Good evening, everyone. I first want to thank all those who attended tonight. My name is Shannon Strader and I'm this year's RFC research representative. I'm also the moderator for the AAP and AR3T Orthobiologics and Regenerative Medicine series. The goal for the webinar is to provide comprehensive education for physicians in training and physiatrists interested in regenerative rehabilitation while reducing stigma, misinformation, and encouraging responsible advancements for the regenerative field. If during the session you have a question, please write in the chat box and we will answer as many as time permits. It gives me great pleasure in introducing our brilliant speaker tonight, Dr. Wiles. Dr. Wiles is an assistant professor of dermatology and pharmacology and newly the assistant professor of regenerative medicine at Mayo Clinic. She is the program director for the longitudinal regenerative medicine and surgery curriculum for graduate students, medical students, and residents. She completed her undergraduate studies at Barnard College, Columbia University in New York. She then pursued a research fellowship at the Harvard Stem Cell Institute. Dr. Wiles attended the Mayo Clinic Medical Scientist Training Program in Rochester, Minnesota and attained her PhD in biomedical sciences, clinical and translational science. Her expertise in stem cell biology was further developed with workshops and internships at Liverpool, John Moores University in England, in Italy, and Trust and King's College London in England. She completed her residency in complex medical dermatology and participated in collaborative research projects focused on developing regenerative solutions for wound healing and hypertrophic scarring. Her current laboratory studies cellular senescence as related to cutaneous wound healing secondary to age-related chronic disease. The goals are to understand the role of senescence clearance for wound closure, excuse me, in the chronic non-healing wound bed. Her laboratory also explores the science of skin appearance, evaluating the role of exosomes or extracellular vesicles in skin rejuvenation. In recognition of her work, Dr. Wiles has received many honors and many awards, including the White House Project Eugene Farber Award for Young Investigators and Women's Dermatological Society Mentorship Award. Thank you so much for presenting to us tonight. Thank you, Shannon. Thank you for that nice introduction. I would also like to thank AAP and AR3T for kindly welcoming me into this forum. I too share the objective of educating our next generation of learners and providers in regenerative medicine. As a dermatologist, I'm looking forward to sharing with you these applications as related to skin wound healing. Our learning objectives today will be to review the underlying pathology of wound healing. We will then go into talking about advances in tissue engineering and ways that we can use methods and scaffolds to understand building new skin. We will also discuss clinical examples of tissue engineering skin substitutes and have a discussion or an engagement about some case-based research ideas for novel biomaterials used for wound healing. The topics discussed here can be translated into other area of orthopedics, spinal cord regeneration, and other fields of interest. These are just fundamental principles of wound healing and ways that tissue engineering has contributed to an example of the skin. Let's start with the skin. Before we get into the specifics of how and why we regenerate the skin, we should really understand the bread and butter, which is what is the skin. It's the largest organ of our human body. It comprises about 15% of our body weight. It really is a barrier between internal and external environments, our first line of defense. This is an important thing to recognize and understand as we build these different models or replacements for skin wound healing. Another important aspect to recognize is the role that the skin plays as a thermoregulator. It's our way to regulate if it's too hot or too cold. The skin can sweat. That is a very important function that we need to think about and maintain as we consider these regenerative therapies to address form and function. Let's go into the anatomy of the skin. The skin is comprised of three layers. It has the epidermis, or the top layer of the skin, the dermis, which is the mid portion of the skin, and the hypodermis, or the adipose tissue, or everything below that dermis. As the top layer of the skin, the epidermis has most of our proliferating cells, or the stem cells. It resides along that basal layer, or the bottom layer of the epidermis, highlighted in dark purple over here. It also resides along the hair follicles in an area called the bulge, where bulge stem cells reside. When we talk about adult stem cells, or stem cells in the skin, this is where they can be found, in that top layer epidermis. Melanin is also produced by the epidermis. The epidermis is composed of different cell types, including keratinocytes, or the epidermal stem cells, Langerhans cells, which kind of act as recognizing foreign materials to trigger the immune responses, as well as melanocytes. Melanocytes produce melanin. In addition to contributing to skin color or pigmentation, it also filters UV radiation. The second from the top is the dermis. This is the thickest layer of the body. This is where the fibroblasts live. When you're thinking about the ultra structure, the collagen ultra structure, what gives our skin that scaffold to stand up and lift up, and that skin elasticity, the tone, texture, tightness, that comes from that dermis layer. Then finally, the hypodermis provides insulation. This is that adipose layer. It gives that insulation. It separates the layers from the bone and muscle that lie underneath it. Let's discuss the stages of wound healing. Wound healing initially begins with bleeding or blood clot to prevent that acute damage. Then it's the fibroblasts that play that primary role. Fibroblasts are recruited into the wound bedside. They deposit a temporary tissue matrix. It really triggers the phase shift into that inflammatory phase. Inflammatory phase then goes into that re-epithelialization. This now shifts from fibroblast involvement to keratinocyte involvement. Really, that wound becomes revascularized. That extracellular matrix gets deposited. You see new angiogenesis or blood vessel, and then remodeling of that tissue. These four phases are important and critical to recognize. Here's a more complex overview of that wound healing process. The reason I bring this up to you and share the complexity is to recognize that wound healing really varies. When we talk about wound healing, we really have to think about it between two phases. Are we talking about acute wound healing, the short-term, or are we talking about chronic wound healing, the long-term? Acute wound healing, you