All right. Good morning, everyone, and welcome to today's AOSSM Research Showcase Webinar. This new webinar series will showcase previous winners of AOSSM Research Grants in their studies. I'd like to thank Lynette Kraft and Alexandra Campbell from AOSSM for helping to facilitate this session. A lot of work's gone into it, and it should be really exciting. I am Drew Lansdowne. I will be serving as today's moderator. I am an assistant professor in the Department of Orthopedic Surgery at the University of California, San Francisco. I'm an orthopedic surgeon, and I specialize in sports medicine, as well as surgery of the knee, shoulder, and elbow. I have an interest in cartilage restoration surgery, and I'm especially excited for our talk today for that reason. I have also been a previous winner of the AOSSM Steven P. Arnosky Young Investigator Grant. But today, we're pleased to have Dr. Carolyn Dealy as the first presenter in this new webinar series. She's a former winner of the AOSSM JRF Ortho Allograft Grant. We thank JRF Ortho for their ongoing support of the AOSSM research efforts. Her talk is titled Biologic Approach for Articular Cartilage Repair, and she will share findings from this funded study with us today. Before we get started, I'd just like to share a few housekeeping items. If you're having trouble hearing or seeing the webinar, please hang up and join again. It's most likely a problem with your connection. Our presenter will speak for about 30 minutes, and then we will have time for a question and answer session at the end. If you have questions throughout the presentation, please type them into the question area on the GoToWebinar platform, and then we'll look to address as many of those as possible during the Q&A session at the end. With that taken care of, I'd like to tell you more about today's presenter. Dr. Carolyn Dealy is an Associate Professor in the Department of Orthopedic Surgery, Department of Biomedical Engineering, Orthodontics, and Cell Biology in the Schools of Medicine and Dental Medicine at the University of Connecticut Health Center. Dr. Dealy's research objectives are to understand disease mechanisms that underlie arthritis and post-traumatic osteoarthritis, as well as to develop clinically relevant preventative and treatment approaches for these conditions. Her focus is on the cellular response of articular cartilage to injury and ways to leverage these responses towards cartilage health, self-repair, and regeneration. Dr. Dealy is active in research translation at UConn. She is an inventor, a startup founder. She's an honoree of the Connecticut Women of Innovation. She's a founding member and mentorship chair for Connecticut Women in Bio, which is a bioscience sector leadership organization. She's also an alumnus of the ABCT eLab NYC Startup Accelerator and a faculty liaison for UConn's NSF I-Corps site Innovation Accelerator Program. She's a founding director of state and university programs that have engaged over 300 students in biotechnology, innovation, entrepreneurship training, and she teaches courses for medical, dental, and graduate students on regenerative medicine, clinical innovation, and scientific writing. She has mentored 60 scientific trainees in her lab from undergrads to junior faculty, and she is an author on over 50 journal publications. So at this point, I would like to welcome Dr. Dealy and turn it over to her for her presentation. Thank you so much, Drew. It's really such a pleasure to be here, and I'm so grateful to have the invitation to speak and share this research, and again, grateful also to JRF Ortho and to AOSSM for their support for this research. So with that, I will open up my presentation. All right. Drew, does everything look good? Looks great. Okay, terrific. All right. Well, thank you once again, everyone, and I'm so pleased to be talking to you today about the studies we've been doing that were funded by JRF Ortho and AOSSM through the JRF Ortho Allograft Grant, and I'll be talking to you today about biologic approaches for articular cartilage repair. So the overall, you know, overarching issue that we're trying to address here is really post-traumatic osteoarthritis, which I don't need to explain to all of you is a form of osteoarthritis that has a very rapid development. It comprises about 14 percent of all osteoarthritis cases, which translates to over 10 million adults per year in the United States, and it's marked by a known injury event. So typically, these kinds of injuries happen in elite athletes or in military service personnel who are engaged in active training and active athletic activities, which cause an injury and lead to an articular cartilage defect, as shown in the arthroscopic image. And again, these typically have a very rapid disease progression to overt osteoarthritis spread throughout the entire joint, and at that point, you know, we know there's not that much we can do for these patients, but can we intervene earlier on to try to prevent post-traumatic osteoarthritis from happening? And yes. So pioneers in this field, including, I'm honored to be one of my collaborators, Bill Bugbee, have, and colleagues, pioneered osteochondral allografting in this country, using donor living tissue generously provided by the families of deceased. The patient's damage is debrided, cleaned up, and then the two pieces are fitted together, and then, you know, inserted into the joint. And osteochondral allografting, some large-scale trials now, it's been around for quite a while, and we're beginning to accumulate quite a bit of clinical evidence, has really good success. At five years, and even at 10 years, things tend to drop off after that, and eventually, osteochondral allografts do fail. So the question here is, can we improve osteochondral allografting so that we can reduce the failure, have more individuals be eligible for this approach, perhaps some individuals that currently are not eligible because of their age, or because they have other injuries to their joint that preclude them being an ideal case? So can we improve this, delay PTOA, and increase accessibility to osteochondral allografting to a larger patient pool? Well, in order to figure