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Methods of Tissue Engineering

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Video 1: Approaches to Tissue Engineering
So, today we will continue with our discussion on the Introduction for Tissue Engineering.So, yesterday I had post a few questions.I hope you had gone back and read a little bit about it.So, we will start with the first question; which tissues do you think would be the easiestto engineer?So, yesterday you answered with a few.Did you find out why you thought those were the easiest?Or did you have a reason for that?Growing rate, how much time it takes.There is one good reason.So, it actually is, generally they regenerate well.So, it might be easier to do and you had mentioned skin, right ok.So, what else?Easy to implant.Ok So, somebody had also mentioned cartilage.Do you think cartilage regenerates rapidly?No.No, then why cartilage?So, what did you identify from literature?I thought the function performed would be much simpler than something like may be heartvalves.Ok So, functionally cartilage is simpler tissue.Yes, that is one reason why you would think it is easier to emulate that, ok.Any other tissues which you thought of?No.So, what about bones?No sir.Bones are also reasonably easier to regenerate compared to most other tissues like that.So, what do you think is the common factor which you saw between all these tissues?They are basically just barriers or supports; they are not functional tissues, right.So, they are, they can skin is just a barrier; bone is just a supportive tissue; cartilagesis also a supportive tissue.All those things are, as you said the function itself is simpler compared to other more complicatedtissues.So, which tissues do you think would be harder to engineer?Heart?Why?Because of the growth rate is.Regeneration is very slow.That is one limitation.Functionality.The function.The cells are already highly differentiated.So, functionality of a heart tissues is very high; along the along that lines can you identifyother tissues, which would be even harder than a heart tissue?Nerves, brains.Nerves and brains ok.So, brain as of now, I do not think anybody is trying to engineer a brain.So, people try to look at nerves and Lungs.Like peripheral nerve system, nerve systems and things like that.Lungs ok.So, why?why do you think it will be harder?Just asking whether is it harder.Ok.So?Lungs are very specialized right?Yeah.So, if you are talking about lung as an organ it is very difficult; obviously because ofthe architecture and there are too many things which else changing there.But, smaller tissues?Yes, it would be about the same level as a hard tissue.It would be the same level complexity as a hard tissue.What about something, think of something which is which has little more biological functionality?Pancreas.Ok.Why pancreas?Because it is actively performing.So, pancreas needs to secrete insulin and glucagon and it, while it senses the glucoselevels, right.So, it has very specific functionality.So, that this is going to make it harder tissue to engineer, so, similarly liver tissue.So, liver would be another tissue where you would still have similar challenges.So, these kinds of tissues which actually have to respond to stimuli are harder to engineerbecause there are some functionalities which you do not really, which you cannot easilyrecreate in an in vitro setup, ok.So, what has been the most successful clinical application that has been commercialized?First, I want you to identify which are all the commercial products that you are ableto find.*inaudible* I want to know the name of the product.Dental implant.Ok, I want to know the name of the product.I want to know what they were, derma graft, ok.Carticel.Carticel; carticel is for what tissue?Cartilage.derma graft is for skin.I something called OP1 putty but I did not know what that was.What?It is.What is it?Something related to the bone.OP1 putty?Yeah.Ok, I am not aware of it.So, o p.So, just OP number 1?OP number 1.Putty ok.So, it is spinal fusion from striker ok.So, basically has a recombinant human bone morphogenetic protein ok.So, what else?Have you seen any other commercial things?So, the commercial ones kind of align with what you thought would be the easiest tissuesto engineer, right.So, another major reason cartilage is actually easier to engineer is it is avascular tissue.So, you do not need to worry about vasculature.Creating vasculature is one of the limitations in tissue engineering.So, that can actually be overcome when you are working on avascular tissues ok.So, we have looked at some of the commercialized ones.Actually, the most established commercial product is the skin.The first skin, tissue engineered product which approved was a skin graft.So, I think it was called Apligraft if I am right.So, or no, integra.I don’t know, there were multiple things.So, the integra was probably the one we just commercialized first and approved by FDA.So, even with that it is still not a full success.Can you think of why it is not a full success?Sense of touch in that area.Ok, So, that would be one problem because innervation has to happen for the sense oftouch, sensory things to have be there.That is one thing.So, what else?It is not yet fully biocompatible.Ok, why do you think that is the case?May be due to immune reactions.So, dermagraft; Ramya you identified dermagraft, right?You know what it is made of?I did not write it down.Ok.So, it basically has a matrix on which cells are seeded.So, it is not an acellular thing.So, there is already you are going to have some cells from other sources which can causeimmunological reactions.So, there are acellular tissues as well which are used.So, I think MatriDerm is probably one product where you use a acellular matrix which doesnot have cells with it, but even with all that your reinnervation is one thing, yoursweat glands and this perspiration is one thing, where aesthetic aspects where you haveto have the hair growth or the matching the skin tone of the person, all these thingscan be a challenge.So, because of these things it is not a complete success, however it has been reasonably successfulto provide enough of a healing effect.And another major limitation you would see with most of these things are they are ridiculouslyexpensive.So, it is not like it can easily and readily be used by everybody for general use ok.So, we already labelled and identified a few other commercial tissue-engineered productsand all of them have their own limitations.One major limitation is the size of the tissue engineered construct itself which is becauseof the vascularization or the lack thereof; which leads to very little nutrient supplyor its supply has to come only through diffusion and that is not the most efficient way fornutrient supply or toxin removal ok.So, let’s move on.So, currently there are different ways to engineer tissues.So, these are the I have segregated them, but as of now people do not try to do themindependently.People try to prepare combinations.So, as I had already discussed the tissue engineering triad, it follows the same pattern.You could either use a material or cells or molecules which can trigger the regenerationof tissues.So, biomaterial delivery is where you use scaffolds or implants and the host cells willhave to migrate here, adhere to the material and help in the healing process.People use cell delivery as well, where you either use a transplantation of cell population.So, the cells will basically produce their own support matrix and integrate into a hosttissue.So, this is where you have the stem cell therapy and all other things where you just harveststem cells and supply it to the site of injury hoping it will help in the regeneration, right.So, those kinds of things are done for cell therapy and you also can have molecular deliverywhere you either use gene delivery or single molecule or multiple molecule delivery.So, the product which Ramya identified what is it?OP1 putty, right.So, that is recombinant bone morphogenetic protein.So, bone morphogenetic protein is a growth factor which helps in the formation of bones.So, delivering such a molecule would help in regeneration of the tissue.So, currently people focus on combining these things.So, people have seen that using them independently has its own advantages, but it can only helpin regeneration to a certain extent.Beyond that point you need to have multifunctional material which can provide different avenuesfor regeneration itself.

