that total nucleated cell counts were similar between our bone marrow concentrate as well as our post-neupogen harvest as well. We found many more hemipoietic progenitor cells with the post-neupogen harvest. But we found in terms of what we could grow, we couldn't quite grow anything with the post-neupogen harvest as well. And it may be that the techniques have been optimized for growing from bone marrow as opposed to hemipoietic progenitor cells. But I'll have to talk to Arnie about that later. So with that, I wanted to briefly talk about why we care about harvesting and optimizing our harvest. And here's why. If you look at the literature of bone marrow aspirate performing for osteoarthritis, it has not to date. This was a randomized controlled trial published in 2016. Twenty-five patients, bilateral NeoA. One knee got saline. The other knee got bone marrow concentrate. And at six months, there was no difference between the two groups. We similarly have completed a randomized controlled trial looking at leukocyte-poor platelet-rich plasma compared to bone marrow aspirate for the treatment of osteoarthritis in an elderly population. And our six-month scores to date have shown no difference either. Likely, our 12-month scores will show no difference either. So at this point, I think we have to learn more about bone marrow concentrate before we get ahead of our skis. Now I want to switch gears for the other subject I'm listed to talk about, and that's augmenting ACL reconstruction. And you notice that I've got an asterisk there because this is under development. So what I'm going to show you today, definitely take with a grain of salt and give some patience to this subject. Now, this is really a story about us trying to participate in building this pyramid of evidence, starting with some preclinical studies and then beginning a case series in a randomized controlled trial. Our goal is to expedite graft incorporation because we know this ligamentization process is variable in terms of what we've known clinically as well as in the literature. Now, there's preclinical animal to support this concept. Stump tissue studies from 2012 showed better histology when stump tissue was integrated into grafts. There's also studies looking at cultured cells showing better histology as well as biomechanics when integrated into grafts, whether they'd be loaded with a fibrin carrier or whether they've been placed into sheets. So there's histologic and biomechanical basic science in animal models to tell us that augmenting ACL reconstruction has legs. Now, whether they be cultured or whether they be point of care, carrier sheets seem to matter. This is a study from 2013 looking at cultured human CD34 cells and the cells that were loaded onto a sheet and placed at the ACL graft performed better when just injected into the joint. So these are lessons learned and I won't belabor them too long because we want to get to the clinical stuff. Martha Murray and colleagues have been developing this technique or this concept as well. She has a number of different studies predominantly looking at porcine models that have evaluated different biologic additions. She's found that differing, she has compared differing platelet concentrations to cultured adipose and cultured buffy coat products and her group has settled on whole blood alone. They've also looked at augmenting these structures with internal splints or with stabilization of the preliminary constructs and they've moved upon basically placing a whole blood in a collagen sponge with an internal splint. Her early results in the animal models are very encouraging, showing excellent biomechanical histologic healing in pig models and they've recently started clinical trials. Now in terms of the early clinical performance, I'll quickly go through a literature review that guided us with our study. When you think about two ways to apply these biologic with ACL reconstruction, you can apply them without a scaffold or you can apply them with a scaffold. Briefly, without a scaffold, randomized controlled trials that have been met analysis showed no difference in terms of placing it injected into the joint in terms of platelet-rich plasma. Now it may technically benefit graft maturation but there's been no shown difference with clinical outcome studies. Now platelet-rich plasma placed at the patellar harvest site alternatively has three randomized controlled trials to show that it has been a fit harvest healing site. Now bone marrow aspirate injected to the joint at this point has one study that's known to show no benefit with MRI evaluation. Our anecdotal experience however was that these patients would have less pain, less swelling and a faster progress with physical therapy. So we think we are altering the inflammatory response when we use these in some of our cases. When you look at the literature with a scaffold for support, there's one study that really caught our eye. That was from arthroscopy in 2010. We're