Pro-Regenerative Biomaterials for Treating Heart and Skeletal Muscle - A Closer Look

Pro-Regenerative Biomaterials for Treating Heart and Skeletal Muscle - A Closer Look

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This program contains graphic images and discussion of medical procedures. Viewer discretion is advised. [MUSIC] Good afternoon. Welcome to a closer look at pro-regenerative biomaterials.

Thank you for making it to this session today, we're looking forward to an exciting session. With that, I'll introduce you to our first speaker, Karen Christman. Dr. Christman completed her PhD in bioengineering at UCSF in Berkeley, in 2003, and then did a postdoctoral fellowship in polymers and nanotechnology at UCLA.

In 2007, Dr. Christman joined the faculty at the bioengineering department at UCSD and became full professor in 2016. In 2020, Dr. Christman became Associate Dean for

Faculty Affairs and Welfare at the Jacobs School of Engineering, as well as being appointed as the Deputy Director of the Institute of Engineering and Medicine at UCSD. Dr. Christman is also the co-founder of Karios Technologies. Dr. Christman has received numerous awards and honors in recognition of her multiple significant contributions to the development of biomaterials to promote tissue regeneration. Today, she's going to tell us about some of that work and the progress that she's made on it. Karen.

Thanks so very much for the kind introduction, and welcome everybody. Thanks for being here virtually. I'm going to kick off the session today and talk about polysemy. A little bit different for the series, which is focused more on stem cell technologies. We do work in the field of regenerative medicine, we take a little bit different approach, which I'll tell you about today.

We work on pro-regenerative biomaterials and particularly, injectable materials that could be delivered minimally invasively. For disclosures, as Sylvia mentioned, I'm co-founder of two companies. I will mention some of the work of Ventrix today. Before I get into the main topic, I just want to give a brief overview of what my lab does.

As I mentioned, we focus on a little bit different aspect of regenerative medicine. We focus on biomaterial technologies and we work with a variety of different biomaterials. Some that are naturally derived coming from animal sources, or extracellular matrix derived, which is what I'm going to focus on today, since we've had the most success in terms of regeneration and as well as clinical translation with those type of materials. But we also do work with synthetic polymers as well as synthetic nanoparticles. As I mentioned, everything we do is injectable with a goal of having minimally invasive procedures to speed up recovery time, reduce chance of infection, and basically make it easier on the patient.

Our main focuses traditionally have been on cardiovascular disease, so treating myocardial infarction or heart attacks as well as peripheral artery disease. That's what I'm going to focus my talk on today. Then a few years ago, we also started working with Dr. Alperin, who's going to talk about some of our joint work on treating pelvic floor disorders. But in all these cases is basically treating either cardiac muscle, so the heart muscle or damaged skeletal muscle.

We'll start off with the heart, which is something that I focused on for a couple of decades now. I'm sure everybody is well aware of the problem we have with heart attacks and heart failure in this country. Sadly, it's still the leading cause of death in the Western world. Two-thirds of heart attack patients do not make a complete recovery. There are about a million in the US, there's about a little over a million heart attacks each year, so those who survive, 2/3 don't make a complete recovery.

What happens is the heart goes through what's called a negative left ventricular remodeling process, which basically means it goes through remodeling or adaptation process, where the heart actually dilates and expands. I'll explain that a little bit more on the next slide, and that leads to heart failure where the heart can no longer pump blood effectively to support the body. Right now, there are no therapies that prevent this negative. It's basically a negative feedback loop from happening, and there's also no therapies for end-stage heart failure really other than heart transplantation or left ventricular assist devices, which are mechanical pumps. Of course there's lack of donor organs, and then the mechanical pumps have a lot of other downsides as well. Really, there's a strong need to develop new therapies to treat the heart, especially regenerative ones that could prevent this entire process that leads to heart failure from happening, or treat heart failure patients who are already in heart failure.

To give you a little bit better idea of what happens when you have a heart attack, how that leads to heart failure. When you have myocardial infarction, that's the term for heart attack, what happens is you get a coronary artery or one of the blood vessels that supply the heart, it has a blockage, and that leads to lack of blood flow to a specific region of the heart. What happens is you get death of the cardiomyocytes, so the cardiac muscle cells. That's typically what people think about that you have a heart attack, you get death of yourselves. However, there's also something else that's really important that happens is that you get degradation of the ECM or the extracellular matrix. For those who don't know, this is basically the structural scaffolding that all your cells and each of your tissue sit inside.

You can think of it like scaffolding for a house and then the cells are sitting inside. So when you have a heart attack, not only are your cells dying off, you also get degradation of this extracellular matrix scaffolding. I'll talk about why that's important in a second.

Because the heart is really not a regenerative organ, this leads to scar tissue formation. Probably most people when they were a kid, had cut themselves really badly and then you have a scar on your skin, it doesn't heal completely. Basically, that same process happens in the heart. Since it's not regenerative, you're going to get a scar inside of the heart.

Then it leads to what I mentioned, this negative left ventricular remodeling process. Here is the left ventricle, which is the main pumping chamber of the heart, and it goes through essentially a negative feedback loop that's trying to compensate for this weakened region where you have the heart attack, and eventually that entire ventricle dilates or expands and the volumes increase, and then the heart can no longer pump blood effectively and that leads to heart failure. There have been a lot of therapies, including stem cell therapies that people have tried injecting into what's called this infarct region, the heart attack region. But the problem is they're injecting cells into a very abnormal environment.

