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Scaffolds: Natural Polymers

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Video 1: Natural Polymers
Good morning, we will start with the next topic. So, in the last class we looked atextracellular matrices right. So, we primarily talked about how extracellular matrices canactually be used for tissue engineering applications; where we talked about what are the componentsand we discussed what the structures are and what their roles in the ECM are.So, we will move on to other materials which can be used as scaffolds. So, first we willtalk about natural polymers today. So, I have also uploaded the slides, for the last lectureand I have actually edited it to give an older paper, rather than the newer paper so, whatI did was I went back and looked at the papers and I realized that the older paper had moredetailed materials and method section. So, I have put the older paper; it is a 2008paper. It is about decellularizing a heart and they have given an extensive, extensivedetails on the protocol. I think I have also uploaded the reading material for the protocolsif not I will upload that if you; if you are interested you can look it up, I will givethat to you. So, the group had actually published a protocolspaper. So, people tend to do that when they have some innovative protocol; so, they willgive the standard operating procedure for a protocol. Step by step it will be described.So, that will help you with your questions whereas, in how exactly decellularizationis done and the protocols paper actually you would say every step that can be done so thatit can actually be reproducible. Ok now, we will talk about the next classof materials which is studied for scaffolds, it is the natural polymers.So, you have different types of natural polymers which are commonly used. So, we will talkabout what they are and how they are used in tissue engineering.So, a natural polymer is something which is derived from renewable resources which couldbe plants, animals or microorganisms. They are complex structures with different physiologicalfunctions. So, depending on which organism they are found in, they would have their ownphysiological functions. Some of their properties are where they wouldhave pseudoplastic behavior, they would have good gelation ability and water binding capacity.They would most commonly be biodegradable; not always. See it will usually be degradablein the organism which we are looking at, but if you are talking to, going to say in humansnot all of them are going to be biodegradable. So, they may possess many functional groupswhich can be used for chemical or enzymatic modifications or conjugation of other biomolecules.So, which is an important property you would want in a base material, because you wantto impart bioactivity to the material. So, the base material you use should have functionalgroups to which you can actually conjugate these molecules.So, advantage of using something like a natural polymer would be; it can interact favorablywith cells through specific recognition domains, because they do tend to interact with cellsin the host organism where they are present. So, they have domains which will help in thatand if it is a molecule which is already present in your human body then it can actually verynicely integrate and interact with the human body as well.So, hybrid materials have been used to mimic ECM. So, basically you take 2 or 3 naturalpolymers and blend them or conjugate them in a way that it would form something whichwould chemically be similar to ECM. So, these kinds of research has been done extensivelyover the past 20 or 30 years and there has been varying levels of success with doingthis. So, the limitation would be; they can be degradedby naturally occurring enzymes which means controlling their rate of degradation becomesa challenge. So, if it is something which is going to have a, if you are going to putit in a wound site for example, you are going to have some matrix metalloproteases there,which can actually degrade some of these compounds. So, you would want to crosslink them in away that they do not get degraded more rapidly than your desired rate of degradation, right.So, that would be a challenge when it comes to this.So, another problem would be an undesirable immune response due to the presence of impuritiesor endotoxins because you are going to be, many a times extracting it from another place.So, there can always be some amount of endotoxins remaining. Even if it is a bacterial cultureyou can still have some of the bacterial cell wall or something which is remaining afterpurification, which can trigger immune reactions and lead to rejection of the implant.Another problem is their property can actually vary from batch to batch. Depending on whereyou actually get the material from, you will have a different molecular weight, differentfunctionality and so on. So, it will be, it will, it can actually be a problem. If youare looking for consistency with every step, it will be a problem.With synthetic polymers you would not have that right; you have control over the chemicalsynthesis which is happening. So, you can control their molecular weight and other propertiesto a very large extent whereas, that is not possible