David J. Mooney, PhD - Mechanotransduction and Bioadhesives

Research that has captured um several of our um imagination and uh as surgeons, um, who, um, as far as translational engineering research. He is the um uh Pincus Family Professor of Bioengineering in the Harvard School of Engineering and Applied Sciences and a core faculty member of the Bees Institute. He received his BS in chemical Engineering, uh, from the University of Wisconsin, PhD in chemical Engineering from MIT, and as he's just sharing with us, um, he spent, um, some time, uh, uh, training here as well under Doctor Bacanti. Um, his, uh, lab, um, researches and designs biomaterials to make cell and protein therapies effective, um, for practical approaches to treat disease. And I think, as I mentioned, um, uh, some ways that, uh, apply to the way we treat disease as surgeons. Um, he's won numerous awards including the Clemson Award from, um, the SFB Merit Award from the NIH, Distinguished Science Award, um, from the IADR. Um, and the Phi Beta Kappa Prize for Excellence in undergraduate teaching. Um, so he, uh, um, I look forward to seeing what you have to say. So, thank you. So thank you for the introduction. It really is a pleasure to be here. Um, as I just mentioned before I got started, I actually spent 7 years of my life in this building. I did pretty much all my research training here in the lab of, uh, Judith Folkman and before that, for a little while in Jay Vacanti's, uh, excuse me, uh, Judith Folkman's lab first before Jay Vacanti got his own independent laboratory. Um, so I've been to a lot of seminars in this room and it's really an honor to come back and be able to participate on ground, grand rounds today. Um, I'm gonna talk today about a range of work that we're doing in just one aspect of the lab, which relates to using mechanics to try to enhance regeneration. And before I get started, I just wanna make some acknowledgments of some of the collaborators for the different projects that I'll talk about today. A number of these collaborators are here at Boston Children's Hospital, as well as scattered around other places. Um, uh, some of the latter work that I'll describe is funded through a research grant from Novartis. So I have that, um, declaration to make. Um, to get started, Um, not to scare you, uh, I'm not gonna talk about detailed, uh, mechanistic pathways. Um, but this slide just is intended to represent the idea that we all know, well, uh, certainly as surgeons, you all appreciate, and as scientists, we appreciate that physical forces, stresses, and strains are present throughout the body. And scientists have done a fantastic job of understanding how, for example, fluid flow or pushing on tissues or pulling on tissues gets translated inside the cell to altered gene expression. That's something that we understand really well at this point in time, in terms of some of the mechanisms that underlie some of this behavior. Now, I'm not gonna go into this today, which we are interested in the lab. Instead, I'm gonna talk about how we exploit that understanding of the role of stresses and strains. In cell behavior and tissue behavior to try to develop new types of strategies to promote tissue regeneration using mechanical cues. And this is just an overview slide showing some of the different ways that we think about doing this. Today, I'll talk about just a couple of these, uh, which includes the idea of using mechanics as a way of regulating the behavior of transplanted cells when we're trying to do regeneration. And the idea of trying to develop systems that can directly provide mechanical stimulation to tissues and organs to control regeneration. Now, along the line of trying to develop these types of strategies, uh, we've come to appreciate the importance of adhesives and being able to put things in a particular place in the body and have it stay there. So, the last part of my talk today, I'll describe some work we're doing with the development of adhesives. And this is just a kind of a preview of what I'll be talking. Talking about the last part of the talk where we have developed new adhesives that in this case, we're putting on a, a beating pig heart. We put some blood there to mimic a bloody surgical field. And the adhesive is that uh clear hydrogel that we just placed over the heart. And now we apply compression for a minute or two, to get it to stick. And now we'll pull off the backing here. And now we'll do a simple peel test on this adhesive. And what you'll see is that this hydrogel sticks exceedingly well, even in this really demanding situation where we have the blood there, we've got the beating heart, and you can see as we're trying to pull it off, it's staying highly adherent. So, some of the mechanical studies that I'll describe to you have led us to the development of these types of adhesives. And as I said, I'll finish the talk by describing how these actually work and what the design of these materials is. To a little better understanding of kind of where they're coming from. OK. So, now back to the beginning of the story though. That's gonna, that's gonna be the end. The beginning of the story for us is based actually in my time here in the Enders building, became very interested in the idea of transplanting cells as a means of promoting regeneration of tissues and organs in the body. Now, as many of you are very familiar, there's been a lot of effort clinically to transplant a wide variety of different cell populations into the body. This is typically done by simple infusions or injections of cells suspended in saline or in other carrier fluid. And the common result here is shown in some rodent studies, but exactly the same data in human studies, is that very few of the cells actually stick around for very long. You maybe get a couple percentage of the cells that are there after a day, maybe 1% after a couple of days, and it's probably not surprising that most clinical trials of cell therapies have shown very marginal effects. And it probably is due to the fact these cells are simply not around for very long, and we have little control over what happens to them once they're in the body. So, our entryway into this area was the concept which was a very common one, which is that if we took the same cells and we put them in a biomaterial carrier, we could dramatically enhance their effectiveness by localizing them to a particular place in the body and then being able to maintain them at that location for extended periods of time. We use a variety of different materials, and I won't go into the chemistry too much today, but one of the materials we use quite a bit is a polysaccharide called alginate, and you see the chemical structure of it. Uh, it actually, the background here is an atomic force microscopy view. So you can see those strands are actually individual polymer strands, and these are cross-linked by the addition of a divalent cine like calcium. So, we can mix cells with these polymers in solution. We can add calcium and the gel will form around the cells. So, it's a very gentle way of putting cells inside materials. And we can readily control the mechanical properties of these gels by simply changing how much we crosslink them, as here indicated by a metric of stiffness, the elastic modulus, and you can see we can get different mechanical properties simply by varying how we formulate the gels. So, Ellen Roach, a very talented PhD student, uh, who's now running her own lab at MIT and we'll talk about her later, more in terms of some of her work, did some really early and very simple studies of taking cells, in this case, mesenchymal stem cells, uh, putting them in a biomaterial carrier. In this case, you can see, it's on one of these carriers that's been attached to a beating, uh, port, uh, in this case, rat heart. And she showed this allows us to dramatically enhance. The durability of the cells at that location. If we put them inside one of these gels and inject them in the myocardium, we get about an eightfold increase in the number of cells that are around