this out, we really need to know first, what is the root cause for failure of the graft? And again, I don't need to remind you that failure typically doesn't mean the graft actually falls out. What tends to happen is it becomes a site of osteoarthritic changes, and, you know, changes within the structure of the cartilage that lead to pain and, you know, coming back in the patient, loss of mobility, and a need for either a vision surgery or some other kind of approach to get them to the point where they can have a total joint replacement later on in life. So some of the reasons that allografts, you know, fail then, we can get some clues by looking at animal studies. So I'm shown here two animal studies, one that was done in goats and examined six months after surgery. And in the panel on left, in the black circle, we can see that the grafts are still visible. You can pick out the two, there are two grafts implanted there, and you can see that they're, you know, they don't look like the rest of the cartilage. You can pick them out really easily. So they're not integrated. And histologically, we can also see that there are gaps between the cartilage edges at the graft and at the host site. Now, in contrast, the bone integrates quite well, and you really can't pick out, you know, where the border of the bone portion of the graft was, but certainly you can in the cartilage portion. Now, if we look at the bottom panel, this is done in a rat, I'm sorry, a rabbit, a little bit earlier after the surgery implant. And what we can see that kind of presages some of these changes that are seen later is a formation of a seam in between the allograft, or in this case, it was an autograft, and the host tissue. And that seam, as indicated by the large arrow, is acellular. It has no cells in it, and it's made of fibrous connective tissue. Essentially, it's a scar. So the idea is that micromotion around this scar develops over time because it's not a firm and integrated repair, and that this leads to the changes that then cause clinical failure of the graft. So in order to get an integrated repair, we really need to knit the two pieces of the tissue together. And that's a cell-mediated process. It requires coordinated breakdown of matrix, proliferation of cells, and then synthesis of new matrix. And this all has to happen along the entire, the two surfaces of the integrating tissues. And the problem with adult articular cartilage, healthy articular cartilage, is that it's a really stable tissue. I mean, that's what it's designed to do, but it doesn't work for us in the case of cartilage repair. It has very little regenerative response, very little growth response. And by growth response, I mean proliferating cells, the ability to turn over matrix, degrade matrix, and synthesize new matrix. And oh, by the way, that new matrix needs to be articular hyaline cartilage matrix, not fiber cartilage or a fibrous matrix, because we all know that's not going to be durable. That's not going to do the job for patients. So if we could stimulate these growth responses in articular cartilage, might it be possible to have cut edges actually knit together through this active plus-minus, you know, some degradation of tissue, some synthesis of tissue? Could we have the tissues knit together in this active way? Well, there's a couple things going against us. So we know that healing, seamless healing of the tissue, is going to require cells because it's a cell-mediated process. But graft storage causes chondrocyte cell death. So the top panel shows human femoral condyle allografts that were stored in a standard storage media with FBS, fetal bovine serum, and sectioned and examined by a live dead stain. So I'm not sure that the contrast is showing up super well for everyone, but basically living cells are green and dead cells are red. And the red cells are a little bit hard to see. But over time, at 28 days, there was 30% cell death in all of the zones of the cartilage. But interestingly, other studies have shown that the majority of that death occurs in the superficial zone or the top zone of the cartilage. I'm just going to flip back just one second to here, just to remind everyone that the superficial zone is the region in the upper, say, 10% of the articular cartilage layer that contains a less mature cartilage matrix and an immature chondrogenic population. So that population particularly seems susceptible to cell death. So around the time that these allografts are available for patients, 30% of them are dead, and a vast majority of the cells in the superficial zone, which has these progenitors, is also dead. So again, I mentioned the superficial zone is a source of immature chondrocytes. The bottom panel shows a study that was done kind of to illustrate this capability. And again, I apologize if the images are not showing up super well, but basically what we've shown here, what I'm showing here is, if you're looking down on the articular cartilage and all of the living cells are labeled green, and then an injury was made in this particular study by Sol et al in the shape of an X, which might be able to make out a red X here, and it's red underneath because the cells are dead. And then over time, over a period of a week to 10 days, the superficial zone cells around that area actually proliferate and start to migrate in, and you can see their elongated shape as they're migrating, and they'll actually fill in some of these shallow defects. So the superficial zone is an important zone in articular cartilage. It may not be the only one that has healing potential, but it's certainly one that we want to engage if we're going to get allograft healing in site. Now, we have other problems as well in that surgical cutting also causes cell death at the implant site. So both in the process of preparing the implant site and in the process of cutting out the donor allograft piece, we generate cut edges that are also sites of cell death. And we can see a section through a sample that was using the oats harvesting system, again, showing the edge where there's a zone of cell death forming at the cut edge, and in the live dead staining, again, the green cells are the living ones, but