with natural polymers.So, natural polymers are actually classified to 8 major classes based on chemical structures;so, polysaccharides, proteins, polyoxoesters, polythioesters, polyanhydrides, polyisoprenoids,and polylign, sorry lignin and nucleic acids are the eight classes.So, out of these, polysaccharides and proteins have actually been extensively studied. Becausethose are the components which are present in your ECM right. So, they have actuallybeen explored in depth. So, the polyoxoesters which would be the polyhydroxyalkanoic acidshave also been studied to a reasonable level. They have, because they can actually be synthesizedby bacterial route; so, by bacterial fermentation. So, that is also, that has also generatedsome amount of interest in this domain. So, in today’s lecture we will talk aboutpolysaccharides and only about proteins that we did not look at as part of ECM, right.So, I do not want to again talk about collagen and say that collagen can be used, right.So, I am not going to do that, but; obviously, collagen is something people do use.So, please remember that although I am not talking about it here, it is a natural polymerwhich is being used in tissue engineering applications. So, are other things like elastin,laminin and all that. We will not go into details of those aspects. We will talk aboutpolysaccharides, because we have not talked about them primarily when we talked aboutECM. We will also have a small introduction on what polyhydroxyalkanoic acids are.So, natural polymers can actually be, where you get it is basically isolate from plantor animal sources. You can also get it from algae. So, sometimes microorganisms whichare capable of producing these polysaccharides can be cultured or you can use it in fermentationprocesses, and produce these. So, biopolymer production by fermentation has actually beena growing field. So, people try to use microbial cultures toproduce different types of polysaccharides. So, cellulose is one common example, wherebacterial cellulose has been extensively studied. In our own department, Professor Guhan Jayaramanworks on developing metabolically engineered strains for producing hyaluronic acid.So, there are different ways people do it and there are also enzymatic processes whichcan be used as fermentation processes. Instead of using the whole microbe, people can tryto use enzymatic processes for creating these polymers. So, there are different such processeswhich are studied for so many different materials; and you are ultimately looking to get consistency.So, this kind of fermentation where you may have better control over the production, canlimit the batch to batch variations which you are always worried about.So, we will first start talking about polysaccharides. So, they are also known as glycans. Polysaccharidesare nothing but bunches of some monosaccharides which are linked together. The monosaccharidescould be aldoses or ketoses and there are linked by glycosidic linkages; and monosaccharidesare basically classified based on the number of carbons. So, you would have triose, tetrose,pentose, hexose, heptose, octose and nonose and so on. So, these are the monosaccharideswhich are the building blocks for the polysaccharides. Polysaccharides can be classified based onthe composition of monosaccharides as homopolysaccharides or heteropolysaccharides. Homopolysaccharidewould basically have only one monosaccharide as a repeating unit. A heteropolysaccharidewould have multiple monosaccharides as repeating units.It can also be classified based on the structure, whether it is a linear chain or a branchedchain. So, if you have a branched chain then it can have better mechanical properties insome cases. So, but the degradability will probably be lesser when you have a branchchain. So, you would have to find optimal levels for using in your application.So, there are different factors which affect the physical property of a polysaccharide;obviously, the monosaccharide composition is one thing. So, you can also have linkagetypes and patterns, chain shapes, like the linear chain or branch chain and so on andthe molecular weight of the material itself. So, you might have these polysaccharides whichcan start from maybe 50, 40, 50 kilodaltons to all the way to a few megadaltons. So, ifyou are going to have that kind of range, based on the molecular weight there will bemany physical properties which will change.So, this is a general structure of a polysaccharide. So, you have a what you look at here is thereare different monosaccharide compositions that can be there, and you also have differentlinkage patterns. So, you have 1-6 glycosidic bond here, you have the O-glycosidic linkagegeneral and beta 1-4 glycosidic bond and so on; and you can also have different substitutionsfor the, in the position of the hydroxyl group. So, you have a hydroxyl group here. So, hereyou can have different substitutions with the R, which could be any group and you canalso have different degrees of freedom because of these glycosidic bonds. So, this will create,this is a general structure and you can actually keep changing this based on the groups whichare there in the monomers which are there. You will get the different polymer structures;so, different polysaccharide structures.

