Today for our nano, nano tech seminar, we have a professor. You don't want to briefly introduce him. Professor Shah is the Brock Family Chair and Georgia Research Airlines eminent scholar in nanomedicine at the Georgia Tech. He received his BS degree in chemical physics from the University of Science and Technology of China. I MS degree in inorganic chemistry from the University of Pennsylvania and a PhD degree in physical chemistry from Harvard University. He joined the Department of Biomedical Engineering at Washington University in St. Louis in 2007 as the james McKelvey professor and then moved to Georgia Tech in 2012. So he's here about ten years, ten plus years holding a joint appointments in the Wallace Culture Department of Biomedical Engineering and the School of Chemistry and Biochemistry. This group has invented a myriad of nanomaterials with Controlled properties for widespread use and applications related to plasmonics, electronics, photonics, photovoltaics display catalysis, energy conversion, nanomedicine, and regenerative medicine. Dr. as co-authored more than 830 publications in peer reviewed journals with a total citations of over 176,000 and then hatch index of 208. He has been named a top ten chemists and materials scientists based on the number of citations per publications. He has received a number of prestigious awards, including Materials Research Society Medal in 2017, American Chemical Society, National Award in Chemistry of Materials in 2013. And I had to Director's Pioneer Award in 2006, David and Lucile Packard Fellow in science and engineering in 2000 and NSF Career Award in 2000 as well. This is very important. This is not there in this bio and the website. It was just announced that Professor Yunan shadow will be receiving the 2023 ACS Award for creative inventions. Let's congratulate Sean that. Let's give a hand to welcome him. Professor Sharp. Alright, thank you. Okay. Thanks for coming to my seminar. Probably I made a mistake by choosing this not so popular topic. Anyway, I've tried my best to give you some idea about one other research projects in my group. And the research I'm going to talk about today has been supported by NIH through to our one grants. And we've just got this two grants renewed, our most recent delays. So we are going to be able to continue this research for another five years. Of course, I want to acknowledge supports other than those two and nitric grants because it will pass 25 years. My researcher has been sponsored by many foundations and organizations. And in terms of research, at the moment, we have five quite different research projects. And the first three are mainly related to chemistry or chemical engineering, including synthesis of nano materials and the structure property or investigation, and also our application of those nanomaterials in catalysis and the fuel cell technology. Then, for those projects related to biomedical engineering, we have two different flavors. One is about nanomedicine, are in that case, we develop some carriers or like, or like nanoparticles per se for the controlled release and diagnosis of cancer, e.g. and the last one is what I'm going to mainly focus on today is about the design and fabrication of novel scaffolds for manipulating stem cell differentiation, control neurite, outgrowth, and then for purpose of tissue repair and regeneration. In terms of types of tissues, we are interested in quite large number of different classes, as you can see over here. As the limited by time today, I can only talk about two of them. The tendon to bone insertion site in orthopedics, and then a peripheral nerve, cyatic nerve repair. In that case. At the very end, I will show you one slide about dura mater or in urine or repel. And tendon to Boeing and social media is very important in many, like, well, let's say this way, okay, It's a very important connective tissue in particular related to like a rotator cuff in the shoulder x-ray? The major issue over here, Yes. This rotator cuff could be easily torn actually, dealing sports or maybe when you get older, this is going to be or become a major issue. And whenever you have problem, when you go to hospital or the surgeons typically will just attach the tendons to the bone. Okay. And then suture them together. And in many cases you can have a temporary recovery. But eventually our disjunction is going to rupture again. Okay, so that's going to be reflected over here. The failure rates of rotator cuff repair could be almost like 94 per cent. Okay, So that means majority of this repels will fail actually in a few years. And this, of course, there's a reason why that's the case like this. If you take a look at the tendon to bone etch rate these two quite different the materials if you tried to attach them together, of course, whenever you are involved in motion, strain is going to be concentrated at the interface because our tendon is relatively soft material boundary is kinda hard material. So string isn't going to stress is going to be concentrated at the interface. So that will rupture the interface are very easily and quickly. And in the native system actually are. Our bodies solve this problem by developing their so-called insertion site or junction, in which we don't have a very sharp interface. Instead, we are going to have a gradual transition from tendon to bone side. Okay, If you look at this junction carefully, you are going to observe are quite gradual