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Module 1: Hydrogels, Bioceramics and Scaffold Fabrication

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Video 1: Approaches to 3D Bioprinting
So, today we are going to talk about 3D Bioprinting. So, this is one of the more advanced technologieswhich is out there where people are trying to fabricate scaffolds using this. So, thisgives a lot of advantages. You can actually get more precise microarchitecture, if youwere to use 3D printing appropriately. So, here in this lecture we will actuallytalk specifically about 3D bioprinting. So, 3D bioprinting is slightly different from3D printing. So, in 3D bioprinting you will be using cells along with the ink whereas,in 3D printing you would just print with a regular ink and then you will seed cells onit. So, we will talk specifically about 3D bioprinting today.So, when you are talking about designing of a scaffolds and tissue engineering constructs,there are two approaches to it. One approach is that top down approach where you basicallyprepare materials which resemble the shape of a tissue and then you seed cells on topof this and then let the cells grow and culture them for a period of time and then you implantthis tissue engineered construct into the body.The problem with this is you do not have control over how the cells are distributed and theECM micro environment may not be created when you actually do this. So, because if you wereto culture the cells along with the ECM being formed then there might be restructuring,remodelling of the ECM, which might not be present in a top down approach. Whereas, bottomup approach is where micro or nano size blocks of cells and the biomaterials are actuallyused to build that tissue. So, what you have is small, very small structures which containcells themselves and you combine these in a way to provide, to create the ECM whichwill act as a scaffold. So, this can provide a better control overcell distribution and also the ECM micro environment. There are many techniques to do this. Someof the techniques are soft lithography, self assembly and 3D bioprinting. So, today wewill talk about 3D bioprinting. In tomorrow’s lecture we will be covering self assembly,ok. So, the other techniques which we looked at earlier where all the top down approach,where we were talking about freeze drying or solvent casting and salt leaching.All these techniques are top down approaches where you create the scaffold independentlywithout using the cells, ok. So, self assembly can be both ways. Just like how 3D printingcan also have a top down approach and a bottom up approach, self assembly can also be topdown or bottom up.So, we will talk about 3D bioprinting today. So, this is an additive manufacturing or 3Dprinting technique to form complex functional living tissues. So, this would include a bio-compatiblematerial which is used along with the cells and you might even add supporting components.For example, growth factors could be blended along with the material and the cells andyou could print it along with that. So, it is possible to do that people have tried todo that. So, conventional 3D printing is used to createcell free scaffolds. So, that is what the 3D printing club here is, right. So, thereis a 3D printing club where people just print 3D structures. And those are just cell freematerials. You can print it in any shape you want, and you can actually print it in theshape of an organ if you choose to. But in bioprinting you would want to print it alongwith the cells, you would want to load the cells and then print it.So, the complexities which are compared to non biological tissues are much higher andwe need to understand some of the challenges associated with 3D bioprinting, so that wecan actually adopt the 3D printing technology to print biological tissues. So, we need toknow what materials can be chosen, what cell types you are planning to use, whether youare planning to use growth factors and differentiation factors. So, all these things will regulatewhat type of printing technology you can use and what would be the conditions you can usefor printing and so on. The sensitivity to living cells should alsobe taken into consideration. Mammalian cells have, are sensitive to shear stresses, soyou would want to use something which is, which will not cause destruction of the cell.And you felt ultimately need to understand the construction of the tissue is going tobe much more intricate than a macrostructure which you would try to print. Printing a penor a gun is not the same as printing a tissue, right. So, you are, the microarchitectureis very different. So, that makes it a quite complex to actually develop 3D printing technologiesfor biological tissues. Currently people have tried to print different tissues using 3Dprinting.So, some of these have actually been reported in literature. You can look up the literatureand you will be able to find all the applications which I have listed here skin, bone, vasculargrafts, tracheal splints, heart tissues, cartilage have all been printed using 3D printing. Thetissue models for research and drug discovery and toxicology have also been printed. Theseare just models where you would just do studies or research on it rather than will take itup for implants. So, the advantage of this technology as Iwas saying is there is a potential for accurate cell distribution and high-resolution celldeposition. And it could be a very scalable process depending on the type of 3D printingtechnology you use. And it could also be cost effective, again based on the technology use.But the major challenges are sensitivity to living cells and vascularization and innervation.The aspect of vascularization and innervation is a common problem for any aspect of tissueengineering; however, the sensitivity to living cells is more unique to 3D scaffolds, right.So, if you are going to have something like self assembly which is again a bottom up approach,but if you are, self assembly will use materials which are, which the cells are used to, andyou