Natural polymers and hydrogels

Okay, hi everyone and welcome to the fourth lecture of biomaterials, one we're going to continue our conversation about polymers by discussing, naturally direct polymers and also hydrogels hydrogels can be prepared from natural polymers and synthetic polymers. But I just thought so that's why I thought I'd introduce natural polymers first and we can discuss hydrogels in the context of you, can pretty much make one out of both types of polymers? Okay. So what is a natural polymer? A polymer? If you remember, a polymer is just a long chain of repeating units, and so that's also what a natural polymer is, but it exists in nature. So DNA is considered a polymer, it's a polynucleotide, but really when we when we're talking about natural polymers, it's generally either proteins or polysaccharides like proteoglycans or glycosaminoglycans.


So the common proteins are silk. That'S a really popular, buy material, that's being used now and college, and that's probably the most common natural DirectBuy material, but there's there's a lot of different kinds. These are just some cut. Like I said, collagen is the most the most widely used. It'S found in connective tissues, naturally in mammals and other animals, but in humans as well. So you find a lot of different types of collagen in the body and its main function is mechanical, and it's also, you know it has a. It is important in swelling the water, the water properties of tissues, but it's mainly mechanical. It also does have some properties in in letting cells transmit signals from the external or matrix, and I guess what I'm thinking here is Meccano.


Transduction elastin is a common one. When you're trying to mimic an elastic tissue like ear, cartilage or ligament, fibrinogen is really a really popular one for making hydrogels, because you can make a fibrin hydrogel out of from it just by combining from it and fibrinogen solutions and that's involved in blood clotting. So those are your major proteins as far as polysaccharides, that's just repeating units of sugars and so alginate and chitin and glucosamine of lichens are examples of polysaccharides and we'll learn more about alginate in the labs for this class. But those are algae's. Actually, alginate is actually derived from seaweed, and algae and chitin is derived from the protective shell around insects and crustaceans, and so they are so. The chitin is definitely used for shape and form and mechanical properties. Algae use alginate for energy storage, but we just use it as just just a hydrogel just to make hydrogels that are have various applications we'll get into later. Glycosaminoglycans are found in connective tissues, especially cartilage, so they're really important part of the extracellular matrix in these tissues and they're very important for resisting forces either in tension or compression. And then you have your poly poly nucleotides like DNA and RNA, which are mainly used for protein synthesis by the cell. But you can incorporate you, can use all of these polymers directly for biomedical applications or you can incorporate them into materials that have other materials such as synthetic polymers and that would confer biological function because these materials are biomimetic and because they're found physiologically. So it's a way to add bioactivity your bio functionality to a synthetic material, okay, so the advantages of natural polymers and using that as a biomaterial is because they're natural they're biomimetic.


So they have physiological mechanical properties in theory. But because they're organization in tissues is really specific, we actually can't fully mimic the mechanical properties of tissues and that's an active area of biomaterials and tissue engineering. Research is trying to actually make you know, for example, a hydrogel that has the same mechanical properties as cartilage. For example, but they are bioactive, so that means that cells have receptors or they can interact with the with sites in the proteins or in these natural polymers. So that's nice on the flip side that makes it more complicated to manipulate them and because they're being synthesized in the body we're not synthesizing them they're, more they're, more complicated to manipulate and for the same reason you have natural variability from different sources and especially interspecies Variability but just patch to patch availability. So if you buy collagen that was isolated from you know, bovine tissue, it will be different. Every time you buy it. So, there's that something to consider a nice advantage is that it's degraded by naturally-occurring enzymes and typically it's at alized into safe and natural byproducts and that's nice, because for a lot of goals and biomaterials and tissue engineering or try are trying to grow. We'Re trying to get the body to regenerate tissue that otherwise would not so that's nice that it could be slowly replaced by healthy and regrowing tissue. But that means that if the mechanical or if the degradation profile is not what you would want it to be, you're going to have to physically modify that or chemically modified in order to make it a better Debra Dacian profile. And then that will, of course affect its mechanical properties well and its bioactive properties. So just by changing the degradation properties, you could get rid of all the reasons why you decided to use a natural polymer um, because they're biomimetic, you have less of an inflammatory response. Sometimes, okay, this is my qualifying statement there. I know, of course that depends on the polymers that you're talking about um, but because the body doesn't think - or I guess not to anthropomorphize - you know the inflammatory response, but it doesn't it's as foreign as much as a foreign body as say a synthetic polymer, because It is similar to the tissues to what you would see in the normal body, so you might have a less of an inflammatory reaction initially, but a lot of times you'll have a an immune response from the adaptive arm of the immune system that you know when You develop antibodies to this non-self material that you're putting in, especially if it comes from a different species which it generally does and there's also the possibility for disease transmission, since it does come from different species, but it's close enough that our body can recognize it. So that's definitely to think about, but the nice thing about. But you know the in conclusion - for the advantage of natural polymers they're, just like synthetic polymers with all the properties that we talked about last week in terms of visco-elasticity and everything. But they have the possibility for bio activity, so that's useful, okay. So the first group of natural polymers that I want to talk about is proteins and peptides, and I'm mainly going to talk about these in terms or in the context of collagen, because it is one of the most widely used natural polymers. So, like I said it's found in skin tendon cartilage and bone there, they keep videos, keep discovering different types of collagen that exist physiologically so right now, there's at least 15 different types and they're degraded by collagenase and by other matrix metalloproteases. All collagen are shared that triple helix configuration, which is important for its structure and their structure is really important for for their properties from the primary level to the quaternary level. So I think it's helpful to know you know to start from there, so the primary structure is the amino acid sequence and that's actually, you know how that's actually the repeating units of the polymer and so, of course, that's important and they have they all have their Own functional groups that come up and they all have their own all the amino acids have their own different ways that you can modify them chemically. Just these simple chemical reactions, the secondary structure is the chain configuration which is generally determined by bond flexibility. So, just like, we talked about with normal polymers, whether or not a bond can easily rotate, determines their mechanical properties and a lot of a lot of their properties. So that applies to natural polymers, like proteins as well, and then the tertiary structure and the quaternary structure are extremely important for protein bio activity. And so that's something that's a little bit different from synthetic polymers for bio activity of collagen.