change in terms of structure and cell phenotype and compensation, so on, so forth. So e.g. or the tendon on the collagen fibers on the tendon side isn't going to be well aligned. And also they are going to just be collagen fibers without mineral coating on it. If you move to the bone side, of course, this tendon organ, this collagen fibers tend to be mineralized, will be covered by our hydroxyapatite is kind of mineral materials. And they are in the order of cost is going to be, are going to be lower compared to those collagen fibers on the left or on the tendon side. And then you're going to have a gradual transition. So basically, by building this gradual change or gradient, actually that can allow us to accumulate or to accommodate that stress that could be accumulated at this interface eventually make this geometry and very robust. So here just show you some additional data just to convince you, or if they do have a transition, we have a gradient in this disjunction. If we take a look at the mineral composition, this is done using a Raman spectroscopy. You can see the gradual change of mineral phase from tendon to bone side. You have a graduate, almost a linear change. And this is like a tissue structure. So you can see the mineral size and the M&O side is the region you are going to have this kind of seamless transition of all. So in order to address the current clinical challenges, we had to find some scaffolding materials that can replicate this conduct transition zone over here. So that has been our goal. And alpha peripheral nerve are, as we know, our body is really a network of this peripheral nerve system. We have basically 43 pairs of sensory and motor nerves that connect from the brain and the spinal cord to different parts of your body. So that will control the sensation, motion and of course, our This kind of like functions of your body. So it's a very important, okay. So in this case, if he does injury, like damage, of course, that part of the body will lose their function because the brain won't be able to give them instruction on anymore. So in this particular case, we are trying to focus on the anatomic structure of that nerve, tried to replicate the specific vascular structure of the node. Basically, if you look at NOV is not just a single nerve, is going to be divided into multiple or no fiber saturate. And they are also going to be bundled together form these are so called fascicles. This is particularly important for big new Fosdick nerves. Okay, so you don't have just one bond that you had multiple wonders arrayed in this way, I can easily provide the function over long distance. Let's start off with this rotator cuff repair or a tenant to Boeing social. When we started this research, I was there at the universe are at Washington University in St. Louis. We find a collaborator in orthopedic surgery or professor some opera rose Azure. We are continuing our collaboration nowadays. At that time. Our idea was kinda like naive or simple. We just think we can develop some patches. This patchy where I have gradual change into or in terms of mineral content. And then this patch can be applied to the top of the tendon when tendon is a suture that to the bone, okay? And the gradient in this patch will be able to guide the differentiation of ourselves and eventually will help us to build a strong our transition over here. That was a really simple and naive idea. So I'll wait, develop a method or to fabricate those kinda of like mineral gradient patches. And the idea is quite simple as shown over here. You just take your scaffold, you just positioned in this orientation. And then into this container you are going to add a solution. This is going to be ten times concentrated, stimulate the body fruit. It's the solution That's already highly concentrated in terms of mineral composition. So that's why when you drop them into this container etch rate, they can be nucleation and growth on the surface of the scaffold. So you can have mineral deposit onto the scaffold. And by adding this solution drop a wise and the liquid level of costs will increase gradually from top to the bottom. So you can imagine the region at the bottom. We'll have our longer deposition time than the region at the top of the scaffold. So naturally you are going to build a gradient along this vertical direction. That's basically the setup. As you can see over here. You have some scaffolding materials. Put the ulna across substrate, and then you add the solution from the top. And then you're going to build a gradient. Here, just show you SEM images we took from different regions of the scaffold. This is from the bottom region, okay, very bottom one. And then you move upward in this direction so you can see the top. Eventually, you are going to have very little deposition of mineral. So in this particular case, we are using electrospinning nanofibers as the basic structure of what the scaffold and then onto the surface with deposit. These are hydroxyapatite mineral phase in the gradient, in the spatial gradient. And, um, we also tested their mechanical properties, as you can see over here, are depending on the average stress. We upright. And basically you can see this is a region with a higher level of mineral coating, and this is a region to the right, you have least amount of mineral coating. So as you can see in particular in this case, are at the highest stress level. The string is going to be