Video 2: Alginate, Dextran and Chitosan
So, we will start with alginate. So, alginate is something all of you are aware of, right.So, you would have used it even if not for biomedical applications, you would have doneit, used it in some enzyme entrapment experiment. So, you would have always taken alginate solutionin sodium salts and then mixed it with calcium chloride to get alcium beads, sorry alginatebeads. So, these are very commonly used in differentapplication. So, biomedical applications also they started looking at this for, for cellencapsulation and techniques like that. So, alginate is basically a polysaccharide whichis derived from sea algae and this is a linear block copolymers of 1-4 linked beta-D-mannuronicacid and alpha-L-guluronic acid. The divalent ions can actually form crosslinks in alginatesby binding to the guluronic residues. So, what happens is during gelation and crosslinkingthe sodium ions which are there, get replaced with the calcium ions and results in the formationof something like the egg-box structure which is shown here. So, these links which are separate,then get crosslinked because of the presence of the calcium groups, the calcium ions resultsin the formation of crosslinking and thereby it forms a strong gel.So, the advantage of something like an alginate crosslinking is it is relatively inert aqueousenvironment. So, you do not really need harsh conditions to create these kinds of crosslinkingwhich means it would be conducive for biological materials like cells and enzyme and so on.It also has very high gel porosity. So, it is a very porous gel which means, so, therecan be very good mass transport. So, that means material can actually come in and leave.So, cells which are entrapped would actually get enough nutrients to survive in when encapsulatedby calcium alginate beads. So, as I said mild encapsulation process whichis also free of organic solvents makes it conducive for biological applications. So,this is used for encapsulation of cells. People have studied encapsulating different typesof cells using calcium alginates so that they can deliver cells to a site and other bioactiveagents you can try to load growth factors and other molecules also using these crosslinkingagents.So, crosslinked alginate can actually immobilize and also later recover cells from the cellculture matrix and it is, because of this, it is been used in delivering cells; basically,it is used as a vehicle for delivering encapsulated cells. So, people have tried to use alginatebeads as a bioartificial matrix for cartilage regeneration and also for engineering livertissues. So, there are different papers on these I am not going to go into the detailsof each of these things I am just telling you these are the applications which are,which have been used. So, you can always go back and refer to literature,try to figure out how exactly people have used this. so obviously, most of these wouldnot just be taking alginate and using it. There will be some level of modification,some other polymers being blended and so on. So, you would want to go back and read upon that if you are interested ok. But just as a, introduction for you to understandthat this can be used, this is sufficient. So, as I said it is commonly used as a compositewith other polymers and also with ceramics for tissue engineering applications. See,most of the polymers will always be tried, blended with ceramics when they want to useit for bone tissue engineering, because the polymers themselves will not have the desiredbioactivity and the mechanical properties. So, you try to blend them to form compositeswith ceramics.So, dextran is another bacterial derived polysaccharide. So, you have alpha-1,6 linked D-glucopyranoseresidues with a small percentage of alpha-1,2, alpha-1,3 and alpha-1,4 linked side chains.So, this is the structure of dextran. Dextran is commonly used for different applications.So, can you think of a common application, where dextran is used; alginate you all know.Similarly, you have also used dextran somewhere, not in obviously, not in medical. you wouldnot probably think of that, but I am talking about something where some experiment thatyou might have done; protein purification. Sephadex.Sephadex. Sephadex.Sephadex is actually made of dextran.Ok so, dextran hydrogels can be created either by physical or chemical crosslinking, takingadvantage of the, all the hydroxyl groups that are present. See, as you see here, thestructure shows a lot of hydroxyl groups that are present, right. So, you have hydroxylgroups everywhere. So, because there are so many hydroxyl groups, it is actually easyto crosslink them. You can actually have simple hydrogen bonding as physical crosslinkingto form nice hydrogels. So, we will talk about what hydrogels areand how they are formed in a later lecture. So, right now I am just