They need to be in a specific conformation, so the tertiary structure is sort of just like how the chains pack together, but the quaternary structure is super molecular. So it's how different molecules of collagen interacts and that's really important, for you know protein receptor recognition and for basically so cells can interact with the proteins. So the quaternary structure is what you actually need to retain. If you want there to be bioactivity, the cells might still be able to react with the polymer, but if it doesn't have, if the protein doesn't have that quaternary structure, then it's not. It won't have the physiological effects that it would natively in the body. So types of collagen there's a more than 15. They all have different functions but they're mainly mechanical and involved in transmitting signals between cells and the extracellular matrix. I worked from my doctoral research. I worked really closely with cartilage and so, for example, collagen 2 is a huge, huge component of a cartilage and it's main it swells with water, and so it's, but it's also somewhat cross-linked in cartilage. So it's responsible for resisting the changes in swelling or resisting the forces and the swelling at you, it's responsible for resisting the forces which is also aided by swelling and that's why cartilage come with Stan such high blows in compression, even though it's mostly just protein and Water, it's mostly water, okay, but there's other different types of collagen and they're all together in fine fibrils. They all they all pack together in a triple helix and then that's packed together into fibrils, and that's why it's really strong tension for a lot of the colleges. Like the collagen, that's involved in tendons, so collagen based biomaterials are extremely diverse, they're used as sutures as a as a tape and that's why the tension. Potential properties are really important. They'Re used as hemostatic agents to promote blood clotting and stop bleeding, and so then you'll have you can have collagen in a powder or small or sponge they've also been used to make tissue, engineered blood vessels or just to replace blood vessels, which I guess it's also Addition hearing depends how you define it, and so it's interesting that so a lot of these say that the physical state of the collagen for these applications are processed blood. Vessel process porcine tissue, so a really big area of biomaterials is actually taking animal tissues and decellularized them to remove anything that might cause a adverse immune reaction and then using it for a different application so or for the same application that it was so porcine is On the most common, I guess because they're pretty their genome is pretty Mulligan's to humans, and so I guess their tissues are like the closest to to humans that we could get, and so for heart valves and tendons and ligaments. You have these decellularized tissues that go through all these washing steps of it. Just they just implants it right back into the into the heart valve to have a replacement heart valve, and it's used pretty successfully but they're still undergoing optimization in research and then see this. The burn treatment is a pretty good application of of collagen, so a porous collagen sponge will be placed, will be hydrated in sailing and then placed over a wound which would aid in the healing process and of course, it's really useful in tissue engineering as well, because Cells really attached to collagen like to produce tissue on collagen, so but you'll learn more about that in the tissue engineering sequence. Okay, so, like I said, the quaternary structure is really important for the for the for all the properties of collagen for the bio activity of its well, it's the specificity of its bio activity, but you can also use collagen if you remove the quaternary structure, just since Different properties, so if you actually heat it above 37 degrees, it turns into gelatin and what we know is gelatin, and this can actually happen in the body as well as it's only that at 37 degrees and that's what collagen will be degraded. It'S originally turned into that randomly coiled structure hope that I guess could be called gelatin and then it's more usually degraded by MMPs. So gelatin is really common in tissue engineering and also in drug delivery. So you will learn more about that in the next sequence of this course and the third and it's much more rapidly degraded in collagen. So that's you know, so that's how you, if you wanted to control the degradation of a collagen sponder at college and scaffold. This might be one way that you that you do it, but when you another another way that you can physically modify the structure is that that banding that's characteristic of collagen. So that's a picture of collagen that has that characteristic banding, and that means that the quantum structure is intact. If you lower the pH below around 4.25, that banding will disappear, but the tertiary structure of collagen is still there, and so that is how researchers discovered that it's the quaternary structure of collagen, that's really important for for hemostasis and and clot formation. So when they found out that the quaternary structure is important for confirmation, if you wanted to make something that does not form claws, so you want it to be less. Bravo genic by lowering the pH to get rid of the banding. That'S how you would do that. The next most common way that collagen is classes for biomaterials is a porous collagen sponge and so a really common way to do that is to make a dilute collagen solution, just an aqueous solution of the collagen protein and then freezing it and freeze drying it. So lay off allowing it under low temperature, back vacuum and the ice crystals form, because it's an aqueous solution and then when they sublime they leave porous behind, and so you can actually control the pore size and orientation based on well the. So if you lower the freezing temperature, you get smaller pores, and so that's so because you'll get smaller ice crystals and then, when they sublime, you have smaller pores. So that can be varied between. I think it was like one eight hundred micron so that that's actually a really big difference and you can actually have directional pores. You can have channels or directional pores by controlling the direction of the freezing. So if you have, if you have it freezing from one side and going up or in one direction, that's the direction that you'll have pores aligned, and so that could be really important in tissue engineering applications. So that's good to know and the interesting thing about these collagen sponges when a cue freeze-dry them is, they retain their Kuantan airy structure, so they're intrinsically hemostatic. So that's why they're pretty good for wound healing, because they'll stop bleeding that way. Okay, um! Then a lot of a lot of biomaterials engineers will cross lengths of collagen matrix, which will really affect their it's a degradation profile. So if you