have inflammatory responses, which is actually thought to be beneficial. You do want that inflammation. They act as cytokines. They recruit different cell types to that wound bed. This then causes the different phases that we talked about of proliferation, angiogenesis, inflammation, bringing that re-epithelialization process together. However, in chronic wounds, this inflammatory response is considered inhibitory because it lasts too long. The timing of wound healing is critical because if you have delayed inflammatory phase, delayed proliferative phase, then really you're getting into a shift of now all of these being beneficial critical components into an inhibitory part where you do have delayed remodeling and delayed maturation phase. We'll talk about that of the differences between acute and chronic wound healing because that really varies in our patient cell types too. Chronic wounds are a massive burden to our healthcare system. Many of you interact with patients with chronic wounds as well. It affects about 6.5 million patients and more every year. About $25 billion are spent annually. It does change with aging. As we are approaching that critical mass of people that continue to age and living longer, but not necessarily achieving health span or lifespan, we do see that there is an increased burden of chronic non-healing ulcers and chronic non-healing wounds. That is an aspect of why we are considering looking at avenues or new fields like regenerative medicine to address these problems. As an outline, we will start by first going over conventional treatments for wound healing. What are we currently doing? How is a normal wound closed? What are the options for skin graft? I'll talk to you about a clinical case as an example. Then we'll shift over to artificial skin, so newer approaches. What has been traditionally done? What is not good enough with that traditional way of doing root skin regeneration? How can we address that through new skin substitutes? We'll discuss another case as an example. We'll talk about some limitations to these approaches. We'll talk about regenerative therapies, specifically cell-based therapies and stem cell opportunities that we can utilize for wound healing. Let's go into a little bit about skin grafting. Skin grafting is the idea of transplanting skin. That can be derived from various sources. There's autologous, which is essentially taking it from your own skin from a different site. This is most commonly what's done in our practice when we do skin cancer surgery, also known as Mohs micrographic surgery. We sometimes need to close that defect with a graft. We take that from a different site in the same body to help close that wound. There's also isogeneic donor grafts. These are from monozygotic twins, so genetically identical sources. Allogeneic, where it is the same species, so human to human. Then xenogeneic. These are also used in different aspects of medicine for heart transplants or valve replacements, I should say. Then there is pig skin that we use when we do grafts to close different wounds that are in tight spaces or convex spaces. Then there's also prosthetic materials. These are synthetic materials that are not from the species origin. The history of skin grafting really dates back to Indian surgeons who are performing different plastic surgeries. They recognized that you can transplant skin from one part to another. These were really the foundations that led to what we do in our practice today in regards to grafting. For skin autograft, these are performed for deeper dermal wounds. Typically, what's used is split thickness graft. When you are talking about grafts, there is split thickness and full thickness grafts. Split thickness graft really takes that top layer, the epidermis of the skin, and a portion of the dermis, so just part of the dermis, whereas a full thickness graft includes the full dermis layer. There are advantages and disadvantages to both, but primarily what we use is a split thickness graft because it allows the donor site to also heal well while preserving some of those vasculature in the recipient site to be able to heal as well. Here's an example of what a graft procedure looks like. You have grafting surface that gets prepared initially. The graft is harvested typically from the anterior thigh or in that type of anatomical location. There's a skin mesh creator that then creates these perforations through that that allows for gas exchange, essentially, and allows for permeability so that the barrier can be freely accessible to the outside environment, but you do have to keep it protected because, like we said, the skin typically acts as that external barrier. This mesh work allows for free flow of oxygen and better wound healing. What happens is that this graft actually gets embedded, and then eventually that fenestration or the perforations in that area will get filled. Sometimes a graft will not uptake, and you have graft failure, and these will fall off. Currently, what does a split thickness graft look like? I just want to take your attention to, over here, an uninjured skin. You see different types of cells. We talked about keratinocytes, hair follicle cells. Sebocytes are the oil glands, sweat glands, melanocyte, and immune system. The reason why I point this out is that the ideal skin substitute or the ideal skin replacement really has so many multifaceted features or different types of functionality that the skin contributes. Hair growth is an important part. Sebocytes or making oil is an important part, or you have that dry skin component. Sweat gland is an important part because you need to have that perspiration, that thermoregulatory aspect. Melanocytes is an important part to produce melanin for pigmentation, but also, we talked about that UV protection component, and then immune cells to really create that barrier between the internal and external environments. If you direct your attention to the split thickness skin graft, which is currently utilized, we do have the keratinocytes, but there's a lot of cells that are missing, including the hair follicle cells, sebocytes, sweat gland, and melanocytes. It is not an optimal source, but just to show you an idea of what it compares to from an uninjured skin. Skin allografts, so these are non-self-human donors. This was first described in Branka of Syslin 1503, used in World War III. Cadavers were typically used as donor sites. We have used cadaver tissue for replacement, but this is a temporary cover. The immune system typically rejects that. It's used with caution. Some practices do not use this because there is that risk. Things are screened now, but there is that risk for viral transmission, so there is a lot more caution that goes into that. Xenograft, we talked about this. These are absorbed, and these essentially help that wound to re-epithelialize. Porcine skin or pig skin is very