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2021 AOSSM-AANA Combined Annual Meeting Recordings
Development of Immune-Directed Cartilage Bioprinti ...
Development of Immune-Directed Cartilage Bioprinting Platform
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Video Transcription
Ina is very well represented here, so many thanks to the panel for considering this proposal, and kudos to our other competitors. Now with those kind statements set out there, let me tell you why ours is better. Really, we're looking at the development of a novel immune-directed cartilage bioprinting platform, which you'd say, I'm not even sure what that means. Let's kind of dive in a little bit, but first, I want to draw your attention to the team that's down at the bottom. I'm really benefited by being with a great list of collaborators and co-investigators, including Gary Paling, who's here today, and really our team at Wake Forest Institute of Regenerative Medicine, or WFIRM, has really been the group around a lot of these ideas that we've coalesced. And that's led by Anthony Atala, who's actually a urologist, but is very intimately involved in our work within bone and cartilage restoration. Gustavo Moviglia is our basic scientist, and he's from Argentina, and he has really been instrumental in driving forward this novel cell concocter technology. Carlos Cangla is our bioprinting expert. Johanna is our cell line expert, and then Gary and I kind of provide the vision. So you're not really necessarily just investing in me, because I wouldn't do that either. You're investing in this entire team, which is a 200,000-square-foot facility. It's five floors, and just really incredible synergy that's coming out of Wake Forest. So buckle up. There's a lot to take in. I'm not sure if you were able to watch this. This patient was having some connectivity issues this past weekend, but he was able to take down quite a bit, and what we're going to try to do is cram as much of this as we can into this talk, but first we've got to define the scope of the problem, which is that chondral defects, as well as PTOA and kind of the arthritic cascade is really something that is quite considerable. We can see at the time of arthroscopy, Gary has certainly shown us that up to two-thirds of our patients have chondral defects at the time of knee arthroscopy, but only about 5% of those are really ones that are candidate for focal cartilage restoration. And certainly we've sort of laid out this a la carte menu of things that we can provide to individuals, but as the lesions get more complex, as the sizing gets more difficult to treat, really we are left with two primary options in this day and age for medium to large defects, and those are fresh osteochondral allografts and cultured cell-based techniques, both of which do have their limitations. In reality, when we look at the data across the United States, and you see all of these procedures, you know, close to about two million, the broader majority of these are just, you know, palliative in nature. They're chondroplasty, they're marrow stimulation, and so really we've continued to innovate in this space, but we are still no better at treating these types of lesions, these large, medium to large defects with potentially a little bit of bony involvement, a compromised joint surface, and you compound that with an active individual, that really puts you in a very difficult problem. So I want to talk to you briefly about our approach to addressing this, and we really have an incredible amount of data, both from in vitro and in vivo, animal on wood to human, and what we'd like to do is try to export this to the next line of inquiry, which is look at bioscaffolds. I want to dwell on this slide for a minute and just kind of set the stage. Obviously, we've talked a lot about MSCs as a potential vehicle for regenerative medicine, and this is coming under increased scrutiny, both by the FDA and within our facilities, but for MSCs, these can come from a variety of different sources. We've certainly looked at fat, at bone marrow, and that is a core ingredient to our cell co-culture technology. In our proposal, we're recommending using fat-based or adipose-derived cells and then combining it with this effector cell population. The effector cells are really drawn from the peripheral blood, so you just obtain a peripheral blood sample. You can spin that down, isolate the buffy coat, and through a process of apheresis, you can get this mononuclear cell lineage. That mononuclear cell is then exposed to cartilage fragments, and what we're trying to do is we're trying to deregulate the system. Most times when we get these damaged particles, say from a chondral defect, these recruit inflammatory cells, the inflammatory environment takes off, much like John is describing, and what we're trying to do is trying to tweak that balance so that we can tip this more into a pro-regenerative pathway. Those mononuclear cells represent only about 0.5% of that cell population, about 1 in 10,000, but when you identify those and expose those to cartilage fragments, then they'll subsequently be expanded. They'll develop into the cells of interest, which are these regulatory cells, and they are then exposed to the MSCs, and that, in turn, will create N cell differentiation, in this case cartilage, but it can be used for bone, it can be used for tendon, it can be used for muscle, and so we've got parallel inquiries into this as well. And really, we've seen this work in fairly granular fashion. One of the ways that we wanted to assess this is obviously by looking at the various different growth factors and expression antigens throughout our process. And so you can see the MSCs are really the cell line that we're looking at, and we're following those through, but those mononuclear cells that ultimately develop into effector cells are really the vehicle by which we get this terminal cellular differentiation. And so this is really what we're going to set forth here in the next couple slides. You can see that initial mononuclear cell culture is then expanded, and after 72 hours, really you've got this self-isolated effector cell population that can be confirmed with these markers shown here. You then expose that to the MSC co-culture, and that's subsequently expanded, and it can be either introduced as a cell fluid introduction, or it can be placed onto a scaffold to localize it to the area of interest. And so