If you look at a section of a heart attack under a microscope and this is what's called an H&E stain section, you can see this pink here is basically collagen, which is scar tissue, is the major protein of scar tissue, and this is not a normal environment or normal microenvironment for the cell. So when your people are delivering stem cells into this area alone, they're seen as very abnormal environment that basically is providing disease cues to the cell. It's not too surprising that the cells alone haven't worked very well. What we focus on a lot is that extracellular matrix and the lack of extracellular matrix, and trying to stimulate regeneration of the body's own tissue using the extracellular matrix. Why the extracellular matrix is so important is that, as I mentioned, your tissue or the heart is not just cells, it sits in this extracellular matrix scaffolding, has this nice fibrous network.

Here's if this is your cell, you have basically this fibrous network that the cells sit inside. The extracellular matrix, all of these fibers actually provide significant cues that influence essentially all aspects of cell behavior. So self-survival, death, division, differentiation, etc, can all be influenced by this extracellular matrix, so it's incredibly important. Again, I think that's been an oversight in the initial type of regenerative strategies that people had tried for the heart. Then the other thing I'd like to point out in terms of the importance of the extracellular matrix is that each tissue has its unique extracellular matrix. The extracellular matrix of your heart is very different than say, your intestine or your lung.

What we've thought about especially initially in trying to regenerate the heart, we've thought about, instead of injecting cells into this abnormal environment, why not inject an extracellular matrix replacement that provides more natural healthy cues for the body's own cells or the endogenous cells to migrate in and to try to regenerate the heart. That's what a lot of our projects have focused on this extracellular matrix replacement strategy to provide a new template for healing and to stimulate repair, and in this case, in the heart. I'll show you later on in skeletal muscle as well. The technology we developed for the heart is called a myocardial matrix hydrogel. We wanted to design an injectable material that you could deliver minimally invasively, but would replace all of those cues of the original extracellular matrix.

That's really hard to do from a synthetic strategy, because the extracellular matrix of your heart as well as other tissues is really hundreds of components. A lot of different types of proteins, but also sugars or polysaccharides. What we decided to do was take a more naturally derived approach and really take advantage of what nature gives us already. In this case, we take pig hearts.

We isolate just actually the left ventricle which we're trying to treat, we chop it up into small pieces and then stir it around in a detergent like you see here, there's what we call decellularize it, which is we've strip out all the cells and isolate just the extracellular matrix. Then we dry it and mill it into a fine powder and then use an enzyme to partially enzymatically digest it, which basically is converting this from a solid into a liquid that can be injected. Then what's really cool about the materials when you inject it back into tissue, [NOISE] right here is our myocardial matrix hydrogel, this purplish tissue here is what a rat myocardium looks like. Here, once you inject this liquid back into tissue, it reassembles back into a porous and vibrous structure that's very similar to the original extracellular matrix. This inset here is actually the extracellular matrix from this step before we've done any other processing. You can see in terms of the pores, the holes as well as the fibers are quite similar, and if you look at it with a scanning electron microscope, you can see these fibers are actually on the nanometer scale, which is very similar to the original extracellular matrix.

We optimize this whole process so that the material could be delivered via catheter, and then in the heart what that means is you're accessing at the femoral artery and you're growing, and the catheter is staked up through the aorta, major vessel in your heart, and then it goes inside the left ventricle and the catheter has a little needle at the end that can be deployed or retracted, and then you can inject the material. Just to give you a visual on that process, this is a quick video that one of my former students- who's actually a little old now because she's a cardiology fellow at Vanderbilt, but it's still probably the best video we have of describing the process. [OVERLAPPING] Hi. My name is Jean Wang and I'm a first-year.

I'm a PhD student in the Christman Lab and this is how we make our therapy for treating heart attacks. We get heart tissue, and the first step is we chop it up into tiny pieces and we put it into a bath, like a beaker right here. We stir it up with detergent to remove all the cellular contents. After a couple of days, we rinse it out to remove all the detergents and all we really have left are the structural proteins that make up a tissue.

We take that and we freeze-dry it into this styrofoam-like substance. It's really light and crispy, so it's freeze-dried. Then we take this and we mill it into a fine powder that looks like this. Then we take that and digest it with an enzyme to liquefy it.

You could see that there's liquid at the bottom of this, and this little bead is just stirring it up and keeping everything well mixed. Then finally we take the liquid form of this and we inject it into the damaged heart tissue. Once the liquid hits body temperature, it forms a gel that looks like this and then no longer flows anymore. I believe that gives you a little better idea of what the material looks like.

The process is, relatively speaking, quite simple to make it. I always say I usually teach in the undergrad how to do it in just a couple of weeks. We've taken that material, we've studied it in a lot of different pre-clinical models. I'm going to show you briefly some of the data from the more translational large animal model that was needed before going into patients. We injected this into a pig heart attack model where you first give the pigs a heart attack, then we deliver the material through that catheter system that I just mentioned.

We delivered that two weeks after the heart attack and then we took the animals out to three months. What we saw were significant improvements in global function, really preventing the heart from dilating. We had reduction in volumes, both in systole, which is the end of contraction, and then in diastole, which is the end of relaxation, when the heart is relaxed. Here, showing ejection fraction, which is a measure of cardiac function, basically a fraction amount of blood that is pumped out of the heart.