over time. And if we put them on a, take the same material and form it into a patch and attach it to the heart like that video just showed us, we get about a sixty-fold increase in having the cells stick around. So, we can change how long the cells are there. Now, in terms of the mechanics though, we're very interested in seeing, can we actually control what these cells do by the physical properties of the material. And the scientific background for this idea goes way back, and actually probably one of the first papers I'm aware of is this paper from 1980, where they took cells, in this case, fibroblasts, from a person and they put them on a very flexible silicon membrane. And what you're looking at here is not a bad picture, but actually cells wrinkling the material they're attached to. So, they reach out, they grab, they attach, and then they pull, and they're actually wrinkling the substrate because it's really soft. And so, this is a great example of the fact that when cells interact with their environment, it's not a chemical interaction only, it's also a physical mechanical interaction. Now we now understand that cells do this by having receptors in their membranes that bind to specific molecules in the surrounding matrix. But once people began to understand this premise, they then asked the really logical question. They said, well, cells grab hold and pull on whatever they're attached to, does the resistance that they feel change their behavior? So, to address this in a relevant context, we've taken mesenchymal stem cells, which are very widely used therapeutically today or attempted to be used therapeutically today. And we put them in these gels that we're transplanting the cells and that I just showed you. And just varied the stiffness of the gel, keeping everything else constant. And if you focus on the um upper left hand pictures there, what you're seeing is some different colors. And what this represents is if we take the same cells and we put them in a really soft gel, as indicated by the 2.5 kilopascal elastic modulus, the cells become fat cells. If we take the same cells and put them in a gel that's a little bit stiffer, about 22, the blue color means they're becoming bone forming cells. And if we have them in a really stiff gel at 110, they're not differentiating at all. And all the other data on this slide just as a molecular confirmation. So we can transplant the cells in these types of materials, we can control how long they're there, and we can control how these stem cells differentiate based purely on the mechanical properties of these gels. Now, this is in vitro. We've also shown this in vivo, where we've taken a very small number of these cells. We've transplanted them on, in gels, in this case into calvareal defects in rats. And what we see in the middle panel with the microCT or the quantification down below is that we get from the same number of cells, more bone formation if we transplant them in a gel that has a stiffness that we think should be optimal for making the cells differentiate and become bone forming cells. Now, this was a striking observation when we made it. And actually, the first observation that one could control the fate of stem cells in the body simply by regulating mechanics. Uh, but this approach actually ignored something that I think all of you appreciate quite well from your interactions with soft tissues in the body, which is when we talk about a stiffness of a gel, we're assuming that the material is basically purely elastic. So, it's like a rubber band. If we stretch it, It stores energy. If we let it go, it goes back to its original size and shape. But all of you are aware that many of the tissues in our body, if not most, are what we call instead viscoelastic, which means they act partly like a solid and partly like a liquid. In this case, what this graph represents is a simple test to demonstrate this, where if we pull a tissue and then hold it and measure how much force we have to apply to keep it stretched. If something is purely elastic, like a covalently cross-linked um polychrolamide gel, the top red line, you have to keep the same stress. But if you do this on adipose tissue, liver, brain, What you find instead is they deform over time and dissipate some of the energy. So this then led us to ask whether or not kind of this visco properties, this fluid-like properties were also important in cell behavior. And so, um, actually Ovi, um, Chaudhuri, who's now at Stanford running his own lab in Luo at Johns Hopkins, running his own lab now, did a really simple experiment where they took the same cells, they put them in gels, and they kept the stiffness the same, but they changed how fluid-like or solid-like. And what you can appreciate from the blue stain in that middle panel is as the materials became more fluid-like, the cells actually differentiated more to become bone forming cells. And this is just quantified down below. So, we could control the fate of the cells by whether they're more liquid-like or solid-like and how stiff they were. Now, you may ask whether this is any real relevance in the real world. Um, to address this, we went back and we characterized the mechanical properties of the environments in which bone regeneration, what, what, well, we thought representative environments in which bone regeneration might occur. So, we took coagulated marrow from rats, and we actually obtained fractured hematomas from patients going into the Cherite Hospital in Berlin for revision surgeries, and we mechanically characterize them. And when we do the same kind of test that I showed you a minute ago, and this is work from Max Darnell and Evie Lippins, uh, what we found is they exhibit exactly the same kind of stress relaxation behavior that I just showed you. And actually, the time constant is very similar to what we empirically found was optimal for driving osteogenesis or bone formation from these stem cells. So, in essence, I think what we're doing is we're rediscovering the properties that our body uses when it creates a material to induce regeneration in this context towards bone. Now, um, to actually nail this down a little bit better, we also went back to these calveral defect models again, took a small number of cells and transplanted them into a gel that had the same stiffness, but one that basically relaxed slowly, so it's more elastic, or one that relaxed more quickly, so, more fluid-like, and we found that we got much more bone formation in the one that, that was more fluid-like. Again, showing that this actually has relevance not just in vitro, but also in vivo. Now, we've looked at this concept of the role of stress relaxation and viscoelasticity in many contexts now, uh, not just mesenchymal stem cells. We've looked at it, for example, in the context of neural progenitor cells, which we're interested in using in the brain for a variety of applications. And this is just a, uh, an RNAsEQ analysis. We're looking at gene expression. For those of you familiar with it. And this is a really simple experiment where we put cells inside gels where we vary the stiffness, the stress relaxation, or the number of attachment sites for the cells. And the circles are just represent the number of genes that are regulated only by that, by one of these variables. And what you can appreciate is these cells don't really respond much to stiffness, as, as the circle for soft versus stiff is small. They don't really respond very much to the ligand density because that circle is small. What they really respond to is how viscous the material is. So, they really care about this property quite a bit. Other contexts include in the context of cartilage formation, where here we're taking chondrocytes, and you can appreciate across the top, we're showing the, basically the rate or the time scale for relaxation. And when we put individual cells inside of these gels and look for how they form a cartilaginous tissue, we get much better cartilage formation when the gels are exhibiting this more rapid relaxation. So altogether, what