All right, my name is Katie Whitney and I'm an MPH student in epidemiology at CU Anschutz and a research specialist at the Stem and Filpon Research Institute. And today I'm going to be talking to you a little bit about innovative methods to improve the cell function of bone marrow aspirate concentrate and bone marrow derived stem cells. Okay, the incidence of post-traumatic osteoarthritis is dramatically increasing in active young adults with the onset of post-traumatic osteoarthritis 4.2 times more likely to develop after injury. Orthobiologics have been used as a biological adjunct in surgery and for pain management to help reduce chronic inflammation. My work over the past seven years has been primarily focused on characterization of cellular molecular constituents in bone marrow aspirate concentrate, BMAC. However, due to the lack of clinical evidence and variance in patient outcomes, there is currently no consensus on the efficacy of BMAC for the treatment of musculoskeletal tissues. There's emerging evidence that aging and injury significantly impacts regenerative potential of tissues through cellular decline and cell cycle arrests, also known as senescence. Senescence has several factors that contribute to cellular decline, including anti-apoptosis, telomere shortening, senescence-associated secretory phenotype factor secretion, also known as SASP, organelle dysfunction, and DNA damage. Specific cell populations that are important for musculoskeletal regeneration repair demonstrate senescent characteristics with aging. In fact, Dr. Buckwalter's group recently found that chondrocyte proliferation reduces with age while the presence of beta-galactosidase, a known senescent marker, increases in chondrocytes with donor age. It has also been found that cultured bone marrow, mesenchymal stem cells, MSCs, have dramatic morphological changes at higher passages and correlates with senescent markers. These senescent features may be a potential biological link when it comes to regenerative decline and development of post-traumatic osteoarthritis after injury. In an effort to intervene in the spectrum of this disease, our group is investigating therapeutic approaches to improve musculoskeletal healing and delay post-traumatic osteoarthritis by a combinatorial approach of senolytic and xenomorphic agents to improve the beneficial effects of cell-based orthobiologic therapies such as bone marrow aspirate concentrate by the removal of senescent cells and inhibition of SAS factors. Our group and collaborators have extensively worked on defining senescent mechanisms and interventional strategies for the removal of senescence in preclinical and clinical models. In a preclinical model, our collaborator found that interticular injection of senescent cells accelerated the progression of osteoarthritis in mice, while our group has found that the senolytic agents facet and incursitin can delay osteoarthritis in a progeria model. Our group currently has two active federally funded randomized control trials assessing outcomes with facet and treatment of senolytic agent in symptomatic knee osteoarthritis alone and also in combination with bone marrow aspirate concentrate. In one of the two trials, we are aiming to treat patients one month prior to the bone marrow harvest to reduce senescence and also SAS factors. This work is to help support our current clinical trials by validation and development of a C12 FDG mechanism to look at senescence not only in bone marrow but also in peripheral blood. In an effort to understand the role of senescence in bone marrow aspirate concentrate, we aim to detect cellular senescence in non-expanded bone marrow samples and also culture-expanded bone marrow mesenchymal stem cells using an autofluorescent marker called C12 FDG. After detection, we will treat bone marrow cells and bone marrow MSCs with facet to remove senescent cells and inhibit SAS. We will also determine the cell function improvements through proliferation and differentiation assays. As part of the experimental design of this IRB-approved study, residual bone marrow will be collected to detect senescence and treat expanded and non-expanded bone marrow cells over various time points with facetin. The C12 FDG detection modality is currently being validated by our lab in different cellular samples, including peripheral blood mononuclear cells and bone marrow concentrate samples. With this detection method, we have observed distinct senescent phenotypes that express high as well as moderate senescence. We've also been able to successfully measure SAS factors within these sample types. With some of our preliminary data, we have also shown that facetin can reduce senescence in culture-expanded bone marrow mesenchymal stem cells. To recap, our goals of this in vitro study are to define the differences between high and moderate senescent phenotypes, to determine the optimal dosing regimen, to reduce senescent cells in SAS with facetin, and to improve the functional activity of bone marrow cells and bone marrow MSCs. Our hope is to contribute this work to the healthy aging program in orthopedics by improving clinical outcomes through senescence reduction. I would like to thank the organizers for the invitation to speak today and also for my organization with their support. I'm open to questions. It's a great idea and a great proposal. One question, I might have just missed this. Who's your patient population? I know you're going to do 20 patients undergoing bone marrow aspiration, but who are they and what are they undergoing at four, and what are their age limits, and will you be able to control for different variables, including age, health, BMI, and sex? Since this is the first time that we have really, truly looked at the detection using a C12 marker, we've opened this up to ages 18 through 80. It's individuals that are coming in for pain management purposes, maybe post-operative, and also individuals that have been diagnosed with osteoarthritis too. I think that's something that we do want to look into, specifically what their conditions are, and also any types of comorbidities that they have too that could potentially affect their senescent levels. To follow up question, for a study like this where age is likely an important variable, would it make more sense to limit your patient population to either an older or a younger age, given that the sample size is so small? Because the challenge I can foresee is a type two error once you get your data. No, I completely agree with you. I think because this is just the validation of the detection of senescence, we do want to look at younger age groups as well to see what really that normative scale looks like. I do think that that's a great suggestion for this to keep this really close to potentially somebody that's above the age of 40 or 50. If I may ask a question, did I understand this correctly? You are trying to dose patients with, I believe, Fisetin or another senolytic in order to then determine whether you can reduce senescent cells in your BMAC aspirations? That's correct. Do you have any evidence at all that senescent cells in BMAC are a problem? That is a great question. We currently do not have the evidence to support that all senescent cells within bone marrow are detrimental. That would be a future direction of ours. With Dr. Kirkland's group, they have shown that in mice, they were able to isolate and expand mononuclear cells. They induced them into senescence and they