just, well, you're going to see the gradient in terms of strain on the surface, okay? That's also going to be reflected by the gradient of the mineral phase on the surface. And here you can see the correlation. Here is the conversation of the mineral content. From the left to the right side, you have more mineral coating than the right side. And the modulus, of course, we measured on the left side, you're going to be higher than the side on the right side, right? This difference could be almost like 100 times or two orders of magnitude in terms of difference. And then we couch you are stem cells. These stem cells are isolated from adipose tissues. And then we cultured as steel onto this scaffold and were captured them will compare again two different for different regions or raise the mineral coating increased 1-4 these different regions. Okay, So basically this is just the auction. In this curve, you see the calcium content increase in this way. And after the couch or for seven days and this is 14 days. We can use two different types of markers to see how the cells have differentiated, right? Specifically, we expect our formation of osteoblast actually add in the region with the highest density of mineral. Okay, So this is r from this run X2 is a random related transcription factor to that bow. It's like an early marker for the osteoblast activity. So you can see from position one to position five along this curve, indeed, you are going to have a gradual increase in terms of differentiation of stem cells into osteoblasts. This is also consistent or using another stain. In this case, we use a osteocalcin is the protein naturally that will bind to hydroxyapatite. It's like a little marker actually for osteopaths actually. So again, you can see the gradual change from position one to position five. So this is just a consistent, when you have more mineral coating on the surface, you are going to have higher level of differentiation into an osteoblast. So that's really what we expect to have. And then we applied these two are some in vivo studies we are using. We were using like a rat model actually, okay? In that case, you are going to create a defect by detaching the tendon from the bone. And then we're going to suture them together with the scaffold. We call this a patch based scaffold, were compared to four different groups. The first group is just a controller, just use suture, just what we are doing at the moment in the clinics. And then we're going to have a cellular or scaffold. You only have the mineral coating ingredient without adding the stem cells. And then you have a cellular group, or you're going to have a patch plus the stem cells. And the last group actually are, is also our cell-based scaffold. But in this case the cells have been further transduced with the AMP2, It's bone morphogenetic proteins. It's kinda like proteins that can, it's kind of like a cost factor actually that can increase in hence Ostia differentiation. Unfortunately, when we compare the performance of these four groups at REI by different measures, we didn't see much enhancement when we apply these patches and with the cells. And like 28 days of 56 days in, if you compare these four groups, actually the cellular group, there are some margin improvement compared to like a acellular group. But overall compared to the control of is just the suture actually don't see too much difference. And then we analyze this problem. We realized maybe this kind of patch is not a good idea actually introduced at all. And when you apply this patch onto this repair side and the stem cells in the surroundings can only really see the surface, okay? They don't really see the region between the tendon and the bone. So that's why eventually the junction is not going to be repelled. As we would expect. Knowing there's a problem with switched our directions, or we are now developing a second generation of scaffold. In that case, the scaffold is going to be something like a disk. And this can be inserted between tendon and the bone and then you suture them together. So in this case, the scaffold away, I have a direct influence on the cells between the tendon and the bone. And we think this may probably give us better performance in terms of efficacy. So that's the second degeneration of scaffold that we are working on. In order to make. Of course, this scaffold again, has to have a transition in terms of compensation and the mechanic strands along this vertical directions. Okay, That's basically the origin of concept. You want to bridge the tendon and the bone with this gradual transition in terms of our mechanical properties. And in this case, we rely on the formation of something we call a density gradient of nanoparticles. Nanoparticles are just hydroxyapatite nanoparticles. You can commercially get from arches and you mix them with the polymer like Piaget PCR, this kind of polymer. And by using some tricks, actually, you can control the density of these nanoparticles. So eventually they can form a linear gradient, almost like kneeling a gradient along these directions. And now we are exploring two different approaches or to achieve this kind of gradient. One of them is just the brush coating, okay? You can take it the solution of this hydroxyapatite polymer mixture and you coded them. Okay, Just like we can use like even like a nail brush actually for cosmetics. The first layer will contain the highest concentration of Minerva and the