You can see pre-MI or pre heart attack. You have a normal ejection fraction for pigs. Two weeks after the heart attack was given, you can see it has this decrease that continues to decline in the control animals. Then with our matrix hydrogel-treated animals, we had significant improvements in cardiac function. The reason why we think that's occurring is that, when we looked at histology or tissue sections, here is an example of our matrix treated, and here's one from our control using what's called a trichrome stain which stains muscle and bread and collagen in blue.

We saw this thickened band of muscle that also had vasculature supporting it. If you quantify that compared to control animals, that was significantly greater. Also, we quantify the collagen content in the scar, and that was significantly less.

So essentially more cardiac muscle and less scar tissue. We think that was leading to these improvements in contraction or cardiac function. We've done, as I mentioned, a lot of different preclinical studies both in terms of looking at cardiac function but also understanding the mechanism action. We really do find that these materials act as a new template for healing. In multiple studies, we see that this material's pro-survival decreases cell death, increases the immunomodulatory response, so really shifts.

Instead of a very pro-inflammatory environment that you have after a heart attack, you actually get more of a pro-remodeling and pro-healing environment and you shift the immune cells to be that more pro-healing phenotype. We also saw increases in blood vessel development as well as indications and increases in heart development. Like I said, it is a new template for healing. That work, as well as a lot of other, especially significant safety studies to look for potential for arrhythmias, biocompatibility, hemocompatibility, which is compatibility with blood, all of which looked great, lead to Ventrix, which is the company I co-founded conducting a Phase 1 clinical trial in heart attack patients using this same catheter delivery strategy I mentioned. Here's what the material looks like. Commercially, it just comes as a dried lyophilized cake.

You add sterile water right before you're ready to inject and then you're ready to go. The trial was a small just Phase 1 trial because people have used decellularized extracellular matrix patches in surgery actually in millions of patients, but nobody had used this hydrogel form of an extracellular matrix in any tissue. Looking at a small safety study was a really critical first step.

Use the catheter technology I mentioned, and then half the patients were treated between 60 days and one year after their heart attack, so about 15 patients in total, half the patients were treated 1-3 years after the heart attack, so an early and a late group. We looked at baseline in three and six months at some measures mainly for safety and feasibility, but also some measures of efficacy. Even though it was definitely not powered and there's no control group, but we wanted to see whether there are some potential indications of improvement. Overall, as I said, this is primarily designed for safety. We found that VentriGel was well tolerated, which was an exciting result since this was the first of its kind, an ECM hydrogel going into any tissue.

Of course, the heart is a high-risk organ. But we also did see some encouraging secondary endpoints in efficacy, significant increases in six-minute walk tests, which is how far a patient can walk in six minutes, decreases in New York Heart Association heart failure class, which is basically decreases in heart failure symptoms, and then 80 percent of patients either maintained or improved their volume. Basically, we were helping to prevent further dilation or expansion of the heart in these patients. Encouraging for a Phase 1 trial in Ventrix is continuing clinical development on this. Next, I wanted to switch just briefly to talk about skeletal muscle instead, but still a cardiovascular disease. Peripheral artery disease, probably many of you have heard of, but many of you may not have because it's not as talked about as, say, heart attacks or heart failure, but it still affects a very similar number of patients.

PAD, peripheral artery disease affects about 27 million patients in North America and Europe. It's a huge patient population. What this is, it's also caused by atherosclerosis as opposed to, though, a heart attack where you get this acute-like initial event where you get a clog in an artery. This is more of a progressive narrowing of arteries, so it's a little bit more of a chronic condition. What happens is, because you get this narrowing of the arteries, you get decreased blood flow to the limbs and you also get muscle atrophy.

The skeletal muscle in the limbs atrophies or decreases in size and so it becomes weaker. Initially, the earlier stages or the less severe seizures called intermittent claudication are things when patients say have pain upon exertion. But eventually, or in the severe case, you have critical mesothelioma where you have pain at rest, and you get things like gangrene and needing amputation. You can see about 120,000 patients in the US each year need an amputation because of this, and right now there are no effective therapies. Because we had seen increases in vascularization in the heart model and overall anti-inflammatory pro-healing effects, we wanted to see if the general strategy of ECM hydrogels could be used to treat PAD patients or in a model of PAD.

We used what's called a rat hindlimb ischemia model where you surgically remove one of the arteries and veins in the leg to create this region of low blood flow down to the one limb. Then we look at percent perfusion as a proxy for blood flow, and we either injected saline as a control or we injected the ECM hydrogel derived from skeletal muscle, so porcine skeletal muscle. The identical process I showed you for the heart, but in this case, we're using skeletal muscle, so just what we would consider meat as opposed to cardiac muscle. If you look at saline, basically, that plateaus, high 60 percent, whereas we get significant increases in blood perfusion, again, a proxy for blood flow with injection of our skeletal muscle extracellular matrix hydrogel. We did histology and looked at tissue sections and found we in fact did increase blood vessels, particularly arterioles.

Then what was also interesting is that we found an increase in fiber area of the skeletal muscle cells or the skeletal muscle fibers. As I mentioned in PAD patients, peripheral artery disease patients, you get muscle atrophy, which is a decrease in size. This also happens in the rat model. Here's a healthy muscle.