commonly used. Just a couple of weeks ago, we were doing a procedure where the patient had an injury on their foot, and it was in a concavity that could not be filled appropriately, so we just placed a porcine graft on there and allowed it to heal. The mesh healed really well and re-epithelialized really well. Again, something to consider about wound healing is location. Lower extremity wounds tend to heal very slowly, as you may have encountered in your practice. How do we address that? Porcine graft is our current one solution to one problem, but it's not a very good solution or a permanent one. Amnion has also been used as dressing for burn injuries. This is a biological skin substitute, but not commonly available. These are rich in collagen growth factors, so these are from the placental tissue. When available, they do act as a great source of nutrients and healing. Just something to consider, but again, access is a limitation. I did have this as a group discussion. I will open it up to the participants here if they do want to share. Otherwise, I'm happy to keep going. Essentially, it's a 65-year-old male patient that came into Mohs for skin cancer surgery for basal cell skin cancer on their scalp. After we removed the skin cancer and got clear margins, this was a large defect, an open wound that was left. This commonly happens in our practice. Mohs surgery is to clear the skin cancer. If you keep going and take these different layers, you see you end up with this large defect. Based on what we discussed, do you guys have thoughts or options for closure here? I can share the different options that we would typically consider. One option that we consider is second intent or just leaving the wound open. This wound is too large. That would not be a feasible option for him. It would take too long to heal. It would expose him to different infections. It's not ideal. For smaller wounds, for smaller areas, that's definitely considered. That's just taking the course of time. We do heal naturally. As long as they're not immunocompromised, the vasculature of the head and neck is really great. People do just fine for wounds that are left open in those areas compared to other sites of the body. In this case, he has a large defect, so that would not be considered. Our second option for him, a couple of different things of bringing that closure together, but certainly for his case, because of the large nature of it, we did end up grafting the wound on his scalp. Similar to the photographs that you've seen before, we took the graft from this man's thigh and then we created different fenestrations to allow for that porous nature to allow oxygen and vascularization and then embedded that in his scalp. Here's the patient returning after several months of having this graft placed and healed well. You can see there are a couple of things happening here. First, the pigment variability. It's repigmented in different spots in a very patchy, uneven appearance. He also lacks hair in most of the graft sites. As we talked about, these split thickness grafts do not have the hair follicles, so that is a major barrier. He does not sweat in this area of the scalp because sweat glands are absent. There's also more dryness or dry scalp. The reason why dry scalp is an important part to note is that dry skin leads to itchy skin. When you have itch that accompanies as a symptom, chronic itch can be equivalent to chronic pain for some patients. Itch is a very important factor to consider and it is often due to that dry skin aspect because we don't have the sebaceous glands that are transferred here. These are some of the results. In terms of his defect, I would say pro, he's got his skin cancer cleared and he has that wound closed. Those are fantastic, but can we do better is what we're asking. Going on to new approaches for skin regeneration, cell co-cultures are important to consider. These are the different cell types. Human keratinocytes, four skin-derived keratinocytes, and fibroblasts have often been used either by themselves or co-cultured with different other cell types to care for burn injuries. This was actually done for the first time in 1981. They did epidemiological autographs. They used keratinocytes for burn victims and use that to help heal that injured skin. Typically these skin grafts or the co-cultures are made from biopsies from different locations are expanded for several weeks in culture before they're ready to be transplanted. Now there are off the shelf options and I'll talk to you about that, but it's limited by its difficulty to manufacture and high costs that comes with it. So here's that example of where you have a patient with an injured area of skin, a small biopsy say from their lateral thigh is used. We can isolate and expand the keratinocytes in culture and then we form sheets and sheets of keratinocytes that then can be used to replace back into the injured site. But the process of expanding can take several weeks. So caring for these sites, having the patient in hospital during this time, those are all considerations that we should think about. Is this the most cost-effective option? No. So how can we do better? Here's an example though of the promise of that therapy. This is a condition called epidermolysis bullosa or EB. And this happens due to a mutation in the skin basement membrane. So the basement membrane is an important part of the skin. That's that top layer epidermis and the mid layer of the dermis. So the basement membrane really attaches the epidermis to the dermis. And that's important because that's the part of the skin that keeps the barrier intact. If you don't have that barrier intact, you can have blisters, you can have skin lesions. So very minor things like just rubbing the skin, gently bumping against a desk or even washing baths and showers. And if the water hits your skin too hard, you can have blisters and you could have significant injury. So this leads to infection, scarring. There's more predominance of skin cancer in these patients. So very debilitating condition. This was a patient in Italy. He was a seven-year-old patient, a child that came in with LAM3 mutation. So what they did was they cultured keratinocytes from his skin in the uninvolved areas and they added cDNA of LAM3. So using a retroviral vector, they were able to introduce the healthy copy of this basement membrane protein and created these epidermal autographs or sheets of epidermis that were then transplanted on his skin. So you can see that from here, the skin turgor is intact. He had wonderful results from doing this. This is that concept of him, of what exactly was the damaged skin, skin taken from his body with that LAM3 cDNA reintroduced and the corrected cells are then used for transplantation. There was a recent BBC article, I mean, I should say five years ago. So in 2017, this boy was then nine years old. So two years out from when