cross link a material chemically, especially a natural polymer, it can no longer be naturally degraded by the body, because those cross links are chemical and the cells or the enzymes won't cut, won't cleave in the places that's but they used to. So that's really useful. It'S for some applications for others. It'S not. I mean if you don't, if you don't want it to, if you need it to degrade, of course, the cross-linking would not be a good thing and it is permanent. So there's that, but this is an example of the integra life sciences. Violent or wound matrix is an example of a cross-linked collagen matrix. That'S actually used as sort of like a skin graft or something that's used to encourage skin regeneration for burns and other dermal weapons. Oxygen is another cross-linked collagen matrix, and that is actually minerals like hydroxyapatite, just first in collagen fibers and so it's biomimetic of bone. It doesn't have the same structure, which is why it doesn't have the same mechanical properties, but it does. It is somewhat osteo inductive in that it would cause it might help to cause bone and growth. In the you know, defects, but so the indication for these sponges is that you would film you, take a bone now aspirin in the surgery, room and and use the sponge to suck up the bone, marrow aspirin, which has stem cells and other good healing things in It and then stick that in the bone defect and it feels better. So that's a pretty good use of collagen matrix okay, so those are some physical modifications of collagen. You can also chemically modify collagen, so I briefly mentioned that you can chemically cross-linked them which will decrease the rate of degradation or not. Let them feed you, they won't be degraded at all. It may reduce the immunogenicity, but that is only if you, if you cross-linked the antigenic sites, the sites that you've got your body would produce antibodies. In response to so, there's no guarantee that just simply cross-linking with a nonspecific cross, linker like cooler, aldehyde or other aldehydes, would reduced in the energy density. But if you targeted those antigenic sites on purpose, then you that you would the problem with cross-linking is that any residual, glutaraldehyde or other aldehyde is toxic, precisely because it cross-links proteins. So glutaraldehyde works by by just having a chemical reaction with with free amine group. So the aldehyde cross just chemically binds to the amine groups, and so, if you have a dialdehyde with two aldehyde groups on either end it acts as an actual physical linker. So if any of that gets into gets released the cells, then, if that would cross like proteins in the cell membrane and kill them, because you need gluten or something and also there's some research that has shown that the inflammatory response to cross-linked materials is much much Worse than if you don't have cross-linking and that's probably because it's less natural, you know it's, that's you don't have that chemical cross-linking in the body. So you just sort of took away the whole reason why I use natural polymers and that there biomimetic so, but you know, I think, if you I think that depends on the degree of cross-linking and it might, it could be a really useful tool for modifying the Properties of collagen sponges, okay, so I talked a little bit about xenograft. So that's when you take a tissue from one species and implant it on the other. So, of course, we're talking about applying into humans because of your medical problems, and so another example of a xenograft is decellularized porcine, small, intestine, submucosa or sis, and that is basically take some of the lining of the small intestine in a pig and you decelerate it And, like I said earlier, and then it's actually used to repair soft tissue talk to Jews and on maybe like abdominal wall reconstruction, so it's used internally and also for stronger tissues like tendons. So it works reasonably well, but you have the same issues of sometimes it needs to be cross linked in order to make the properties stronger and more longer stability in the body, and then you might have a more planet or response to that. But we are going to talk about that more in the lecture about the farm body response, but you also have religious and cultural issues because there's a lot of religions and cultures that don't want other animals and planet into their body. So that's definitely an issue for trying to make something that would be translatable to humankind, little huge populations that the court wouldn't want: pig or cow okay, so that was mostly College and that we talked about there's a lot of other common natural polymers, like so Tyler Onan brenigan and sin Agora thousand and they derived from multiple sources, but a lot of these are used to make hydrogels. So I'm going to go into hydrogels now, then we can come back to more specific examples of one. You would use these polymers. Okay, so hydrogels are 3d cross-linked networks of of hydrophilic polymers so that they swell with water but they're cross-linked, so that they have elastic responses and also so that they're not soluble in water. So, even though they're hydrophilic polymers - and they probably they start from a solution of polymer and water, when they're cross-linked they're not soluble, so you can see it and feel it in that and it's got a 3d structure. So that's how you know that it's I fill so this is a picture of a really cool hydrogel that was just published in nature like last week. So I encourage you to go check out that video. This is just a screenshot of the video where the hydrogel is super-strong, but it can deform like a thousand times. It'S some original shape or something. So it's super strong and super stretchy, that's pretty cool and then the other pictures are examples of using hydro gels for cartilage replacement. So if you have an area cartilage that is damaged, it's from proposed that you could replace it with a synthetic hydrogel or even a hydro made from natural polymers, which is you know so man-made, and I'm going to talk about that. A little bit more detail later in this lectures, I will wait on that. Okay, so, like I said, hydrogels are cross-linked networks of hydrophilic polymers that are highly swollen with water. They can be made from natural or synthetic polymers. It can be physically or chemically cross-linked and they can be environmentally responsive. So there's a lot of different ways to categorize hydrogels and that a lot of different properties that you need to consider when you're talking about hydrogels and they can swell with I set up to 95 percent water. But I that numbers always turn around with different. And in at sea 99 percent water and 90 percent water, so it's just a lot, so the nicest thing about hydrogels is that they have similar material properties to tissues, or at least more similar than other than other materials. We have like. Just you know, dry polymers or you know sorry, I'm accent coming wellactually is right. Answer back no