secondary layer you are going to reduce the content of mineral phase. Then you gradually reduce and the top layer is going to be just a pure polymer. So you can see by divide them into like multiple layers, each layer can contain just the concentration of this hydroxyapatite and nanoparticles. So eventually you can create a gradient, or even though this may not be a continuous green, there might be as an, looks like a step function, but overall you're going to have this gradual transition from a high mineral content to low mineral content. And this, of course, you would argue, is not a highly reproducible method that brush coding after all, depends on the pressure you apply in the amount of liquid you apply in. Eventually the thickness of the film may not be so well-controlled. So that's why recently with switch to a spin coating, that's really the most widely used technique in microfabrication lab to make very thin, poor American films. But in this case, we are using a mixture of hydroxyapatite and this polymer solution. And you can do spin coating layer by layer again, okay, so from bottom to the top, the concentration of the mineral phase is going to decrease. And in this way we can easily control the thickness and we can have a very robust method, okay, to control the burst compensation and the thickness of this. Here just show you the cross section of the film. We fabricate either using brush coding or spin code. And actually it's relatively uniform, but you're going to see some gradient in-between etch rate over here. I'm going to show some more details. And first just show you how the reproducibility actually you can have very good way, very high precision to control their thickness. This is two layers of coating, these six layers. So every layer is going to be able to give you about ten nano micro meter of coating, or either using brush coding or spin coding. And when we do spin coating in particular, there's another advantage. Actually, you can spin coat on a pretty larger wafer and then you can punch them into small disks. Actually, each one of them could be used for one repair entry. So that's why this kinda like a parallel fabrication. You can generate this kind of identical scaffolds are just in one fabrication. This could be like a study 20 or this kind of almost identical or scaffold for our next step of applications. So let me show you one example. This example was developed are like four or five years ago. At that time we are trying to combine this kind of scaffold with another type of scaffold we called inverse all but it's like a highly porous structure that could be made of hydroxyapatite nanoparticles. So eventually this red region will be inserted into the bone. Actually this gradient and region where B the intersection and this green region where we basically into the tendon, okay, So you can suture them together. As I mentioned earlier, this transition is the most important things there. So you can see you do have this gradient formed by using a brush coding. And you move when you measure the concentration of calcium, e.g. you do see more or less linear or transition. And we also measure their mechanical properties. If you cut it this scaffold, and in that particular region you can do elemental mapping. You see the gradient As same time. You can use the AFM indentation to measure the local mechanic strands, the Young's modulus actually, as you can see when you go from the polymer side or to the mineral side, actually, you do have a gradual increase in terms of Young's Modulus that, that could be increased by more than like almost ten times actually in this way or over a distance of only above. This is not the distance unit, actually, it's just the position along these directions. This is about 10 μm entry is relatively thin regions. The top layer is going to be patterned using micro machining actually are in the IEA naturally they have this nice capability to machine this raid channels into this film. At that time actually we only had this CO2 laser X-rays. So the precision and the comorbidity was not perfect at it, but you do get a pretty small size, about 100 micro meters above the surface of it become very rough. Okay? This could be improved on now by using a different type of laser. Okay. But at that time, this was a pretty rough this was the top surface and this is the cross-section I've shown over here. This is going to be the pause in this inverse Arbus. Careful. Then we are seed these scaffolds with our adipose derived stem cells and then we'll look at their differentiation naturally. In this particular study, we look at two different types of markers. One is wrong too, That's for the indication of osteoblasts. And then we also are located sclerosis. This is a different type of marker. This is the marker for tender naturally. So this is just the four different regions. Along the cross section. So you can see when you move from the polymer side to the bone side actually, you are going to see gradual increase in pterosaurs were occurring in Tennessee, that's osteoblast actually are numbers. And this red one, you can see the opposite trend actually. Ok, so the tendon side, you're going to see more tendons are the polymers that you see more tendons and bone side you're going to see very few tendon cells. And most recently, we further simplify this kind of idea. To fabricate this gradient scaffolds, we find that you can still call it take a polymer solution, in