Then if you look at the saline group, the saline control, you can see a decrease in fiber area, which means that the muscle is atrophing. Whereas we basically prevented this with our skeletal muscle extracellular matrix hydrogel. The reason why we think at least this is partially occurring is that we've got a significant increase in what are called Pax-7^positive cells, which are the skeletal muscle satellite or skeletal muscle stem cells, compared to saline that we think was helping with the regeneration. Interestingly, in a subsequent study in a different model, not a hindlimb ischemia model, but using what's called a notexin injury model. You essentially inject a toxin into muscle and watch it heal. It's very common model in the muscle physiology field.

Using that model we looked to see if you inject saline versus ECM hydrogels that are derived from the skeletal muscle like I just showed you or a cardiac muscle ECM hydrogel or a LNG ECM hydrogel, all porcelain derived using very similar processing, but only the skeletal muscle, only the tissue specific extracellular matrix hydrogel got that significant increase in Pax-7^positive stem cells. We do think having tissue specificity in terms of the ECM hydrogel and then the tissue type you're trying to treat is important. Then just for the last really couple minutes, I just wanted to briefly mention some of the work we've also been doing towards trying to develop new therapies for COVID-19, which of course has been dominating all of our lives and why we're on Zoom right now. Because our extracellular matrix hydrogels have been shown to be anti-inflammatory and pro healing, as many of you have probably read in the news, the infection from SARS-CoV-2 not only infects and causes a problem in the lungs, but it's really a systemic inflammation.

An inflammation all over the body in multiple organs, that's an issue. Given what we knew about these ECM hydrogels, we wanted to see if this strategy of pro-healing anti-inflammatory biomaterial strategy might be able to use to dampen inflammation once somebody develops severe COVID-19. What we wanted to do is then develop a hydrogel though that could be delivered systemically.

Ideally, you deliver it in the blood. It could go to all the organs because the ECM hydrogels that I showed you before require injections into the specific locations. That really wouldn't work with something that's really systemic like COVID-19.

We developed a version of the hydrogel. Same processing where you take in this case hard, process it to the liquid stage and instead of relophyilizing it and having it ready to store it and inject, we actually fractionate it into the low molecular weight fractions and the high molecular weight, basically, the more soluble or very small nanoparticles to the greater sizes or the insoluble components. Then you can relyophilize this, what's called the supernatant, this liquid, and then it's ready to go unrehydrated. What we found is you can actually inject this intravenously or into other blood vessels and it will target areas of inflammation. In areas of inflammation, you actually have leaky vasculature and you have gaps in the endothelial cells that line each blood vessel.

Here in this image, green is endothelial cells, red is our matrix, blue is nuclei of cells. You can see it actually seems to bind to and potentially gel within the gaps of the endothelial cells. We found this in multiple models so far, that you can inject it into the blood and then it will target to areas of inflammation. We found that in fact it does in a model we don't use the virus since we're not equipped for that, but we use what's called an LPS, a lipopolysaccharide systemic inflammation model, that it causes severe inflammation across the entire animal, in this case a mouse model. Then what you're looking at here is basically profiles of inflammatory genes.

You can see the M animals here are our matrix, the S here are our saline and control. Hopefully you can appreciate the difference in colors between the two groups. Basically what we found in doing this inflammatory gene panels, we saw a dampening in this case I'm showing the heart and lung, but we found it really in all the organs that we've tested so far as well as decrease in the inflammatory cytokines and the blood too.

We're still studying this further. Hopefully COVID with all the vaccines won't be such an issue anymore, a severe COVID-19. But we do think this could be also used for other causes of acute respiratory distress syndrome and things like sepsis. With that, just to briefly summarize, hopefully I've convinced you that there are other regenerative medicine strategies and we think ECM hydrogels really are versatile, minimally invasive platform for treating multiple different types of pathologies including myocardial infarction, peripheral artery disease, I mentioned COVID-19, and then Marianna is going to talk about her expertise, which is in pelvic floor disorders.

With that, I'd like to really thank all the members of my lab, both current and former, especially my current lab, everybody's had a rough time this year and they've been chugging away in the lab but trying to get things accomplished, particularly the COVID-19 team, which is Ray Wang, a former member, as well as Ryan Middleton who have been working on the COVID projects. The skeletal muscle project was really by Jessica Ungerleider and Todd Johnson, both former students of mine. Many of the names that you see up here have contributed to the cardiac work as well as I thank Ventrix and our clinical investigators, as well as the funding sources. Thank you very much and then I'll be happy to answer questions later on. Thank you so much, Karen, for that very intriguing presentation.

Sounds pretty exciting. That phase 1 trial, Dr. Alperin received her MD degree at St. Louis University School of Medicine in 2001. Then she did her fellowship in female pelvic medicine and reconstructive surgery at the University of Pittsburgh, where she also did a Master's of Science in clinical research and design. Dr. Alperin then did a residency in obstetrics and

gynecology at Brigham and Women's Hospital at Harvard. Then from there, she went to Kaiser Permanente in Los Angeles to become the founding chief of the division of urogynecology. In 2012, we were lucky enough to recruit her here to the Department of Reproductive Medicine at UCSD, where she is now associate professor.

Since 2019, Dr. Alperin has been the fellowship Research Director for the Division of female pelvic medicine and reconstructive surgery. Dr. Alperin has been the recipient of

many awards and honors in recognition of her work which has focused on understanding diverse aspects of female pelvic skeletal muscle function and disorders, which she's going to talk to us about today. Marianna. Thank you so much Sylvia for this introduction. [NOISE] Today, we will talk about how the materials that Karen described can be applied to the female pelvic soft tissues.