medic, so bit more than metal. So it's mostly because their water content is so high, just like tissues that we see that the corresponding properties so in terms of the mechanical properties, they're, lubricating properties - and you know, transport of nutrients and oxygen through hydrogels they're, all really similar to tissues, or at least You could modify the hot result to make it really similar to tissues, so Hydra dolls are really useful in drug delivery. You can, because you can control release of drug or protein based on diffusion, which you can control based on properties of the hydrogel Network and they're. Also really useful into showing hearing, because encapsulated cells interact with the hydrogels in a similar way to their native extracellular matrix, depending on the type of pound. That is used to make the hydrogel, of course, but it also allows a hydrogel. Encapsulation of cells also allows nutrient exchange because diffusion is really good for your hydrogels, so because of the high diffusion of gases and liquids through hydrogels, it was one of its first uses was for contact lenses because you can the problem with contact lenses when they are. First invented in the 50s was that oxygen could not diffuse through the glass that they were using to make contact lenses so that didn't work, but when they switched so and then I was also, I think I think you guys saw in the first lecture, which was About the history of biomaterials, there was also a generation of of contact lenses that were just PMMA plastic, which was also not really perma with oxygen. So they had holes in it to allow oxygen to go through and that didn't really work because it still wasn't sufficient oxygen diffusion. But when Hydra dolls were invented in about the 70s and they and they had excellent oxygen diffusion that really revolutionized contact lenses and now they're widely used. Okay, so hydros can be classified in a number of different ways and you'll read: there's a lot of review articles out there and Hydra dolls. Annuals you'll read a lot of different ways that they're classified as even classified based on the nature of their side groups. So they're classified as either ionic or neutral. I'Ve also seen them classified based on their network morphology, and this is a lot more kind of confusing, so they can be amorphous. I mean crystal and hydrogen bonded, based on some super molecular structure like collagen, and then I've also seen them based on their pore structure. Micro, pores macro pores are not pores, that's probably the most confusion, because there's no such thing as a non porous hydrogel. It just means they have very small pores, so I don't like any of these classifications, but the way that I like to classify hydrogels is, if they're, physical or, if they're chemical, / covalent, so physical hydrogels are there's still 3d cross-linked networks. But the cross links are not permanent, so even the hydrogel might stay permanently as a hydrogel, but the actual cross links are not chemical they're, not covalent, so they're either hydrophobic interactions, hydrogen bonds or ionic horses, and if it's a chemical or covalent hydrogel, then that's actually Permanent cross that are chemically bonded covalent bonds between different polymer chains, so every single handle can be classified as either physical or chemical. So that's why I think that that is a good method of classification. Okay, so we can talk about physical relation in a number of ways, so there's hydrophobic interactions, and so that's when you might take a hydrophobic polymer and make it more hydrophilic by changing the side groups to be more polar and 100 Billy. You know by hydrolysis or oxidation, so then you have a polymer. That is maybe it has a polymer. You know a polar backbone or a non-polar backbone, because it's a carbon backbone, but then it has polar side groups and so the non-polar back backbone wants to be together because of hydrophobic interaction. That puts the polar side groups towards the liquid, because it's in it's in an aqueous solvent. So we're always talking about about these polymers dissolved in water. So just that that interaction between the hydrophobic groups within polymers is actually what causes the in solubility of the polymers. In the winter, but since the polar groups are still swollen in the letter it becomes a solid, so it's actually an insoluble polymer that is swollen with water. So that's physical, that's physical generation by hydrophobic interactions. You can also have ionic gelation, where you have ions that are holding together a charged polymer, so you're going to work with calcium alginate in the labs, and so alginate is a poly anion and when you add a divalent at cation like calcium, you have the the Cat eyes hold together to put different parts of the polymer because of ionic interactions, and just I wanted static interactions. So that's a pretty common one. These hydros are interesting because you have to assume that the ions are going to exchange with the ions in the body. So this is something that you're going to implant it, the ions, the ion composition, might change, which would change the properties of the Haifa del Sol. That'S why it's really important to understand the cross linking of ionic patch drills and then, if you're, going to study these hydrogens in vitro. So in lab experiments like this, this hydrogel might dissolve in something like PBS. That only has mono valent ions. You need to have the divalent ions to keep it together. So that's just something to think about. Okay, so those are your your primarily your physical IP bells. I actually didn't. I didn't put a diagram or a schematic of the third most common type of physical hydrogel, which is hydrogen bonds, and so our hydrogen bonds hold together the chains and friends. What I forgot to do that, because my research was really heavy on PVA hydrogel is polyvinyl. Alcohol hydrogel, so that was what my doctoral research was on, and that is a long carbon polyvinyl. Alcohol is a long carbon chain with high with hydroxyl groups coming off of it, and so what happens with aqueous solutions of polyvinyl alcohol is that you can actually freeze them and the ice crystals cause the chains to align and cause crystallites to form in the polymers And then, when you thought hydrogen bonds form between the between the hydroxyl groups on adjacent chains and so the more you freeze it and thought the more alignment you have and the more information of crystallites and aggregates and the stronger it gets with you pretty small cycle. So the most famous example of the physically cross-linked hydrogel - that's cross-linked by hydrogen bonding, is PVA hydrogels. Okay, so those are your physical hydrogels. We also have chemically cross-linked hydrogens, so there's a couple different ways to do it. They all result in just covalent bonds between polymers and you know, there's a little flutter, so this schematic is showing that you might have a bi functional, monomer or polymer. And