this case the PCL. And you place it on top of another film that's made of a composite of film is to have hydroxyapatite and the PCL. You just put this together, you heat up a little bit and not hear that, I'm sorry. You just take the solution of this polymer on this film. You pre cast on the content on the dish and then are dealing this aging process. The solvent will be able to penetrate into this PCL composite of film underneath. So same time, the PCL polymer from the bottom can diffuse into this solution phase. So at the interface, eventually you are going to form a gradient in this way through the inter diffusion of hydroxyapatite particles and the polymer chains. Okay? This is like a piece of film basically. And then we can come to IgM to do micro machining so we can generate a rate channels all the way through this film. So eventually we are going to just use this film as the scaffold. In this at the bottom, you're going to have a high concentration of hydroxyapatite and pop. You're going to have polymer and in-between, you are going to have a transition in terms of mineral content. So that basically allow us to control the differentiation of stem cells you seeded onto the surface of these channels. And this kind of fabrication technique. There's another advantage, actually, okay? If you look at the cross-section of these channels, it's not a kinda of like a straight walls actually they're going to have this particular like a funnel shapes. This is going to be unique when we apply this between tendon and the bone, they can form Stato like interdigitated like tissue structures that can further enhance the mechanical strength of the repair. Okay. So here I'll show you what we are trying to do in the next step. So basically we are going to have this kind of scaffold that will be basically a suture, the bitten tendon and the bone. And this side with Polymer dhamma in dominance, they are going to just control the definition of stem cells or into a tendon ourselves. And the bone side will become osteoblasts at the in-between, you are going to have a gradient. So that's really the control, the nice control we can have. Here, just show you some characterization. Okay, We made, this is a cross-section. You can see the gradient from the EDX mapping, the calcium concentration will increase. And in particular, it's very nice Now we can generate this kind of junction with the relatively like things stickiness that can really match the native transition side. Okay, That's typically about 50 micro meters. It's very thin, very thin layer actually of all. Here is the SEM image of the top and the cross-section of this film. And in this case, we already switched to the femtosecond laser in the I j and accurate that can provide much better resolution when we do this kind of laser micro machining. And then we can also categorize this cross-section using our Rama mapping, okay? You can map e.g. two different functional groups. One is this CO2 group, okay? That's for the PCL, this polymer. Then you have phosphate group that's for hydroxyapatite ash tray. So you can do mapping. Eventually you can generate this kind of map. You can see how gradual change or mineral content from the bottom to the top and the PCL concentration also decrease from top to the bottom. So it's like opposite, like a gradient for these two different materials. So when we are again stem cells onto this scaffold, and first we can see the cells are pretty happy or on the scaffold. So you can see increased our differentiation and proliferation of these cells as a function of time. And then we also look at their differentiation. So in this case, we are using a different type of marker for the osteoblast is the alkyne or phosphate marker. So you can see the gradual change when you move from the top to the bottom region, you have gradual increase in terms of ALP levels. So that indicates. Or increased osteoblast formation. And here, again, this is like an early marker at you for ARP. In here we are using labor market HOCN, osteo, calcium marker. So in that case, you also see this similar trend actually from top to bottom, you're going to have increased in terms of osteoblast generation. So now we're moving into a secondary face. I will study or we just got the grinder renewed. We're going to apply this kind of scaffolds to a different animal model. In this case, we are using our rabbit instead of rats. Are there in collaboration with a professor, some opera rose at Columbia University. He moved to Columbia a few years ago. And in that case, our rabid will provide a much better clinically relevant animal model because the size of this tendon to bone insertion site isn't going to be much bigger than rats. Rats is just that difficult to do the surgery. Okay. Now let me switch gears to talk about the second type of tissues we're trying to repel. That's basically going to be the peripheral nerve repair. And currently in the clinics, autograft is really the standard. Okay. If you have damage due to trauma, accident or whatever in the damaged nerve can only be repaired by suturing segment of at different than nope, harvested from the same patient. Okay, so that'll basically eventually we're able to regenerate the connections through this. Nope, an autograft, of course, is good. In a sense, the nerve is harvested from the same patient. So you don't really have like immune rejection or this kind of problem. But at same time, It's a self-destructive