he originally presented for that transplant. And they called him the butterfly child because of the fragile nature of the skin and how he's now here playing soccer with his dad, doing so much better, you know? And so this was a significant impact and significant lifestyle change for him. And the promise of this treatment is huge. But I think given the cost of what it had taken to achieve that, I think we really have to keep thinking about alternative options to help provide these options and accessibility to everybody. And I'm gonna digress here just for a minute because this is an important topic to me, which is addressing healthcare disparities as we get more into regenerative medicine and these emerging therapies and technologies. It's very important for us as physicians and scientists to be thinking about how do we make our care accessible to everybody? And that is all patients. In dermatology, I work closely with a lot of skin of color patients and minorities. And even when we start at the top of thinking about regenerative medicine and these different new therapies that are coming across, it is important that we build clinical trials to have an equal representation of skin of color patients and underserved patients so that we have appropriate results that can be shared with everybody. So I would just encourage you as a side note, as we talk about this amazing treatment option that helped him, you know, and as you all venture into your fields and research, I would just encourage that inclusion. Okay, so coming back to our different skin substitutes. So we just talked about co-cultured options and cellular options such as keratinocytes and fibroblasts. And now we'll kind of switch gears into skin substitutes. So these are actual graphs that are synthetically made. And so these can be various options. So they can be cellular or acellular. They can be epidermal, which is just that top layer of the skin. They can be dermal, which is that mid layer or a composite including both. They can be acellular, meaning they can just have collagen or other extracellular matrix factors. They can be biological or source biodegradable. So those are the different options. And I'll touch base on a few different options that are currently available. So here's EpiCell, which has human keratinocytes embedded. Apillograph is that composite option that I was talking about with the epidermal and dermal layer. Dermagraft is another one. And that's got a polygalactin scaffold, which is then embedded with fibroblasts. So really more structure for that one. Integra is very commonly used in dermatology and plastic surgery practices. And this is more of a silicone with collagen scaffold. Okay, so here's another picture of Integra. This is the oldest available dermal equivalent. So it's 3D, it's composed of, it's acellular as you saw previously. It's bovine collagen. It also has sharp cartilage and an outer silicone sheet. So typically this is a quick option. If a patient comes in, they have significant full thickness burns. This is what a lot of practices, if they have and have it available, they use Integra to cover up that wound or a surgical wound that's really hard to close. Limitation is that you do have a limited healing response that can be variable. There is a smaller risk of increased infections. It is a bovine product, which some patients may not prefer. So looking at, so that was an acellular option, looking at cellular substitutes. We did, these are autologous keratinocytes like EpiCell. There's Dermagraft or neonatal fibroblast culture composite EpiGraft, I think is now five to $10,000. So this is one of the more expensive ends of composite substitutes, but it's that dermal epidermal product. Has multiple cell types, keratinocytes, fibroblasts and collagen as well. Immunologically inert, meaning it doesn't have that antigen presenting cells. So it's not going to be readily rejected. Okay, so we'll switch gears now. Here I have a case of a 45 year old patient comes to the burn unit with severe third degree wounds. She's had deep debridement and topical standard of care, but thinking about these newer ideas for management, what could be considered? And here, I would just encourage you, whether it's this or thinking about your problem related to orthopedics or related to other injury models, spinal cord injuries, et cetera. I would think about what is needed and how to fix that void, right? So what is needed should, in our case would be, well, an ideal skin substitute would be sterile. It would act as a barrier. It has that low inflammatory response. So it's not rejected right away from the immune system. Not cause for systemic or local toxicity. Allow for that water vapor transmissibility so you can have that simulated thermoregulation and these topics that kind of get it closer to that normal skin. So how can we achieve that with the different types that I talked about to you with the acellular options, with the cellular options and the skin substitute options? So going into that, it's kind of fun to think about because it's like a recipe. So it's like we have these different components that we can add together, but the pieces don't, at the end of the day, you'll see that the pieces don't fit together perfectly. There's always something you wish you had from here or wish you had from there. With the option for a cellular component, if we do the cell isolation, keratinocytes, fibroblasts, they do create that monolayer. They do create that closure, but there are different sources that are, so the patient variability, where's the source that this is coming from? Does the keratinocytes, the time that it takes to expand these keratinocytes and fibroblasts and culture. So those are all some of the limitations to that. Now, if we kind of think about the tissue engineering preparation, we have a lot of different options, epidermal, autologous dermal, that composite and synthetic ones too. So if we look at our recipe, like I said, so these are some of the ideal skin substitute. We covered that. And from a manufacturing standpoint, a long shelf life with low storage requirement, and that's ideal for most products we kind of think about for clinical grade manufacturing, but something to consider and then user-friendly in cost as well. So these substitute I've listed as acellular and skin substitutes, but I just wanted to direct your attention to this figure right here, which kind of shares with you some images of the acellular and epidermal options. So as you can see, you can have acellular options like Alloderm, which is more just that top layer of the epidermis and part of the dermis. You can have BioBrain Integra, which we talked about, that oldest dermal equivalent epidermal layer. So when you hear things in the media about spray on skin, so this is what they're talking about. It's the keratinocytes. So these keratinocytes could either be autologous and