then, when you put in cross linkers you have the monomers. You know they form a polymer, but then there's of water and the multifunctional cross linkers connect different chains and that forms a hydrogel network. You can actually even do this now in the presence of water, and then you can swell it later and still considered a hydrogel. You can also have poly functional polymers with them where their pendent side chains are reactive, and then they form a cross-linked. You don't need a cross linker to actually connect them if they have their own long enough side chain that could connect to another one, and then that would form a network. Other ways are that you might polymerize a monomer in the presence of a cross linker into a polymer and because there's also a cross-linking agent there. That would cross lengths different polymers together forming a 3d network which is a hyper bill once it swells or you might have longer monomers that are referred to as MacRumors and it's the same concept there or you might have full-on water soluble polymers. And then you add a cross linker there. Just you know like a shorter chain, one that actually forms the cross links with the actual cross links and that's a high drill Network inter penetrating hydrogel networks are kind of interesting. Those are abbreviated to IP n networks or IP n hydrogels, inter penetrating network. So you might have heard about them. And that's when you have a hydrogel Network and you add in the monomers to a different polymer in the hydrogel Network and then you cross like them and they form a network all dispersed between around the hydrogel that you that you started with. So you have two hydrogen networks that are all inter penetrating, so that's why it's called that and that's cool, because the properties that they have is like a composite between the two different hospital networks. So that might be a really interesting way to take two advantages. Advantages of two different materials and combine them: okay, so that's how you make Hydra dolls and how you classify them. There'S properties of the network of the Hydra network is what is going to determine its material properties, including its mechanical properties, its structure, swelling behavior and diffusion, which is going to be really important for drug delivery. So that's we'll talk a lot more about that in biomaterials and really important in tissue engineering if you're going to encapsulate cells and to be diffusion of oxygen and nutrients. So that's really important to show in here so we'll talk about that by charles 3. But for now i want to talk about porosity, so I was saying that I don't like this as a classification, because non-porous hydrogels are still porous. They just have pore sizes that are less between 1 and 10 nanometers microporous. I really don't like that name because they're, not micro, sized pores, they're, nano sized they're between 10 and 100 nanometers same reason. Why don't like macro pores, because they are micron micro sized and for my doctoral research? I actually worked on super pore size dolls, which is sort of like not really an official classification, but it's something that we used the term for, and so we made very large pores in the hydrogels. I think that scale bar is 500 microns. It must be 100 microns, but I have a graph of the pore size there and the pore size ranged from 40 to 100 microns depending on the processing parameters, and that can be useful if you want. I wanted to have cells from the surrounding tissue grow into the hydrogel in order to have some integration between the surrounding tissue and the hydrogel. So that's why I wanted large pores, so that's basically the porosity and so when you're thinking about the pores of the hydrogel. So super porous is kind of obvious why we call it that because they're really big pores but they're sort of different from the other porosity classifications that I have up here, because within those super pores hydrogels, the walls of the pores are made of hydrogel. That itself could be classified as non porous, micro, pores or macro pores. So the reason why is that that pore size is actually the mesh size of the network? So if you look at the literal distance between chains in the hydrogel between polymer chains, so that actual distance is the mesh size also known as the pore size. So if we go back to the picture of my super porous, Hydra does so because the hydrogel walls had wasn't, they were networked themselves, they had their own cars, find a mesh size and within their network, okay, but so the mesh size and the cross-linking density determine Almost all properties of hydrogel, so they're, really important kind of intuitive to understand the mesh size is just the distance between the distance between cross links. So that's kind of like porosity, so you can see how that would affect properties, especially swallowing properties, because if that pore size is bigger, then it can accept more water into the Pikeville Network and that it will swell a lot form cross when you density is very Related to mesh size - and that is the distance while cross-linking the molecular weight between cross links, so MC in this diagram is the actual distance between cross lengths. So if you have, you know multiple polymer chains, cross-linked the average distance between two lengths is the molecular weight between cross links and you can calculate the degree of cross-linking by dividing the molecular weight of the polymers repeating units over a molecular weight reaching between cross links Times two, which is derived from rubber elasticity theories, I'm not going to get into why there's a two there and then so degree of cross-linking is one parameter that you could use to describe hydrogels and then another is cross-linking density, which is extremely related, and that is Just one over the specific volume of the polymer times the molecular weight between cross links and the specific volume of the polymer is just the inverse of the density of the polymer and it's an amorphous state. So that is so. The cross-linking density is really important because that's going to determine a lot of their properties, so in what way does it determine there properties like I was saying if you increase the pore size, which means you decrease the cross-linking density, you are going to have a if You decrease the crossing against. You have an increase in gel swelling because more water can go in there if you increase the cross-linking density, so you make the mesh size smaller and then molecular weight between costs like smaller, so that the like, so that the cross-linking density is increased. Then you have an increase in mechanical properties like modulus or stiffness, and that's because they can withstand more forces because there they can't move as much in response to an applied force. So that's why it's stiffer there so because the cross-linking density is so important. I want to go through the derivation of how you can calculate the molecular weight