process. You had to sacrifice some node or maybe from some less important parts of your body. But still you are going to sacrifice in those functions. And also when the damage of the nerve is thick, you had to harvest pretty long on light and less important nerves from your body. So that's really going to eventually create some secondary damage actually. Okay? And so that's why p bar interesting. I interested in developing a different type of concept in that case, or tried to impose no guidance conduits through this gap. And then somehow promote extension of nerves and covered the cap and eventually recover the function. So this concept of nerves, guys, dense conduit that has been around for several decades. There are some commercial products in the market already, but most of them don't have a good outcome. Because right now this conduit is just like a piece of polymer tube. Actually. In many cases, they don't really support the growth of all extinction of excellent nerves entry. So that's actually hasn't been problematic. In particular, when this gap is large, like typically like 40 mm and this kind of tube death rate, it will fail as a result of P by interests in developing second degeneration of nerve with guidance conduit. In that case, we want this conduit to be porous. On the surface of this war that allowed the transportation of like nutritions, waste molecules, and so on, so forth. So you do really want this to be a highly porous Actually for the wall. At same time, you want this war to be decorated with some physical features that can guide the extinction of neurites, okay? And also you want to present some cellular curious or like bioactive molecules. This could include in a Schwann cells, e.g. that's very important for nerve regeneration. And also you want this to be multiple channel instead of just a single channel. This is particularly important when you deal with a thick nerves. When you treat the thickness or if this lumen is too big, actually, eventually the impact from the surface where be lost. So you want to have any guidance at all. So in that case, there's nowhere become like a tapered when they grow into the middle point of this conduit. So all of these are just basically some general ideas or we have to follow when we design on this conduits. And specifically, we are using our electrospinning to fabricate the scaffolding materials. Electrospinning is kind of simple. Technique, has been around for like more than 100 years. Probably. The idea is simple. You just take a polymer solution, you put in a syringe. And when the polymer solution coming out from this metallic needle, of course, you are going to have droplet it like more or less spherical shape because of surface tension confinement. However, if we apply high voltage to this needle, that needle where be able to inject charges on the surface of the liquid droplet. And eventually you are going to create a very strong repulsion on the surface that will lead to the formation of a liquid jet due to the electrostatic repulsion from these charges. This Jad will be basically continuously ejected from this liquid droplet. And Monday are size becomes smaller, smaller because of the stretching force coming from this electrostatic repulsion. And eventually the solvent, the very fabric. Quickly the polymer will precipitate out as fiber. So it's a very simple idea, can be applied to many different types all polymers to generate this very thin fibers could be from 20 nm all the way to several micrometers. It's a very broad size range. So you can control the voltage used Polymer weight or molecular weight of the polymer, polymer concentration and the many parameters. You can have a very good control over the diameter of this nanofibers. Specifically, quite a long time ago actually we find that we can also have these fibers are aligned in a way rather than have this random morphology. And the idea is to use a two electrodes to Claire the fibers. So they are going to be inserting gaps. So across this gap, you're going to have fibers coming down to create a very strong stretching force because these fibers are still charged. So they can induce opposite charges on these two electrodes. So you are going to induce a very strong are stretching force. So the fibers will be aligned basically across this gap or in this perpendicular directions. So here just show you some idea. This is the fibers being aligned in this way. This is an SEM image. You can see they are pretty well aligned, okay, even though it's not perfect, but oboe, the fibers are going to be uni-axial aligned. This is a region on this electrode. You can see fibers TO take a random orientation. Here, just show you the R, like this edge. Okay, so you can see the fibers do fear the strong stretching force when they're moving close to the edge. The fiber was along this direction and then suddenly feel the stretching force. There are stretched across the gap. This is the cap over here. And now we can easily generate align the fibers of long distance by using a very simple device. This simple device is fabricated using a piece of metal. You take a piece of a mad at you just the bend it into a U-shape. So these two edges basically can serve as the electrode to collider fibers. The fibers will be stretched across these two edges. So you can have multiple of them are stack together. So eventually you can make a line. The fibers are pretty long distance. This is about 