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Video 1: Scaffold and its Properties
So today, we will actually talk about the Scaffolds.So, we looked at the three different arms of the Tissue Engineering triad, right.So, we saw materials, cells, and signals.So, we looked at a brief introduction of all of these things.So, today we will start with aspect, which I work on, which is the designing of scaffolds.So, we will talk about different materials that can be used for scaffolds and what arethe desired properties and so on.So, the first aspect which we are going to talk about is, the first type of scaffoldwe are going to talk about is the Extracellular Matrix itself.So, as you know ECM is present all through your body and that is where the cells actuallyattach and grow.So, there are two aspects to understand this, to be gained from this lecture; first is tounderstand what is the ECM? and actually how it is relevant for tissue engineering?right.So, for us first we need to know what are the components of ECM?How it is structured and so on?So that we can try to mimic it.So, that is a crucial aspect which we need to start with, and then we will talk abouthow ECM is currently being employed in tissue engineering, and there will also be a smallreading assignment.So, which is just an research article which you would have to read, which talks aboutusing extracellular matrix for tissue engineering applications ok.So, before we go into details of extracellular matrix itself, what are scaffolds and whatare their roles?Scaffolds have a very critical role in tissue engineering, because they actually can directthe growth of cells, it could be either the cells which you have already seeded.So, you could either place the cell seeded on the scaffolds and use this cell seededscaffolds as a tissue engineered construct or you could use only the scaffold, then youmight have cells migrating towards this or migrating away from this right.So, it can regulate these factors.So, mammalian cells as I had already mentioned are anchorage dependent, they are actuallyadherent cells.So, they can, they need some substrate on which they can attach and then grow.So, scaffolds are the matrices, which provides this type of a surface.And they also provide the mechanical support for the tissue.So, depending on the tissue the mechanical properties and other physical properties canbe imparted by the scaffold.So, the cells which are delivered, you might want high loading and you might want specificcell attachment to the sides of which you are interested.So, for these types of things you need a scaffold, which needs to be engineered properly.So, having said this, what are the desired properties of a scaffold?Can you think of properties, which you would want in a scaffold?I will write down as you shout out the answers you think of.Compatibility.Ok.Mechanical properties.Ok, Compatibility, mechanical properties.Biodegradable ability.Biodegradability.Ok, Biodegradability.So you had something?Same.Ok, What else?It should not be immunogenic.Ok, Part of compatibility ok.Higher cell adherence.Ok, Cell adhesion.Porosity, high surface area.Porosity ok, why porosity?Nutrients.So, the other ones are more direct you know why, but porosity why?Because we will be adding molecules that should diffuse and reach the tissues.Ok, so, it will help in diffusion that is one reason.Can you think of any other reason?Cell infiltrate nutrients.Cells will infiltrate.Oxygen; oxygen and nutrients.Oxygen transfer ok.Toxic byproduct.So, cell also.So, all these are diffusion.So, diffusion in and out that is one aspect and then cells in infiltration is one aspectok.Porosity also will give you the real structure of an ECM ok.It is closer to what an ECM would be, ECM has these porous matrices which, in whichthe cells can actually adhere and grow ok, anything else?Nontoxic.Again part of compatibility.Ok, is that all, can you think of anything else?I do not know word for it, but like there is a possibility that a scaffolds can be infectedby bacteria or foreign pathogen so.Ok.The that property I do not know what is the word?So you want the scaffolds to be.Ok, to be bacteriostatic or bactericidal.Yes.Ok, so that could be a desired property, but again that can be part of compatibility right.So, it will again, just like how your own cells rejecting it, again having some infectionscan also come as part of compatibility, but you can less it as a separate property aswell.So, we will just call it antibacterial; how that antibacterial property is given can bediscussed and it might also depend on what type of tissue you are talking about right.So, if you are talking about wound dressing, which is for skins then you might want a veryantibacterial material, where as if it is going to be for something else it might notbe that crucial.Ok.Interaction with growth factors like adherence with growth factor with the ECM.So, ability to deliver signals, can I say that way, signal delivery.Anything else.Light weight.Light weight.Ok, why?So, it is.So, that if it is a implanted in long; like, it won’t restrict our movement or something?So, I would say it needs to be of similar density as your natural tissue.So, saying light weight is probably not the best