between cross lines so that you can then in turn calculate the cross-linking density from swelling expectance, because you could calculate the molecular molecular weight between cross legs from mechanical models. You could do mechanical testing, but because it's so related to swelling properties, you can also do swelling experiments to relate them and by swallowing experiments I mean you just take the hydrogel put it in PBS or water or some other solvent and see how much water it Up it takes up, and that's really related to the molecular between cross links in the food of following derivation, so equilibrium swelling theory is what's used to derive this equation. It'S kind of complicated, I'm not going to go through the entire derivation, but I'm going to just show how you can how it works. So the flory-huggins theory is was developed in 1953 and it just states that the degree of cross-linking of a or the degree of swelling of a cross-linked polymer is dependent on the elastic retracted force. Because of those cross links in the polymer network. And it also depends on the interactions between the polymer and the solvent, which is water. That makes sense. That'S because the the amount of swelling, of course depends on the interaction between the polymer of the water. If it's you'll have more swelling at the interaction stronger. So to go through the equations on how you might relate molecular weight between cross links and swelling Florian Huggins started with the free energy of the system, and they said that this is simply the sum of the elastic attractive forces and of the in the free energy Of the mixture of the polymer and the solvent, which is water, so each the free energy, their change in free energy, due to the elastic retracted forces, can be determined based on rubber elasticity theory, which is assumed that hydrogels are perfectly elastic for small deformations and we'll Go through that in just a little bit, but I want to start with how you derive the free energy for the for the actual swelling of the cross-linked polymer in water. And so that's the Delta G of the banks, and that is based on the chemical potential of water inside and outside of the gel which at equilibrium will be, will be equal. So if you differentiate this free energy equation with respect to chemical potential, you get that the difference in chemical potential is the sum of the chemical potential of due to the elastic retracting forces and to the polymer water interactions. So, in order to so I guess that it's important to know that the chemical potential change at equilibrium is zero. So that means that they, you can make them the two sides equal to each other or to the negative of each other. So you can basically plug in zero for the chemical potential change and set the change in chemical potential of the elastic forces to the mixing forces. So we're going to go over those two terms. So the changing chemical potential due to the polymer solvent interactions is based on this equation, which the derivation is out there and you can look it up if you want, but I'm just going to go really quickly through that, and so it's based on the gas constant And on temperature, of course, it's based on temperature, because the energy that you add to the system is going to affect the interaction between the polymer and the solvent, and that, of course, is going to affect the change in free energy and then v2s you'll see a Lot, that is the volume that is the volume swelling of the swollen palomar, and so you have that you can get that by dividing the volume of the polymer divided by the volume of the swollen gel, which is also known as 1 over Q Q. Being the swelling ratio, and so the swelling ratio is the volume of the swollen gel divided by the volume of the polymer without being swollen. And so you can think of that. As like the full change of how much the volume changes when it swells in water and that's often approximated as wetness over dry mass, so if you swelled a hydrogel for five days and you assume it's at equilibrium at five days, you could say you could just Divide the mass of the swollen hydrogel, divided by the one that you originally had and that's Q, that's the swelling ratio, and so that can be really useful to tell you. You know how much that you can relate that to cross-linking density, and you can also relate it to diffusion within within the gel. So that's a really useful property. Okay, so that is the equation for the changing chemical potential due to the mixing of the polymer and the solvent. So now we just need to get. We need to go into rubber elasticity here in order to get that second term, which is where you would get the molecular weight if you cross links and then that's how we relate molecular weight between cross links to swelling. So that's like we're going through this. So the rubber elasticity theory assumes that the response of hydrogels to applied loads is approximately elastic under small deformations. Actually, this theory assumes that is perfectly elastic under small deformations. So is that a legitimate assumption? This is J that I took a really long time ago of pba hydrogels during my doctoral research, and so this is the stress strain curve from 0 to 50 %, and it is not linear, which means it's not elastic. However, if we look at small deformations, so less than 20 % and I cut off from 0 to 10 percent because there was noise there, that would made it harder to see, but from 10 to 20 percent they are linear. So that means that they are elastic under 20 %, so it actually is a pretty good assumption to make in rubber less to speed right. So, as the name suggests, rubber elasticity theory was developed for rubbers and to describe the mechanical properties and the elasticity of rubbers. Without going into what this is or how this was derived, you can see that the stress in the system is inversely proportional to the molecular weight between cross-links of a rubber, which is a cross-linked polymers, just not really swollen with water. So to modify this for it. Being swollen with whatever, because that's not hydrogel is you just add the volume swelling ratio of of the swollen polymer, so that's that term at the end there so we can write, we can rearrange that a little bit and add in that volume swelling term, and you Find that you can describe the molecular weight, you can describe the change in chemical potential of the elastic forces based on the molecular weight between cross lengths and M, and there is the molecular weight of the polymer. And then you have the swelling of the pack of the polymer there, but this is all in terms of the elasticity theory. So this is not actually so you have the swelling ratio, but it's just it describes it some mechanical properties. So if you combine those two equations that we had before you have the one that was chemical potential of the mix and chemical potential of the elastic forces, and then you solve for molecular weight between cross legs. This is how you can calculate it. So