all these ones here on the left are actually red, and that means that they died in response to being cut. So we can imagine that when we make this construct inside the patient, that we've actually now have two opposing faces where there is a zone of cell death. There's no cells are present on the two adjacent faces, or soon there won't be very many cells present. So the chondrocytes disappear, but the matrix is still there. So there are no chondrocytes to proliferate or respond to signals, and the matrix remains and is a barrier to integration. So in order to achieve integrative cartilage healing, what we're going to need to have, of course, it's cell-based, so we need to have cells present. Those cells will need to proliferate and perhaps have signals that, and or signals will be needed to recruit cells to that region, and they'll have to be matrix breakdown and matrix synthesis. And if all those things happen, then healing should occur. And I say should and hypothetical because this doesn't happen in vivo in either animals or in people with either autographs or allographs. Can we ever get it to happen? And the answer is yes. So there are studies that have shown that in vitro, it's possible to stimulate articular cartilage healing. And these studies were done quite some time ago, thinking about the idea that when the dead zone forms, and we can see it here in the left-hand panel, this absence of cells adjacent to the two opposing faces and the formation of the seam down the middle, the matrix is still hanging around. And well, maybe if we digest away the matrix, we'll provide room for new cells to come in that can then proliferate and knit the edges together. So in this study, a model was used that kind of recapitulated the OCA surgery model with an inner core being implanted into an outer ring of cartilage. And the inner core was treated with enzymes that digest away matrix, hyaluronidase and collagenase. Then the new recombined graft host unit was then cultured. And what we can see in contrast to the control, which I mentioned has a dead zone around it and forms a seam in between the two opposing faces, that the core that was pretreated with enzymatic digestion has lots of cells at the place where the two faces of the cartilage are meeting each other. And it also appears that there might be some healing going on, although you can still see a seam where the two faces are pressing against each other. But this is certainly an improvement. There are lots of cells present, they're proliferating, and there is new matrix and turnover. So it's definitely a step in the right direction. So this indicates that seamless healing should be possible if we can provide proliferation, matrix degradation, and new matrix synthesis, basically cartilage growth responses. So what other kinds of signals stimulate cartilage growth responses? And we ask this because we probably don't want to be treating allograft cartilage with enzymes that can digest away structural components of the tissue and then using that and expecting durable repair out of that. So we need to come up with another kind of signal that can stimulate these growth responses, but without modifying the structural capability of the matrix itself. And we've done studies in the lab for quite some time. I've been interested in signaling from the epidermal growth factor family. This is a very well-known family of tyrosine kinase receptors and growth factors that is, of course, misnamed because they were all named according to the first property that was observed and happened to be stimulation of the epidermis. But the EGFR does lots of things. And we've done some studies showing that it's important in skeletal tissue and especially in cartilage tissue. And I'll just really briefly describe what's going on here in this slide. This was a study in which we used a transgenic mouse model in which EGFR family signaling was activated. It was not activated through an overexpression, but rather it was activated by removing a natural suppressor. And that part's not that important. What's important is the result, which is that in contrast to the control knee, which you can see in these sections, frontal sections of the knee of the mouse, where the articular cartilage is marked by red saffron staining, that the samples or the animals that had activated EGFR signaling have a dramatically increased thickness of the articular cartilage. And in fact, it's nearly double the thickness of the regular control mice. So this indicated that at a gross level, activating this family could really increase growth responses in articular cartilage. Again, increasing the thickness, matrix synthesis is increased, remodeling is also increased, and it's more cellular. So there are more cells present, proliferation is increased. So we wondered, could we use some of these signals to stimulate cartilage growth responses that might be useful for improving allograft cartilage integrative healing and wondered also well which one so the EGFR family is a complex family there are four related receptors and over 12 ligands and some of those families of ligands have sub isoforms so it's a large family with a lot of diverse functions to it so there are a lot of options here and I'll mention that there's there is a nuance to the signaling in that the mechanisms involve formation of either homo dimers so you need two receptors to get a signal and those can be two members of the same receptor or they could be heterodimers forming between two related but different receptors in this family and depending what ligand activates that you can generate different kinds of responses often they're pro mitotic pro survival sometimes they can be pro differentiative or pro migratory etc so there are a lot of options here so how do we narrow this down so one way we went about that was to go back to our mouth where we overexpressed or rather we removed expression of the suppressor of family signaling so in this animal all of the receptors if they're present will be active okay it doesn't mis-express them if they're not originally there and it doesn't constitutively express them in a in a non-normal way but if they're present in the tissue they will