this is telling us is that we need to really control the properties of these gels, not just to localize the cells, but to control their behavior and allow them to form new tissues. Now, all of this is done in a bulk way. Where at the end of the day, we have a gel, let's say in a syringe with cells in it, and we have to inject it someplace or, or surgically implant it someplace. We'd like to also be able to do things as they're done clinically today, which typically involves infusion of collections of cells in a fluid carrier, typically an IV infusion. So, we've been exploring ways of translating this concept all the way down to the single-cell approach, and we're exploring this using microfluidics. And so for those of you not familiar with it, the idea of microfluidics is we have a liquid stream coming from the top in this video that contains cells and the polymer that will gel. And what happens is the kind of the top of the tea you see there, we have another fluid coming in and flowing, and it basically cuts off droplets of the first fluid. And the idea is each of those droplets would then contain a cell. That then we could gel the polymer around it, and then we'd have an individual cell in each of these little droplets. When we went down this road, people had already been doing this, but they've been making particles that were about 60 microns thick. And we wanted to get it down to about 30 microns, and that may not seem like a very big difference, uh, but in a 60 micron gel, which was the state of the art at the time, the cells comprise 3% of the volume. So, 97% of your payload is actually not the cell, it's actually the carrier. But if we get it down to a 30 micron gel, it's about a third of the volume is actually cells. It actually is very similar to tissue densities of cells. So, what these look like when we ended up making these is shown here in this confocal image. So, this is an individual cell, an individual stem cell. The blue is the nucleus, the red is the actin. So, that's the edge of the cell and that green halo you can see around the cell, that's actually the hydrogel. So, we can now coat these with a really thin layer of gel, and we can have, play all the same games that I was just describing to you, which is we can control the properties of these gels to control the cell behavior, but now we can do this on an individual cell basis. So, in this context, we've taken this as a proof of principle into what we consider to be a pretty harsh setting, which is an IV infusion of stem cells, which is very frequently used, actually, the most common approach to transplant these cells today in the clinic, in this case, in a rat model. And the idea is we're still gonna do an IV infusion because the gel is so thin, it doesn't prohibit us from putting them directly in the bloodstream. Now, these will end up getting lodged in the lungs, which is what always happens with an IV infusion of these cells. And the hope is that from this having this coating, We can protect the cells from sheer forces and protect them from immune clearance for at least some period of time. And what the images here in the bottom are showing you is cells being IV infused inside these microgels, and we can image these actually, and the intensity, the color tells us how many cells are there. And you can see that at 24 hours, when we have the cells inside the microgels in this allogeneic transplant model, we have high numbers of those cells still being present. But if the cells are outside of microgels, they're basically completely cleared by this point in time. And this is just some quantification of this behavior. And to put it in a little bit more specific terms, here's the half-life of the cells. And when we do it a simple infusion, as is done clinically for these cells, most typically, the half-life of these cells is under 2 hours. It's about 2 hours is how long the average cell lasts. When we optimize the system, the bar on the far right, we get it up to about 200 hours. So, we can have about a two-log extension of these cells sticking around in the body, even in this really complex setting. So, we think that this general approach may be very useful to do IV infusions of cells or local deliveries or implantations to control their behavior. Now, uh, up to this time, all this has been pretty esoteric, kind of some tools that we're using. Um, so, I'd like to spend a few minutes now talking about how we're trying to use this and related technologies to do things that are a little bit closer prior to things you're interested in. So, one of the areas that we're interested in the lab is heart failure. And we think that there's opportunities for new technologies in the space. Uh, in particular, there's two areas that we're interested in innovating around. One is the idea of mechanical strategies. You're all familiar with the LVADs that are used to provide, uh, ventricular cysts and replacement of heart function. Um, as you're aware that those can cause blood clots and there's a variety of limitations of those. And then, There's obviously, as I've been talking a little bit about, there's a tremendous amount of activity using cell therapies today where we inject or infuse cells into the damaged myocardial tissue to try to promote regeneration. Um, those actually are again, really limited by the fact that the cells don't last very long. We need them to stick around longer. And it's become clear from a variety of clinical and pre-clinical data that we probably need multiple infusions of the cells, not a single infusion to be successful, even if we can keep the cells there for a long time. So, the, the approach we've been taking and this work has all been done in very close collaboration with Connor Walsh, who's an expert in soft robotics at the engineering school at Harvard, has been one to create mechanical assist devices and the, the device here is called HAD, which is a ventricular assist device. The idea is it's a sleeve that goes around the heart, so it's non-blood contacting, and it would provide uh assistance for the beating heart over acute periods of time. And then to deliver therapy more effectively, we've been thinking about something we're calling therapy, which is it also is a device that would go around the heart, so it would not be placed within the heart tissue, and it would allow replenishable administration of therapy. And I'll describe each of these over the next couple of minutes. Before I do that, I should mention that all this body of work was really done by a simply incredible PhD student, Ellen Roach, um, who, as I mentioned earlier, is now running her own research laboratory. And this project is a big one. It's not a single PhD student working on something like most of the projects in the lab. And put together a whole team, both at the VIS in my lab and associated activities, and then in Connor's lab, and then a whole group of physicians here at Boston Children's Hospital as well to pursue this project. So, what I'll show you in the next few slides is really a culmination of work of all these individuals. So, she's first of all, designed, and I'm gonna be very brief here, a device that would fit outside around the heart. And the idea is that this would mimic the normal motion of the heart, so it's not simply squeeze the heart to get it to basically pump more blood, but would actually have a twisting motion as well. So, it's intended to be biomimetic and really mimic the natural contractile motion of the heart. You can see one of these actually on a porcine model here in this video. It's pneumatically driven, and you can see it beating here and driving heart. Um, in terms of cardiac output, uh, you have a certain baseline level. In this model of acute heart failure, we have a dramatic decrease. If we apply this device, we can actually drive, uh, the cardiac output back to the normal levels. So, at least in acute phase, this has some potential promise as a way of providing short-term mechanical support to promote regeneration, which is the long-term solution. As I mentioned, we're