do not have to provide very toxic environments or like very extreme in conditions for creatingthe self assembly, whereas, here you might have to provide that kind of an environmentfor the printing to happen. So, that makes it very unique challenge to be overcome.So, when you are talking about 3D bioprinting, these are the things which you have to lookat. So, we will talk about pre-processing and processing. Post processing is kind ofcommon for all tissue engineered constructs and application is also the same.So, pre-processing involves imaging and a 3D modelling and slicing. So, you first needto understand how the tissue architecture is and that you get from images. So, you canget images from X-rays, MRI, CT scans, ultrasounds, optical microscopes whatever is at your disposalyou can use them to create images which you can process to get the 3D structure. And this3D structure would have to then be modelled. So, that it can be fed to a computer to beprinted, right. So, that is the pre-processing aspect.And during the processing aspect which is where you are trying to print the tissue,you, there are two major things which you need to work on, one is the bioink. So, whatwould be the ink you use, right. So, that is basically the material on which the cellsare going to be cultured. So, identifying the bioink is a major challenge and then thatprinting technology itself which printing technology would you want to use. So, thebioink preparation will also depend on the printing technology. So, depending on thetechnology you use, you would have to design a bioink which will be suitable for that printingtechnology. Post processing is common, so anytime youcreate a tissue engineered construct you are not going to have enough cells on it, right.You are probably just going to have cell seeded on it, so you might want to expand the cellsby culture, cell culture. In cases of using stem cells you might want to differentiatethem to form the specific cell functionalities and cell phenotypes. You also might want maturationand, of tissue and organ which would require vascularization and so on, so which mighthappen in vivo. So, these are post processing things which will be done for any tissue engineeredconstruct. So, it will also be done for a 3D printed scaffold.So, finally, you take it to the application whatever the application could be. Here theyhave just listed two applications, one is being transplanted to the patient and theother is being used just as a model system for studying in vitro cultures and so on.So, this is the general idea of 3D printing. This is the process in which 3D printing istaken up. So, let us go through some of the steps and see what exactly can be done.So, first thing is imaging. So, there are different ways you can image a tissue. So,we can use CT scan, MRI, ultrasounds, X-rays for bone tissues and so on. And you can usesimple video systems can also be used and then you can process these things. All ofthese have their own advantages and disadvantages. A 3D scanner is something which can be usedand it is quite simple to use. It is less expensive to use this process; it is veryfast to create this image it can scan something very quickly and you would be able to reconstructthe image very quickly. Devices are commercially available you can buy that and use it.But the disadvantage would be you will get only superficial data. You will not get theintricacies of that tissue. So, for creating that you might want to create multiple crosssections, then process them together which will add to the complexity of the processitself. And oh sorry. Sir, is a 3D scanner is same as ultrasoundscanner. No, no, no it is not. Ultrasound. So, 3D scanneris I do not know where you would have seen something. So, it is something like what yousee in an airport. Airport, it has scanner which is used to detect what is in, it islike, more like an X-ray scanner, but this is a 3D scanner which would look like that.So, it just scans tissue or an organ all over and comes up with the 3D structure.It is like sir, actually I have seen it, it is a portable device you hold on hand. So,now, let us say a object. So, you, it is like program such a way like you move it, you justscan it through all the direction it will build a image accordingly.Right. So, that. These type of scanners and there are alsoscanners where like it is a scanner and you put a object inside. So, it will scan accordinglyfrom straight way. So, it is it is like a regular scanner whichscans a surface, here it just does it for 3D structure. So, you can have it in any sizeand shape, so I. And the problem with this is its very low accuracy. It will give onlylike 5 millimetre accuracy, but you are getting such a fast processing, so you will, you willhave to compromise on the scanning ability. So, if you are going to use a 3D scanner foran, for a tissue microarchitecture you are probably not going to get any information.Like 5 millimetre is actually too large resolution to actually use it for biological tissues,but the acquisition speed is very high. So, this can be used primarily for non-livinglike structures. So, if you are going to have only macrostructures which are there, thenthis kind of a scanner might actually work. So, CT scanner has high resolution in bonetissues and it is more accessible than MRI. So, it is commonly used for scanning. So,MRI is quite expensive and it is not very easily accessible. But the problem is, thisis also expensive, it is not that it is very cheap to perform a CT scan. And ionizing radiationwould be used while performing CT scan. So, the resolution is better, it is, it has about150 micrometre resolution and it can take about 30 minutes to acquire the image andyou would be able to get enough information for organ constructions. So, if you are lookingat smaller tissues even the 150 micrometre might actually not be sufficient for gettingall the information about the microarchitecture. So, you might want to do, you can do MRI orultrasound. MRI is very high resolution and using a contrasting agent in soft