talking about thematerials that can be used and then we will talk about how they are being fabricated.During that time, we will talk about hydrogels. Ok so, these are widely used in separationmatrices which is the Sephadex. Sephadex is one common example where dextran is used orit is also used as cell microcarriers, its commercial product called Cytodex; is usesfor cell delivery. It is also been explored as drug delivery vehicles.So, these dextran has actually shown that it has very good hemocompatibility; so, ithas been used for reducing vascular thrombosis and reducing inflammatory responses, or toprevent ischemia and reperfusion injury during organ transplant. So, dextran actually hasbeen extensively used in biomedical applications even before tissue engineering. So, becausethere is so much promise and with biocompatibility, people have tried to explore dextran for tissueengineering applications as well.So, another polymer which is very commonly you studied is chitosan, right. So, I thinkjust behind collagen, chitosan is the highest researched material. So, if you search forpublications related to chitosan in tissue engineering, you would find thousands of them.So, there are actually many studies which have worked extensively on chitosan; simplybecause it has very similar structure to naturally occurring glycosaminoglycans in humans. Andit is a lot easier to get, compared to other polysaccharides which are not readily available.Hyaluronic acid you can get it, hyaluronan you get it, but hyaluronan do you know whereyou get hyaluronan from? If you have attended seminars from Guhan’s lab you would know.Hyaluronan is actually taken from rooster comb. So. you can imagine the quantity youwould actually be able to get from that, right. It is not very easy to get that. Its extractionprocess is quite painful. There are also commercial processes where streptomyces is being usedfor production of hyaluronan. Like Guhan’s lab works on, be trying touse Lactococcus for production. Because I do not, no, they use streptococcus, not streptomyces.Streptococcus is used in commercial cases, I think. But Lactococcus is a grass straincompared to streptococcus. So, they are working on, trying to use Lactococcus.So, anyways so, chitosan is not like that; chitosan is actually found in arthropod exoskeletonsand fungal cell walls. So, you can actually very easily get chitosan right. So, all theshrimp shell which you throw away has chitosan. So, it is, that has chitin and chitin canbe processed to get chitosan. So, because of this there is actually a lot of abundancewhen it comes to chitosan because of this it is actually reasonably inexpensive.It is also biodegradable, bioadhesive and biocompatible. So, for these reasons peoplehave actually explored chitosan extensively and it is basically a linear polysaccharideof 1-4-linked glucosamine and N-acetyl-glucosamine.So, the molecular weight you get can actually range anywhere from 50 kilodaltons to 1000kilodaltons and also you have deacetylation of these, chitin. Chitin is acetylated; so,when you deacetylate chitin you will get chitosan and the degree of deacetylation can vary from50 to 90 percent. So, it is a semi-crystalline polymer and the degree of crystallinity isactually a function of the degree of acetylation; deacetylation.So, it is actually very high at both 0 degree; 0 percent deacetylation and 100 percent deacetylation.In between it is a very semi crystalline material. So, as I said it is a biodegradable material,because it gets degraded by lysozyme. Lysozyme basically cleaves the glycosidic bonds anddegrades the chitosan. So, that kinetics inversely related to thedegree of deacetylation. So, higher the degree of deacetylation lower will be the degradability.So, it is soluble in aqueous, it is insoluble in aqueous solutions above pH of 7 and itis fully soluble in dilute acids with pH less than 5. So, you can increase temperature,you can play around with the solubility conditions to dissolve it even around neutral pH. Itshows a cationic nature and has a high charge density in solution.So, these are the major properties of chitosan. And this chitosan molecule is actually crosslinkedwhen you want to prepare gels. So, there are different ways you can crosslink it. So, glutaraldehydeis a very common crosslinking agent, which is used extensively for crosslinking becauseit has two aldehyde groups right. So, you can easily form crosslinks.So, genipin is other crosslinking molecule which is also been used, UV irradiation andthermal variations can actually cross, physical crosslinking. So, these are some of the techniqueswhich are used for crosslinking and chitosan is primarily processed using freeze dryingtechnique to prepare scaffolds. So, what is freeze drying?Lyophilization. That is another name for freeze drying. So,what is the process? Like the water molecules get