there's some terms here that I didn't go over. There'S the molar volume of what are the volume of polymer and the relaxed state volume of polymer. The swollen state we already talked about that and Chi is the flooring, interaction parameter. That describes. That'S like a constant that describes the interaction of the polymer on the winter, so this is a really ugly equation, but you can use it to just use simple, swelling experiments and calculate the molecular weight with your cross links. So I wanted to go through this because if you invent any new hydrogels you're going to need to describe the molecular weight between cross links and the swelling properties, because it's very important for comparing these hydrogels well, first of all, for comparing it to other hydrogels. But also for predicting its properties and in a variety of situations. So that's why I'm going back through that? Okay, so rubber elasticity theory is one way to describe hydrogels, but you can also describe them with visco-elasticity theory, and that is because, when you apply a load like a compressive load to a hydrogel, what is actually happening is the fluid is moving through the porous matrix Or and there's different models that can describe it, I'm showing a model here which is called the biphasic theory, which is just what I just said, is fluid flowing through a porous matrix, there's other things that that there's like a poro viscoelastic Theory, there's a whole lot Of modeling that people have done to better describe the behavior, but suffice it to say that when you compress a hydrogel fluid flows through the matrix, so this that graph there is an example of creep testing of hydrogels in comparison to cartilage. So, like I said, my doctoral research was on trying to make a biomimetic hydrogel that had the same properties as part Allah, CH operative word being trying, because cartilage is a really unique material. That has a lot of things that make its properties very different from just a from a hydro. That'S made it one type of polymer there, hydrogels and cartilage are both pretty well described by the biphasic area of fluids flowing through a porous matrix and the equation up. There is stress as a of or strain as a function of time and there's some terms in there. That are like the stress that you apply in a creep experiment, and you remember that creep is when you just hold a constant stress, and you see how much the material Kreutz over time, and so that creep is sort of like how much fluid was moving around And so the aggregate modulus is sort of like a measure of its stiffness in this sort of fluid flowing situation. So that's H, a that's how you can get that there's, there's an interaction parameter there and there's time showing us strain over time and that's how if you and there's also permeability which is K in that in that term there. So if you fit your creep data to a model like that, and you can actually calculate out aggregate modulus and permeability and that's how you can you know in a physiological test, you can compare a hybrid, a leap that you made to something you're trying to mimic. Like a tissue, so that's what I did. I compared a PVA hydra delta cartilage, and what i found is that, even though the the water content of the hydrogel and the car and cartilage were pretty similar in this experiment, their permeability to water was way different. So the permeability of cartilage was a lot lower than the PVA hydrogel, and that's because cartilage is not just a hydrogel of one polymer. It'S got a lot of sulfate proteoglycans in there that have charges with it with a fixed charge, density that attract water, which is polar, and so as water is flowing through the matrix. It'S not. It doesn't flow as easily through cartilage as it does for a hydrogel. So that is so. That was the reason why this hydrogel would never behave the same as cartilage, because it didn't have that fixed charge density, and that's why cartilage is so stiff because it can resist it can resist the mechanical forces, because that fluid can't flow as much as it would. If it were just like a hydrogel so because the fluid is an incompressible fluid and because it can't flow, you have major resistance to applied forces. So that's a really interesting material that we had not been able to replicate in the lab, like anyone has its odds of Lila. Okay. I talked a little bit about PVA hydrogels earlier prepared by freestyle cycling and how that works. Here'S why I wanted to use them for cartilages, because they have been extensively characterized and found to be really similar to cartilage in terms of a lot of these mechanical properties. That permeability, which is which are just showed, but they do sort of there. I think that there would be a way if you modify the backbone of the hundred L chain to get that permeability to go down, and then maybe you could get it a lot closer to cartilage. But the problem with PVA had two goals for cartilage. Is that when you implant it in cartilage, there's a complete lack of integration with the surrounding tissue, and so that image is of hydrogels that were planted in human knees and the flat image was taken to three months? And you can see. There'S just holes around the hydrogel they're just sitting there in a hole and there's no interaction. There'S just a complete gap filled with fluid between the hydrogel and the cartilage, and so my in my doctoral research identically showed that the reason why that happened is that cells cannot attach to PVA, hydrogels and so they'll never integrate because they can't migrate through or anything So i found that the only way that you could get the hack the cartilage to interact with the hydrogel is one if you added something that the cells could attach to like a hydrophobic polymer. So that's why I added in a phase of a hydrophobic polymer that could absorb proteins that cells could attach to, and I also had your release. I do a controlled release of proteins to actually have a chemoattractant concentration gradient that the that the cells with migrate towards. So, if you just have the hydrogel, if I just had the PVA hydrogel, that would not have any interaction with the body. So I did some in vitro testing to see. Why didn't the cells attached to the high details, or why didn't you have this? Why didn't you have that interaction, and I did this was actually just confirming what a lot of people have shown that if you so the in this image, you can see the pores hydrogels and you have the Hydra dolls that were porous, but but it had large Pores but no hydrophobic polymer in this case PLGA. Those are the only cell that I found after seeding like half-a-million chondrocytes, but when I added in PLGA the cells could attach and then they could produce proteoglycans and cartilage tissue. So I confirmed with lots of people have found that cell attachment will not happen on a hydrophilic polymer network. If you have, I guess, there's some hydrogel that you can have some attachment to. It depends