I'm Jonathan Rebo, for those of you who don't know me, I'm a surgeon at OrthoCarolina, and I have a faculty appointment at Atrium Health, which is a big hospital system in Charlotte. And I'm here to try to fix a big problem. And this is all the cases that I've included, pictures of your patients of mine that I've seen in the last two weeks. Okay? So these are real problems that happen. This is a 15-year-old girl's knee. This is her lateral compartment, referred to me by one of my partners after ACL surgery, multiple meniscal injuries, for consideration of meniscus transplant. And what you can see is that this girl is 15, and she's in trouble, right? And I would pretend to anybody in the room that an ACL injury is an irreversible biologic event to the joint, right? And I think you can go to the meeting next door and see how good we are at reconstructing the ligament. We know where to put it. We know how to put it there. I would argue that's not enough. Does that really restore a healthy joint? Does that really restore a healthy athlete? Does that really give somebody a good outcome? And if you look critically at the data, and of course, Brian, I'm sorry, I had to put in a Duke lacrosse picture here, the outcomes aren't as good as we think they are. Seventy percent only return to sport, and if you really look at the data, only 50 percent go to sport at the same level. The re-injury rates are exceptionally high, particularly if you're taking care of adolescent athletes like I do. One in three young athletes will have another ACL injury in their lifetime. This is terrible, if you think about it. And this is a guy I saw last week, he's 35, okay? Post-traumatic arthritis after ACL injury is a real thing, and up to a third of patients will develop this within 10 years, and I think this is the biggest unsolved problem of our generation in sports medicine, is putting in a ligament's not enough, how do we avoid this? And I say to everybody here, what if, okay? What if, as orthopedic surgeons and as sports medicine physicians, we do what every other field of medicine has been doing for years, which is precision medicine driven by biology? So this I took from a breast cancer journal, and these are survival rates based on molecular markers for patients with breast cancer. When you have breast cancer, they take a biopsy, they figure out what your receptors are, you get different treatment depending on the receptors, and that has led to dramatic improvements of both prognostication and also treatment. And why can't we do that in our field? So what's the solution? You've got to understand the biology, and what I'm going to propose to you is the way that we do that in ACL is we focus on synovium. Why the synovium, you might say? Well, first of all, it is the only tissue that has really been proven to be a driving force in post-traumatic arthritis, osteoarthritis, and rheumatoid arthritis, so there's actually a lot of literature from sort of the non-orthopedic world on the role of synovium in joint degeneration, and it's easy to biopsy. I remember a great study that Rob did a couple years ago where he took a part of the ACL and they did gene analysis, I think they did gene chip analysis, and it was great, but the problem is if you're trying to get ligament tissue or bone tissue or cartilage tissue, you're causing harm to that patient when you're getting the biopsy. We cut out the synovium on 100% of patients that we do an ACL on, so this is a freebie, right? The other thing, too, is it's a dynamic tissue. It's very responsive to the status of the joint, and so if there's acute inflammation, you're going to see it in the synovium. If the joint is quiet, you're going to see it in the synovium, so it's really this dynamic tissue that we can harness to understand what's going on with the joint, and any solution, I think, particularly for a funding mechanism like this, has to have strong foundation in existing methodology, so I'm not here to try to pitch you on something that's brand new that's never been done before. I'm actually modeling what I want to do after, that you can see this is something published in Nature Immunology, so a reasonable journal from a large consortium of rheumatologists on rheumatoid arthritis, and this is really the key slide, and I'll show you what this all means, but this is what I want to do and what, to some degree, we've already started to do down in Charlotte, so every ACL that I do, a research assistant comes in. Before I do my fat pad resection, I just get an arthroscopic grasper. I take some synovium, and I give it to the research person, and they go off to the lab. This one, I think we have 20 samples that we've already gotten just in the last month or so that we've been doing this. In the lab, we can then dissociate the synovium. We can get cells out of this, so that's sort of step one on this. We can then do flow cytometry, so I can figure out where the B cells, the T cells, the fibroblasts, the macrophages, every type of inflammatory cell that you might find in synovium, and we can basically sort them into separate tubes, if you will, and then we can analyze what are these cells doing, what is their gene expression doing, and we do this with RNA-seq, and there's sort of two different ways that you can do RNA sequencing. For those who aren't familiar with RNA sequencing, that's sort of the modern-day way that we do gene expression analysis across an entire set of genes, so I'm not just looking at one gene or two genes or three. We're