tissuesand it can actually help you in studying the hemodynamics as well. So, you can actuallyidentify where the vascular network is and try to emulate that while you are trying toprepare the scaffold. So, however, it is expensive and it cannotbe used in patients. So, if you are going to use it for, as a personalized medicinething. So, you cannot actually use it on patients for who have a metal implant, so that wouldrestrict the usage. But it gives very high resolution. So, to about 1 nanometre. Andit can take about an hour to do this. And not everybody is also comfortable going throughan MRI. So, has anybody gone through an MRI, ok. So,like if you have claustrophobic. Yeah, you have done, ok. If you have claustrophobic,you would not want to do an MRI. You will actually get really scared, you have put inthe tube and you are trapped in there for like an hour and you are expected not to move,your, it is quite a daunting thing for a person who is claustrophobic.So, ultrasound is a more widely used technique it is quite easy to use. You just use a smallscanner and you can scan the person and you will be able to see the tissue. It has lowresolution. Depth perception can actually decrease if you increase the resolution. So,it has about 10 micrometre resolution and can take in few minutes, but it can againgive outer structures, like inner structures can actually become very difficult becausethe resolution will get poorer as you do that. So, video systems have also been tried tobe used, so a real time video and images are processed. You take the superficial data andyou process it and try to build a 3D structure out of it. So, the image processing aspectof it plays a major role in constructing the 3D structure.So, these are the different imaging techniques which are used. But after you get the imaging,you still have to come up with the internal architecture. You might not have all the detailsof the internal architecture with just the imaging that we have done. So, you would stillhave to fill the voids using the architectures for which building the 3D model is done throughdifferent approaches. So, some of the strategies which are used in designing the internal architectureare CAD based designs, image-based designs, free form design, using implicit surfacesand the space filling curves. So, I will not go into details of all of these.So, I have actually given a couple of references at the end of this lecture. So, these aretwo very good review articles which were very recently published. I will try to upload themto moodle as well. You can go through them, if you are interested in getting the detailsof all these processes. So, the current strategies which are beingused have limited design flexibilities. It is not like you can get exact microarchitecture.So, that is the problem. So, although 3D printing theoretically can give you the exact microarchitecture,you still have to draw that microarchitecture onto a computer. That has to be some way todo that and those aspects are still not very accurate. People are trying to work on it,people are trying to use different things to get something which would be close.So, CAD-based design, there are three major approaches which are used in CAD-based designs.So, this is one of the more commonly used strategies for designing the internal architecture.So, constructive solid geometry modeling is one, which where you use solid primitive structuresand Boolean operations to build the actual structure.So, if you were to take two structures, let us say you have a cube and a sphere. So, nowyou can actually combine these two in different ways to get different structures. You canactually add these and you would get something which basically has a sphere here or you coulddeduct this, delete this and you will end up having something like this where you havethis as the structure, right. So, it is possible to do either of this. And you can also havean intersection which will only be a portion of this sphere and so on. So, this is justfor sphere and cube. You could have other solid primitives which you can bring combinationsthereof using Boolean operations to get different internal architectures, so which, which canclosely resemble what you would see in a real tissue.Another strategy which is used is a boundary representation. So, here the boundary elementsare used. So, it is basically the surf, the line between the solid and the non-solid istaken as the boundary and this is used to define the geometry. And spatial occupancyenumeration is another strategy which is used. So, this basically represents solid objectsinto cubic elements. So, you know pixels, right. So, what are pixels? The tiny squareson a 3D, on a 2D surface, right. So, if you take an image, you look at how many pixelsare there. So, your screen resolution is usually given in terms of pixels per square inch orsomething, right. So, similar to that, for 3D structures, thereis something called voxels. So, these are 3D cubes which are used. And you can organisethese 3D structures to get the desired shape, ok. So, if your voxel is very very small thenyou can get very nice 3D structures. So, these are just some of the strategies which areused for designing the scaffolds using 3D printing.There are also other modeling aspects which can be incorporated into 3D printing. It isnot just for bringing in the internal structure, you can have, you can apply find, apply finiteelement analysis method to evaluate the mechanical and fluid flow properties and diffusivityof a scaffold which you would be printing. You can also investigate the impact of matrixdegradation just looking at how the, how the scaffold will actually change due to this.So, computational fluid dynamics can also be used to study the permeability, and itcan also be used to design scaffolds based on the shear stress, mass transfer and theother microarchitectural parameters which should be there because of the fluid flowinto the system, ok. So, these are some of the modelling aspects which people have triedto incorporate as part of the 3D printing technology.