re-injected them into a joint. They found that it did accelerate osteoarthritic conditions. The point that I'm making here is that the next step for us would be to evaluate that, to basically sort out some of those cells from the bone marrow aspirate concentrate, as well as senescent cells from bone marrow mesenchymal stem cells, to isolate, expand them, take those senescent cells, and then re-inject them back into a joint in an animal model to see what would happen to assess that. Does that answer your question? Yeah, it does, but it concerns me because I get the impression that this is really kind of looking for an application of a tool rather than finding the background as to why. I still have no evidence and I see no evidence that a BMAC injection, for example, you would assume that it causes harm if that was true. We've had an entire symposium yesterday about that and there's actually very little harm that we see unless you go through an effort where you actually intentionally concentrate senescent cells in it, it sounds. So, where's the clinical application here? Right. So, the overall clinical application of this is that we're evaluating the mechanisms of action with senescence and basically trying to re-insure that there are specific SAS factors that are being expressed by those senescent cells and to compare those to other donors too. One of our major aims out of this is to really establish what the norm really looks like in potentially younger donors versus older donors too and why those two different phenotypes look differently. The other side of the clinical application of this is that this technology can be performed within a matter of hours. And so, the turnaround time with this to tell us, okay, can we find or detect senescent cells within these bone marrow aspirate concentrate samples for an individual that's undergoing this? Can we have a quicker turnaround time so that we can say, okay, we're finding that there is elevated senescence within your bone marrow sample versus somebody else's? And so, we can look at the functional activity of those bone marrow senescent cells separately, but that would have to be done in an in vivo model and an animal model. Yeah, that was a great presentation. I think you mentioned a couple other studies and how this is part of a combination of things. Would you mind just talking about how this study fits in the overall translation plan and incorporates into some of the other stuff that's already ongoing? Sure, definitely. So, the overarching plan and kind of what I reiterated to earlier is that we're really just trying to detect senescence within this group or this population that's undergoing this procedure, number one, and then number two, it's looking at the mechanism of action too with facetin. So, there's three genetic or gene expression markers that we're going to be evaluating with this. And some of the previous literature has really shown that with facetin, it should target P53, which is a gene that's being used for cell cycle arrest, and it modulates that. And so, as part of this study, we're going to look at the gene expression there, and that's going to fit back into the rest of our clinical trials that are currently ongoing to say that, okay, we are very much aware that there is some kind of antioxidant properties within facetin and also is able to inhibit that specific gene, but what else does that inhibit too, such as P18, P21, P16? Those are other genes that we're interested in evaluating as well. So, it's to help support the rest of those clinical trials, if that addresses your question. All right, can we open it up to the international crew? Thank you for the interesting presentation, and it's quite an innovative approach. My question is, please correct me if I understood wrongly, but you are modifying the bone marrow harvest to inject more juvenile cells finally, but don't you think it's more important to rejuvenize the recipient side, either intra-articular and how you want to try to even address the subchondral bone with this rejuvenation strategy? That is a great question. I actually had this on a separate slide. I really went back and forth of whether I wanted to include this, but no, you make a really great point there that we should also be really assessing what the local mildew within the joint looks like. So, with our group, what we've started to do is not only just in an animal model, but we have currently a trial ongoing in which we are taking synovial fluid from patients that have acute knee injury, and we're also using this exact same detection methodology to measure and compare senescence within that local mildew. Another future direction of ours would be to essentially take a baseline of that synovial fluid, manipulate the bone marrow cells, potentially either systemically or locally for banking purposes, and then we would re-administer the manipulated bone marrow cells, and at that point, we could potentially do a post-injection or a post-operative aspiration of the synovial fluid to see whether or not we're actually reducing some of the senescent cells that we're initially detecting. Does that answer your question? Yeah. So, finally, I see it's a first step now. Yeah. Thank you. Hello. Congratulations, of course. I understand that you're working on the expanded MSCs as well, right? That's correct. Is this a process which is FDA approved? So, we're going through a process that's not necessarily FDA approved, GMP approved. I think that's the question that you're asking. So, for this basic science study, we're just trying to look at, essentially, when we expand, isolate and expand these specific mesenchymal stem cells, do they respond to facetin at higher passages? And so, because we're just looking at this from more of a translational standpoint than anything else, and because we've just started this project out, we're not going through FDA or GMP guidelines for this specific study, because we're just trying to look at the overall assessment of how this treatment really is responding with these cells. But that would be the overarching goal and future direction of ours for banking purposes, because right now there's about 15 different companies, different types of mesenchymal stem cell products that are currently on the market. And so, that's something that we're really looking into. Can we take some of these mesenchymal stem cells and treat them with a senolytic agent, such as facetin, prior to administering it into a patient's joint or whatever conditions that they're using it for? So, your goal is to use facetin right from the beginning, after you realize what is going on with the juvenile part, then to use facetin or not? Right, exactly. But it's always good to know that part of the study is to be able to detect these types of cells as well. So, having a baseline from these samples is really important, because we haven't ran into this yet. There's certain varying levels of senescence within individuals' peripheral blood, as well as bone marrow samples. And so, as part of this study, we're just trying to establish what that real norm looks like and what's an outlier, essentially. And do we need to come up with a different dosing regimen for those individuals with senolytics or cinnamorphics? All right, Katie, I think we're going to have to call it there. Thanks very much for your presentation. And we'll go back to you.

Katie Whitney

epidemiology

bone marrow aspirate concentrate

musculoskeletal tissue treatment

senescence