interested in this device called therapy. And the idea here is this would be a device that we place around the heart. Um, it would have a depot within it where we could put the cargo of interest. This might be low or high molecular weight drugs. It might be a biomaterial carrying cells, which is what we typically use. And the idea is that it will have a refill line. That we will then make available for, through a subcutaneous port. So we can do a minimally invasive introduction of refills of the therapy into the depot. And then the bottom surface of this that's sitting on the heart is a semi-permeable membrane that allows easy communication between the cells or the depot in general and the underlying tissue. This is what a rat-sized version of this looks like. You can see the semi-permeable membrane on the bottom. You can see the reservoir on the top. Here, there's a dime to give you a sense of the scale of this because obviously, rat hearts are pretty small. Uh, these can be compressed and loaded into a catheter for a minimally invasive delivery into the heart tissue. And in the middle, what you're seeing is the, um, refill port that we would make available by snaking the, the refill line out into the subcutaneous tissue. To give you a sense of the potential of this strategy, if one does a simple injection of mesenchymal stem cells into the heart tissue, as I mentioned to you, they get cleared quite rapidly, as you can see in the top. In this case, we use a biomaterial carrier, so they stick around for a little bit longer, but they still get lost over a period of multiple days, excuse me, on the bottom. Uh, but if we actually introduce refill, at this case, at 4 days, you can see the, the intensity of the color increases again at 4 days and then fades again. So, we can refills, you know, 1 time, 2 times, multiple times to keep reintroducing the therapy in a minimally invasive manner with this kind of strategy. In a model of myocardial infarction, we see that we can have a dramatic impact on the efficacy of the cells. When one does a single introduction of the cells in this model, it's very similar to what one sees clinically that there's very little effect. So, if you look at the graph on the bottom right, the ejection fraction, no treatment, a single injection, you get a very marginal effect. But if we introduce multiple times using this device, we now get significant increases in the ejection fraction. So we're actually promoting an enhanced functionality of the tissue in this context. So how we see this going forward is that we'd be interested in exploring the idea for end-stage heart failure patients of placing the HAd to provide the mechanical support, as well as the therapy with it to try to promote regeneration in this context. While for the acute MI patients, uh, the premise would be that the therapy alone might have utility as a way of inducing regeneration in these patients. So, this was our entryway into this idea of using mechanics as a way of regulating biology. And we've been taking this to the next step to try to use the mechanical system, not just to provide replacement of function, but also active regeneration. And we're exploring this in the context of skeletal muscle injuries. And this is work that's being led by Bo Ricio, a really talented postdoc in the lab. And you're all familiar with skeletal muscle or soft tissue injuries and the limitations of the current clinical strategies. Obviously, if we're going to do transplants, we have to then take the tissue from someplace else and create damage. There's limitations in its availability. If we try to replace with synthetics, we don't get replacement of function. And cell therapies, while we're actively exploring them in the lab today, have a lot of limitations, including the fact they're going to be very expensive and very cumbersome from a regulatory perspective. So, what we're exploring is whether we could use these kinds of mechanical systems that I just described to provide mechanical stimulation in a really controlled manner. So, I have the exact stress, exact strain that we're interested in for the exact period of time and to be able to adjust that in real time if needed. So, what this looks like right now, pre-clinically in rodent models are shown here in this movie, you can see one of these mechanical actuation devices from Connor Walsh's lab in the bottom that we're applying actually to the skeletal muscle tissue, in this case, on a mouse, and the image on the right, you can see using ultrasound, the deformation that we're inducing in the muscle tissue. So, we can apply exact or precise loads for precise periods of time with these strategies. And what we're now finding is this allows us to intervene and actually regulate regeneration. So when we optimize the mechanical stimulation, if we look at the muscle tissue over time, in this case, first looking at early regenerative events, so looking at the number of PAC-7 positive cells, which is an indication of satellite cells or the stem cells, we get increased numbers of those with the mechanical stimulation, which is MS as versus control. And this is in the context of a severe injury to the muscle. And it's similarly, if we look at the number of centrally located nuclei, which is an indication of an active regenerative process, you see that the, that is significantly enhanced with mechanical stimulation as versus the control. So we up regulate the stem cell population with the mechanical cue, and this is a simple device that's applied from outside the body. If we look at calcification, which is a result of this injury, we see we dramatically diminish the extent of calcification. And also the amount of fibrosis by having this defined mechanical stimulation in this context. At the end of the day, the result of this is we get enhanced functionality of the muscle, where here we're measuring the total force that the damaged muscle can apply. And you can see initially, it's very low post-injury. It recovers very slowly in the control, but we get a dramatic acceleration of return of function with these mechanical devices. And as I mentioned, we can control precisely the amount of force we apply. In this case, we're not seeing a tremendous difference between forces that range from 0.15 to 0.6 Newtons, um, but we're beginning to explore and broaden this range inside and figure out where the optimal stress might be. Now, I don't wanna spend a lot of time today talking about mechanism, um, but in all these types of injuries, obviously, the immune system is playing a major role after injury and likely in regeneration. And so, we've begun to explore whether there is an interplay between these mechanical sig sim uh sym systems that we're developing and the immune cells that are present at that local site. So, as you know, following injury, you get neutrophils, monocytes, macrophages, and ultimately cells adaptive system coming in over time and participating in regeneration. So we've been asking whether or not part of the effects of this loading might be due to changes in immune cell populations at that site. So, we actually eliminated a lot of things that I won't tell you about that we're not impacting. What it looks like we are having a significant impact on is neutrophils and the duration of time at which neutrophils stay at an injury site. And this is again in the context of a skeletal muscle injury. And what you can see from the facts state on the top or the quantification at the bottom is when we apply the mechanical stimulation, we don't change the number of neutrophils that are there early on. But we rapidly, uh, induce their depletion from the tissue site. So, we're able to change the time course of the presence of these cells. And particularly, if we look at, uh, basically a measure of the activated neutrophils, we see a significant decrease in the activated neutrophils over time. So, we see change in the neutrophils. If we look at the cytokines, the signaling molecules in the tissue is a function of time. And this is a broad screen of many, many different