expanded, or they could be allogeneic or things that have been cultured or companies have already made. So there's also, you may see like 3D printed skin, spray on skin. So all of these are kind of looking at different epidermal equivalents there. Now, why is that dermal equivalent important or why is it important to consider? It depends on the patient type. So for some patients, it will be important to have that structure. So patients with, oops, I'm sorry. So for patients with that chronic wound that is deeper and has more of that dermal layer missing, that's something to consider for them as an option for a graft. And then you do have other types of grafts, including that epidermal dermal composite graft as well, we talked about. So some of the limitations, it's poorly vascularized, meaning the cells and the substitute die and they can slough away from the host tissue. So there's a few commercial skin substitute that allow for true longer vascularization. So that's an important thing to consider is these are temporary options. They're not a permanent fix. So you may invest in selecting the perfect skin substitute for them, but these won't last beyond a certain time. And if you're talking about a patient that usually has chronic wound, so our chronic wound patients also have diabetes, poor vasculature, and these are often the lower extremities. So in that situation, it's a very tricky situation to find the ideal substitute that will kind of take them to a state where their body can now contribute to healing on its own. So other things is poor mechanical integrity, failure to integrate, as we talked about scarring. So not all of the tissues will have that perfect assimilation. Immune rejection is another thing to consider. It's a time-consuming process. So it can take two to three weeks for the cell culture to be expanded. Perhaps the patient, if it's a burn victim, may not have that time. And then, you know, thinking about things like hair and sweat glands, like we talked about. The other thing I wanted to bring up was epi cells. The cost is important to consider. And as we talk about accessibility and kind of addressing healthcare disparities, thinking about if it costs to cover 1% of the body's area, the size of the palm, is about $13,000. You know, if there is a significant wound or significant injury in our largest organ, how do we address or lower that cost? So future perspectives, you know, lack of blood. We talked about the importance of blood supply and to refrain from immune rejection. So thinking about an option that would utilize that standardization of storing and preserving these different types of graft. 3D bioprinting has really taken off and there's a lot of great opportunities and collaborations that you can do. A lot of companies are actually doing this where they're doing 3D skin organoids and looking at different patient tissues and they kind of assemble the different cells to put that together. So it's really exciting. Stem cell-based skin substitutes. So I'll kind of cover briefly about the different stem cell types and options there that are available. But it's truly a collaborative effort. Engineers, biologists, entrepreneurs, physicians, physician scientists. So this expands beyond the scope of the skin. So looking into your practice and your interests, bringing forward regenerative therapies really requires the symbiosis with the ecosystem. So thinking about who's in that ecosystem to make it possible. It's going to be people that are biomanufacturers. It's going to be engineers and biologists, entrepreneurs. It's also going to be insurers. How do we think about getting these treatments covered by our medical insurance beyond everything else that you're doing and I'm sure doing prior authorizations for many more things. How do we reduce that additional burden that we may encounter? So, okay. So now kind of switching gears, I will pause for questions. Let's see if there's, it looks like there is a question on the chat. What is the duration between removal of the thigh flap to attachment of the scalp? It's done same day. So we do that on the same day. So typically when patients come in for Mohs surgery, it's all under local anesthesia. So that scalp defect was actually done under local anesthesia. So the patient's actually talking to us. They don't feel anything. And if they do, they let us know and we give them more anesthesia. And the same thing is done from where we take the graft. Sometimes it's kind of on the neck area or other times it's behind the ear, but for that patient, it was the thigh. So we do that on the same day. So typically it can be long days because we also read the slides and make sure that the borders are clear. But say, if it is a long day, especially for that large defect, and we're not able to do it, we can sometimes have them return the following day or the day after. And we would do a temporary closure for that wound. I also had a question. Versus cellular versus acellular, do you find that like infection rates change with different like of the components that can flourish infection or maybe even prevent one or the other, I guess? Absolutely. That's a great question. So we find that the biggest factor that contributes to infection is actually patient quality. So looking at who's the patient, have they had infection rates in the past? Are they immunocompromised? Do they have diabetes? Do they have other comorbidities? Those factors actually contribute the greatest to infection rates over things like cellular, acellular. I'm sure that certain types of acellular products, even things like Integra, just know it has a little bit of a higher infection rate from where it was sourced, where it was sourced, things like that. So there are certain products that have a little bit of a higher infection rate than others. But overall, the biggest factor that contributes to infection rates tends to be patient characteristics. Thank you. Perfect. Well, great questions. So now I've kind of shared with you about the different types of skin substitutes. Now I'm just gonna switch gears and talk to you about the different options for cellular replacement from our regenerative toolkit, specifically about stem cells. So this lecture, I typically teach a course for medical students and residents, and you guys are all well into, I'm sure, your fellowship and practices. So I apologize if this is a little bit too rudimentary, but bear with me while I cover some of these topics. It'll be fast. So overall, stem cells have two main properties that we try to highlight. So first, they are able to self-renew. So if you were to take one cell into a dish, they proliferate, and that's that quality of self-renewal. They also have potency of differentiating. So what types they can become. So that's called differentiation. So those are two critical factors. So this is a stem cell parental advice of a cell saying that