looking at every gene in the genome, and you can do that either on all of the cells that you've clumped together, or you can literally do it what's called single-cell-seq, where you sequence every single cell that you've harvested, and you then create these data sets that are quite robust, and we have the capability to do all of this at Atrium, particularly through our cancer center lab. So there's a couple fundamental questions that if I were reviewing my proposal, I would want to know. Number one, can you actually get the tissue that you say that you're going to get, and the answer is yes. These are the first seven that I collected. We looked at how much we get. We get between 150, 300 milligrams, and it's really easy to do this, and we get plenty of cells out of this, and that's the second question. Can you actually get the cells out of this stuff? The short answer is yes. So you don't have to worry about the details, but what we did is on our first 10 samples, we went through and we tried a bunch of different enzymes to get the cells out. We figured out which ones work best. I can tell you it's a collagenase 4 and a dysphase, and we get somewhere between 500,000 and a million cells from every sample, so we can do the analyses that we say we're going to do. Can you get good quality RNA out of this? The answer is yes. I was hoping to have the final slide for this, but the samples are actually in the bioanalyzer today. I was trying to get it in for this talk, but it didn't quite happen in time, but if you look at the literature using the exact technique that we do, on average, you get good RNA with something called an RNA integrity number. It's a 0 to 10 scale of how good your RNA is, somewhere about 7 or 8, which is plenty sufficient to do the studies that we're proposing to do. What are we going to do with the money? Step one, our goal is to actually biobank the synovium. We're not going to analyze these. The second they get into the lab, we freeze the samples. The whole goal, and I'll get to this in a minute, is to actually create essentially a large, almost publicly available repository of tissue of ACL patients with synovium, not just for me doing research, but for anybody in this room who has questions biologically that they want to answer. We've got to make sure our biobanking works. We already have the allotted freezer space. We have the protocols, and what we're doing is we're taking some of our samples, we freeze them, and then we're just going to analyze them after we freeze them to make sure the cells are still there, the surface receptors are still there, the RNA hasn't gone bad. Step two, and this is sort of basically I'm showing you the figures that I'm going to put in the first paper that I write with this grant, this is figure one. Just looking at the abundance of different inflammatory cells in synovium in an ACL population, and this is easy. You do this with simple flow cytometry. This costs a couple hundred dollars and is very straightforward to do, but it's never actually been looked at in the ACL population. This is where the science gets a little bit crazy, so bear with me for just a few minutes. But the next step is to understand not just, okay, there's T cells and there's B cells, but specifically, what about subpopulations? And this is where it gets really cool. For example, in rheumatoid arthritis, they found that there's one particular type of synovial fibroblasts that really drives bad acting rheumatoid disease. And what if we find that there is one particular type of synovial fibroblasts that helps me predict who's going to get arthrofibrosis, or who's going to have bad patient outcomes at six months, or who's going to not be able to return to sport, or even better, who's going to get PTOA? So the way that we do this is with single cell RNA sequencing, and I kind of blew up that figure, and this is obviously something that is done by a statistician that we have that has expertise in this, but you look at all of these genes that are being expressed in these cells, and you figure out which cells are acting like each other, and you cluster them. In this particular example, this is in rheumatoid arthritis, they found four different types of fibroblasts based on their gene expression patterns. And then you can say, okay, well, how are these different types of cells expressed differently in different groups? Here, they compared osteoarthritis versus rheumatoid. For me, it could be arthrofibrosis versus not, reTER versus not, PTOA versus not. So there's a lot of questions that we can answer with this technology. We just have to prove that it's feasible. And then the final step, and this is where it gets really cool, is where you use something called time-of-flight cytometry, or CyTOF. Most people have heard of mass spectrometry. This is sort of a fancier variation of that, and this is available to us through our collaboration with Wake Forest, who actually Atrium Health bought just recently, and so we have access to all of their labs and their cores at sort of a facilitated price. So this is really exciting, and what you're doing is, here, you're using cell surface markers as opposed to gene expression to cluster different types of cells. And what's really cool is you can then correlate these two things and figure out which clusters from your mass spectrometry correlate to your clusters from sequencing, and you may ask, why does that matter? Well, it's actually fairly expensive and fairly time-consuming to do single-cell RNA