It's almost certainly not, but thank you very much for your time and attention. Thanks for the opportunity to present this in this fun format. I definitely want to start by saying, well, first of all, my name is Adam Yankee. I'm at Rush University. I did all my training there, which included a doctorate in biochemistry and continue to do some basic science work. And my co-investigators, which are more importantly, my mentors are Dr. Cole and Dr. Shubhanskaya, who really need no introduction. Dr. Cole is the president of ANNA, Dr. Shubhanskaya, the president of ORS. I was the president of the orthopedic interest group in my medical school. My disclosures are online. We also recently got an NIH grant for some cartilage work, which is really honoring too. So the knee is what it is because of many tissue types working together and functioning as an organ. And it only works correctly by all of these pieces working correctly. So if you have an orchestra playing the garage, it's not going to sound the same as if it's in a symphony hall and you need to take into account the environment. And that's why normal articular hyaline cartilage has the structure and function that it has because of everything that led to its creation. Similarly, the milieu that you heard about a little bit already in the synovial fluid is really what maintains this because it has only nutrition through diffusion and what the chondrocytes do to maintain homeostasis. And the synovium itself is a huge factor here, which is seen by the fact that rheumatoid arthritis is almost non-existent in total knee arthroplasty surgeons office now because of treatments of the synovium and helping this environment. When you have acute traumatic injuries, such as fractures or impactions that come along with ACL tears or similar injuries, this results in a cascade of inflammatory factors, which is usually led by IL-1 beta followed by TNF-alpha and other cartilage breakdown products. This has been shown in multiple labs and similar numbers to what you just saw. There's about a 10% risk of development of post-traumatic arthritis after these acute events. PTOA affects approximately 700,000 adults in the United States and costs $6.4 billion to treat annually, include lost wages, as well as total knee arthroplasties. So if we can have a treatment that even reduces the rate by 5% of total knees from PTOA, that's $2.5 million of annual savings. And I think that's even a small goal. I think it could be much more than that. So the question is when to intervene and when can you stop this cascade and prevent the development of arthritis, which is really all the work that's being done in post-traumatic arthritis models. This is not the first, we're not the first people to look into this. Dr. Latterman, as well as Dr. Krause have looked at this in a clinical study looking at steroid injections and they show decreased cartilage breakdown products when you do an acute steroid injection compared to placebo. Similarly, Dr. Krause looked at IL-1 receptor antagonists, which we heard anakinra, which was also injected and this improved patient symptoms even two weeks after. And she has a similar finding in her mouse model, looking at post-traumatic arthritis and preventing the onset of these negative cascades and catabolic cascade essentially. We did some work in our lab where we impacted human normal articular cartilage. We then looked at cell viability with normal cartilage alone, and then with the addition of normal synovium. So it was a co-culture model with just regular synovium from that same patient after the impaction was done. We saw sustained cell death without any treatment and with the synovial treatment with the synovial membrane just in the same culture well, we saw mitigation of this viability response and significant improvement at days 2 and 14. The only factor that was significantly upregulated in the media, so this is control again, looking at IL-1RA, was IL-1RA with significant increases from day 7 and day 14, which is coming from the interaction between the normal synovium and the cartilage itself. So we thought this was very interesting and this led to some of the ideas of the current work, which is really an extension of some prior work looking at amniotic suspension allograft as well as dehydrated IRAP. The reason we're looking at these two potential therapeutics are their potential clinical roadmap where ASAs should be available now based on FDA regulation after hopefully being indicated for knee arthritis. And the one that we're choosing specifically is because it's the only one that has level 1 clinical evidence looking at its use and effectiveness for knee osteoarthritis compared to hyaluronic acid and saline, as well as some early work that we did in another co-culture model with arthritic chondrocytes where we applied bone marrow aspirate concentrate from the patient as well as ASA and found that we had no anti-inflammatory effects or reduction of IL-1 with BMAC whereas we did with ASA. And so this is an interesting therapeutic target and it has been shown to have a high IL-1RA concentration that's relatively conserved across donors. We also are investigating dehydrated IRAP, which was really brought about through a collaboration with our institution and Colorado State University. And David Frisbee has been working on this to a significant extent. It's similar to the orthokine or rigenokine process where it takes ACS and develops an IL-1RA to IL-1 ratio that's very favorable and is approximately 10 nanograms per milliliter. And there's already some equine studies that have shown its benefit in preventing lameness in horses. So these are the targets of what we would like to investigate and essentially we want to see is what is the effect of IRAP and ASA on chondrocytes in an in vivo co-culture human model of post-traumatic arthritis. We're taking five distal, again, human femurs using an explant of their cartilage at eight millimeters doing a controlled impaction that we've used in our lab in the past as well as synovial explants. These are then going to be co-cultured for 24 hours and then we will subject them to saline, dehydrated IRAP, or amniotic suspension allograft at clinical concentrations. These would then be cultured for up to 14 days and along these timelines they'll be evaluated for pro-inflammatory and cytokines in the media as seen here as well as growth factors and cartilage breakdown products in the synovium and the cartilage will be evaluated with histology as well as gene expression to see really what's driving the changes that are seen in the media that lead to the downstream hopeful benefits of the application of these products. So the goals of this study are to determine the ability of ASA and IRAP to mitigate the catabolic cascade that follows impact injury to human articular cartilage in a co-culture model