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Module 1: Hydrogels, Bioceramics and Scaffold Fabrication

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Video 1: Overview of Scaffold Fabrication Techniques
Good morning everybody, today we will be talking about Fabricating Scaffolds for Tissue Engineeringapplications. We have looked at some of the materials which can be used, we have lookedat the natural polymers and synthetic polymers. So, we will also be talking about other materials;however, today we are going to talk about how scaffolds can be fabricated. So, as youall know from the previous lectures, we want to prepare a scaffold which can actuallymimic the extracellular matrix. So, the extracellular matrix support cellsto adhere, grow and it also provides cues and signals which help in the cells to performthe way they do. So, this helps in making sure that the cells integrate to form a tissue.So, when we are engineering this tissue, it is crucial that we prepare a scaffold whichcan mimic the functionalities of the extracellular matrix. So, let us try and recollect whatare all the properties that we look for; first thing is it needs to be bio-compatible.So, that is decided based on the material you choose. So, that would not be a part ofthe fabrication strategy itself, if you are looking at what parameters have to be broughtinto fabrication, then we have to see what else are there. One of the things which iscrucial is the surface area to volume ratio. If you have a high surface area to volumeratio you can load more cells, which means there will be higher cell density and thereis a higher chance of the tissue functioning as it should. So, creating tissues which havethis kind of a high surface area to volume ratio would be very effective.Another factor is pore interconnectivity. So, when you create pores in a scaffold, youneed to have them connected to each other. Why is this crucial? When you have good poreinterconnectivity you will have better cell infiltration which means the cells can actuallygo inside the pores and populate the tissue to form a living tissue. So, creating an interconnectedpore would be advantageous, this can also help in formation of blood vessels. So, thisway you would have sustained nutrient supply and toxin removal. So, this is an importantfactor which needs to be considered as well. Other than this, you also need to make surethat the material is fabricated into the shape and properties which fit the tissue whichyou are looking for. So, taking all these into consideration, you have to design strategieswhich can help in fabricating scaffolds. So, today we will talk about some of the morecommonly used strategies for fabricating tissue engineering scaffolds.These are some of the strategies which we will be talking about today. The first oneis called as a leaching method. So, this is a broad category within which there are multiplestrategies which can be used for fabricating scaffolds. So, the three strategies whichare commonly employed are solvent casting and salt leaching or ice particle leaching,gas foaming and salt leaching. So, what is done here is, a scaffold is prepared and gasforming salt, which is leached out to create porous structure.So, we will see how it is done and how the scaffold would look like if this strategyis used. So, the next type of scaffolds fabrication strategy is using microspheres. So, you cancreate microspheres, using different strategies it could be just a simple thing like preparingalginate beads. So, which you might have done as part of one of your undergraduate lab programsor you can create macroporous beads using different strategies and you can also havethese beads aggregate to form a scaffold creating a porous structure. So, these are differentstrategies which have been employed to create a scaffold using microspheres of differentpolymers, it could either be synthetic or natural.The next strategy is called as a phase separation strategy. So, here you have two techniques;one is the freeze-drying method and there is also a thermally induced phase separationmethod. So, by creating a phase separation, you can actually create porous structuresand this also will provide good interconnected pores. So, we will see more about freeze-dryingmethod and see how exactly the scaffolds would look like and what is the principle behindthe freeze-drying method. So, you also have fiber-spinning strategies;so, this is where you have different textile-based fabrication methods which are employed intissue engineering. So, nanofiber electrospinning, microfiber wet spinning and nonwoven polymerfibers are all some of the commonly prepare, commonly used methods for preparing fibrousscaffolds. In many of the extracellular matrix you have a fibrous structure.So, creating a fibrous structure using these kinds of spinning techniques can actuallymimic ECM very effectively. In case of electro spinning where you create nanofibers you wouldalso have a significantly large surface area to volume ratio which can help in cell adhesionand proliferation. So, we will talk about electrospinning strategy and look at how thosescaffolds will look like as well. So, next strategy is called as decellularizedmatrix. So, this is one of the common; one of the