way to describe it.Because see, if you are going to talk about a bone tissue, that is obliviously going tobe heavier than a soft tissue and weight is not the parameter you are looking at densitywhich is a more uniform.So, what is density compared to, so what is the fundamental difference from propertiesstandpoint between density and weight?Weight is volume based and density is material based.Correct, what is that property.intensive.Intensive properties and extensive properties.Intensive properties are ones which depend on size sorry, which do not depend on sizeand extensive properties depend on size.So, when you are talking about something like this, you are talking about the property ofthe material itself, not the, it should not vary with the size right it.So, obviously, if you are replacing a large tissue, it is going to be heavier anyways,ok, what else?Ok.So, let us look at what I have; so, many important things you have given.So, support and deliver cells; so induce differentiate and channel tissue growth, target cell adhesionsubstrate and stimulate cellular responses would all come under providing signals.So, these are just different signals which we are looking at.Biocompatible, biodegradable; so again, compatibility is crucial, because you need to make surethere are no immunogenic responses and so on.Degradable depends on the type of application.So, how fast it needs to degrade or slow it needs to degrade will depend on the rate ofregeneration of the tissue we are talking about.And then you have to talk about the large surface to volume ratio, which is one of theaspects which porosity will provide you.And when you are actually having this kind of, this large surface to volume ratio, forthe same volume of implant you can actually have a lot of surface on which the cells canattach.Right, the cells are only seeing surfaces, they are not going to see the scaffold asa whole right.So, when you provide a lot of pores or if you make it into nano fibrous matrices, thenyou have a lot of surface area, come for the same volume.So, this will help the cell adhesion and growth.Mechanically strong, structurally stable.So, these are all the properties which we think of from biological stand point.From a final production and manufacturing stand point, we need the material to be processableand malleable and it should also be sterilizable.So, there are different techniques which you can use to sterilize, but whatever techniqueyou use should not affect the properties of the material right.So, if you are going to use protein on top of these scaffolds or like collagen; it shouldnot get denatured, while you are processing, while you are sterilizing it.So, these are important factors which you need to account for and choosing the techniqueused for sterilization would also matter and you can actually design things appropriately.Ok so, these are the desired properties, you are looking for.How you provide these desired properties is the challenge.So, you need to look at what materials can be used, what chemistries can be used forcrosslinking.So, depending on the level of crosslinking, your mechanical strength can actually varyand so on.Might be a silly question, but do optical properties matter, when it comes to scaffolds?Depends on which scaffold right.So, optical property of a scaffold would matter if you are trying to engineer or cornea right.If you are going to engineer a liver I do not really think optical properties matter.If you are going to engineer nerve tissue then your electrical properties would matter.So, depending on the tissue you would have to design the properties.Physical properties will primarily be drive a, driven by the type of tissue you are tryingto engineer ok.Mechanical properties; bones are going to have different properties, muscles are goingto have different properties and so on.So, what are the, what is the role of scaffolds in vivo?So, it helps in constructive remodeling of functional tissue, engineered tissue.So, this scaffold has to degrade and cells have to adhere, migrate, proliferate and differentiate.3D organization of the site to form appropriate tissues should happen and it should also helpin vascularization.So, this is what would happen when a scaffold is placed in vivo ideally, right.So, if these things happen you know that the scaffold is doing its job.So, what factors will control these processes; blood supply, pH, oxygen, carbon dioxide,concentration, mechanical stresses, host surface interaction are all some of the parameterswhich can actually regulate these things.So, for getting these type of scaffolds, people use different materials.So, we will talk about some of the major classes of materials and we will also look at howmaterials can be processed to get these types of materials.So, first today we will talk about extracellular matrix.So, extracellular matrix would be the gold standard, because that is what is there inyour body right.So, if you can get the extracellular matrix to do its job, if you can use that directlyfor tissue engineering that is what you would do, but what would be the limitation of doingthat?So, if you want to use the extracellular matrix, what do you think could be the limitation?Availability.Availability