you can see the MSCs, not to belabor some of these points, but you can see the expression features from time zero, and then with exposure of the MSCs in the expansion process as you go from 24 hours on up to 96 hours with the expression of SOX9 and a variety of different pro-regenerative factors. Additionally, you can see shown here, again, our slides, just showing proof of concept of what I've just shown with you on the cartoon model, showing that this is effective. So we've shown this in vitro, and then subsequently expanded this to several small animal models in order to convey that this is effective. And what we did is we started with looking at a chemically-induced OA model. There's a lot of interest in PTOA from post-ACL to just degenerative osteoarthritis in our military population, post-traumatic arthritis from blast injuries, from repetitive overuse. So there definitely is a lot of interest in both focal chondral defects and arthritis. And so we started with an arthritis model, and so we used monoiodoacetate in order to induce this osteoarthritis model, and then subsequently applied our treatment. So you can see that shown on some of these slides here. You can see our control. This is a section showing healthy cartilage initially on the top left, and as you progress along, you can see the natural damage that's brought on by introduction of MIA. Well, then we can see the control at day 28, and then when we apply the effector cells, we apply the MSCs, and then we provide that cold culture down there on the right. You can see those yellow lines carve out those cartilage cells that have been directed towards terminal cell differentiation. And you can see these cells continue to convey the similar concepts, which is that you're getting N-level hyaline cartilage in an area that was predisposed to fibrosis and ultimately abnormal bony reaction. So our question has been, can we resolve this chemically induced OA? And what we have continued to show on several different levels is that it's effective. You can see the ORSI scoring. I'm not very good at histology. You can certainly evaluate these pictures. But what you see is with that cold culture, we can really regress that knee back to its native state. You can see the LCM blue is really isolating those GAGs on the top right, and then on the bottom you can see expression of those collagen type 2. So it's really quite impressive. And again, these are just more samples, again, conveying that. You can see the high expression of collagen type 2, the paucity of collagen type 1, which is expected in some small component. And then you can see human response. So we're fortunate to have pilot data from Dr. Moviglia from his initial pilot series, and you can see the regeneration of this nice hyaline cartilage in an otherwise compromised articular environment. So briefly, what about focal chondral defects, which I think we're all still continuing to try to innovate in this space? We have a pretty robust bioprinting division within WFIRM, and they've done everything from trying to regenerate cartilage for the ear, trying to regenerate renal tissue, and really the next frontier for us is looking at osteochondral defects, both for OCDs and also the focal chondral defects. This is challenging. The technology requires a pretty significant buy-in, and we have all that contained within our facility. So our goal is really, with our three aims of this process, is one, we're going to try to expose this cell co-culture with use of micronized porcine cartilage tissue. So we're going to use that as the vehicle to prime those effector cells, then to expose it to MSCs, and then try to demonstrate proof of concept that we can print this so-called bio-ink. The next step is really to try to strip that down. So just try to get more purified proteins, glycosaminoglycans, and use those as the priming antigen for our mononuclear cells. So something that's commercially available, try to work it from the bottom up rather than the top down. And again, just try to show that we can generate these scaffolds. And then lastly, it's really to generate these scaffolds using those purified components, and then re-implanting them into our Lewis rat population to really assess some parameters for their implantation. And subsequently, the next phase of this would be placing this into the articular environment. So with that, that will kind of lead to the ask. What's the ask? With the direct costs, it's really looking at our Lewis rats and housing them, ultimately upkeep, all the consumables related to our cell culture and the reagents that we would use, our lab wear and reagents for acquiring that decellularized extracellular matrix powders, the isolated biomolecules for mimicking that cartilage, and then ultimately just the other consumables for the rodent surgical care. A very modest amount would be utilized for partial salary support for our staff and postdoctoral team, but really we're just committed to this next line of inquiry and this science. We think that this will allow for development of a new tissue engineering platform based on a novel stem cell or signaling cell differentiation paradigm, and really will enable us to develop novel strategies for further research and expansion within the broader musculoskeletal system. So thank you, and happy to address any questions. Brian, thank you very much for an awesome talk there. When I looked at the proposal, I kind of broke it down to kind of two different really concepts. One is how you guys make the cells, and then the other part being the customizable scaffold. So I have a question for kind of each. In terms of the cells and being able to produce cartilage, chondro-like cells in this manner, how do you think this technology is different compared to how we're able to do it currently with MACI? It's an interesting concept, and truthfully, I don't know that I entirely believed it until I saw some of this data. It's completely different in the regard that we're using a immune cell to direct end cell differentiation, which is a novel concept. The way Dr. Movigli explains it is we have a variety of autoimmune diseases, and so we know that we have our own immune cells that will attack native tissue. Well, the counterpart to that is that when they are exposed to these levels of damage, are there a cell line that you can use to build that back up? And so, to my awareness, there's been nobody to really look at that avenue of trying to