you can be anything you want when you grow up. So they have a lot of potential. So just briefly, in this field, there's a lot of hope as well, as you may have encountered and seen in the other orthobiologics lectures. So it's so crucial, and I really value everybody in the room here for taking the time late in your day to be educated about this, because it truly is a field that holds a lot of promise. And I think by coming together as a group and sharing what is true hype and hope, we can really discern the truth for our patients. So, okay, what are the types of stem cells? So really I highlight four key concepts of embryonic stem cells, adult stem cells, umbilical cord blood cells, and induced pluripotent stem cells. As we go through each category, I'll kind of share about the clinical translation and applicability for wound healing. So embryonic stem cells, these are from the embryos, the inner cell mass specifically. They have the potential to form into the three germ layers as pluripotent stem cells, the ectoderm, mesoderm, and endoderm. They have potential for immune rejection. So these are typically never used in clinical trials. And if they are, they're usually done internationally and where there's not a lot of tight regulation. So I just encourage patients to kind of proceed with extreme caution of what is being advertised in regards to these stem cells therapies. Now with perinatal or umbilical cord blood cells, these are initially harvested at the time when the baby's born. So these have kind of mixed features in terms we talked about the self-renewal and differentiation as the two potential for stem cells. These have the embryonic-like potential and the adult stem cell-like. So they're kind of an in-between where they have more of a proliferative, youthful kind of nascent without a lot of genetic variation and accrual with age. so that is a benefit. But the con is that if you do do a medical core blood stem, so banking, it can be quite expensive and the fees to maintain it for years to come will be significant. So anywhere ranging from a thousand to $2,000 for that initial banking, and then a yearly fee of three to $500, depending on the company that you go with, of course, to kind of maintain it. There are still limited studies, although there has been promise to show that the amnion and things like that have been useful for wound healings, but this is just something to consider in terms of applicability of looking at the types of applications that you're doing, and if this is really worth that investment. And then adult stem cells, this is what is more from that bone marrow or adipose tissue. So these have, these are usually autologous, although the allogeneic option is available, but these are older cells, so there's potential for mutations. Here's a study that's looking at chronic wound and bone marrow derived cells for that. There are three patients here that they have treated the wounds, again, variable wound size. This is a case report, the number of treatments varied. So for one patient, it was one treatment for others, depending on the wound type, it was multiple treatments and multiple different concentrations, as you can see here. So this here, part of it is more of an art than a science, and that's because there's not enough validated science to prove benefit or to standardize it. This comes at the cost of these bone marrow stem cells are variable from patient to patient, so perhaps a direct comparison is not plausible, similar to PRP therapies. And then there's also the wound variability. Wound healing can be one of the most difficult and rewarding things to study because there was so much variability, interpatient variability, interwound variability, and a lot of different factors contribute to wound healing. Therefore, it's hard to standardize studies. So even with this study of three patients, you see a variability in age, variability in sex, number of treatments. But with that said, they did find improvement in this wound. I just wanted to share briefly, so we're primarily covering stem cells, but I thought I would just share briefly about PRP, which is an acellular, as you're all familiar with, platelet-rich plasma, and how it contributes to wound healing. So it has multiple growth factors, and we talked about the cytokines that are involved in wound healing. So these growth factors are responsible for different aspects of the wound healing, specific to the skin, but otherwise bone as well. So that's just how it plays a role. There was this great review article that I would encourage you to read. It's a PRP for the treatment of chronic wounds. It was published in Chronic Wound Care Management and Research, and they just kind of go through an algorithm for different types of PRP options. So there is two types, so you can do it intralesional, injecting it into small wounds. And then, so there's the type of PRP, so sometimes there's activation of the PRP, or they can kind of use it in a dressing if it's topical, and apply it directly on the wound. So they kind of talked about PRP application on a weekly basis. This review evaluated 10 clinical trials. About 400 patients were totally compiled into that, so that's where these recommendations are coming from. So looking at things like PRP weekly application and that duration of four to 12 weeks, and then doing standard of care in between, and certainly some of the metrics on the side here of when to stop the PRP option. So I see a question in the chat. Does the research for PRP concerning wound care have stronger evidence than it is for its current use in MSK world? I don't necessarily think that it's a stronger evidence, and in fact, I would say it's more of a variable evidence, just like what you are seeing in MSK, and that's just from the lack of standardizability. They are using different, everything from the minor aspect of different centrifugation systems to what is their even protocol to spinning and collecting the PRP, and how are they isolating the PRP, and it's patient variability that contributes to it. When is it applied? How is it applied? So here we see, even in this article, it talks about interlegion or topical. So I think overall, I'm glad that we're reporting these studies, but it's still, and that might be just the nature of PRP studies. There's not a way to standardize it because we do have so much variability, but that's what I would say, is that I don't think it's stronger. I just think it's possibly just variable, just like you see in MSK. Okay, and then lastly, bioengineered stem cells. So these are induced pluripotent stem cells. I'll just, I'll skip therapeutic cloning and kind of focus on nuclear reprogramming. So in 2009, Shinya Yamanaka in Japan was awarded the, well, he published and discovered ways to turn the skin back and turn the clock back into a more stem cell state. And he was awarded the Nobel Prize for that discovery. So