sequencing, and that will never be something that's probably clinically available. But a quick flow cytometry with markers that you've identified can be pretty much a point-of-care test where I could take a synovial biopsy from my patient, give it to my lab, and tomorrow, I get a predicted risk of their developing PTOA or arthrofibrosis. And so it's really important to get these cell surface markers, and this is how you do it. So what about the finances? We're talking here on the orders of tens to twenties of thousands of dollars. What I'm proposing to do is to do this study looking at one cell type, meaning synovial fibroblasts, because there's a lot of interest in that in the arthritis community, and that's something pretty novel, particularly in the ACL world. And we could do this entire set of experiments on probably a dozen patients with the allotted funds, but looking at just a synovial fibroblast. Once we've done this, we've published this, my goal is to then apply for a bigger, larger federal fund to do this on all the different cell types. You can do this on the T cells, on the B cells, on the macrophages, and in this study that I was citing from the rheumatologist, that's exactly what they did. What types of questions can we answer, and I hinted at this a little bit. So each one of these could be an NIH grant proposal, and this is not even an exhaustive list. And so what I'd portend to you is I'm not proposing one project to you, I'm proposing to you the next 15 years of my career trying to answer a lot of these questions using a particular technology. And it's exciting because these are questions that are really broadly applicable across not just sports medicine, but arthritis, and as such I think they're highly fundable from a federal standpoint because PTOA is a big source of interest amongst national agencies. Bigger picture, what do I want to do with this? Well, if one of it is just risk stratification, can we better educate our patients about look, you're really high risk of having a bad joint, you might want to pick up golf, right? And this has a really high clinical value and honestly is something that could even be commercialized if you have a system where you use mass spec or flow cytometry to do a risk profile, and it's almost like 23andMe where you send a swab of your cheek and you get your own genetic information. You could do this with the ACL world. But probably more importantly is this is how you find molecular targets for disease modifying drugs, particularly for PTOA, right? If you don't understand the biology, you can't find out what drugs are going to help. The studies that have been out there like Christian Latterman's study looking at steroids are great, but if you think about it, that's pretty rudimentary in terms of mechanism, right? We're just throwing in a generic anti-inflammatory. Can we be a lot more precise in the way that we do this? And ultimately, can we do precision medicine? If I find that a certain cell profile leads to bad outcomes when you do a BTB or bad outcomes when you do a quad or bad outcomes if they go back to sport too early, we can really start to tailor our rehab and our surgical decisions for patients. And ultimately, I would envision this if we could get large funding, is this really being a resource to the AOSSM community? Perhaps the research committee is sort of a vector through which you guys approve requests to tap into this biobank. Somebody says, hey, I want these number of samples of these types of patients. And if the AOSSM research committee says go, we do it. Why us? Why now? Well, we have great institutional support. The AOSSM is a very, very financially secure health system that's very committed to their young clinician scientists. I've been one of their named young clinician scientists. What does that mean? That means I have 20% protected time to do this, okay? So while I'm busy clinically, I have the time and I have the support from my institution to make this happen. I have a PhD who has the expertise in all the molecular work. And most importantly, she is not tied to her own grants. She has protected time to help the people like me, which is really critical. What's very cool and why I'm proposing this now and not five years ago is that the cost of this stuff was prohibited five years ago and the cost has gone nosedive. So now we can actually do this cool stuff without massive NIH-level funding. And ultimately, I think that both AOSSM and the AERCAS Foundation, as well as national organizations have identified PTOA, youth sport reinjuries, and return to sport as really important areas of study. And this kind of addresses all of them in one fell swoop. So with that, thank you. And I'm happy to entertain any questions. Great. John, that was great, and I think this is such an important area. And one question that I have is, you know, the ability to link these markers with the development of PTOA and, you know, it's a process that takes 10 to 15 years. So these samples that you're collecting now, if you use just traditional measures, you know, we're looking at like 20, 35 before really understanding what that link is. And what would you propose for like how to connect these markers to development of arthritis? That's a great study, and it's sort of fortuitous with where you're located. You guys have done a ton of work on joint health using MRI after ACL surgery. And that's, I think, where really the answer lies is that you're not going to propose, you know, a 10-year time point