regenerate tissue. Cell-based technique, like autologous chondrocyte implantation, is, one, it relies on a process of native regeneration and reorganization that I'm not entirely sure always occurs. I've done several second-look arthroscopies to look at my MACI, and some have integrated in well, and truthfully, some haven't. And also, when you biopsy that, that is definitely hyaline-like cartilage. That's not really hyaline cartilage, and I think what we've shown here is you can see native integration, and it's also reconstitution of that native bone-cartilage interface that is directed by this technology. And then my second question was just, do you envision the scaffold being able to address some of the subchondral disease that comes with cartilage processes, as you know? And that's really where our goal was initially. We've, you know, Gary and I have really been very invested in a lot of different populations that are unique to North Carolina and our area, but we see a very heavy amount of OCD lesions. And so, in that younger patient population where you're maybe a little hesitant to place an osteochondral graft, knowing the long-term survivorship of those lesions, is there something else that we can use to direct regrowth of bone? Which we can. It can be used for both cartilage and an osseous lineage. And then how do you address those more irregular shapes, right? Not everything falls into an oval and a circle, at least not in my practice. And so, trying to address those that are multi-compartmental involvement, as well as these fairly irregular lesions, that's what this would provide. Not to mention, you know, arthritis. We're focusing on focal chondral defects, but we all know that where the money's at is treatment of arthritis using endogenous sources. I don't know about you all, but in North Carolina, you know, the birthplace of Krispy Kreme, we've got a lot of adipose cells to donate. Everybody's got a little bit of blood. And so, if we can export this, I think we'll have several willing participants. Hey, Brian. Thanks for a great presentation. Very important need, certainly. And you seem to have a wonderful team. You know, for me, while I'm assessing everyone's presentations today in a true Shark Tank fashion, I'm looking for a couple of things, return on investment, as well as timeline for deliverables. And the impact that the Playmaker Grant Award will have. Meaning, if Amazon comes to us and says, hey, we want to try this new thing, and we want to get the Playmaker Grant, it may not be as impactful. It just seems that, I'm just so impressed by the Wake Forest Institute of Regenerative Medicine. The question I have for you is, if you don't get the Playmaker Grant, how will it derail this project? Or is it going to continue on with Dr. Maviglia's work? It just seems like he's done fantastic work. So I want to hear about the impact of this Playmaker Grant, whether it's $20,000, $25,000. And we also want to support orthopedic surgeons who are involved in research. And so I want to find out exactly what your role is going to be, because I want to support you, rather than to support Dr. Maviglia, where you're a secondary investigator. Is that fair to ask? I know that's absolutely fair. Welcome to the Shark Tank. Full disclosure, I will not be pipetting. I will not be performing the prosections on the Lewis rat bodies. Am I intimately involved with the project? Yes. And that's the value we bring to a lot of these multidisciplinary work groups, is we provide the clinical perspective that's incredibly value for pushing forward these important problems. So Gary and I have a weekly meeting with our team. And then we direct treatment with a variety of our employees. I mean, there's 400 employees there, and they're all at the direction of Dr. Atala. He is committed to developing this forward. And so you're supporting me, because previous to this, they were working on things that probably are more targeted towards a very small niche population. We've really taken this a step backwards, started looking at really compelling problems like rotator cuff disease. How do we ultimately develop muscular tissue that can really allow for that muscle to hypertrophy in a cuff-deficient model? And then, obviously, in this case, we're talking about focal chondral defects. The impact of this funding and whether we would be able to proceed forward with our research in the absence of this, yes, but we'd seek other grant funding mechanisms. I think this is a concrete project that allows us to take the technology that we're already working on in parallel. We're submitting all that through other different DOD and NIH-funded grant pathways. But we really want to look at scaffolds to, one, help to localize the tissues, and two, to show a proof of concept that we can take this from the stripped-down components and develop it and implant it. So we would just seek other measures, but it would be incredibly impactful for this focal area. Great. You know I love you, Brian. Thank you. Thank you, Brian.
Video Summary
The video transcript is a presentation by Brian Cole, who is part of a team at the Wake Forest Institute of Regenerative Medicine. The team is proposing a novel immune-directed cartilage bioprinting platform for the treatment of chondral defects and post-traumatic osteoarthritis (PTOA). They aim to use mononuclear cells and mesenchymal stem cells (MSCs) to direct end cell differentiation and regenerate damaged cartilage. The team has conducted in vitro and in vivo experiments to validate their approach and has shown promising results in chemically-induced osteoarthritis models. They also plan to use bioprinting technology to create customized scaffolds for the regeneration of focal chondral defects. The team is seeking funding to support their research and believes that their approach could revolutionize tissue engineering and lead to improved strategies for musculoskeletal restoration.<br /><br />Credits: This video presentation is given by Brian Cole from the Wake Forest Institute of Regenerative Medicine, with acknowledgements to the team collaborators, including Dr. Gary Paling, Dr. Anthony Atala, Dr. Gustavo Moviglia, Dr. Carlos Cangla, Johanna, and Gary.
Asset Caption
Brian Waterman, MD
Keywords
immune-directed cartilage bioprinting
chondral defects
post-traumatic osteoarthritis
mesenchymal stem cells
customized scaffolds
tissue engineering
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