cytokines and the, the specifics aren't so important. But basically, blue means that when we apply mechanical stimulation, we down regulate the cytokines. Red would mean we up-regulate. And what you can appreciate is on the early times when we applied the mechanical stimulation, we down regulate a vast number of cytokines and these cytokines are involved in things like lymphocyte hemotaxis, neutrophilchemataxis, TNF alpha signaling. So all things that are involved in the early stages of the immune response. So, it does look like we're having a, a dramatic impact on the immune cells, immune cell populations, and activities. And if you now go in vitro and look at what's happening in terms of uh how neutrophils might impact skeletal muscle, which was an unknown when we, when we started doing this work, and we're surprised to find the neutrophils. It's kind of potentially a key player. This is some cell culture data where we take primary muscle progenitors and culture them with conditioned media from freshly isolated, um, and activated neutrophils. And what you can see from the graphs. is that when we expose these progenitor cells or stem cells to neutrophil conditioned media, we dramatically enhance their proliferation and we retard their differentiation. So, the premise is that by applying this mechanical stimulation, we're keeping the injury site in an earlier state, allowing more cells to proliferate, slowing down differentiation to allow a more, uh, a larger number of stem cells to be present and participate in regeneration. So, kind of the, the conclusions from this and a variety of other data, I don't have time to go through today, is that when you have a dramatic injury to tissue, an ischemic injury, a, a tear injury, um, uh, basically resection to a skeletal muscle, as we know that basically, you get a lot of inflammatory response to come in and clear up the dead cells and the dead tissue. Mechanical stimulation, while allowing that to occur in the early stages, will dampen the pro-inflammatory environment over time. Um, it'll decrease the neutrophils within the tissue. Um, it'll enhance the number of stem cells and the regenerative muscle fibers, regenerating muscle fibers and allow them to regenerate for a longer period of time and at the end of the day, enhance regeneration. So, hopefully, I've given you a sense of, you know, how we're actually interested in mechanics and using mechanical cues to regulate regeneration either of cells that are already in the body or cells we transplant. One of the things, as I mentioned at the beginning of the talk that, as we've gone down this path that we've realized though, is that all of this work involves placing a material someplace in the body. And oftentimes that's a challenge, right? So, you are all very familiar with this idea that adhesion of devices onto wet and dynamic tissues is a really big challenge. It's actually very hard oftentimes to get our systems in place and get them to stay where we want them to be. So, um, actually a very talented postdoctoral fellow, Jane Lee, shown here, uh, came to the lab about 2.5 years ago, and he became interested in this problem, and he started looking around to say, OK, you know, biology had to solve this problem before. We're not the first people that ever wanted to do this. And he came across this slug, you see the picture of Aaron Sofufusca. And this slug is actually quite interesting in that it secretes a mucus that it usually uses just to slide around and basically move around on. But when it's threatened by a predator, it changes the composition of its mucus, and it uses it to stick really tightly and make it hard for predators to pull it off. And when you look at the composition of that mucus, what you find is it's um consists of uh polymers, and it's a hybrid network of ionically cross-linked polysaccharides combined with covalently cross-linked proteins. And those, uh, that combination seems to give these really interesting properties. Now, what was really striking when Jane you made this observation. is that a few years earlier, we had started to get really interested in these hydrogels we're using for transplanting cells and trying to understand how to improve their mechanical properties. There's one of the things I didn't mention to you before is when we take these gels, and for example, if we inject them via a needle into a tissue, hydrogels are 98% water. So they tend to be really weak materials. And some of you work with, you know, for example, fiber and gels, you know, these are pretty weak materials. And you can measure something called the fracture energy, and it's about a value of about 10. So, they're really weak and this limits their performance in many settings. So, with Johnny Zha, uh, postdoc at the time, now running his own lab at MIT and son, who's now back at Seoul National University in Korea, in the labs of my collaborators, um, over at Harvard, they had proposed a way of creating much tougher hydrogels by creating what's called an interpenetrating network, where you'd mix together two different polymers. And the basic idea Is that when you have a hydrogel, and if it's covalently cross-linked like polyacrylamide, and you start to apply a force to it, you start to get it failing, you see the crack that's beginning on, in part A. Well, all the energy of that crack gets concentrated in a very small volume of the gel, it becomes really easy for the crack to keep propagating and move through it and for the material to fail. Now, so that's for covalently cross-linked. Now, these alginate gels that I described to you earlier are cross-linked by divalent cations, and those bonds are actually reversible. If you pull on the gel, you'll actually disassociate and then the bonds can reform. Well, in the process of breaking these bonds, you actually dissipate energy. So, as you start to have, apply a load to an alginate gel, you'll get decrosslinking. And then you'll end up getting dissipation of the energy. However, the gel has failed at this point in time. There's nothing that's going to bring it back together. So what they realized if they made a combination of these two types of networks, made a combination of the polyacrylamide and the alginate, the polyacrylamide could transfer stresses within the gel. The LGHL could decrosslink, dissipate a lot of energy. And then if it started to fail, the polychrylamide would bring it back together and provide an elastic return. And they thought by doing this, they could make a much tougher material. And they're actually quite successful in this. And to give you an indication, I'll just show you this video. So, this is an example of one of their gels. It's 1 millimeter thick. And they've dropped a steel ball, which is 1 inch, from a height of 6 ft. So, you can see that they dropped this steel ball from about here to the floor on this really thin layer of gel, and you can appreciate this gel, even though it's 98% water, is not failing. It actually can sustain the blow from the steel ball hitting it, and now it's actually going to do, have an elastic return and send that steel ball back. So, here we have an incredibly tough gel, and it's formed of this ionically cross-linked polysaccharides combined with a covalently cross-linked polymer. And so, Jane, you realizes this actually sounded an awful lot like that mucus that, that slug that I described a minute ago secretes. He said, that's almost the same thing from a physical perspective. And he said, so I think that actually this could work really well as an adhesive, but we needed some way of getting it to actually stick to a tissue. So, here is, you know, the blue is basically the schematic of the gel, and we consider this to be a dissipative. Matrix means it dissipates lots of energy and allows things to stick. But we needed it to actually then be able to interact and bind to tissues. And so, the trick that Jane you came up with, which ended up being very effective, is to use another polymer, in this case, the green lines you're seeing that he put at the interface. And