is one problem; ok, can you think of something else?Compatibility.Compatibility why?For immunogenic reasons, you have to get from same tissue or it elicits immunogenic responses.So, that is true for cellular ECMs, when you decellularize it, it would not be a problemthat is what I wanted to get to.So, if you actually have removed all the cells and cell debri then the matrix itself willactually not be immunogenic, because all these proteins are reasonably well conserved.So, they are conserved across species ok.So, it will not cause any problems.Sir, so how will it is, we developed in vivo, in vivo.What do you mean?So, I have.In vitro or vivo?In vitro.In vitro, how would you create a decellularized ECM?So, there are techniques for it.So, the last few slides on this lecture will be that, we will talk about it and the readingmaterial will actually talk about that extensively.So, so ECM function is to support cells, regulate polarity, cell division, adhesion motility.It is also involved in tissue development through cell migration, differentiation andgrowth factor delivery ok.So, ECM features which we need to understand are, they are stable with the ability to bereorganized right.So, extracellular matrix can actually get reorganized by activation, by action of enzymes,like metalloproteases, we will talk about them but it, we also need to understand thatthe features of the ECM are going to be different for the different tissues, it is not goingto be the same.Even though we just call it as collagen, there are actually different types of collagen andwhich collagen is present in which part of the tissue matters ok.
Video 2: Composition of Extracellular Matrix (ECM)
So, ECM composition can be broken down to three major classes; so, basically two majorclasses; proteins and proteoglycans.So, the amongst proteins there are actually two things; one is a; one is a structuralprotein, which provides the support for the tissues to grow and the other are specializedproteins, which have special biological functions.So, the structural proteins also have some biological functions, but we will for simplicity,we will classify them as only structural proteins.And proteoglycans are basically, molecules where you have a protein core to which longglycosaminoglycans are attached.So, these are complex high molecular weight components present in the ECM.They provide the viscosity and the like, fluid like nature of the ECM itself.So, amongst the structural proteins, the common ones are collagen, elastin and fibrillinsand amongst specialized proteins you, the common ones which you see are fibronectinand laminin ok.So, we will talk about these in little more detail.So, collagen is the most abundant protein by weight ok.So, when you are asked this question please remember, this is a very common question,which is asked in many places where you say what is the most common protein in the bodyand inevitably most people say hemoglobin, which is not correct, ok.The most common protein in your body is, most abundant protein in your body is collagenok.This is the structural component, it provides the support, it also can have some functionalproperties.So, there are actually 46 different collagen genes in the human genome and it generates28 different types of collagen fibrils.So, these are just identified using Roman numerals.So, you would have Type I, Type II, Type III and so on.So, the collagen fibrils are the ones which provide the strengths.So, the fibrils assemble to form collagen fibers and these bundles which are the collagenfibers actually impart the mechanical strengths.So, they are very strong structures.Types I, II and III are the most abundant; amongst which Type I is by far the most abundant.Type I, actually constitutes of about 90 percent of all the collagen in your human body.So, primarily Type I is present in all over, in all the tissues.So, these are primary, predominantly synthesized by fibroblast.So, if you culture fibroblast and you maintain certain environment, you can actually makethese fibroblasts secrete collagen.So, there are labs, which actually do that, and in our own institute Professor Verma’slab does that.So, they culture fibroblast to secrete collagen.And the yield of it is always a problem.So, depends on the cultural condition you would not know whether, how much of a matrixyou would get ok.So, this is one way to get it, get the ECM type of a scaffold.So, epithelial cells can also synthesize some of ECM collagen.So, as I said there are 28 different types of collagen fibrils and these are some ofthe major ones.So, Type I collagen is seen in skin, tendon, vascular, ligature, organs, bones.So, this is the main component in bones.So, collagen component of bone is primarily Type I.And you have Type II collagen, which is mainly seen in cartilage and Type III collagen isseen in reticular fibers along with Type I, and Type IV is seen in the basement membrane.So, basement membrane is the one which separates the tissue from the matrix.So, you would have that in the basement membrane, and Type V is seen in hair and nail ok.So, they have different mechanical properties, they will actually have different, significantlydifferent physical properties and they are seen in different