become activated and we found when we used immunohistochemistry to identify which receptors were activated i.e. phosphorylated we found that the predominant members that were activated were EGFR or in mice it's called ERB1 and ERB4 a related member in humans it's called HER4 so these were the two most upregulated members which meant that these are the ones that are endogenously present in articular cartilage and the ones that correlate with the increased thickening of the tissue so this suggested that maybe the most appropriate candidate to look at in terms of stimulating a cartilage growth response might be a ligand that activates both of these receptors and so we chose oh sorry we also confirmed that in healthy human articular cartilage those two receptors are expressed so not only in mice but also in humans and we picked out a growth factor that activates both of those receptors and it turned out to be heparin binding EGF can activate both EGFR and HER4 so that makes it a candidate for promoting cartilage growth responses so now we went about developing an in vitro model in which we could test the hypothesis that exogenous HPEGF might promote cartilage growth responses in human articular cartilage and I'll mention that none of this would have been possible without J.R.F. Ortho, David Wilkie the the director of research there and their generous provision of tissue samples for this study so we used talus for our study because the femoral condyles and tibial plateaus were generally being there's very high demand for those in the clinical pipeline so by using talus we were able to you know pull samples for study that wouldn't detract so much from the clinical pipeline and again we're just incredibly grateful for this generosity because I know how valuable these these samples are. What we did was we would immobilize the grafts when they when they arrived and then extract out the osteocondyle explants. We used an oats harvester to take out 10 millimeter explants because we wanted them to be of a clinically relevant size and then we created an injury in them so this was an established model where using a dermal punch it's lowered down on top of the explant and punched all the way through the cartilage but not through the bone so it stops when it hits the hard tissue of the bone. This creates a full thickness defect through the cartilage but leaves it still attached to the bony platform so it's an easy model to study because things are not moving around in vitro. And then we placed these explants into organ culture using a base media similar to storage media with 10% fetal bovine serum and ascorbate etc and we maintained them for 28 days or four weeks and then subjected them to histological analysis. So first I want to explain how we validated the culture system. This is a control explant so from a healthy talus and these are individuals who range in age from around 15 to 30 years of age and after 28 days it still looks pretty healthy there's lots of saffron and no staining but we can see the two injury lines and for reference this is what the explant looked like so where that injury is corresponds to the ring here which goes all the way down through the cartilage okay and those are still present so there was no healing okay and a higher magnification again shows there's no healing. We compared the general appearance of the articular cartilage after 28 days to that of freshly harvested talus and they're virtually identical at the gross level so the tissue does not appear to be deteriorating but it's certainly not healing. So the culture system reproduces the stable homeostasis of healthy adult articular cartilage. We also confirmed using mechanical strength testing that the healing that there was no healing in these control explants. We used a push-out test in which a load cell was used to lower a rod which was four millimeters in diameter down onto the core which remember is partially held in place by bone we cut off the bone so now the only thing holding it in place would be any integration that's occurred between the core and the surrounding ring of cartilage tissue. So we lower that rod down over that region and then it pushes through and then record the amount of stress it takes to push it out and this is shown in the graph here this is increasing stress stress stress stress and then pop it pushes the core through so you see this break and we found that explants that were freshly prepared or cultured for 14 days or cultured for 42 days had no difference in the amount of force required to push out that central portion so that again confirmed that there is no spontaneous healing happening no substantial integrated strength happening in control explants over at least 42 days. Now one thing that's interesting about that is that this model was originally used in boving articular cartilage this was done using calf so two-week-old and four-week-old calf articular cartilage and the authors of the study found that there actually was pretty full integration so you know here on the on this graph 60% is right here so I cut it off but 100% is about here so there's virtually a hundred percent integration happening in the calf articular cartilage at two weeks and four weeks. This is showing the scale using histology of how they scored integration so this is not the time course but this is the scale so full integration in their study looked like this and we can see some of the same features that I showed before about cartilage that's showing a healing response including increased numbers of chondrocytes that appear to be dividing you see doubled cells there so evidence of division maybe some heterochromatic staining suggestive of matrix turnover but interestingly and again I apologize if you can't see this really well in your image but there is still a seam all the way down so it's not seamless integrative healing the two edges are pushed together but they're not integrated like like the loops and and tags on velcro which is really what we want to achieve and it's quite interesting that that this happens in that there is some kind of healing response in the cow but not in human and we're not sure whether this is the result of a difference between