these green polymer chains would interact both with the gel, and we'd covalently attach them to the gel, and they would also attach to the underlying tissue. So, form a really strong chemical bond between the gel and the tissue. And now when you pull on the gel, pull on the tissue, a lot of the stress gets transferred to the gel. Bonds break within it, dissipate the stress, and the adhesive can stay stuck to the tissue. It's basically the premise. So, what it looks like is we have a, a hydrogel that we pre-form typically. We then simply coat it with this solution. When we want to apply it to a tissue, we then apply it to the tissue, we push it, hold it in place for a couple of minutes, and then we form this very strong adhesion. So, uh, in terms of the kinetics, this is what the adhesion energy looks like as a function of time. So, you can see that to get full strength, it takes a long time. Um, you know, basically takes about an hour to get full strength. However, for comparison, um, we're showing what superglue looks like uh here in this adhesion test of porcine skin. So, you can see within about 1 minute, we actually have a strength that's equivalent to cyanaracrylate that most of you are probably familiar with. By 3 minutes, we're up to about 800 joules per meter squared, which is 3 times greater than cyanoacrylate and we eventually get to even higher levels. So we can actually form adhesion, and we actually get stronger adhesion than superglue. To put this in a broader context, if you look at the adhesion energy and compare it to all the different types of adhesives that are out there, you can see this dramatically outperforms all of them. So, the adhesion energy in about 1200 joules per meter squared, um, CA is the cyanoacrylate. Um, then we have coal, which most, many of you are probably familiar with, uh, the peg-based, uh, um, We have DPA, which is experimental. We have some of the fibrin-based commercial products, and then we have nanoparticles which people have also proposed. And you can see in terms of the adhesion energy, this dramatically outperforms all of those. Now, one of the nice features of this as well is it's a hydrogel. It's filled with water, and it's aqueous chemistry we're using. So, having some exposure to blood shouldn't be a problem. So, if we take these gels, in this case, attach it to porcine skin, and then use an instron in the lab just to pull it apart, you can say, even though there was blood on the surface when we did the adhesion, we actually still got a very strong adhesion between the skin and the adhesive. And if we quantify this, So, the gray is without blood. And so I already showed you this data that we get a much stronger adhesion with these tough gels than we do with cyanoacrylate. If you expose cyanoacrylate to blood, as many of you are probably familiar with, it actually doesn't set very well and you actually get very poor adhesion. You lose a lot of the performance, but we don't lose any of the performance of this adhesive, even in the presence of contaminate, contaminating blood or other aqueous fluids on the surface of adhesion. So, it looks like it actually works very well in skin. We've begun to explore it in a wide variety of other settings. So, we've looked at how well it can adhere to things like tendon and muscle, cartilage, meniscus, skin I mentioned, liver, uh, basically, a variety of oral applications, and the heart. And so, I'll go through a couple of these examples over my last couple of minutes to show you where we think this might be useful. And this is something I'd greatly appreciate input. We're now trying to figure out where we really should be pushing this, where it might be useful clinically, and where we shouldn't waste our time because there's already good solutions. Um, so one of the obvious things to do is to use it as a skin adhesive. Um, this is just an example, a movie showing a, um, A laboratory mouse where we've actually created a couple of incisions on the back. We've attached these tough gels and you can appreciate that these adhere well. Here, the mouse is running around and the gel stays adherent very firmly over time. So, I think it could potentially be useful in that context. Now, these gels, in addition to staying adherent and sticking really well, we can also design them so they contract over time. And in particular, what we've been doing. As we've been creating what we call an active tough adhesive, that after you place it on the tissue and it heats up to body temperature, it'll actively contract and wanna close. So, it'll actually then pull on the tissue that it's attached to, to try to close as well. So, that's shown schematically here where, you know, if we put it on a skin wound, the idea is after we place it, not only does it stay there, but it will actually start to pull the wound closed with time. Um, and so we've begun to explore this and, you know, some pretty preliminary uh rodent studies at this point in time, and the AAD is the active adhesive dressing here. But you can see that with that, we actually get about 40% wound closure in a splinted rodent model of wound healing just by applying the adhesive by itself. No active, just a mechanical force to try to pull the tissue closed with this adhesive. So, I think it could be useful in these kind of contexts where you want to have an adhesive, you perhaps want to have a force that the adhesive applies to the surrounding tissue. We think it may be useful in the context of things like hemostatic agents. Here's a really simple study where we've gone to a rat model of liver laceration. So we basically lacerate the liver with a standard injury. Uh, we allow it to bleed. We apply the tough adhesive. In this case, we compare to surgiflow, which is a, uh, typical hemostatic agent. And if we, uh, quantify the blood loss, you can see the control, we get a substantial loss of blood in this model. If we apply either our adhesive or surge of flow, we reduce the bleeding down to a very low level. It's actually similar between the two. So we think it could be useful in that context. We're interested in using it in a variety of orthopedic applications. Um, Ben Friedman in the lab right now who's heading this project up currently has a lot of interest in soft tissue injuries in the context of orthopedics. In the context of tendon rupture, which is particularly what he's interested in, as you are very familiar, sutures can be used for the repair. There's a lot of limitations with those, um, you know, in terms of, um, the outcome, things like cyanoacrylates or fiber and glues have been explored to be used in place of suturing. Um, but they also have, uh, inferior properties and do not, are not able to replace the idea of using suturing. Um, so we're beginning to explore, and this is a very early stage, whether or not we could use these adhesives, either as an adjunct to suturing or even to replace suturing. And so, this is some simple studies where he's taking these adhesives, he's putting them first in vitro on planks of tendon that he's created. And uh what, see if the movie will play. Nope. Oh, there we go. So, you can see here, the gel is, or the adhesive has been attached to tendon. So white is the tendon, the blue is the gel, and he's pulling it off with the Instron. And you can see that the adhesion energy is quantified on the right and compared to tissal, and you see that within a matter of about a minute, we actually get much stronger than the fibrin gels and able to get reasonably high strengths. We've begun to look at the biocompatibility of these systems in a variety of settings. So in this, for example, we're taking the gels, we're putting them on injured tendons in rats, and then following what happens over time. When we place these, uh, in this case, on a tendon site, we can use ultrasound to follow the gel over time. And this is just some imaging of the gel, um, at a particular time point. But when we monitor them, we can see that they're stable over multiple weeks in vivo. In this case, over a 3-week time period, we