these are self embryonic like stem cells. These are, this achieves the ability to create that pluripotency that we see in embryonic stem cells without going through that embryo process of isolating them. It's, and it also has the flexibility of having that patient specificity. So it has a potential to generate all skin types. We talked about how it can differentiate into all different cell types and layers. And there's no immune rejection because these are self derived cells. However, these are pluripotent stem cells. So that means they have that potential to form that teratoma and that risk carries through. So it's really important to make sure that these cells are truly differentiated or achieve that ideal cell state before transplanting them back. And the other concern is just like in our keratinocyte cultures that we discussed, these cells also need time to be expanded from a skin biopsy into that pluripotent stem cell state. So again, this is a great opportunity to study diseases for drug discovery, but I think these, this is quite limited yet, but although there's studies going on to get it to translate into clinic. And I just wanted to highlight this really great potential for utilizing the induced pluripotent stem cells. So we talked about that, the patient with the butterfly type of skin, the epidermolysis bullosa condition. So it is a genetic mutation and there is a lot of potential with these iPS cells is that you can use gene correction. So you can use CRISPR-Cas9 to correct and see if those keratinocytes are able to be transferred back and, or just study them from a basic science perspective. But what this paper unfortunately reported was that the graphs from iPS-derived epidermis didn't survive longer than a month. So these are, again, cultured in vitro, so there could be some variabilities and differences that we don't see naturally. And with that, I'll kind of summarize the potential and promise of regenerative medicine that everyone in the room here is familiar with. The idea of replacing cells, tissues, and organs to, again, restore that tissue functionalities. Today, we talked about the skin and I really wanted to highlight some aspects that you don't normally think about with the skin, like thermoregulation, dryness, and that oil gland preservation, even things like nerves, because the sensation could be very different. And the aspect of hair, hair-bearing skin is another important thing to consider. So as we go into regenerative medicine and kind of thinking about new solutions, really identifying the different aspects of each organ or cell system that you're trying to build and seeing how it functions and how do we restore that form and function is an important part to contributing to that longevity that we hope for. I'm coming to you today from Rochester, Minnesota, where it's a balmy 32 degrees. And we do have two other campuses as part of Mayo Clinic. We have the Scottsdale-Phoenix campus in Arizona, where it's a little bit warmer than here, as well as the Jacksonville campus in Florida. So if you all are interested, I would highly encourage you to apply to the AR3T fellowship, or we offer a course in February and in June, and we would love for all of you to consider it. And with that, I will take questions and thank you for your time. Thank you so much. While people may be writing their question, I'll ask one question I have. On the call, we're all very familiar with spinal cord injury. And so I think most of the people are probably really interested in wound care in that sense. And we know the number one cause for mortality in those patients are respiratory compromise and infection. But wounds and infections and wounds are not far behind that. I think it's like number three for those patients for mortality. So my question is with the functional impairment, do you see a solution with, I guess, regenerative options for these patients? Because I know in the MSK world, we talk about PRP or we talk about acellular options and that you need pressure, you need exercise, you need physical therapy. And in those patients in spinal cord, where the wounds are, they're not necessarily going to be able to, you know, functionally have those extracellular, extra epigenetics telling it to grow and regenerate. So I guess it's very complicated, but do you see? Oh, that's a great point. I think it's really important to think about regenerative medicine as an adjuvant. And exactly as you pointed out, I think standard of care and current therapies have been extremely well studied and we have experience with them. And I would kind of look to regenerative medicine to serve as an adjuvant to what can we supplement these different options that we're offering to patients. And in regards to wound healing, I think it holds a lot of promise, but wound healing is very complex. I had somebody once tell me about the way wound healing is looked about is as if you were looking at a giant elephant and everybody in the room has blindfolds and they're looking at the elephant and they're describing, oh, it has this great tusk. And the other person saying, no, it's got hair. And they're talking about the tail. And the other person's talking about the skin of the elephant. But it really, you know, everyone's got their blindfold on and each person kind of looks at it from a different siloed view. And I think looking at wound healing, whether you're an MSK or in skin, you know, it's really all of us working on the same problem. And, you know, while you're finding these different things for spinal cord injuries, you'd be surprised at how relevant it is for skin as well and for other organ systems. One thing that I didn't address in my talk today is that concept of cellular senescence and that's what I study in my lab. And especially for chronic wounds, we're starting to understand that it's part of the reason why these cells and wounds are not closing is because, and they go into the stalled wound phase, you know, where standard of care is not working there. And that is because a lot of the cells are turning into senescent cells where it's actually harmful. And the senescence are secreting this negative SASP and inhibitory environment. But that's what we're finding for skin in diabetic foot ulcers and otherwise. But I think that concept is certainly applicable to spinal cord injuries and other injuries as well. So looking at the picture from multiple angles, I think will always be key when studying wound healing. Brooks, do you have a question? Yeah, sure. Thanks. And Dr. Wiles, that was an awesome talk. Thanks a lot. I learned a lot. You alluded to it a little earlier about that there are different predictors for failure for wound closure, and usually it's patient characteristics. I was wondering if you knew some of the top, you know, predictors for failed wound closure, and if, for example, you or others are trying to co-treat those underlying