The female pelvis has a big aperture that is filled with different structures including connective tissue, skeletal and smooth muscles, and the nerves that innervate them. As the humans became obligate bipeds, the interplay between structural components that are critical for the proper function of the female pelvic floor became even more important, because when these components fail, one or the group of them, pelvic floor disorders can occur. Pelvic floor disorders is an umbrella term and unlike a myocardial infarction, potentially less familiar to the audience.

I will describe it. It consists of pelvic organ prolapse. Here in the picture you see the uterus that is coming out. It's certainly shouldn't be there and the urinary and fecal incontinence. These conditions are very prevalent.

Approximately 25 percent of the US community dwelling women are affected. Even the projection that by 2050, close to 44 million US women will suffer from pelvic floor disorders. Unlike myocardial infarction that can directly cause death, pelvic floor disorders impair quality of life. They impair quality of life so much that in a recent Journal of American Medical Association articles, where the participants were asked to rate various conditions on the scale; much better than death, somewhat better than death, little bit better, or worse, astoundingly, approximately 55 percent of participants rated bowel and bladder incontinence as worse than death. I just want to point out that the next runner up was relying on the ventilator.

This is a devastating condition that's been known since antiquity. The first description has been encountered in Egyptian papyruses in 1800 BC. At the time of Hippocrates in 400 BC, you can see here a woman depicted hung upside down in order to correct her prolapse. The first surgical intervention for pelvic organ prolapse was performed by Soranus of Ephesus in 120 AD in a form of vaginal hysterectomy. There are multiple risk factors for pelvic floor disorders, including family history, genetic predisposition, pregnancy, vaginal delivery, mechanical factors such as obesity, hormonal deprivation associated with menopause and aging.

However, vaginal delivery by far is the leading risk factor for pelvic floor disorders, increasing this risk by 4-11 times and therefore it represents the most important potentially modifiable risk factor. Despite this high prevalence, that's almost epidemic proportions currently available preventative strategies are limited to cesarean sections, which of course have a host of side effects that we want to avoid. There's a lot of risk factors where women should not undergo cesarean sections undesirable. In terms of the available treatments, they're all pretty much delayed, compensatory, and don't really address the underlying pathophysiology. In fact, we really haven't evolved very much from ancient Egypt, where they used pomegranates as a form of pessary, which is a device to support the prolapse. In Europe in the 1800s, a bronze pessary was used.

Our patients benefit from the silicon-based materials, but it's still a pessary. As you can see in this timeline, we haven't really made a significant leap forward in our treatments of these devastating disorders. This represents a significant scientific disparity that is especially pronounced when it comes to the female pelvic floor disorders.

We're always fortunate to have a program like Galvanizing Engineering in Medicine or GEM program, which is a partnership between UC San Diego Altman Clinical and Translational Research Institute and The UC San Diego Institute of Engineering in Medicine. This program's goal is to identify clinical challenges for which engineering solutions can be developed and implemented to improve healthcare. Through this program, Karen's and my partnership had fostered, and this program is very much aligned with the NIH vision.

That multidisciplinary approach to clinical care and research is the most fruitful paradigm for the development of significant advancements within a specific field. Today, out of all the pelvic soft tissues that can get injured during vaginal delivery, we will focus on the pelvic floor muscles. Here you can see a superior view of the schematic of the human pelvic floor muscles, which consists of them, coccygeus muscle in orange, iliococcygeus muscle in green, and pubovisceralis muscle that has two parts, pubococcygeus and puborectalis. Together, the latter two represent something called levator ani, it's only that might be familiar to some of you.

The reason we're focusing on the pelvic floor muscles is because pelvic floor muscle dysfunction has been recognized for a long time as the major contributor to the development of various pelvic floor disorders. As all skeletal muscles, pelvic floor muscles also contain contractile myofibers that are organized into fascicle, bundle, fibers, and extracellular matrix, which is also hierarchically divided into epimysium, which surrounds the entire muscle, perimysium surrounding fascicle and bundles, and endomysium surrounding individual fibers. Individual fibers are composed of the functional muscle units called sarcomeres, which I arranged the carts on a train.

In turn, sarcomeres are comprised of the myosin and actin, which are called myofilaments. The contractile myofibers are responsible for the active mechanical muscle properties, while extracellular matrix is responsible for the passive mechanical properties like load bearing capacity and muscle stiffness. In order to prove why vaginal delivery is the leading risk factor for pelvic floor muscle injury, a lot of groups including University of Michigan group whose work is, I'm going to discuss briefly here, perform computational modeling of human parturition. Here you see a picture of fetal crowning with this net-like structure representing the fetal head, and then the green like structure representing a levator ani complex. The different colors is how much strain is imposed on different components of the pelvic floor muscle complex. It was identified that tremendous strains impact these muscles during parturition and especially this specific point called entheseal region of the pubovesceralis muscle, which is where the muscle is attached to the pubic bone, that can achieve strains up to 250 percent.

Those strains are so large. There's certainly an acute access of 60 percent strain that is enough to permanently injure the muscle. We wanted to know what is the impact of mechanical strains associated with parturition on the pelvic floor muscle myofibers? In order to do that, they used a widely used simulated birth injury model. We specifically use rat, but it can be done in a mouse model or other animal models. We use a Foley catheter balloon that is somewhat modified and induce vaginal distension, which simulates the circumferential and downward distention associated with fetal crowning, as in rats and humans, as you can see here. Here is that growth picture of the pelvic floor muscles specifically pubocaudalis portion of the rat levator ani, where on your left, you see a normal intact control muscle, which is outlined in blue with the star, asterisk identifies the entheseal region, the one that undergoes tremendous stretch during human parturition.