and then look at the individual contributions of the cartilage in the synovium with hopeful future directions to utilize this in an in vivo randomized trial of acute knee injuries such as the ACL model that Dr. Latterman and Dr. Krause have used and apply this to cartilage transplant patients to try to improve that post-operative environment that we're asking cartilage to integrate or grow that's really filled with a hemarthrosis or pro-inflammatory cytokines that we looked at through an aspiration study post-cartilage transplant and those are elevated for up to six months after transplant. Thank you very much for this opportunity and we think this is a very realistic project that we could accomplish with the funding involved and we'd be honored to receive it and thanks for allowing us to present. Yeah, Adam, that's a great study. Can you talk to us a little bit more about the control of impaction and how do you make sure it's uniform across your samples? Yeah, it's a great question. We had a custom-built jig from I think about 12 years ago that is a pneumatic impaction device that allows us to take the cores, load them in, and then do direct perpendicular impaction at one Newton second. We have a controlled impulse and a controlled force and each plug is done individually. Adam, nice job. Are there any concerns with getting your amniotic suspension allograft due to what's happening in that market right now and then furthermore, even if you were able to get it for this benchtop project, then going potentially to market with that or going into a clinical trial? Yeah, it's a great question. Certainly the FDA component has waylaid really expanding the indications for this. It hasn't stopped the ability to examine it in a laboratory setting, so it wouldn't have any direct effects on this project. It is in a phase three clinical trial due to the FDA regulation, so it has already enrolled its first patient, and so it is along that process, but to use it clinically, it would require potentially an off-label usage once it's already indicated for knee arthritis. So it hopefully is just a delay, not something that is not going to be possible. Adam, great work, and this is obviously something that is close to my heart. When you're dealing with IRAP, and the problem is when you're using the purified protein, it is a single-time shot, right? So you basically get an effect, it gets bounded, it goes away. The consequence after an impaction injury, if you're creating your model correctly, will go on for slightly longer. How are you planning to deal with the dosing effect? Because this is a system where the balance, the ratio between IL-1 and IRAP plays a role How do you mitigate that in your system? Yeah, again, great question. This system that was developed and helped produce at Colorado State University, they've modified the beads and such to try to really increase the ratio of IRAP to IL-1 to have it be as high as possible. It's hard to know how high is adequate and what's appropriate. There's no delayed response or treatment here. The only thing I can say is that we've done a similar model where we were able to see significant changes and reversals within the timeframe that we're suggesting currently. So it's just a matter of how different is it when you have an acute impact? Can you actually just do something over a short period and reverse it versus trying to reverse osteoarthritis that's taken a long time to develop and has resulted in gene expression changes that may be harder to overcome with a single treatment? I would have another question if I may. ASA is an interesting kind of construct, right? It contains a lot more than IRAP. Another important factor, for example, when it comes to inflammation and a potential response of synovial tissue and synovial monocytes is fast ligand, for example. So there could actually be a response of the synovial tissue to the ASA that will affect your outcome measures. Are you planning to look into that at all? Yeah. If I understand correctly, we are analyzing the gene expression profile of the synovium as well as of the cartilage so that we can see which component is upregulating genes to render the effect. Adam, Kevin Stone here. Nice study. I just want to point out to a study that Dan Grandy and I published because we wanted to understand how paste grafting works when you smash the paste, articular cartilage and put it back into the knee. And so in his lab, we smashed articular cartilage into a paste and demonstrated upregulation of the cellular behavior on all the factors, proteoglycan production, extracellular matrix production, gene expression at 24 and 48 hours. So you don't kill the cells, you actually upregulate them for a while. They try to reproduce their own matrix. They try to produce healing. I don't have any data past 48 hours, but you might want to take a look at that just so it doesn't screw up your concept of, hey, you're causing harm right away. Because at least we showed you're not causing harm right away. Maybe later. Yeah. Thank you. I wonder if it's a difference in the force and the impulse to apply. I think that it's certainly dose dependent. So the model that we have, we have shown decreased viability, but it could be that we're hitting it a lot harder. Okay. Thanks. Thanks. Can we go to the international group? Yeah. Thank you for this very interesting approach. You claim that it closely mimics the complexity of a knee joint, but I honestly doubt this point. Could you tell me a little bit about the limitations from this in vitro approach to a real clinical scenario? The challenges for translational kind of steps? Yes. Sorry. Closely is a loose subjective word, I guess. But the thought is that if you have a cartilage explant utilized alone in vitro, the response is definitely different based on our work as well as others than if you did a co-culture with synovium. Since synovium is probably arguably the more biologically active tissue in the knee joint affecting the trajectory of what happens to the synovial fluid in that environment. So the idea is that it's a more close estimation of what could happen in a joint, but certainly that model has never had a head-to-head validation with an actual in vivo model. Thank you. One more guys. Yeah. Thank you. Do you take the osteocontral track or just superficial cartilage only? It is the full thickness cartilage and we try to have a small component of the calcified cartilage layer included. So we try to be right at the junction and we can usually harvest enough that if we have some that are too superficial, then we don't include them. So it does have to have the full thickness and with a small bit of bone potentially on the back, but no marrow elements. Thank you. All right. Thanks very much, Adam.

post-traumatic arthritis

PTOA

treatments

amniotic suspension allograft

dehydrated IRAP