factors while delivering this regenerative medicine, and if that seems to help or not quite yet. Yeah, great question, Brooks. So we actually did a study on this and we are just working on writing it up, but I can tell you what we found. We looked at 70 patients that had chronic wounds, diabetic ulcers mostly, and looked at factors that prevented wound healing. So we followed them over 10 years just from literature search and, you know, times that they've been into our wound care clinic and just seen how they respond to standard of care and did they respond and how long did it take and different things like that to see if we could have predictors for wound closure. We did find several things. It didn't achieve statistical significance, but something that we're looking to add more patients to to see, but essentially wound laterality was important. So if the wound was on both lower extremities, that contributed to poor wound healing, which we can kind of allude to. The male sex was also poor at wound healing compared to the female. I think there are also other coronary that goes along with that, including BMI and smoking history. Interestingly, these patients that we looked at also had biopsies, skin biopsies of their wound within seven days of presenting to the wound care clinic, which was my interest because I was looking to see, well, what's underneath the skin? What is the histology showing? And histologically, there was this epidermal, we talked about the different layers of the skin, the epidermis, that top layer of the skin, they had a finding called epidermal hyperplasia, where the cells in the epidermis were essentially hyperplastic or just enlarged. And so that contributed to a thicker epidermis, which you would think actually contributes to the wound that's healing. But what we're finding is that that epidermal hyperplasia has different factors, different cells, and potentially senescent there, and that are contributing to these harmful factors and why the wounds are not closing. So in terms of predictors, I think those are some of the things that we're getting close to finding, but other factors like things we've already know about, smoking history, diabetes, wound laterality, sex of the patient, BMI, and other comorbidities all contribute to that. Awesome, thanks a lot. And just one other quick question. Since you have those biopsies, I'm not sure if you're planning or if you see people in the field really using omics technologies to kind of understand wound healing better and what you think is kind of the way to do it. I don't know if you think, for example, bulk RNA-seq is kind of now too old, or you have to do single cell, or for example, if maybe proteomics or metabolomics might be a way to answer some of the open questions or just any insights on that, if you have any. Yeah, I'll tell you, when I started my interest in science, I was a post-bac and I remember doing a PCR and thinking how expensive that was. And just the other day, I went into our animal facility and they had a vending machine for a PCR. And it was about like a quarter of the cost of what it used to be. And so the reason why I share that story with you is because I'm hoping that single cell RNA sequencing is one day a vending machine option where it is so accessible because I'm interested in specifically looking at the different cell types, because if I tell you these cells are senescent, that's why the wound isn't closing, then your next question should be, well, what cells are you talking about? You just told me there were seven different types of cells in the skin, which is it a fibroblast? Is it a keratinocyte? And that is critical because as you saw in the wound healing pathway, they play different roles at different stages. So the keratinocytes are important in the initial inflammatory component, and then the fibroblasts come in a little bit later for scar formation. And we're just learning that even within the dermis, there's different components and each component has different fibroblasts. Some contribute to wound healing, the other contributes to scar. So they really have a lot of different roles. So I think not just single cell sequencing, but even the localization to say where are they and how does that play a difference, I think is very important to understand. So I'm just gonna wait for the vending machine option to be available so I can afford it. Yeah, so I had a question. It's kind of hard for me to wrap around my head all this because I'm sort of new to a lot of this information, but I'm wondering what's kind of, we've been hearing about stem cells for a really long time, but what in your thought is kind of the next step that needs to happen in the next two, three years for them to get a bit closer to being more applicable than they currently are? Yeah, that's a great point. And I'm gonna answer that question by telling you, I'm not sure if it's going to be stem cells. And that is, all this time we were thinking that stem cells were the greatest thing. Like my whole PhD was in stem cell biology. And then I realized towards the very end that it's actually what they're signaling, what they're communicating with each other, these extracellular vesicles or exosomes that may hold greater potential than the stem cells themselves. So when we are kind of thinking about, what's regenerative medicine look like in the next 10 years? I think the main question is, or the main goal should be accessibility. How do we get these treatments to everybody in a standardized accessible way? Currently our options for stem cells, everything I talked to you today, even options for PRP are not standardizable. They're not accessible. However, where we're headed is to better understand how the stem cells are working, which is through extracellular vesicles and extracellular signaling to communicate with each other. And there are labs, there are groups now who have been able to capture extracellular vesicles at specific sizes, filter them and make them into shelf stable products. So I think that's where we should really be looking at in terms of, how do we get these treatments to everybody and how do we standardize it? And how do we make sure that they get that same benefit from that regenerative aspect? Because if that exosome product is giving you the same result as a cell therapy would in a much faster time and a cheaper option, I think that's really where we should be headed. Thank you. Thank you again. It was so fun. Learned a lot every time you speak and congratulations on your new position. Absolutely. And if you guys are ever interested in coming to Mayo Clinic or want to partner on any projects or just have questions, please send me an email. Thank you. Have a great night. Bye, take care.

regenerative medicine

wound healing

stem cells

skin substitutes

thermoregulation

omics technologies

extracellular vesicles

exosomes

regenerative therapies