see, uh, basically, the gel stays in place and keeps its physical properties in terms of its dimensions. If we look at histology over time, We see that we don't have any negative, uh, inflammatory responses to the gels, either in uninjured animals or in injured animals. Obviously, the injury itself, in this case, creates a lot of inflammation, but we don't see any increase in inflammation with the application of these hydrogels. We've also looked at this in different contexts. So, for example, placing these on heart tissue and then going and looking over time at the inflammatory response and here comparing it to cyanoacrylate, and we actually get similar or better inflammatory response than one gets to cyanoacrylate um with the setting. And overall, if you compare these to what's out there today, what you see, if we look at the adhesion energy and then the toughness of the matrix, here you see the fiber and glues on the bottom left, some of the muscle-inspired adhesives you may have heard about that are being developed, Coseal, so the peg-based systems, uh, the adhesive or cyanoacrylates on the bottom right is adhesive bandages, just the regular bandages you use in the clinic. Um, and then you see the tough adhesive of the red circle on the top. So, it looks like it has some pretty spectacular properties, and we're, as I said, beginning to explore how we could possibly use these. Um, this takes me back to one of the first videos I started with, showing you how these now can be applied. And in this case, now, you actually understand a little bit more of what this is. So, going back to that video, we're applying it to the beating heart. So we have the hydrogel, we've pre-formed. We've now gone ahead and just put on the polymer that will form the adhesion onto it directly for placement. So squirt some of the liquid on the gel, and then we go ahead and apply some compression for about 2 minutes here. We basically pull off the backing, and now we'll see if we can pull off the gel. And I should mention here that with the gel, that we did try to adhere the whole gel. The idea is we only wanted to adhere this part of the gel, um, and then leave the top part. Not adherent and you can see we're basically pulling and extending the gel, uh, and we're maintaining the adhesion. Um, if you actually are more interested in the gels in particular, or this adhesive, uh, this is something Ben actually showed me, cause I'm not savvy enough, uh, that now our cell phones, uh, if you actually take your cell phone and actually put it on that barcode, it'll actually ask you if you want to go to the website that actually has the description of the technology. Um, so, I think we'll be seeing a lot more of that, but it's kind of fun. Um, my students teach me new things all the time. Um, so, that's, uh, the end of what I had to talk about today. Um, hopefully, I've given you a little sense of how we're trying to develop new types of materials to promote regeneration, to solve problems where we see them in the applications of these devices and other kinds of technologies. Everything we do is focused on developing materials, um, that can be useful in a wide variety of different contexts. So, thank you for your time. And if, uh, there's any time remaining, I'd be happy to take some questions. Doctor Mooney, I'd first like to thank you for bringing to us much of this fascinating work that you're doing, and it's hard to think that there aren't gonna be lots of uh um ways where this has very significant surgical relevance. So, so I have two questions. One at the start of your talk, you had a discussion about how you were, um, wrapping a few cells that were being implanted, uh, prior to doing that. Has that been used in Some of the studies were like the, the, I guess the cytoskeleton of the, of the lung has been preserved and it's self-free and then autologous cells are re-injected into that. Has it, has it been shown to be Effective in enhancing the, the cell repopulation. We've not looked at it in that context. So, we've not, we've done this mainly with, uh, using this as a strategy to induce cells back into the body. Um, but yes, it potentially could be used as well as a way of enhancing reseeding of a decellarized tissue. That's an interesting idea. It's something we haven't explored to this point in time. And the second question, um, what are the, um, steps or challenges that you face on taking some of these products to the clinic as far as the FDA is concerned? Yeah, you know, it's, um, there are certainly challenges. Uh, one thing that we try to do as much as possible is stick to known materials and known chemistries. Now, we're using these in new ways. So, they're still looked upon as being a, you know, a new material by the FDA, but at least there is a track record of their use in other contexts. So we try to, you know, simplify things as much as possible. If, if it's, you know, if we came in with completely new materials, uh, new chemistries, then it becomes a very, very long path. Here, it's, you know, it's still, it's substantial, but, um, It's not, uh, it's not crazy. Um, and we also, I, we try to use cells that there's also a track record of using clinically. I didn't talk at all about drug delivery, but all these systems we also use for drug delivery. We try to focus on using molecules that have a track record of use in humans. So, at least parts of it are known in terms of the safety profile and then what we really do is bring them together in new ways, which introduces some additional risks but hopefully not too much. The FDA won't take that slug mucus is safe for slugs, so it's OK for humans also, no. Additional questions for Doctor Moon. Doctor Buckmiller. I'm probably asking this for Doctor Fauza as well, but one of the uh niche areas is fetal surgery. So I'm wondering where things exist as far as the application of the um adhesives in a completely um aqueous environment like in utero. So the field and fetal surgery is looking at adhesives to eliminate the need for suturing patches in utero and whatnot from mid gestational fetal surgery. So I know you can apply it to a moist environment, but can you actually apply it in a completely aqueous environment in a completely, what's the aqueous environment like the amniotic fluid. Yeah, so we've not, I've actually not thought of, uh, trying to, the area you're describing, we've not even considered. It's really interesting. And then in terms of completely aqueous, so, we have that gel, we apply the chitosan to it and it's gonna stick, um, and then we apply it, it is in wet environments, but yeah, if it's completely aqueous, what will happen over time. So it depends basically how quickly you work, um, because the The whole idea of the protein or the um the pollen we put on the surface is that it will diffuse into the gel and it'll actually diffuse into the tissue and then chemically react with both. So, we have to have it be mobile for that to happen. And so, it's mobile, which means it can diffuse just out into a fluid as well. So, if you took this, you prepped it, you threw it in, you know, a, a buffer of PBS or blood and didn't try to adhere to anything, I'm sure a half hour later, the whole surface would be, you know, the, the polymer would be gone. So, the question is, how long could it stay stable and completely aqueous? You know, if it's a matter of, we apply it, you use it, it's exposed to an aqueous solution for 30 seconds, I'm willing to bet that would be fine. If it's 5 or 10 minutes before you can actually put it in place, probably not. So. Doctor Jennings. This is fabulous. Um, I can see it's gonna, you know, modern surgeons don't actually know how to do suturing and tying anymore. They just use staplers and they use other devices. So, I could foresee you turning this even smaller and replacing every stapler that we do, and that would be a gigantic business, you put a surgical out of business. So, but that's, but more interesting, there's this whole problem. For, for me, my, my particular issue is there's a whole issue of tracheal and airway