growing strategies which is being lookedat. So, where you actually take the extracellular matrix from the native tissue this could beeither from a human donor or also from non-human sources from other species. So, what you dohere is you remove the cells and all the cell-based components.So, this is done through different methods, it could be using some chemical treatmentor physical treatment followed by complete removal of all the toxins. So, this way youcreate a matrix which resembles the native tissue. So, here the major challenge is actuallyto remove the cells and its components without damaging the integrity of the extracellularmatrix. So, if you can do that, maintain the mechanicalproperties, physical, chemical and biological properties, then you would have an extracellularmatrix which probably has all the desired functionality and thereby it will be ableto provide all the biological cues as well. So, this would be an ideal strategy to lookat; however, it is a major challenge to make sure that the properties of the scaffold isnot lost during the decellularization process. So, we will not be talking about this today,but you can read about this on many of the papers which are readily available. So, thelast and the up and coming strategy which is, which people are looking at is 3D printing.So, 3D printing is a novel technology where people are looking at how scaffolds can beprepared by bioprinting the scaffolds. So, here an ink is used. So, the ink will basicallycontain the material which you want the scaffold to be formed with, and this can be used forfabricating scaffolds based on the design we need, using auto CAD and other CAD-basedsoftware. So, here you can also load cells along withthem and print the 3D print, 3D structure for the tissues. So, we will be talking aboutthis in a future lecture. So, there is also another strategy which is called as self assemblyso, that we will also be discussed in detail in a later lecture.So, now let us look at some of the more traditional strategies which are the leaching methodswhere we will be talking about the solvent casting and salt leaching and also the gasfoaming and salt leaching method. So, we will first start discussing this.

Video 2: Scaffold Fabrication Techniques
Let us first talk about solvent casting and salt leaching method. So, this is an exampleof how you would use this method to prepare a scaffold. So, in the protocol given youhave three things; first is the solvent which can dissolve the polymer and the salt whichyou are trying to use for this method. So, the polymer chosen here is PLGA and thesalt which is used is just NaCl or sodium chloride. So, these three are poured intoone vessel and they are mixed to create a homogeneous mixture. This homogeneous mixtureis then poured into a silicon mould where you keep it and evaporate the solvent. So,once the solvent is evaporated you now have a solid structure so, which will have theshape as the silicon mould. So, here they have shown a disc like mould. So, you wouldend up with disc like scaffolds. So, now this scaffold which you have containsthe polymer and the salt. The salt because it was a homogeneous mixture while it wasin solution, the salt crystals would have formed in different parts of the polymer scaffold.So, now, the next step is to leach the salt. So, this is done using DI water. So, you placethe salt polymer scaffold into the DI water and mix it for 48 hours, by doing this thesalt will actually get dissolved out and you will end up with a disc shaped scaffold whichis only the polymer. So, now this can be kept in a vacuum ovenafter freeze-drying to remove all the leftover water. So, thereby you now have a scaffoldwhich is prepared through solvent casting and salt leaching. So, how does this createa porous matrix. So, the salt which was present would have crystallized and formed salt crystalsin the polymer mixture in the polymer blend. So, this salt when it dissolves out or getsleached out, the area occupy, the volume occupied by the salt crystals will now become pores.So, these porous structures are used for cell attachment and culture.So, these kinds of cells scaffolds would look like this under a scanning electron microscope.So, what you see here are images of salt leached scaffolds. So, you can see nice structureswhich are basically the regions where the salt crystals were occupying. So, when youlook at this SEM images, you would also see that there are some areas which are dark blackwhile some are grey indicating that there is a 3D structure and there are crystals whichhave been formed all over, creating such pores. However, one of the limitations you wouldsee is in many of these pores there is very poor interconnectivity.For example, you look at this particular pore. So, this pore actually is fully covered, youactually have a region where cells can go and attach, but they cannot penetrate intothe scaffold if they go into this pore. So, this would mean the inter connectivity mightnot be excellent when you are talking about solvent casting and salt leaching method.To overcome this limitation people have looked at gas foaming and salt leaching method. So,this also uses very similar principle; however, instead of using a simple NaCl kind of salt,you use a salt which