humans and cows or whether it's the result in age the samples we used the human allografts you know could have been a little bit older than calf calf would presumably be like an adolescent but it's a it's a market difference on something that I think the field needs to keep in mind that studies that are going to be relevant for humans should focus on human tissue so now moving on to our question can we use this growth factor heparin binding EGF to try to stimulate some growth responses that would accelerate integrative healing in human articular cartilage in vitro so we added HP EGF to our culture media and maintain these X plants for 28 days and first what I'll show you again is this control X plant showing no healing and here is a representative X plant from a group of four each that was subjected to continuous exposure to heparin binding EGF throughout the 28 days and we can see that it's harder to see those gaps I think we've got some higher magnification views here again the control has absolutely no healing present at all but in the treated the edges of the gap are closer there appears to be more cellularity around the region although there's still a seam present you know you can kind of make out little bits there might be a small portion where it kind of disappears maybe a little bit of integrative healing but there is still a seam and it's not closed at the top but this is certainly promising we were very excited to see this we've measured the morphometry of these X plants and showed that there is a significant increase in full cartilage depth so that means from the top all the way to the bottom of the cartilage including both the non calcified and calcified zones was significantly increased so this was really suggestive and indicated that this growth factor can actually stimulate cartilage growth responses in adult human articular cartilage so we were super excited to see this we also tried a variation of this approach using intermittent growth factor treatment so withholding it for five days adding it to the cultures for three days taking it back away again for five days adding it back for three days and so on over the 28 day period and we found that intermittent treatment maybe gave a little better results we're not sure again our numbers are small we have a group of four each so the numbers are small and these are coming from three different individuals but four separate X plants interestingly that when we quantified this similar to the the case where we applied continuous treatment we have a significant increase in in the cartilage depth but interestingly a decrease in the calcified the thickness of the calcified zone and the calcified area we also have a loss of the superficial zone in other words the superficial zone kind of disappears and you can see this in the histological images as well where the control has the traditional or the expected layer of immature matrix at the top that doesn't stain with saffron to know our treated explants stain with saffron and oh right up to the top so what this suggests to us is that we're engaging the population of progenitor cells that are present in the superficial zone and accelerating or converting their differentiation into more mature chondrocytes that are producing highland like cartilage and that stains with saffron I know because it contains lots of agrican our interpretation of the significant difference in the thickness of the calcified portion of the zip zone is also really interesting because we know that activation of signaling by this family is a negative regulator of chondrocyte hypertrophy so this would be consistent with the idea that this growth factor treatment may actually be working on two different populations within the chondrocytes and we think that this is super exciting because not only may it be activating the superficial zone which we know is important in healing but it might also be activating some of the cells of the deep zone and holding them back from progressing into the calcified region where they become terminally differentiated in fact ultimately transition into bone so we have a kind of a combined effect on two different populations that have the potential to form more of the middle zone tissue the weight-bearing portion of the cartilage that is you know full of highland it's it's all highland cartilage so again we're very excited about this but we're greedy we still have a little gap in healing and so we wanted to see if we can enhance this healing response any further so we tried a couple other other possibilities you'll recall that I mentioned that some early studies that were done in the cow had shown that digesting away some of the matrix around the edges of the cartilage could improve healing in vitro in the cow and in fact this was the actual first evidence that suggested that some healing integrative healing was even possible in articular cartilage so we wondered if maybe our approach combined with a pre digestion of the matrix at the edges might be even better so we treated our explants we added hyaluronidase and collagenase together to the media of the explants for a two-day period and then took them out of that and then subjected them either to no treatment or to our growth factor treatment until the end of the four-week period had elapsed and we found that basically didn't work so matrix pre you know matrix pre digestion prior to applying the growth factor did not improve healing in human articular cartilage there is essentially no difference between digest pre digested explants followed by control media for 28 days and those that were followed by HPEGF treatment for 28 days they both have severe proteoglycan loss and there's no no closure at all of the gaps so this this was not promising we had a course of positive control in this experiment where we mock treated in other words the explants were just held in control media for a couple of days during the time that their their peers are being treated with enzyme and then treated them with growth factor and as before there was a very robust response cartilage looks healthy and possibly some integrative healing happening in the cartilage tissue as well so digesting away the