sublimed leavingthe solid state. Ok so, what happens is you basically reducethat pressure enough so that water does not have to evaporate, but it just sublimes. Icejust sublimes to form a water vapor. So, the advantage of doing something like this is,you would be able to create pores. So, the ice which is there, if it immediately goesinto vapor phase, the space occupied by the ice is going to be left empty; leaving pores.So, these porous structures are going to provide the porosity for the cells to attach and grow.So, that is one use of doing lyophilization. So, that is why people try to lyophilize scaffoldswhen they prepare it.Chitosan has been used in different tissue engineering applications. It is identifiedas one of the most promising natural-origin polymers for tissue engineering applications.So, it has been looked at for different tissue engineering things. So, bone it probably,primarily is used along with some ceramics because just using chitosan is probably notgood enough for the mechanical properties. But it has been used for a lot of soft tissueapplications. Again, it would be blended with other materials, composites should be prepared.So, all the materials we talk about are pure component, pure polysaccharides and proteinswhich we are talking about, but current research almost always uses composites. You would almostnever see just one material being used and that is because your ECM itself is not a singlematerial right, it is a mixture of things. So, you would not want to, if you are tryingto emulate and mimic the ECM, you cannot just use one material and think that will actuallygive you the exact property of the ECM. So, it does not work that way ok. So, differentthings which people have worked on are skin, neural, ligament, liver and tracheal tissueengineering.

Video 3: Cellulose, Starch and Hyaluronan
So, cellulose is another molecule which people try to work on. So, this is one of the non-biodegradablemolecules, in the sense that it is not degradable in your body. You can use cellulase to degradeit, if your, if the organism has cellulase, you can, it can degrade it, but we do notand it cannot be degraded in vivo in humans. So, it is the main component of plant cellwalls and it is the most abundant and renewable polymer resource available. It is primarilyavailable as lignocellulosic material. So, you would have to separate the lignin fromthe cellulose to use it for many of the applications. So, which in itself is a big challenge. Thereare actually extensive research going on about how to separate lignin from lignocellulosicmaterial. So, this basically has a linear polymer consistingof the D-glucose residues, which are linked by beta 1-4-glycosidic bonds. So, what happensis these chains are actually stabilized by the formation of the beta-linked glucopyranoseresidues and once these chains are stabilized then the flexibility of the material decreasesand these chains can actually form hydrogen bonds amongst each other to form microfibrilsgiving it the mechanical strength and the chemical stability. So, that is why it isvery strong; it is actually very mechanically strong material.Cellulose, in tissue engineering; people have tried to use it for different tissue engineeringapplications. Although it is not degradable. People have actually seen that partial degradationcan be obtained in vivo. So, there are some literature which suggests partial degradationbut there are no hydrolases in your body which can actually degrade these linkages and thereforeit is non degradable in vivo. So, people have tried to use this for bonetissue engineering and shown that cellulose actually supports bone ingrowth and also inducescell migration. So, showing some kind of bioactivity when it comes, when it is used for bone applications.Cardiac tissue engineering applications have been shown to have promise, because they showcell growth, connectivity and also some electrical functionality while using cellulose as themajor component of the ECM, of the scaffold. People have used it for cartilage tissue engineering;so, bacterial cellulose have been, has been shown to support proliferation of bovine-derivedchondrocytes. So, again these are all preliminary studies; some of them would be only in vitrostudies, some of them would probably be some small animal studies, it is not like theyhave actually taken it for commercialization. So, please do not think that oh, these areall things which are completely proven. No, it is actually just initial studies have shownthis is what its properties are, and there is always a chance of failure at differentlevels, right.So, starch is another carbohydrate molecule which is actually a carbohydrate reserve forhigher plants and it is one of the cheapest biomaterials available that is also biodegradable;it is completely degradable to form carbon dioxide and water. So, because