On your right, you see the parents of that muscle after the injury is performed acutely. You can see that see-through appearance and that see-through appearance and due to dramatic stretch of the muscle and therefore myofibers. In fact, the stretch ratios for pubocaudalis muscle in simulated birth injury model reach the same stretch ratios as were identified in the computational models of human parturition.

Several fellows in the lab, our female pelvic medicine reconstructive surgery fellows identified what is happening at the intrinsic structures of the muscle from these strains. What they discovered is that sarcomeres, those functional units that I showed you, that are arranged of the carts on a train undergo significant hyper-elongation immediately upon birth injury and that is a known mechanical injury of skeletal muscles from the investigations done in the limb muscles. This sarcomere hyper-elongation is associated with something called myofibrillar disruption. Here you see an electron micrographs of the uninjured pelvic floor muscle on your left and injured on the right.

What becomes immediately apparent is that well aligned sarcomere structure of the uninjured control muscle is disrupted. As you can imagine, that would not be conducive to force generation. We know from the limb muscles studies that such myofibrillar disruption that comes from the fullest sarcomere hyper-elongation eventually leads to a significant inflammatory response by the tissue, and long-term manifests as fibrosis, which is a pathological accumulation of collagen in the muscle and myofiber atrophy, which means small fiber size or death of the myofibers. Together, these two degenerative conditions can lead to muscle dysfunction.

We wanted to know, does this acute sarcomere hyper-elongation leads to this long-term pathology of the pelvic floor muscles, because that has never been investigated. Pamela Duran, who is a PHD candidate that Karen and I co-advise as well as one of the masters students in my lab, they perform these experiments where they used a simulated birth injury that I described previously, and then they house the animals and allowed them to recover for either four weeks or eight weeks, at which point the animals are sacrificed and the tissue was procured to assess fiber area, which is a marker of myofiber atrophy, and collagen content, which is a marker for fibrosis. Here, in the first panel you see the control cross-section of the pubocaudalis muscle where all the rats different shapes, but the red is the laminin, and it outlines individual fibers. That is what we use to calculate fiber area. At four weeks after the simulated birth injury, the fiber area was significantly decreased. But at the same time, we noted that centralized nuclei, which is a metric of every generation because when the de novo muscles, they have a centralized nuclei that subsequently migrates to the muscle periphery.

Because it was significantly larger, we didn't know. Is this because the area generation is ongoing, or is that really because of atrophy? This is where the eight weeks animals came in handy. Eight weeks up to the injury, the number of centralized nuclei return to control levels, which means that regeneration was no longer taking place.

However, the fibrosis stayed lower than the uninjured controls indicating myofiber atrophy. Our second outcome of interest was the pathological accumulation of collagen. Here you see a cross-section of the rat pelvic floor muscle. It's a trichome stained where collagen is in blue with the myofibers in pink. You can see just by eye even without looking at the quantification below, that there's much more blue after the birth injury at four weeks as well as at eight weeks.

In fact, the significant increase in collagen content did not differ between four and eight week time points, and was significantly increased compared to the uninjured controls. Then we wanted to know, what are the mechanisms accountable for this pelvic floor muscle phenotype falling birth injury? Specifically, we wanted to focus on the tissue morphology at the very beginning after the injury ensues from one day to 10 days. We will combine it with the gene expression studies that spanned at one day to 35-day time course. We know from the studies of that particular muscles that the first event upon muscle injury that is required for muscle regeneration, is the infant's ration with leukocytes.

This stirs the pro-inflammatory stage, which is characterized by Type 1 macrophages and Th1 helper cells, which are responsible for cleaning cellular debris. Subsequently, this switches under the regulation by regulatory T cells, the switch to the pro-regenerative phase occurs, which is characterized by the Type 2 phenotype of macrophages and Th2 helper cells, which are responsible for tissue remodeling and repair. This all occurs within the first seven days post injury. We know that the inflammatory response of the tissue to injury is very much connected to the muscle stem cells that Karen had mentioned, there the residents down cells that normally present in the quiescent state. However, upon injury, they undergo activation, and then after activation, they can go to different routes.

There's a sub-population of a muscle stem cells that undergo differentiation without proliferation, in order for them to respond very quickly to injury. That usually occurs in 3-4 days in the limb muscles. There are other sub-population of this muscle stem cells, which is characterized by Pax7, undergo proliferation and self-renewal to repopulate the stem cell pool.

This normally occurs at 5-7 days post injury. In our morphologic assessment of the pelvic floor muscles response to burst injury, here you see a cross-section of pubocaudalis muscle with a tightly packed myofibers as we had seen before. At one day, we saw a profound myofiber death. At three days, we saw significant cellular infiltrate, which is the immune infiltrate, and by seven days, we identified centralized nuclei, which as you can recall, is the metric of muscle regeneration. Then with that in mind, that despite these events that were anticipated at the beginning, we know that at the end that pelvic floor muscles underwent atrophic and fibrotic changes. We designed gene expression screening panel with those in mind.

We know that immune infiltrate that induces inflammatory response can impact muscle anabolism/catabolism pathways, fibrogenesis, and myogenic pathways. We focused our custom panel on this pathways. First, we looked at the myogenesis. Myogenesis followed the expected time course that I described.