matrix or you know loosening the matrix it didn't help and again that's interesting because this is what worked in in the bovine so in adult bovines this actually stimulates some kind of end you know almost looks like integrative healing but it doesn't in human so there again maybe some differences that we should appreciate between bovine and human articular cartilage in our studies so what next well we thought we would try another angle to try to amplify this HPEGF mediated growth response so HPEGF which if you recall stands for heparin binding EGF has a heparin binding domain and it binds matrix heparin sulfate proteoglycans or HSPGs. Heparin sulfate proteoglycans are abundant in articular cartilage matrix some of the perlican is one there are many but they're very abundant so the HPEGF is binding to the matrix and interestingly this binding actually happens at a higher affinity than the affinity of HPEGF binding to the EGF receptors so what this means is that the matrix binding of the growth factor actually kind of sequesters it away from binding to its tyrosine kinase receptors so it can't signal the cells so we wondered if we prevent this binding will we then will the HPEGF then be more bioavailable to activate receptors instead of being stuck in the matrix and perhaps we'll have an amplified cartilage growth response so the approach to do this was to use heparinase so heparinase is an enzyme that removes heparin sulfate residues from heparin sulfate proteoglycans this is just showing a diagram of what an HSPG looks like you can see the gag chains attached to the core protein and the heparin sulfation are just these random moieties which I've shown here by the the green the green dots so heparinase removes those so the rest of the molecule is intact but it just doesn't have any heparin sulfate if there's no heparin sulfate there's nothing for heparin binding EGF to bind to so it can't bind anymore and instead the heparin binding EGF will be available for the cell receptors which should potentiate its effect whatever on cartilage growth responses so we treated the explants with heparinase and then removed it and then subjected them to our standard treatment with or without growth factor. And what we found is that, again, control X plants that were treated with heparinase followed by HPEGF had a nice, robust-looking articular cartilage. But those that were pretreated by heparinase in comparison were dramatically thicker and appeared to have better healing. So this is shown here in high magnification. And in this case, it almost appears as though there is almost invisible seam. And healing is starting to go all the way up to the top. And again, the superficial zone is fully engaged because it's making lots of sefrin and O positive matrix. So this suggests that the cells in it are no longer immature. They've been converted to mature hyaline cartilage-producing cells. We quantified these and found that not only was the full cartilage depth increased, but also there was a significant loss in the thickness of the superficial zone, consistent with its conversion to mature hyaline tissue. So again, the numbers are small. These allografts are precious. And we do everything we can with what we get. But we need to do a little more here. But it's suggestive that this might be one way to modulate or amplify the growth factors effects. So in summary, what I've shown is that by activating signaling through HPEGF, we are able to promote cartilage growth responses and in vitro healing responses. The cartilage tissue actually increases in thickness, presumably via the increase in cell number that we see, as well as the robust matrix synthesis and deposition that we see. We're very excited by the possibility that we could be stimulating multiple populations of cells within articular cartilage that have the potential to form true hyaline tissue, which might include not only the superficial zone cells, but also some of the cells that are down towards the deep zone by holding them back from progressing further and calcifying, essentially maintaining them more as middle zone cells instead of deep zone cells. We've also found that heparinase pre-treatment might increase bioavailability and amplify these effects. And again, I want to point out that we're beginning to think we see some differences between what we're finding with healthy human articular cartilage and what's been previously reported in the most often used system, which is either adult cow or calf articular cartilage. So I want to call attention to that because I think this suggests that we really need to be focusing on using human tissue because it may well be different. And for that, I'm so grateful for JRF Ortho for providing us with this tissue. Otherwise, we would never have known that these differences may exist. So looking forward, we're hoping that this growth factor might be useful for maintaining the health and homeostasis of osteoconagrafts during storage, perhaps extending the storage time so there may be more grafts available for use, or making them more healthy or amplifying their innate healing potential so that when they're implanted, they can achieve a seamless healing response that will keep the graft in place longer and improve outcomes for patients. And I wanted to note that UConn has just filed a provisional patent on the applications of this work for this purpose, which is good news because once something is patented, that means that there can be interest from external groups beyond government funding levels that might be able to promote and advance actually into clinical application because that requires deep pockets. And finally, I'd like to thank the people who've done the work in my lab. Two master's students did the majority of this work, Sandra Clouseau and Caitlin Flint. In addition, my research associate Melanie Fisher, a dental student, Edison Altador, and an undergraduate student also contributed to this work. And I can't be thankful enough to my clinical collaborators, Lee Pace, who used to be here at UConn right across the street at the Children's Medical Center and is now at Andrews Institute in Plano, Texas, who was so generous with his time. When we were