Video 2: Bioink
So, these aspects are the, whatever we discussed till now are the aspects where you would needexpertise in image processing and computational techniques and so on. So, the aspect whichwe are covering here is where you would need people with more chemistry and materials background,right. So, you need to desert, design a bioink. So, whatever your printing is basically hasto be some kind of an ink which you print it out, right. So, this bioink is nothing,but a printable material which will consist of various biologics.It could be just the cells, or cells and media with serum, genes, proteins, growth factors,whatever, right. So, that would be the bioink which you have to use. So, this can be cellswith biomaterials or only cell aggregates as well. So, you have scaffold free bioprintingwhere people have tried to use only cells which are suspended to be printed, but theproblem with that is usually the mechanical strength is not very good. So, you would notbe able to get a strong scaf, strong tissue which you can use for replacement; however,you would have very high cell density. So, it is a trade-off between the two.Material properties which you need to consider while you are designing a bioink is printability.So, whether the ink is actually printable, whether you will be able to load it, and whetherits rheological properties will actually help you in printing the tissue. Then, you haveto look at the degradation factor. So, whether how well this material is going to degradeand whether it will degrade during the process, so then and so on, and obviously, compatibility.So, these three aspects have to be chosen carefully to identify the appropriate bioinkfor a 3D printing technology.So, in designing a bioink the major challenge is embedding cells into the bioink, right.So, you need the cells basically encapsulated by the bioink when it is being printed andthat can actually be a challenge. So, an ideal bioink would require no pre or post processingafter printing, you should not have to treat it with chemical agents to crosslink or providesome physical stresses for it to crosslink to form a solid structure. It should be ableto form this kind of a solid structure while it is being printed. So, that is going tobe a requirement for getting an appropriate 3D printed construct.So, you need to have a material on which everything can actually be homogeneously mixed. So, becauseyou are, you might end up with something which is viscous, right. So, these inks are notgoing to be like aqueous solutions, these are going to be viscous solutions. So, whichmeans you need to have an ability to disperse things homogenously, otherwise it would goingto have them loaded in one part and it is not going to print uniformly or print theway you expect them to print. So, you also have, to have reduce cell encapsulationtime; in the sense that it should not take a long time for the cells to actually be engulfor dispersed in this and if that is going to be the case then the cells are probablygoing to die. So, you would want it to have a short encapsulation time.For creating a cell-laden bio ink, cells and other water soluble components are usuallysuspended in an aqueous solution and their concentration in which it is being dispersedand other things which are, other components which are loaded can actually have a seriouseffect on the stability and the strength of the final construct which is printed. It canalso delay the crosslinking time, it will obviously, affect the swelling and gelationproperties. So, while you are preparing these inks, preparing the appropriate aqueous media,aqueous solution also plays a role. It is not just about the polymer you use.So, when you are trying to print, use a bioink, you are actually trying to mix cells uniformlywithin the bioink solutions most of the times. So, when you try to do this, you need to understandthat if the bioink solution actually has many chain entanglements, it will be difficultto break this down and suspend cells uniformly. So, you want the bioink to actually crosslinkas soon as it is coming out of the printer, right. So, only then you will have a solidstructure. So, you would want it to have some entanglementinitially, you cannot expect all the entanglements to happen, right after being printed. So,if you are going to have these entanglements initially, it needs to be in an optimisedway, so that cells can actually be uniformly dispersed, it cannot completely affect thecell distribution, cells, cell distribution cannot be just in pockets, that will be aproblem, ok. Cells do not attach on the. Sorry. Even if any, initially cell distribution ispocketed or fragmented, but later on when cells are proliferated, it will be uniformlater on, right? It depends on how the crosslinking has happened,right. So, if the entanglement is very strong and you have a lot of crosslinking, cellswill not be able to infiltrate. Ok.So, that is the challenge. So, cells do not attach on the surface of the crosslinked networkand are likely to accumulate in certain regions. So, it, it will not be dispersed if you haveto mean, too much crosslinking, but. So, for getting a uniform distribution people haveeven tried mechanical mixing systems. So, while you are mixing it using mechanical mixingsystems, you need to make sure that the shear stress which the cells encounter is lowerbecause the cells are shear sensitive and you would not want to just keep rotating themin a flask for a long time. So, in addition it should also be done verycarefully, so that you do not have any bubbles. If you have bubbles it affects the printability.So, while it is being, while the ink is being printed you know, you should not have anybubbles, it should be a uniform flow of the ink. So, these are things which need to betaken care of while you are preparing a bioink.So, the factors which you need to consider while you are preparing a bioink is the celldensity. So, effect