10 cm, actually in this way, okay, that can be aligned pretty easily. For those of you. If I interested in electrospinning and electrons, carbon nanofibers are, we have a major review articles over here published five years ago. You can take a look at it that this nanofibers are pretty ideal or to guide the eye. Extension of neurites are we use this particular model system. Our dorsal root ganglion are isolated from chick, okay? So you can adjust the culture them on this nanofibers. If the nanofiber has random orientation, the neurites are going to be projected into all different directions. There's no preferential orientation for this new rights. However, if you culture them on unit actually align the fibers are, show you on previous slide. The newly formed the neurites well be extended along the fiber directions. The fiber is aligned in this direction. So you see the physical guidance from the nanofibers. And you can, on the surface of this, align the fibers, you can add another laminin layer actually, that's kinda like adhesion proteins that can further enhance the extinction of neurites along the fibers through a combination of physical and biochemical cues. And you can also use some fibers that has good electrical conductivity, like a party parallel, this is a conductive polymers. And you can also apply electric field. So in this case, you can have electric field and the physical guidance to have this neurites extended over a very long distance. This is like a centimeter of distance. In reality, these fibers are can also be deposited into different patterns. And that can control the neural extinction in such a complex way. They eventually maybe possible to form a neuron. Neural circuits actually are in days, but that's not the current interests in my group right now with just a focus on peripheral nerve repair. As I mentioned earlier, one of the major requirement for peripheral nerve repair is really the Schwann cells that eventually will form this myelin sheath over the nerves. And that's actually is very important to provide them with the right functions. And so that's why I, in one of the earliest studies, we tried to seed the electrospinning fibers with a Schwann cells and then we culture DRG on top of it. So as you can see over here, this Schwann cells and this neurites do interact very favorably. Actually, they can form a layer or unwanted. And so that's why we move one step forward, tried to apply this to in vivo studies. And we are going to fabricate these conduits that can be used to bridge the two ends of the damaged nerve. And we use this bi-layer design at the beginning, we have aligned the fibers and then we have random fibers, will choose this bi-layer structure for a reason because you want your conduit eventually have a good mechanic Austrians to not just the guidance for neurites, right? So that's why when we put this align the fibers on top of this random fibers and then you a roar up and the seal them at the edge. That's basically you're going to be the conduit. This conduit has the porous loss that comes from the natural deposition of the fibers. And on the inner surface, you are going to have this uni-axial align the physical structures that can provide the excellent regrows. Okay, So I'll read this with a rat model for Cytotec nerve repair are here. Just show you what we did. You have basically had to create a defect. In this case, the defect is about like ten millimeter long. And then you are going to just graphed this conduit across this gap. And then you wait for like typically like two months. And then you harvest this, repair the nerve and do analysis. Here just show you the comparison of three different groups. One is for the ISO graft, ISO gravity just sutra back at this graph, this nerve or defect. Okay, So that's basically going to be the original nerve. It just take it out and put it back. So that's gonna be the ISO craft. And one group is going to be the fiber based conduit, and the other group is going to be a conduit plus Schwann cells. So as you can see, for this group, in particular at what the performance is pretty good. Impressive actually compared to the isoelectric, iso gravity is the maximum performance you can expect, right? So in this case, if you counted the number of no fibers in that cross-section, naturally you can see for this group, actually, this is going to be the middle point and this is going to destroy. This though, is this middle is over here. You can see, at least for the middle point, actually, the number of no fibers is almost like half of the autograft or ISOC arrived. Okay, it's not perfect, but it's going to be significant improvement compared to desktop. Know the conduit itself without a Schwann cell. So you do see the positive impact coming from the Schwann cells. The only problem over here are for this particular study are the Schwann cells were isolated from healthy nerve. So again, you had to sacrifice the nerves in order to harvest. These are Schwann cells. So that's again, you have same drawback or as autographed. Okay. So you had to sacrifice some housing nerves. So that's why I'll move forward. Are we develop, are another approach tried to take a bone marrow stem cells and differentiate them into our Schwann cells. And then we can use that as the source for our repelled. It's almost there. So in this case, we are looking at how we can control the different issue, or bone marrow stem cells actually are. We look at