can, which can cause gas to be generated. So, here what they havelooked at is, here is an example. So, where they have prepared a polymer gel using non-solventprecipitation method and the polymer gel paste along with the ammonium bicarbonate salt particlesis mixed and you pour it into a Teflon mould to create a semi solidified polymer salt complex.So, this semi solidified polymer salt complex is then added to the acidic aqueous solutionor to hot water. So, what happens when you do this is the ammonia and carbon dioxidegets generated and starts effervescing, getting released into the water. So, from the saltpolymer blend you now have the gas leaving and so, this leaves a porous scaffold. So,you end up with the macroporous scaffold which can then be freeze dried to actually be storedfor further use.So, the scaffold which you have prepared using gas foaming and salt leaching method wouldlook something like this. So, what you see here are pores which have good interconnectivity.So, because the gas is actually pumped through the pores, you would have nice interconnectivityfor the pores. So, you can see that there are lots of pores,you look at the SEM image. So, you see that there, this is the pore and these are someof the pores. And as you see most of these pores are very deep and even in this particularpore you would see that are smaller pores which are much darker in color indicatingthat they are much deeper and they are actually probably interconnected to some other pore.So, this kind of highly interconnected pore can actually be very useful when you are talkingabout tissue engineering applications. So, as I had already mentioned cells will be ableto infiltrate, you would also be able to get blood vessels forming and also it can helpin good diffusion in early stages of any tissue being implanted, nutrient supply has to comethrough diffusion. So, if you have such highly interconnected porous structures. So, thediffusion will be effective thereby it would not be a limiting factor when you are talkingabout tissue formation.Let us move on to the next strategy. So, one of the strategies which I mentioned is formationof microspheres. So, here we will be talking about one of the three or four different strategieswhich have been used, where microspheres have been used to prepare scaffolds. So, what isdone first is the polymer is dissolved in a solvent and then you start dropping it intoa solution. First this polymer is dissolved and then it is actually dropped through asmall pore using a may be a syringe pump or something. And then it is being mixed, itis stirred and then it is centrifuge to remove these beads, this micropores, this microsphereswhich are washed and then freeze-dried.So, these freeze-dried microspheres can form structures like this. So, as you see theseare nice spheres which are present and you see the cells which have adhered on this aswell. So, these images which you see on the scaffold are actually cells which have beenadhere to the scaffolds. So, because you have these microspheres whichhave assembled to form the scaffold you also have porous structure which is present inbetween this spheres. So, this can help in infiltration of nutrients and also hopefullycell infiltration, ok. So, this is what is done for preparing microspheres.Moving onto the next topic you have freeze-drying. So, freeze-drying is one of the most commonlyused methods for creating a scaffold which is highly porous. So, what you do here isyou basically create the polymer solution and you pour it in a mould and this mould,this polymer blend is actually then frozen and kept for freeze-drying. So, freeze dryingis also called as lyophilization and in after freeze drying it is washed and dried, youcan also re-lyophilize it to make sure that you have a completely functional scaffold.So, when you observe these scaffolds under a scanning electron microscope you would,it would look something like this. So, these are actually scaffolds which we were preparedin my lab where a isabgol, one of the carbohydrate polymers was actually lyophilized to formscaffolds. As you can see it is a highly porous structure and you have huge pores which arevery nicely interconnected as well. So, this kind of a highly interconnected structurewhich you can see in the magnified images which are highly porous this kind of structurecan help in cell infiltration and also with nutrient diffusion.So, this is one of the common strategies which is used, We will look at the principle whichcauses this kind of porous structure. So, in the earlier cases, where we looked at saltleaching or gas foaming, it was quite simple we had the salt crystals, which are presentwhich got washed away creating the pore. You had the gas forming salt which then releasethe gas which could be ammonia and carbon dioxide in the example we showed; so, thiscause the pores. However here where are the pores coming from? We only did lyophilizationfor that reason you need to understand what the process of lyophilization does.So, let us look at the phase diagram for water. So, this is a simple phase diagram where itis between, the plot between pressure versus temperature for water. So, you have threephases, you have the solid phase, you have the liquid phase and you have the gas