With the upregulation of myogenin, the mark of differentiated muscle stem cells by three days, and up-regulation of Pax7 a mark of proliferating, and non differentiated stem cells by seven days. With the help of the postdoctoral scholar Francesca Boscolo in my lab, we wanted to confirm that our gene expression studies correspond to the histological assessments. Using immunohistochemistry, we determined that myogenin did indeed go up starting at one day and with the maximum increased by three days, and then Pax7, which is the mark of those upper self-renewable cells was maximumly increased at seven days post injury. This also corresponded with the large expression of the embryonic myocin heavy chain, which is the marker of the de novo myofibers, which would have the centralized nuclei. All of this was very consistent, showing that the myogenesis followed the expected time course. Again, Pamela Duran partnered up with Lindsey Burnett, another fellow, FPMRS fellow who works on basic science projects.

They determined that their gene expression studies that looked at the inflammatory pathways showed sustained pro-inflammatory response after birth injury even at 35 days. They also discovered impairment on pelvic floor muscle anabolism, and upregulation of the pro-fibrotic genes in response to birth injury. This was responsible for that phenotype we speculate, that we saw basically myofiber atrophy and fibrosis at long-term time point.

Clearly we need to develop an optimal healing environment at an optimal time point to avoid pelvic floor muscle dysfunction associated with that phenotype. We can use it at the time of birth trauma to prevent pathological alterations, or the delay time point to promote constructive remodeling that can revert pathological alterations. We hypothesized the delivery of tissue-specific skeletal muscle extracellular matrix hydrogel that Karen described, will prevent and revert pelvic floor muscles atrophy and fibrotic degeneration, but just as it is not easy to deliver things to the heart, it is actually not easy to deliver things to the pelvic floor muscles, which are structures deep in the pelvis. At the start, we partnered up with our radiology colleague Dr. Eric Chang, Professor of Radiology at UC San Diego. With their resident Vipul partnering with Pamela Duran, they developed a minimally invasive approach to deliver this material directly into the pelvic floor muscle.

Basically, the system that we used allowed us to deliver the materials through transobturator approach, because the muscle here in blue, as you can see, overlays the medial surface of the obturator foramen through which we can introduce the needle and inject the material. We confirmed the reliable delivery of the material into the muscle by staining the material with either India ink or a fluorophore, and you can see here the dissection of pubocaudalis muscle in-situ after the delivery of the material. The black is the India ink that wasn't the hydrogel delivered specifically into the entheseal region of the pubocaudalis, where most strains occur. Then on the right, the rat is the fluorophore there was a hydrogel laden by the floor for us shows the delivery of the material into the muscle belly.

The study designed to answer these questions was divided into immediate injection, where the animals were randomized into salient or hydrogel injection immediately upon simulated birth injury, and then were housed for four weeks, where fiber area and collagen content was analyzed. For delayed injection, the animals underwent simulated birth injury, after which they were allowed to recover for four weeks, at which point they were randomized into salient or skeletal muscle a hydrogel injection, and housed for another four weeks for the same outcome. What we thought is that an immediate injection resulted in both saline and the extracellular matrix hydrogen prevented pelvic floor muscle atrophy, but only the hydrogel resulted in increased fiber size that return the fibrosis to the uninjured controls.

Both mitigated fibrosis somewhat but much more pronounced in the hydrogel group. Both modulated immune response, but skeletal muscle hydrogel promoted an earlier up-regulation of myogenesis pathway and decreased expression of pro-fibrotic related genes, specifically TGF-Beta. In the delayed injection group, here we observed pretty similar result, saline and extracellular Matrix hydrogel, prevented pelvic floor muscle atrophy with the fibrosis greater in the hydrogel group compared to saline. Both modulated immune response as well, and extracellular matrix hydrogel enhanced the myogenesis and down-regulated expression of pro-fibrotic related genes. Interestingly enough, we saw that mitigation of the fibrosis with saline to be a similar extent as it is that the hydrogel.

In conclusion, birth injury leads to significant pathological alterations of the red pelvic floor muscles long-term, impairing muscle anabolism, upregulation of pro-fibrotic genes, myofiber atrophy, and fibrosis were observed. Skeletal muscle extracellular matrix hydrogel, prevents the rat pelvic floor muscle atrophy and mitigates fibrosis. We think by modulating immune response, and impacting myogenesis. Saline injection, interestingly four weeks post-birth injury, also positively impacted muscle regeneration.

I once again want to thank the UC San Diego [inaudible] CTRI grant that really encourage the team signs that allows us to bridge the longstanding gaps in female pelvic medicine, and of course, many thanks to my collaborator Dr. Christman and thank you for your attention. Thank you very much, Dr. Alperin, that was very interesting, especially the observation about the saline was interesting. We'll see now the teams that we have some questions lined up here from our audience.

There's one here from Richard Robertson, it's for Karen. It says, "Impressive difference in porcine hearts with and without ECM therapy. Are those new cells from stem cells or fill-in from adjacent regions?" Yeah. Thanks for the question. Excellent question. I get that question a lot. I'll answer it in two ways.

If you're looking at its skeletal muscle, the answer is yes, at least partially, we do get a stem cell component, as I mentioned, when I talked about the skeletal muscle. The heart, identifying really what a true stem cell is a little bit more challenging, and actually, Sylvia can talk to this a lot better than I can. I don't want to rule it out that it doesn't exist in the heart, but right now we think it's probably that increase in muscle is coming from two things. One is the pro-survival effects. We have seen that it decreases cardiomyocyte apoptosis, so it decreases the death of existing cardiac muscle cells.