the different conditions. The result is that underwriter conditions like diameter of the fibers and the surface are compensation e.g. you put a lemony on the surface. You do observe various significant differentiation of this bone marrow stem cells into our Schwann cell. So that's a very promising. And finally, we will develop new design for the conduit. Basically we are going to take multiple single tubular guidance conduit and assemble them into a ray will make this mortal tubular structures. And what's that advantage? Well here we can easily target of thickness depending on the diameter of the nerve, you can increase the number of this single tubular or conduits actually, so you can really have good match for that. We can also differentiate bone marrow stem cells inside these multi tubular scaffolds. We have demonstrated that there's no difference compared to the single table ones. We also are held start to evaluate the efficacy ways. Firstly, started with this try tubular conduit. Because the model we used, it was just a rat. Its various small, relatively small on nerves. We cannot accommodate the seven candies, we just use three. And as you can see over here, this is the 30k individual conduits and this is the control group is autograft actually you can see the final result. This is a cross-section of the repair the nerve or in terms of density of no fibers are it's very it's not too bad at your compared to the autograph. Hey, can you do see some difference? Overall? It's getting very close. So now we're moving up to larger animals. Or in this case, we are using a mini peak. For these kind of studies. We are collaborating with people at Emory neuron sodium. At Emory. We are trying to apply this model to really just a repair official nerve actually on the face of this pig Patrick. Yeah. So that's the study for the next five years on this renewed grand. Finally, I'm using one slide to talk about wonder dressing and deal with tissue regeneration. That's a very important subject. And in this particular case, we develop a way to align the fibers into these radial fashion instead of this uni-axial alignment. In this case, the fiber is going from center to the periphery. So in this kind of orientation, What's that advantage? Well, you can imagine if you apply this kind of scaffold as a patch on a wound, this one that could be like the defect or during brain surgery. Okay. And the cells from the healthy tissue in the surrounding area where we about to migrate actually across this radially aligned fibers. So you eventually will cross over and close this one very easily and quickly. So that's basically the idea here. As you can see over here. When you apply this patch to ex vivo model, you can see this void actually eventually it could be just eliminated within a relatively short period of time, only like a few days. If you use a random fibers. This could take weeks, actually still have avoid over there. So the reason I want to mention this at very beginning because the student who did this study was an MD PhD student in my group at Wash U, and he started a company or after graduation. And now all of these kind of scaffolding materials are already in clinical use. Actually, they have a company called Sarah surgical x-ray. They already commercializing this kind of scaffolds that condom for applications. So I think that that's the end of my talk. I will be more than happy to answer your question. Thank you. We only have two people left anyway. Yeah. Thank you for Reza Shah. Pull a great talk. I'm interesting in the first part of the page. Yeah, I'm curious. Have your team look at the biocompatibility of the patch. The biocompatibility is not the issue because they are fabricated from biocompatible polymers like PCR PIJ, there's kind of polymers. So we don't worry about those sins. We do use some solvents during the processing, but those solvents are typically removed, actually dealing at the end of the fabrication. So we don't worry about this. So the reason, e.g. the last one, this was so quickly commercialized because the materials we are using, just the Piaget PCR, those kind of polymers. We don't use exotic materials or only FDA approved polymers. So biocompatibility is not going to be issue here. Yeah. The question I had was you used femtosecond laser? Yeah. In that part, That's really nice tool actually, I had to say that's yeah. In fact, it's a heavy heavily used to. Yeah. One other question is that what are the dimensions that you are successfully able to handle using femtosecond laser. Okay, well, for us, because of the size of the cell, right? We want our diameter to be about 50 micro meters to maybe 300 micro meter. It's relatively big Actually, yeah, it's easy to do with the femtosecond laser. We don't want to go to like micro meter or this kind of scale, right? Yeah, yeah. At least should be ten micro meter. Yeah, It's very interesting how uniformly it's it's all that's really impressive. I have one. Yeah. Okay. Yeah. If we look at that, It's pretty nice. It's a very nice actually, yeah. This, uh, kinda like sidewall is what really we prefer. Instead of like a straight wall, we want them to be tapped with like it that way, right? So they're going to form like a self locked like junctions. We have tissues in grows into these channels. It has a pretty nice tool. Yeah. Yeah. Great. Great work. Thank you. Thank you. Yeah. Thank you. Thanks. Alright. Thank you. Yeah, I appreciate it.