on the polymer, but in ghent, but but in general, if the if the polymer is very hydrophilic, the cells can attach because proteins can adsorb. So that was really not good for cartilage, but it is pretty good for a lot of other applications like anti anti biofilm anti bacterial surfaces also called non fouling surfaces, and that's because, since proteins and cells, including bacterial cells, cannot attach to these materials, they can't and You can't have a bacterial colonization and you won't have infection, so they're also called stealth materials, because the immune system doesn't really recognize them. But that's not that's kind of a an ambiguous statement so we'll just stick with the fact that it cells can attach. It'S really useful for something like urinary catheters, which has like a hundred percent rate of infection. If it's kept in there long enough, because it's just really exposed to bacteria, so if bacteria can attach, then it would be more resistant to infection so coding, something like a catheter with a hydrogel that cells can attach to is very useful. This the drug delivery applications are also really useful because, if the immune system can't, if the immune, if the cells of the immune system can attach to these the tool like a circulating drug delivery system, like nanoparticles or micro particles, then they can't clear it from the System and you'll actually have longer residence time of your drug delivery system before it's cleared from the system, so we'll go through that more environment eros, but this is one application of the hydrogels. So I wanted to qualify all these statements by talking about fiber and fiber gels, which cells actually can attach to and they like to so this is a mixed fibrinogen and thrombin. So when you mix solutions of fibrinogen at thrombin, the from includes the fibrinogen and they'll makes it no longer soluble in the water that you dissolved it in which causes it to precipitate out of the solution, but it's still swollen with the water. So it's a gel. It'S a hydrogel and it's a natural, adhesive hydrogel, and so it's marketed and Ciccio by by Baxter and also by tissue call. Or, as did you call - and this is an image of it - being used as an adhesive to attach a metal mesh for hernia repair to the tissue, so it's naturally adhesive. So that's fiber and that's a really common biomaterial okay. So we talked a lot about proteins. Glycosaminoglycans like hyaluronic and clint and chondroitin sulfate and heparin are really important parts of the extracellular matrix of connective tissues and of all tissues. So, but I was talking just a moment ago about how cartilage has the sulfate proteoglycans, and so this is a picture of Tyler Onan, and so you can see that it has the negatively charged sulfate groups on there and that's what causes the resistance to flow that Carla chooses to resist applied forces, so all gags are negatively charged and attract cations and that actually results in osmotic imbalance that leads to swelling, which is what we've been talking about. Some tissues actually swell like hydrogels, do because of the because of these gags, and so hyaluronic is really important for fluid film, lubrication and cartilage of articulating joints, because when you apply a force that will the water will squeeze out like we talked about in the creep Experiment and that actually forms like an articulating a very lubricating layer. Okay, so hi LaRon is actually marketed as a supplement to, I guess, increase it. They inject it into joints that are osteoarthritic in order to increase, articulation and reduce the COPE and a coefficient of friction. So a lot of patients report reduced pain, this way by inject um, but a lot of engineers and clinicians think that it's like a placebo effect, because how long does it stay around? Does it that's not really how high Llorona works in the body? So but again, I think it definitely works in the short term, at least that it maybe puts in some extra cushion there. Okay, so alginate, you guys are going to be working with in the labs. So, if derived from cell walls with brown seaweed and it's a linear copolymer of two different sugars, man, erotic acid and cool erotic acid, the molecular weight can vary depending on you know, processing any cross links in the presence of divalent cations like we talked about earlier. So alginate is really useful for biomaterials and tissue engineering applications because it gels under such mild conditions that you don't really have to worry about it being having any residual cross-linked gears. That would be toxic to cells and you can control the hydrophilic properties based on a lot of different properties of Osment, like the concentration, the type of ultimate like how much cooler on a gas that are man, erotic, acid and that's because of the bond rotation and Health and that affects the stability there and then based on the concentration of the ions that will change the cross-linking density, which will affect the properties that we discussed earlier. So alginate is commonly used as a wound dressing, and so it's used wound dressings in like burn wounds or for diabetic wounds or ulcers. They use hydrogels to keep the wound moost and provide a protective barrier against infection. So that's really just the physical properties of the hydrogels that make it's beautiful there other than that they're, not really interacting with the skin at all, and then the other really cool thing about hydrogels is that they can be environmentally responsive. So the two most common types are ones that are responsible to temperature changes, so they'll be liquid at room temperature and then solid it or they form the hydrogel at body temperature. So that makes an injectable. So if you um, if they're liquid, they can go through its arrangement get into the body they won't take. They'D fill the shape of whatever they were injected into because they're a shape filling liquid, and then they were eyes to draw hydrogel, and so that's very useful and pH sensitive hydrogels might be collapsed at a low pH and then swell at a high pH. So that's extremely useful in drug delivery because you might want something to be to not release drug. If you, if you take a drug like a pill, you don't want it to actually release the drug until it gets past the harsh environment of the stomach. It needs to get into the large intestine small intestine in order to actually release the drug into the bloodstream, so you want it to remain collapsed and not release the drug until you get past the stomach when the pH is low. So as soon as the pH increases it swells and releases the drug, so we're going to go into a lot more about these smart hydrogels in bio, charles two and three. But I just wanted to mention them here, because they're, pretty cool and so in summary, natural polymers are nice because they give you the possibility to confirm by activity into your biomaterial, and you can actually engineer interaction between cells and proteins of the body and your body.