tissues.So, this is what the collagen fibril looks like.So, this is a triple helix which you see.So, this is a matured Type I collagen.And so, basically what happens is they are initially synthesized as pre-pro-proteins.So, pro-protein is one which basically, a protein precursor, when these have a, thesecome along with a signal peptide, they are called pre-pro-proteins.So, you have pre-pro-collagen which is usually synthesized.So, from here, from this, there are actually a lot of co- and post-translational modifications,which happen for the collagen to form the fibrils.So, collagen monomers, which are called the alpha chains self-associate into triple helicalstructure.So, collagen has a triple helical structure basically, which are called the collagen fibrilscontain two identical alpha chains and a third alpha chain, which is different ok.So, the Type I collagen itself is encoded by collagen, COL1A1 and COL1A2.So, there are actually two alpha helices from COL1A1 and one from COL1A2, ok.So, these three form the triple helix, which is shown here.So, the two blue ones are the collagen 1 alpha 1 and the red one is the collagen 1 alpha2.So, from this they form the triple helix, which forms the collagen Type I fibril.So, this process actually shows the synthesis of collagen.So, what you have is collagen fibers are produced from a pro-alpha chain.So, this pro-alpha chain then; so some of it is done intracellularly and some of itis done extracellularly.So, the once you see after secretion, after this points type, after the sixth step isthe all extracellular.So, until then it is intracellular; so you have synthesis of pro-alpha chains, whichis the first step and then you have hydroxylation of selected prolines and lysines.So, collagen primarily has glycine, as the amino acid and the next to that is prolineand hydroxyproline ok.So, you have hydroxylation of certain prolines and lysines.So, the prolines are actually hydroxylated and so, are the lysine, some lysines are hydroxylated.And this hydroxylysines are then glycosylated.So, once these glycosylated hydroxylysines are present, you have self assembly of thethree pro-alpha chains to form pro-collagen triple helix.So, this pro-collagen triple helix is secreted and outside what happens is the pro-peptidesare cleaved and finally, you have a self-assembly to form the fibril.So, these fibrils are above 10 to 300 nanometers thick.So, what happens is these individual collagen molecules or triple helix molecules are crosslinked, using the activation of an enzyme so.Anyway so, by the action of an enzyme you have a crosslinking, which is done.Depending on the crosslinking, you would actually have the strength ok.So, once more and more collagen triple helices are crosslinked, you would have very thickfibrils and these fibrils all aggregate to form a collagen fiber.So, this collagen fiber is what forms the matrix, ok.So, this is the typical process for the Type I collagen and you can actually find similarprocess for other collagens as well, ok.So, depending on the initial pre-protein so, initial chain; alpha chains, which are usedyou would get different types of collagen ok.So, this is from basic cell biology.So, you do not need to know the process or this biosynthetic pathways of collagen; however,what you might want to know is what are the molecules, which are the precursors for collagenok.So, if you have that, you could probably try to use them for your tissue engineering applicationsor you can try to understand some of the pathway where we can push towards the generation ofcollagen.So, you could try in vivo secretion of collagen as a way of regenerating tissues.So, elastins and fibrillins are found in tissues that undergo significant stretching or bendingas you, as the name suggest, they have elastic properties so examples would be large arteries,lungs and skin.So, these actually go through a lot of the stretching and bending right.So, some physical, mechanical stresses are experienced by this.so, you would have a lot of elastin or fibrillins.So, this is, they are found a specialized types fibrils, which are called as elasticfibers, the elastic fibers contain large masses of cross linked elastin, interspersed withfibrillins.So, you, the elastic fibers primarily contained elastin and there is some amount of fibrillinsas well.So, elastin is basically synthesized as a precursor called tropoelastin.And tropoelastin has two major types of alternating domains; one is a hydrophilic domain, whichis rich in a lysine and alanine and the other is a hydrophobic domain, which is rich invaline, valine, proline and glycine.So, the hydrophobic domain provides the elasticity.so, if you are going to design polypeptides to prepare scaffolds then providing thesetypes of amino acid, using these types of amino acids will give you the elastic property,which you are looking for.So, tropoelastin is expressed and then secreted as matured protein into the ECM and aftersecretion and alignment with the ECM, elastin monomers are crosslinked and so, this threelysine derived aldehydes, crosslink with an unmodified lysine to form a tetrafunctionalstructure which is called as the desmosine.So, this is the elastin fiber which would, which you would have.And