of this itis an interesting material to work with. It basically contains alpha-D-glucose units thatcan be organized as amylose and amylopectin. Amylose is a linear very sparsely branchedpolymer; which is linked by 1-4 glycosidic bonds and you have amylopectin which is highlybranched polymer that contains 1-4 bonds and 1-6 branching points and it appears for every25 to 30 glucose units. So, because of this the structure itself isreasonably branched, when you are talking about starch. So, again starch has also beenused in different applications, I have not gone into the details. Starch has actuallybeen used along with other molecules, other polymers for using, for showing some tissueengineering applications.Hyaluronan is highly hydrophilic polysaccharide. So, amongst the polymers which we have discussedtill now, hyaluronan is the only polymer which is actually present in your body.Sorry. Sir, what made researchers think that starchcould be used for tissue engineering? It is biocompatible; so, see any time a materialis biocompatible you might want to explore its potential right. So, if it is not biocompatibleit cannot be used at all, right. So, when you know that it is a biocompatible materialand it is completely degradable, it is, you know for sure it is not going to cause anyharm; so, you want to see whether it can have desired functionalities. When people exploresometimes you see desired functionalities, sometimes you do not; and in some cases, therewill be positive results during in vitro studies which will get published.But eventually when you take it further, you would realize at some point its applicationhas to stop because you cannot take it further for a clinical application. So, I do not know,I do not even think 1 percent of whatever is reported as publications can actually betranslated to clinical products, right. So, it is, you would see publications on everyapplication, you will see thousands of publications on bone tissue engineering, thousands on cartilagetissue engineering and you probably have like 3 products in each.That is because it is not going to translate that well. It is going to be a lot of hiccupsand most of the times what happens is people study things in vitro, right. So, when youstudy in vitro many a times you also use a cell lines instead of using primary cells.So, you are going to have differences from cell line to primary cell and then into asmall animal and then you take it to a large animal and then to humans. There is just somuch chance of failure from what is published to what is actually becoming a clinical product.But it is important to explore every material which has a potential to become a clinicalproduct. That is why people who work in this domain, will always start with anything theybelieve is biocompatible, right. So, that is why in our lab we started working on isabgolright. Isabgol pretty much nobody had worked on.So, it is a polysaccharide. It is used primarily for, as a food supplement. So, you use itso, you might have heard satisab or metamucil. So, these are products which you can buy offthe shelf; it is very cheap and it is soluble in water and people just dissolve it in waterand drink it. It is a dietary supplement; it is a fiber supplement actually.So, in our lab we just started to, decided we will use it and see what happens right,and it showed some positive results, obviously we have a long way to go whether, to knowwhether it can be taken much further. So, similarly glucomannan, so many things arebeen studied because they are, they show promise; not because we know for sure it will workin the final stages. So, and here I am only talking about materials which have been reasonablywell studied. There will always be thousands of papers wherethousands of different materials have been explored. I cannot actually go into detailsof all of them. It is not practical. You can go and look up biopolymer tissue engineeringand you will probably get like twenty thousand hits and I am pretty sure not all of themwill fall within the 6 or 7 materials which I have identified right.So, people work on so many different things, people try to use so many different sourcesfor things. And see, even if I am going to use the same material; so a glucomannan isa glucomannan, but it does not always behave that way because if it comes from one plant,it could be have a certain property compared to another plant. So, then you have to lookat what would be the effects of using that separately. So, you would have different studies.So, hyaluronan is a component, is a key component in your ECM. It has a lot of biological functionality.There is this; a chapter from a book called tissue engineering. So, it was edited by abunch of people and the authors for this chapter was also a bunch of people. so, the chapteris called natural polymers in tissue engineering applications, ok.