phase.So, these three phases are existing in the given temperature and pressure conditions.This point is the triple point; so, the triple point is where you would have all three phasesexisting in equilibrium. So, you also have these curves which are the vapor-liquid equilibrium.So, this would be the gas-liquid equilibrium curve and you would have the solid-liquidequilibrium curve here and the gas-solid equilibrium curve here. So, these are the curves whichyou have in this phase diagram. So, now when we are talking about lyophilizationwhat do we exactly do? See when we prepare the polymer blend and we actually pour itinto a mould. So, you have liquid water. So, this liquid water if it is evaporated wouldbe the process where you have liquid under some condition moving to gas. So, this isthe process of evaporation. However, when you are talking about freeze-drying, whatyou are actually doing is not evaporation you are removing the water at very low pressures.So, what you do is you freeze the water to form ice crystals and these ice crystals aredirectly; so, these ice crystals are directly sublimated instead of being evaporated. So,it is not first condense, it does not actually melt to form water and then get evaporatedinstead these ice crystals directly sublimate to form water vapor.So, how is this accomplished? This is done because at very low pressures in this region,where you see the gas-solid equilibrium curve here if you actually have the conditions wheresolid can move to gas under this condition under these pressures, then you would endwith; then you would end up with sublimation instead of evaporation.So, what is done is you actually cool the water. So, that it solidifies and then you reducethe pressure to such a large extent that this solid ice crystal then becomes a gas. So,by creating a vacuum and reducing the pressure significantly, you can actually convert solidice crystals into gas. So, this way you are actually be creating what you would see withsalt leaching or gas foaming techniques. So, what you have done here is you have createdice crystals within the polymer blend and this ice crystal is then evap, is then sublimated;So, causing something like a gas forming effect. So, this results in formation of the highlyporous structures. So, this is one of the most commonly used methods for forming scaffolds.So, let us move on to the next topic, which is electrospinning. As I already mentioned,some of the extracellular matrix is fibrous in nature. So, it is important to create astructure which mimics the natural ECM. So, for this reason people have looked at someof the textile-based techniques such as electrospinning for creating scaffolds. Electrospinning createsnano-fibrous scaffolds which have been studied extensively for a variety of different applications.So, let us look at electrospinning here. So, what is electrospinning? Electrospinning isa technique where you apply a high voltage to create very thin fibers from a polymersolution. So, what you do is, you create a polymer solution which is loaded to a syringeand this syringe is pumped using a syringe pump. So, that the polymer solution comesout of a needle, this polymer solution which comes out of the needle is exposed to a highvoltage and because of this high voltage it would actually form very very thin fibers,these thin fibers are collected on collector which could either be a flat surface or itcould be a rolling drum and so on. So, here the parameters which can be variedare the flow rate at which the polymer solution is pumped, you can also vary the viscosityof the polymer solution and you can vary the diameter of the needle, you could vary thevoltage which is supplied. There are also parameters such as humidity which can playa role in how these fibers are formed and based on the distance between the collectorand the spinneret you would also have the thickness varying.You would, another factor which can be looked at is the different types of collectors whichare used. By using a flat collector, you would be able to get nanofibrous mat which is notaligned. However, if you were to use a rolling drum or something you can actually createaligned fiber.So, the scaffold when you look it under scanning electron microscope would look something likethis, what you see on the left is non-aligned electrospun fibers and what you see on theright is the aligned electrospun fibers. So, these are some of the commonly used techniqueswhich are used for scaffold fabrication. So, we have given an overview, we have notgone into great detail of each of these techniques. Depending on the polymer which we have chosenand the application for which we are working on, we have to choose fabrication strategieswhich will provide scaffolds which mimic the natural ECM. With this we will move on tosome of the more advanced techniques which are being currently researched for scaffoldfabrication, which would be self assembly and 3D bioprinting.So, self assembly will actually we discussed by Ramya one of my students, she is currentlyworking on her PhD, she will describe how self assembly can be used in tissue engineering.Her research is focused on self assembly, so she would be able to give a very nice perspective.See you all in the next lecture. Thank you.