for a study, but probably something using cartilage mapping, whether it's, you know, T1 or T2 mapping of cartilage, probably at a two-year time point on MRI. And you can get sort of a semi-quantitative assessment of cartilage health. That would probably be your surrogate metric for PTOA. But you know, in the longer term, at 10 years, I mean, if you look at Moon, they're publishing their 10-year data, I would want to follow these people out for that amount of time. But in the short term, I think it's MRI cartilage mapping. And then one other question, too, is I think the idea of scaling this up on, like, society-wide is awesome, but what would be the cost per sample, like, to have, you know, surgeons around the country banking this, storing this, like, how much would we pay per person? It's actually very cheap. It's basic, because the cost of the banking is not where the cost is, because that's basically the cost of FedExing the sample. And it's freezer space. And what we found is that, you know, we ran it out when we're trying to figure out, like, how big of a freezer do we need? With a standard minus-80 freezer that we can get, we could pretty much store more samples than we could ever collect in a lifetime. So cost-wise, per, so for example, if you wanted to join, it'd probably be on the order of, I would say, $30 to $50 per sample for you to participate. Then obviously, if you do something with it afterwards, that's where the cost comes in. But just the banking's exceptionally cheap. So, nice job, John. We should definitely talk offline at some point. The one thing I would, you know, the vision is there, the roadmap is there. You're still taking the first step towards Rome, so I wouldn't put the cart before the horse. And the specific questions I have there are, so when you do your proof of concept, don't just look at ACL tears. You need to look at some other knees. So it's second order in the fat pad, and you want to make sure, I think you, the long term is definitely interesting. You've got a ton of great studies leading up to it, which I think you showed us in terms of that. I don't know if it's on, but I think you need to, some of the proof of concept is looking at it, making sure you see some differences in, say, ACL tears than knees undergoing meniscectomy, or knees undergoing a total knee, or, do you understand? I mean, I don't know if you've done that proof of concept, but you- Well, so what's nice is we have a universal waist IRB, so basically any knee that I do surgery on, I can take synovium from with this protocol. And so it's very easy for us to do that. You know, I could just add in meniscectomy, so that's great feedback. John, I think this is a great, this is a monumental task, you know. How do you translate? You have a suppository basically of, you know, cells that, you know, in particular conditions might be more than others. How do you get that all the way down to the molecular level where you can actually affect, you know, you have all these great questions that you want to answer, but what is the mechanism that you're going to use to kind of, you know, kind of get down to that molecular level pathway? That's a great question, and that's where the, this particular methodology is interesting. It basically allows you to identify of all of these different cells that exist in synovium which ones are different between different conditions. So it depends what question you're answering, and I'm just going to take arthrofibrosis as an example, you know, and I'm going to compare my patients that got arthrofibrosis versus those that didn't. And when you look at the differences, you can actually figure out which cells with which cell surface markers are different. But then what's cool is then moving forward, you can isolate those cells from any given patient with flow cytometry. And you can then, then what you do is you do gene expression analyses on those specific cells. And that's where really you can get down to the molecular mechanisms, because when you do sort of bulk sampling, and Rob, this stuff wasn't available when you did your study, but that was the problem with studies like, you know, what you did with ACL tissue is there's changes in expression, but you don't know which cells it came from. And this methodology allows you to say this change in expression came from this cell in this particular specimen. So this is a vector to get to then targeted studies. And then if you find a gene of interest, you do probably have to say, okay, we're going to do an animal model. And there's an abundance of, you know, animal models of ACL transection-induced PTOA, for example. And you might say, okay, well, now we're going to overexpress or underexpress this gene, and we're going to see what actually happens. Does that have effect? So it's sort of like you use this to go back to the lab to do a lot of work to then come back to the true precision medicine. But this is, you're right, in the big vision, this is step one. And the other thing you should really think about is arthrofibrosis is where to start. Because arthrofibrosis, you can answer in a short timeframe where I build the foundation to look at the long run. So start with arthrofibrosis for sure. Awesome. Because you can, A, it directly relates to synovium, B, you can answer in a short timeframe, and then you can lay the foundation. All right. Awesome. I think that's my cue. Thanks, John.

ACL injuries

precision medicine

synovium

gene expression patterns

personalized treatment plans