A lot of those cells will die off immediately after a heart attack, but it's actually, there's a significant number that will continue to die off in patients actually for years. We help decrease that cell death. Then we've also recently found actually in collaboration with Sylvia's lab, that we have indications of also cardiomyocyte proliferation. I'd say maybe Sylvia can help answer this too.

But I think right now the cardiac field, if you see regeneration, I think a lot of people think it's likely predominantly having a component where you're having more proliferation. I don't want to rule out that there's a stem cell component that we haven't discovered yet, but right now we think those are the predominant two components. Okay. Thank you, Karen. Thank you for that question. Next question is from Zhuangzi. "In the treatment of heart failure [NOISE] is the extracellular matrix of embryos, or younger hearts better than that of adults?" Yeah. We actually, many years ago,

now probably close to eight almost 10 years ago, we really were interested in that question. However, from a translational standpoint, we quickly realized that you really would be difficult to use younger source tarts. One just the access to them and two their small size, just in terms of making a big batch that you could scale would be really difficult. We gave up on that concept, but it wasn't what we were very interested in because younger animals are more proliferative and so potentially the ECMQs would be more proliferative.

Subsequent to that, Lauren Black who's at Tufts, they actually did those experiments using rat extracellular matrix. Got to give his credit to his grad students who meticulously isolated very young, teeny, tiny little rat hearts created material. They did in fact find that they got more proliferation of cardiomyocytes. That was more of an in-vitro experiment if I remember correctly.

Outside the body, but they did see that the younger matrix was better. But the issue from a translational standpoint, like I said, is I don't think you can make a clinical batch size of a small heart even from pigs because it's so small. But I think if you could, the therapeutic benefit might be even better. Okay. Thank you. One more question for Karen from Ksenia Malukina.

I hope I pronounced that right. "Did injection of porcines skeletal ECM into the rat model cause any complications due to differences in ECM between the porcine and rat models? Yeah. Extracellular matrix is actually quite conserved among species. As long as you remove the cells and appropriately decellularize the material, you can actually use generic or different species sources. Actually, as I mentioned before, their surgical patch is made out of decellularize extracellular matrix.

Actually, most of those are porcine-derived or actually, even some are equines or horse or bovine-cow derived. They've been shown to have a proper modeling. You don't get a rejection response unless you inappropriate decellularize it. If you leave too much remnant cell debris, then you get a rejections response, but otherwise, you can cross-species without an issue. Okay. Let's see.

We've got some more questions here. Jinhu, "Could we use cultured human cardiac fibroblast to prepare ECM instead of porcine hearts, which are not easy to obtain?" Theoretically could, and I'm blanking on the group's name, but there's a group I believe it's out of Wisconsin that has tried doing this. I think the issue is that it's actually much harder to obtain large quantities because you'd have to do massive cell culture, which actually porcine hearts are very easy to obtain, especially in this country. We don't use them really for food much in this country, so it's considered a byproduct. It's very easy actually to get porcine hearts just from food hogs because they're not used. It's actually a quite readily available source and you can scale it much easier.

Whereas trying to culture cells it would be significantly more expensive to obtain that extracellular matrix, and then it's also a question. It's not going to be the identical matrix as what's in a heart too. Whether that makes a difference or not, I don't think it has been well studied. Okay. From AC, "Any studies in the heart, ECM therapy with adjunct stem cells added?" We've got a good question.

We've done a little bit of that where more just feasibility. We have found that you can get some increases in survival by delivering cells. But because we didn't think we knew the best cell type to deliver in terms of my lab, what would be the best cell. We just stopped at the feasibility stage, but I think there is potential for that. We have a couple of companies actually that are interested in looking at the combination of the material with cells.

The jury's still out on that, but I do think it's promising particularly with some of the newer cell technologies that are out there. Okay. Maybe this is a question now that Marianna can help us with. From Julian, "In what we've talked about, you were mentioning how interdisciplinary this work has been with the two of you interacting. Is there a strong collaboration between chemists, physicists, and biologists as well as physicians and engineers from different industries? I don't know about specifically with the industries.

We certainly do try to collaborate with our industry partners. But again, in my field, the most collaborations has been about biomaterials or the synthetic materials that I used unfortunately that are associated with a lot of different complications that many of you probably heard in the mesh-related complications. That was our partnerships with the industry by engineers. Also, of course, creating the synthetic materials did involve physicists and chemists that work in those industries. In my Department of Obstetrics, Gynecology, and Reproductive Sciences, there is a strong partnership between reproductive biologists and clinicians who work in the endocrinology world that affects, for example, polycystic of ovary disease and others. In the female pelvic medicine, unfortunately, the collaborations with the chemists and physicists has not been profound.

But I certainly think that given how far behind our field is way behind cardiology, for example, or orthopedic surgery, where a lot of what we do, we look at what the appendicular muscles studies show and then see what's applicable in our world. I think that those partnerships would be invaluable because we really can gain a lot of time and make it economically feasible if we adopt some tools that have been developed in other fields instead of developing them, de novo. Okay. Well, thank you very much, both of you for really fascinating presentations. It looks like we've answered everybody's questions, and we'll just perhaps end here and thank you both very much.

Thank you for having me. Yeah, thanks. [MUSIC]

2021-05-11 08:06

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