looking at platelet-rich plasma incorporated onto a scaffold to a graft. So platelet-rich plasma loaded onto gel foam that was integrated into a hamstring model showed on MRI results better integration in terms of time to graft incorporation. So the concept of the trilogy may be important for this concept. You need growth factors, you need cells, you need a scaffold. And that's what we've moved to in terms of a number of our three specific studies that we've developed this concept into a clinical trial. We started first with an MRI study to develop an objective measure of graft maturation after reconstruction. So what we did is took 30 patients looking at 3-Tesla and 7-Tesla MRI to first establish a method to objectively measure graft maturation. We mapped these tendons that were reconstructed and we looked at inter-observer reliability and found no differences in volumes in T2 maps with age and that this was a reliable method in terms of evaluating ACL objectively. We then took that into another study looking at could we use the ACL injury effusion as a potential source from harvest. So the concept was that if you have an injury and we've immobilized cells to that effusion fluid, are there cells with stem capability in that effusion fluid? So we took 20 patients, average age 18-62, were having surgery within 5 weeks of their injury and we took this effusion fluid as well as some of the surgery byproducts and we looked for viability of cells that would then culture out. And we found about 429 cells per milliliter of tissue. We published this last year in Arthroscopy. So we felt like there are cells with stem capabilities that are immobilized to the effusion fluid. We then took that concept a step forward and said, well, can we harvest them at the point of care? So we took 10 subjects, we took that effusion fluid, we mixed it with blood and then loaded it into a point of care buffy coat based system. And what we found was that we could concentrate cells with stem capabilities with this method. The synovial fluid had about 42.2 where the buffy coat product had a concentrated amount of cells. We then also looked at those cells to see about their flow markers and they had cells expressed similar to cells that had been cultured out for some time. We also found that we could differentiate those into the three lineages, chondrogenic, osteogenic, and adipogenic. We then took that idea to developing a technique. And I'll present that technique to you now. We called it the Bio-ACL for short and our concept was to wrap our graft on the back table as you see here. And once we had that nice sausage, we would implant that sausage, typically with our standard ACL reconstruction techniques, then we would dry the jort and inject a biologic underneath the sausage. Concept was that we would get the cells and the growth factors from the bone marrow concentrate and then keep them there at the point where we want them the most upon a scaffold. Now we developed this first in the cadaver lab. That's one of the first videos that you saw. And then September of last year, we started a case series with Dr. Andrew's patients and a randomized control trial with my partner Steve Jordan's patients and myself. He's a hamstring guy and I'm a bone tendon bone guy. We're looking at patient reported outcomes and MRI outcomes as well using that MRI metric that I just presented to you earlier. In conclusion, I think a better understanding of cellular mechanisms is very key and us getting our heads more into thoughts that Arnold Kaplan is going to present to us next. But we also need to consider what these cells do naturally, not just in a petri dish, but what they do in us as an ecosystem. And that may be the key to leveraging appropriately. Bone marrow concentrate, I call a point of care bone marrow product. I don't necessarily call it a stem cell product. It has cells with stem capability in it, but it's not just a stem cell product. I think semantics are important as you were alluding to earlier. Consider posterior crest harvest. Our experience, the literature would reflect that posterior crest is best. Our experience has reflected the same. It doesn't slow down your OR when you go lateral cubitus. In fact, it speeds your harvest up. We were just finding that anterior crest harvest was taking longer for us. Anecdotally, we've had less inflammation when we just injected it into joint. We've had a couple cases that have basically the fat pads become a little bit tight afterwards and AC augmentation is likely going to require the entire triad. We need to pursue this as a pyramid and building that pyramid to really help our patients. Thank you for your attention. Also, we have a lot of partners. Our fellows are involved. We have a whole research staff and funding from the state of Florida as well as the city of Gulf Breeze and industry as well. Thank you again.

bone marrow aspirate

ultrasound imaging

ACL reconstruction

harvesting bone marrow

clinical trials

Bio-ACL