of cell density on mechanical property should be taken into consideration.So, if you have very high cell density then your mechanical strength is probably goingto be low because you, the more the polymer you will actually have better mechanical strength,right. So, if you are going to have lot of cells you cannot have a lot of polymer too;so, your mechanical property will come down when you have very, a lot of cells so, yeah.Sir, do they really use any reactors or anything for the bioink preparation?I do not think they use it for bioink preparation. The reactors are used for the post processing.So, once you actually have the cell layered in construct then you can use a reactor toprovide signals, physical cues and other signals for the tissue to be developed into a maturetissue, but for preparing inks they do not use any reactors. So, so high cell densitycan be achieved by scaffold free bioink, bioprinting. You need to understand that the shear stresscan affect cell density. So, depending on the technique which is being used, the celldensity which will also be affected; depending on the 3D printing technique used the celldensity will also be affected. So, cytotoxicity of the ink has to be takeninto consideration obviously, when you are putting it. It is not just the polymer youare using; you might have a solvent; you might have other additives which are present alongwith the polymer. So, you would have to take all of that into account, while you are printing.So, the cytotoxicity of all the components have to be looked at.Bioprintability is an important aspect, whether it is actually a printable thing. The liquidform should be able to come out without clogging the nozzle. So, in certain types of printers,so we will talk about the types of printers later. But, in some types of printers youhave a nozzle which means the printer, the ink actually comes out of this nozzle. So,this no, this cannot actually clog the nozzle. So, if it clogs the nozzle then your whateverthe ink which comes out is not going to come out at the same flow rate, so the printingis going to be very different and as it clogs more and more it will completely stop theflow, ok. So, the ink cannot clog the nozzle. So, another thing is, this printability itselfcan actually mean different things for the different techniques which are being used.So, we will talk about the techniques. While we talk about the techniques, we will talkabout what exactly would be a printability for that specific technique, right. You havea question. Yeah. Regarding the printer nozzle is it possibleto engineer the nozzle instead of just the liquid so that, I am not sure that I do notknow anything about it, maybe the material or something or may be the shape, so thatit does not stick in; is not that possible? because it will be in a small scale. So, itwill be dealing with those kinds of problems. Yeah. So, see nozzle, you can design nozzlesin different ways you can actually look at using different materials for nozzles andthings. But the problem usually is you are, if you want very high resolution of printingthe nozzle is going to be very small, right. So, then clogging is a problem which you willface anyways. Ok.If you are, it is, if you are looking at the larger diameter tube then it is not a problem.If you are looking at micrometres kind of diameters then it is, it can be a problem.What kind of scale are they printing at these days?So, to about a few micrometres, resolution of few micrometres people have been able toprint. Are they able to place single cells?Yes. With laser, so laser technology people are able to place single cells. So, each dropletcontains a single cell. But laser printing has its own disadvantages. So, some of theproperties which govern the printability are the rheological properties, gelation kineticsand the surface tension of the bioink. So, all these things have to be very well characterizedand we need to understand how this will actually affect the printing process before we takeit for the printing process.And some of the process parameters that need to be considered are the fusion of the hydrogelswhile it is being printed and deformation due to gravity. So, if you are going to printa 3D structure it should just not collapse, right and there could be and it is quite possiblethat happens. Non-uniform droplet size or extrusion can happen because of how the processis being taken care of and concentration of crosslinker will also play a role in someof these properties. For example, gelation will be depend on the concentration of crosslinking.So, if you have high crosslinker concentration you will actually have a better printabilitybecause it will form like very strong structures after printed, but it will limit cell infiltration.Another thing is a final solidification of this construct after it is being printed.So, all these factors have to be looked at while you are printing a 3D structure.So, when you are looking at the rheological property is, obviously, viscosity plays arole when you are printing it. Another important property you look for is the shear thinningproperty. So, why do you want shear thinning property? So, what is shear thinning property?For non-Newtonian fluids shear force, there is the viscosity reduces.Ok. So, it is a non-Newtonian fluid where when you give a shear the viscosity reduces.So, Newtonian fluids and non-Newtonian fluids are two types of fluids. Newtonian fluid isviscosity is constant. Non-Newtonian; viscosity is not constant. There are different typesof non-Newtonian. So, shear thinning property is desired for 3D printing, why?Because you will be forcing the fluid through the nozzles, so we will be producing shearand it should not increase the viscosity also. It should have low viscosity.Yeah. It should, definitely it should not increase the viscosity.So, please do read this review articles when you get time.And we will take it from there, ok.