doing our first extractions of explants from the talus, we literally FaceTimed him so that he could explain how to use the Arthrex harvester. So video is everywhere. And thank you, Lee, if you're listening. We really appreciate your help then and always. And Bill Bugbee, some of the material I showed you as supporting data was done by studies with Bill. And he was one of the pioneers in establishing osteochondroallografting as an approach here in the United States. His input has been absolutely invaluable. And Bill, if you're listening, grateful, grateful thanks to you as well. Two of my UConn collaborators are biomedical engineers, Tannen Schmidt, who helped us with our mechanical testing, and Sham Nukavarapu, who also is helping us in various ways on this study. And finally, thanks to the American Orthopedic Society for Sports Medicine and JRF Ortho for their innovative idea to team together to provide investigators with the Allograft Research Grant, without which this research wouldn't be possible. And with that, I'll be happy to take any questions. And thank you so much for your attention. Great. Thank you, Dr. Dhealy. That was outstanding and really exciting and impressive work. So congratulations also. And for all of our attendees, if you have any questions, please send them in through the GoToWebinar question feature. And then we'll be able to read those off, get them answered for you. And maybe just to kick it off, so one of the ideas was that this may be able to extend the shelf life for osteochondroallografts. I know that's a real clinical problem because we have such a short window to use these grafts. Do you have any feeling for how long that might be able to extend? That's a great question. So we have definitive evidence that we can get out to 28 days. So that's already a week after some of the grafts would be set to expire. So we're really confident about it. And those grafts are great. They don't show any signs of deteriorating at all. And again, it appears that their healing potential is amped up. So we're pretty confident that we can bring this out quite a few weeks, possibly as long as maybe five or six weeks, which would be tremendous. Because as you say, there would be no wastage because grafts could wait until the patient matching happens. So that would really increase the number of grafts. The industry wastes 20% to 30% of the grafts that are harvested because they don't match in time before they get expired. So if we can remove that, that puts more grafts into the pipeline and makes it more available for people like you to use. We've got work to do before we get to that point. But we're optimistic. Great. I think another question is, so you showed how with treatment the superficial zone changes. Do you think those would have any clinical relevance? And in line with that, do you foresee any other potential safety issues with using this type of treatment? There will always be some concern with introducing it clinically, of course. Absolutely. Absolutely. I mean, safety has always got to be the primary concern. And that's one of the reasons that using this as a media additive rather than something that's implanted into the patient in the form of a growth factor itself is a more natural response. So we're stimulating the cartilage, and then we're taking it out of the stimulus and then putting it in the patient. We've seen, like I said, a pretty amped up response. What we want to do now is actually kind of titrate that back down a little bit. So make sure that the cartilage that's treated has the same characteristics that untreated cartilage has in terms of its mechanical strength properties. So we've only tested a small group of samples that were not treated to kind of basically get the procedure down, because it was new for my lab. And our next step is going to be to rigorously test the mechanical properties. So there are different ways of doing that, just force and compression, to just make sure that it just has the same integrity and properties in the matrix as healthy regular cartilage does. Another thing that I should mention, when people hear EGFR, because it has so many different effects, it's a required, it's an essential factor for life, in fact. But when it's dysregulated, it can cause cancer. So people hear that, and there's a concern there. And in fact, one of the things I study in my lab is actually cancer diagnostics, believe it or not, with a collaborator who's a cancer biologist. So I am a little bit aware of the realities of that. And one of the features is that many of those cancers are actually driven by the receptor signaling. So overexpression of the ligand itself is quite different and doesn't tend to cause. In some examples, you can get transformation by just overexpressing ligand, but it's not nearly the same as activating the receptor, overexpressing a receptor in a mutant fashion. So there's some safety built in there. In other words, it's nature's stimulus, not an exogenous mutant receptor gone wild. So we have some, and also, this factor is a normal component of serum. And it's present in synovial fluid. It's present in serum. So again, if we can back down the concentration to something that's more comparable to what it is in serum, providing it perhaps in an intermittent fashion to phase out stimulation, we think there are some ways that we can get at the safety. And of course, we'll be testing everything rigorously. Great. And another question. In seeing a lot of those images, I think the allograft application is obviously very exciting. And then it also makes you think, what about applying this towards an acute cartilage injury or when patients have symptomatic fissuring or their breakdown? And do you foresee future other clinical applications and how those might be able to be applied and work? Well, everyone's holy grail is let's cure osteoarthritis, right? And perhaps there's applications there. Working with tissue that's osteoarthritis is kind of a case of hyperactivation of all those chondrocytes in all the wrong ways. And sorting out mechanistically what happens and then figuring out how to stimulate the right population of cells to go in the right direction in order to get