the other major component in the elastic fiber is the fibrillin.So, there are actually three fibrillin genes in humans and fibrillin 1 is the most abundantand it serves as the scaffold for elastic fibers after in crosslinking with the elastinitself.So, this gene expression is consistent with the role in the ECM.So, FB1, FBN1, which is fibrillin 1 is secreted mostly by cells from mesenchymal origin, itis seen a lot in bones.FBN2 is secreted highest in fetal cells whereas, FBN3 is expressed in embryonic and fetal tissuesok.So, some of these are not seen very commonly in adult tissues.So, those were the structural components.So, you also had functionalized, functional proteins, so couple of functional proteins,which we had mentioned where fibronectin and laminin.So, fibronectin is a major component of, major fibrillar glycoprotein in the ECM.It has a role in attaching cells to all matrices, except for collagen Type IV; Type IV; whenyou have a Type IV matrix laminin is involved in the cell adhesion.So, what happens is this, these have a multimodular structure with three different amino acidrepeat domains and these are called FN-I, FN-II and FN-III.In the primary amino acid sequence that binds to the integrin, which is expressed on thecells is the RGD domain, so arginine, glycine and aspartate.So the fibronectin actually provides this type of a motif on which the cells can actuallyattach.So, that is why fibronectin is even used for coating of cell culture plates and other things,where you can ensure cells are attached to the surface.So, people also use RGD domains.So, you can create these tripeptides and use them along with your scaffolds to promotea cell adhesion.If you do not, if you cannot use fibronectin to like all of fibronectin, you can createthese short peptides, which can be used and that will help in cell adhesion.So, fibronectin exist as a soluble protein and insoluble protein.Soluble protein is present in the blood plasma, it is involved in blood clotting process andlinks to fibrin during that process.You have insoluble protein in the ECM, where ECM fibronectin actually has the polypeptidesegments, which alters a morphology and helps in the cell attachment.So, you have laminins, which are another set of glycoproteins.They constitute the structural scaffolding for all basement membranes.So, if you remember, we also said that collagen Type IV is involved in basement membranesright.So, along with collagen Type IV you would have laminin existing.So, that it can help in cell attachment.This is a very critical component of the ECM, because it has a lot of functions; it canactually bind with a integrins, and other receptor, many other receptors.And it is involved in cell differentiation, cell movement, shape of the cell and promotionof cell survival even.So, because of this, it plays a crucial role and it is actually present in reasonable abundants.It is a heterotrimeric protein that contains an alpha chain, a beta chain and a gamma chainso, which is what I shown here.So, it can also be called as beta 1 chains and beta 2 chains, which is the traditionalway it was represented.So, there are at least 15 different types of heterotrimers that have been identified.The common structural features, which you would see are having a tandem distributionof globular and rod-like and coiled domains.So, that is what is a laminin and this is the general representation of the lamininstructure.So, other than these we also have a proteoglycans and glycosaminoglycans.So, glycosaminoglycans are the most abundant heteropolysaccharides, which are seen andthey are basically long unbranched polysaccharides with repeating disaccharide units.So, instead of having random chain, you would have disaccharides, which are repeated.And then these are highly negatively charged with extended conformation that can impartviscosity.They also show very low compressibility.So, because of these properties they are used as a lubricating agents in the joints.So, you would see these material like hyaluronan and so on, chondroitin sulfate in the joints.And these, all of these occur in different tissues, you can actually look it up on thenet and you would see that the tables which explain where these can be found.So, some of the common glycosaminoglycans are hyaluronic acid, dermatan sulfate, chondroitinsulfate, heparin, heparan sulfate and keratan sulfate.So, these are all commonly seen in your body.So, proteoglycans are basically, glycosaminoglycans that are linked to core proteins which arerich in serine and threonine.They make sure that the fluidity of this, of the tissues maintained and they provideresistance to compressive forces.So, they play a crucial role in making sure the tissue maintains the gel like structurewhich it, gel like feature, which it has.So, when we talk about ECM, we also need to understand that ECM is not a static matrix,it is getting reorganized.So, you can have proteolytic degradation to remove the ECM.So, some of the enzymes, which are involved in this are matrix metalloproteases and serineproteases.So, they will degrade the tissue and as I already said many different types of cellsare involved in secreting these ECM matrices to actually continuously remodel this tissues.