and vascular stents that are used to keep things open, and the reason they're so long and cause damage far, far away from the specific point that we're trying to fix, is because they move. And so we put in these really long stents, we're trying to fix 2 millimeters of a problem. If we could actually have a 2 millimeter long stent stick to the place we wanted to and not shift all around, that would, one, it would improve the efficiency of the repair or correction, and that would be every vascular stent, every tracheal stent, every esophageal stent would be replaced. I mean, that's just one tiny tiny idea. The third idea I had, it, I've always heard about this, um, gecko feet. Oh yeah, yeah, and that's some sort of an adhesion with some sort of a molecular ionic something or other. I don't really understand how gecko feet work, but How do they work? And does that have any application to this kind of stuff? Yeah, so, the, how those tend to work is you're, what you're doing is they have very fine features, um, and so, you have a very high surface area for, you know, they're weak interactions between, you know, each little area and whatever it's trying to, like, the gecko, whatever it's trying to walk on. But since you have these very fine features, you have a very big surface area. So, kind of the sum of a lot of small things ends up being big enough that you get good adhesion. So, there are efforts to try to mimic that with the, with adhesive materials as well. Um, you potentially could combine that concept with this strategy. Um, we've not tried to do that. You know, here, what we're depending on instead is a covalent bonds between the tissue and the gel, and then having this gel be able to dissipate a lot of stresses. The, the gecko strategies are really the interface, so that could potentially replace the covalent bonds that we're looking at here. I don't know if they would be all of that, um. Robust in kind of the environments that we've been trying to get into, which is more kind of bloody, wet environments. Um, but you, yeah, you potentially could combine the two and just use that as the means for adhesion instead of, you know, the, the, the chemical approach we're taking. Doctor Fauza. David, uh, great talk as usual. Uh, some of the questions I have, have been asked. But one is the, the endogenous adhesive, like a fibrine clot. One of their benefits is that they allow for it even encourage the penetration of certain cells, uh, as well as platelets. Do these bio diesels you're working on allow for cells to penetrate and eventually repopulate the area? So, the, the, uh, original design is no. So these basically, these are intended to be more or less inert and to not encourage, uh, nor allow initial invasion. So, what we're working on RO right now in the laboratory is having biodegradable versions of these that will basically slowly dissolve and allow cell infiltration, cell invasion, replacement. So, so, the first generation are intended to not allow that, but in the future, we hope we have versions that can. Additional questions for Doctor Moody, Doctor Modi. It's a great talk. Um, along with what Rusty was saying, one of the things that we could see as an extension of this to make it even more tailored would be if the adhesive properties could somehow be reversible. Um, so for example, deactivate with a light or something, for example, with Rusty's, um, uh, stents, if they could be applied and be placed in, uh with the adhesive to hold, and then a week later they would want to go and remove those stents out of the airway. Um, to have that adhesive be reversible so that it would be easier to remove without causing tissue damage would be, uh, an even greater advance, I think. Yeah, you know, do you want me to wait to respond? OK. Um, the, uh, so we, we've not explored that to this point in time, but there are chemistries, as you're probably aware, because you've mentioned, you know, light activated. So, there are chemistries that one, that are reversible with things like light. So, we, those could be explored for inclusion here to try to make it reversible. Right now, we've not tried to make it reversible. These basically will stay adherent until the tissue, since they bind to the tissue, as the tissue turns over, they're gonna come off. And so, we've designed it that way, but you could, you know, try to make them reversible either with a chemical means or the other thing that we haven't really started to explore, but a major component of this is alginate, which is degradable by a non-mammalian enzyme that basically then doesn't degrade our tissues. And so, you know, another approach with this may work. We haven't explored it in the body yet, would be to use an alginate solution. You basically wet it, let that start to degrade, the alginate component, which should then dramatically change the toughness and you probably can peel it off. But I think the, the instant reversibility would be much nicer as you're proposing. And one more, so I, um, I was fascinated by that thin gel that was incredibly strong, and one of the biggest problems in cardiac surgery, I'm not a cardiac surgeon, but I work with them closely, is the valves. There's no good replacement for the human valve, and that really thin, super strong deformable gel. You, you might spend a little time working with Chris Baird or some of the cardiac surgeons. About seeing if that would work as a valve leaflet cause that is a tremendous problem in pediatric and adult surgery that that's a very interesting point. Oh, you are, you're already doing it. No, OK, thank you. Final question. That was mine. How do you get it off? Got usurped. Anyone else? Now, Doctor Murray, thank you. Oh, Doctor Lillliha. Maybe I don't understand very well. I thought this is where um uh Doc Jennings was going, but we spent a lot of time connecting things uh together, and it would seem to be that, that, uh, in lieu of a series of sutures, or, or staples, or whatnot, that just laying that uh uh adhesive over the surface of a tube to make that connection, and, uh, uh, would Would speed up perhaps that that process. I guess the question uh I have a little bit is, is whether these, it sounds like these materials themselves then not necessarily degrade but fall off the, the adhesive product so it would be just a period of time and quite frankly for most of these connections, you know, on the order of a week or so, uh, would be. Uh, likely sufficient, and if there were longer, uh, uh, longer time frames, it could be used in different parts of the body as we put layers back together again. I mean, it just seems to be a way of revolutionizing our, what we do as surgeons is we connect things and, and we, we secure that in some way with, uh, sutures that we've been using for hundreds of years, and, and, uh, why not glues and adhes. Yeah, see, that's really interesting thing we've thought a little bit about is. You know, we need to figure out exactly what performance we can get over time and, you know, and how strong they can be in terms of keeping things together. But we thought a little bit about that as a goal. And then also, would there be utility of being able to use this as a way of quickly positioning things as well? So, you might use it with sutures, but maybe you actually use this the way instead of, you know, trying to get something together and be suturing it, use this as a way of bringing it together and then perhaps suture it anyways. But then you actually have already aligned everything and would that be useful as well, or? As kind of, cause I'm thinking as a, it's kind of a big jump for me to think about going from sutures to this without something between the two. That's why we're kind of thinking about maybe we use it with sutures originally and then if it performs well, then we get rid of the sutures. That OK, thank you. Well, thanks so much for bringing your pioneering work to us. It obviously has great relevance to all that we do. Yeah, my pleasure. Hey, it's great to see you here of course. Um, I think it's gonna be 10 by 10. about