repair in that kind of toxic environment is going to be a challenge. And that's why it is a challenge. But sure, of course, we're interested in that idea. We're also interested in something in between more related to allografting, but instead of allografts, you have synthetic grafts. So treating synthetic grafts with the growth factor implanting and then using that approach. Because allografting is popular here in this country, but not so popular elsewhere in the world. So there are a lot of patients that don't have the benefit of that gold standard of cartilage repair and are relying on other modalities that perhaps could be improved by including this growth factor as a component of the manufacturing process. Great. We have a question from Lynette Craft, who's asking, as a basic scientist, how did you go about creating this collaborative team to conduct this work? Thanks, Lynette, for that question. Gosh, I think for me that the transformation was really, you know, starting to work with colleagues in different departments and then becoming interested in working with clinicians and then seeking them out. I think in the case, what led me first to Lee, because he is local, was actually someone who reached out to me, come to think of it. It was actually a clinician who reached out to me many years ago wanting to collaborate about cartilage. And that eventually led me to Lee and to the idea of this, you know, our studies were evolving and this idea started to take shape. First, he was just giving me tissue samples. And then we really started thinking about this problem of osteoconic allografting and kind of realizing that there may be potential here. And I certainly realized that it's so important to have collaborators that are in the clinic, because there are needs that I certainly didn't appreciate, realities of the pipeline of allografting and protocol, as you say, you know, the testing and those kinds of things. There are certain challenges that are important to understand in order to figure out what the solution is and how that's going to work and make it feasible. And some of that, I guess, motivation for wanting to learn all of that also stemmed from some of the work that I had done in the area of innovation and startups, where the same sort of philosophy applies. You know, you really need to figure out what the need is and make sure you have a good match between what you're developing and what the need is. And working with clinicians is the only way that a basic scientist can be sure that what they're working on is solving a clinical research question. And obviously, the work was done in the talus for the reasons that you stated for availability. Do you think the femoral cartilage or tibial cartilage, would you foresee it behaving differently? Or do you think it's transferable? I would expect it to be, but. It's a good question. And in biology, expectations are proven wrong every day. So at least in my lab, they are. At least in my lab, they are. This was one of the exceptions where our expectations were surpassed, frankly. So the talus could be different. I mean, I'd like to think that it will be transferable. And we're going to try that with the blessings of JRF Ortho, of course, in providing the samples to do so. But the talus is a more reactive joint. The ankle's a more reactive joint, as I'm sure you know. Following traumatic injury, if you compare traumatic injury in the knee versus the ankle, the percentage of patients who go on to PTOA in the knee are injuries only 50%. In the ankle, I've seen 95%, 98% of injuries proceed to post-traumatic osteoarthritis. So I have a personal vested interest here because my daughter is a ballerina. And her ankles are already under a lot of stress. So I'm working hard because I know she's going to need something in her ankles pretty soon. But the talus appears to be maybe a more reactive kind of cartilage. It may have more progenitor cells than the femoral or tibial cartilage does. There's some studies that have compared by transcriptomic analysis the collection of genes that are expressed in, I think it was the articular cartilage of the hip, the femoral cartilage, and the ankle, and found that the ankle had the collection of kind of youngest genes. And the hip was sort of the genes that were more characteristic of aging or older tissue. So that almost suggests that more distal cartilage portions, like the ankle, might be more progenitor-like, maybe more responsive to what we're working with. I think there will be some differences. But I do hope that it is transferable because so many more patients have the need there. Yeah, definitely. And I think maybe with that, we'll look to wrap up for the webinar. We're just approaching our hour. But Dr. Daly, thank you so much again for doing this. That was a fantastic talk. And thank you, everyone, for joining today, as well as for watching the recorded session later on. And thank you again to JRF Ortho for supporting this work, and to Lynette Kraft and Alexander Campbell for facilitating, as well as the AOSSM support. We just want to remind everyone that this year's JRF Ortho grant is open on the Research Portal. Submissions are due by August 1. If you want to learn more about it and apply, you can do so through the AOSSM website, which is listed here, sportsmed.org slash research slash grants. And then if you'd like to watch the webinar as a recording or access other AOSSM e-learning, you can go to education.sportsmed.org, education.sportsmed.org. Also, if you forget that, you'll be getting an email in 24 hours as a reminder with that information. And then last point is AOSSM does have open registration for two upcoming in-person educational events. The annual meeting in DC is coming up in early July. And then the AOSSM isosteotomy is around the knee session in October in Rosemont. So with that, thank you all so much. Hope you've enjoyed it as much as I did. And enjoy the rest of the day. Thank you so much, Drew. And thank you all for attending. Bye bye. Bye.

articular cartilage repair

post-traumatic osteoarthritis

HB-EGF

cartilage growth responses

heparinase

thicker cartilage

better healing

acute cartilage injuries

collaboration