Video 3: ECM in Tissue Engineering
So, when we talk about ECM in tissue engineering what people are doing is; people are harvestingECM for tissue engineering applications.So, they take the tissue and so, either it can be allogeneic or even xenogeneic ECMsand they decellularize it, and it is well tolerated by human hosts, because ECM componentsare well conserved.So, decellularization is very crucial, because the cellular antigens are actually foreign.Even if you get it from an allogeneic source you would have to make sure that there isproper tissue type matching, if you are going to have the cells along with it.So, if there is no cells then it cannot trigger immune responses or inflammatory responses.So, for that reason you have to carefully decellularize it, there are many techniquesto do it, to remove all the cells ok.So, decellularized ECM can actually be an off-the-shelf product.So, this can provide a favorable environment for constructive remodeling.These can be seeded with autologous cells before implanting if you want.So, then you have a patient specific personalized medicine right.So, because ECM is common for everybody then you do not have to worry about that.Now, the cells which can create the immunogenic response are from the patient himself, whichmeans there is no risk of rejection.So, you can tailor it that way.So, once you have it inside, then in vivo environment will determine the remodelingof the ECM.So, people have done in vitro experiments to study the effect of the possible stressessuch decellularized ECM can go through.So, there are different techniques to do it, but what is a fundamental goal of decellularization?What do you want to accomplish, I said, why we want to accomplish that, but what do youwant to accomplish?what are all the things which you want to remove when you are talking about decellularizationok.So, you want to prevent any immune response, for doing that what are all that you shouldremove?removing cells is the general thing, but.receptors There can.Cell receptors.Cell receptors, ok.Antibody.So.Antibody.Antibodies so.Cell debris.Any cell debris, ok.So, that is actually what you want to do, but specifically, what you are try to do is;you want to remove all cellular and nuclear material.While you are doing this, you want to maintain the composition and mechanical propertiesand biological activity of the ECM.So, that is the challenge.So, removing all cells is not too difficult.See, I can always take an ECM and dip it in sulfuric acid right.So, the idea is to remove the cells, without damaging the ECM.So, how you go about doing that is the process impart, which makes it unique.So, there are a combination of mechanical, physical and enzymatic processes which aredone.So, mechanical process would be the simple delamination of layers.So, this is nothing, but just stripping of layers.So, you take out one layer and then strip out the first layer, which might just be,it might contain too many cells, and you take that out, and then you can have physical thingslike sonication, freezing and thawing and so on right.So, sonication will destroy the cells, will just rupture the cells and you can freezingand thawing can also do the same, have the same effect.So, these are just cell disruption techniques.And then you can use enzymatic treatment like trypsin.So, trypsin can remove the cells from their adhesion sides and trypsinization is doneeven when you do cell culture right.So, you can so trypsin treatment, and then you can also use a chemical treatments like;detergents and ionic solutions.So, the other commercial materials are just patches you take the skin and you take thecollagen crosslink it and use it as a patch right.So, unlike that this is slightly different, ok.