Video 2: Biomaterials
So, the materials which are used can be classified primarily as four things.So, they are the metals, ceramics, carbons and polymers.So, metals are basically titanium, gold, silver and alloys.Ceramics, it can be alumina, can be zirconia, hydroxyapatite, so many different ceramicsare available.You have the carbons which would be carbon nanotubes, pyrolytic carbons, graphenes, fullerenes.There are too many options where you can try to use them.So, people have been trying to use these for different biomedical applications.Polymer, obviously there is a wide array.Whatever I have listed here is only synthetic polymers.You can also use natural polymers like chitosan, collagen.There are just wide array of options which can be used.So, these are the four major classes of biomaterials and composite is just another class whichis basically a combination of these material which we have said.So, people use polymers along with ceramics or carbons and to provide different functionality.We will go into a little bit of detail of all these things and hopefully we will havemore discussion in the subsequent lectures.So, metals can either be a single metal or alloy.So, some examples for alloys which are used in biomedical application would be stainlesssteel or nitinol.The advantages: they are strong, tough and ductile, however the disadvantages is theymay corrode and they usually have very high density.So, this would mean there could be discomfort for a patient when it is being implanted inthe body.So, that could be a limitation.So, metals are used as biomaterials in many cases.Some of the options are where you use it for structural components like hip and joint replacements,bone fracture pins and plates which is used during surgery, you also have dental implantswhere metals are used.So, other applications would be for cardiac devices, stents, wires and tubing.So, what you see here this is actually a stent.So, this is how stent looks and this is used to make sure the blood vessels do not collapse.So, in case of angioplasty that what is done.So, you actually put the stent there and it helps to keep the blood vessel open.And nitinol is one alloy which is quite popular.How many of you know what nitinol is?Does anybody know what nitinol is?So, this is a superelastic material which is used in vascular stents.Hope this video plays ok.Let’s see if it plays ok.I really did not want the audio.So, that is just a water bath you are looking at.So, what you have here is just a water bath and the wire which they are playing with isthe nitinol wire.So, any kind of deformation it will again go back to its shape provided the environmentis right.Like temperature and conditions, the physiological conditions, it will actually go back to itsoriginal shape.That is way it is called as the shape memory alloy.It can retain shape and elasticity.So, this can be deformed for easy implantation.So, you do not have to open up the patient all the way to put this in.So, and it restores its original shape inside the body.So, that is why in angioplasty all they do is just have basically catheter which is putinside the person body either through the wrist or through some blood vessels betweenthe thighs.So, that is what is usually done.So, it is a simple enough procedure because the material can actually be deformed andit will regain its original shape once it is implanted.So, people are now looking at developing these stents for different applications.So, recently there was an FDA approval for even bifurcation stents.So, when there is a clog in the blood vessel, a stent is placed, so that the vessel canbe can be kept open right, but what can happen is these kinds of clog can happen at the pointof bifurcation of blood vessels?If that happens, keeping one stent is not sufficient.So, what people trying to do is they put one, cut it open and place another.It is actually a very cumbersome procedure to do that.So, now recently there has been some approvals which have come out for bifurcation stents.So, some approvals had already been obtained in European Union, but recently it was approvedin FDA as well.So, major problem with metallic implants is effect of corrosion.So, this would affect the surface and the bulk properties.It also alters the way the material interacts with the host because you are now having adifferent surface morphology even the chemistry is different.So, you do not know how the cells are going to react to it.So, it will, it can cause problems.So, it can also cause serious volume change.So, if you are going to use something like iron and it obviously, you would not use iron.You would use stainless steel or something, but there is going to be a significant changein density.When you have some corrosion and if the density for iron is 7.8 roughly and the iron oxideis close to 6, you have a significant difference in volume which you are going to have andthis will cause major discomfort for a patient.So, the image you see here is actually a new implant works for hip and joint replacement.So, you see the one on the left side is the actual new implant and the one is basicallytaken out from a patient after a few years.So, you can see there is some amount of corrosion and this can actually be a problem, right.So, that is one of the reasons metals are usually an issue and that is why people usevery specific metals which can prevent this kind of corrosion.This image shown here is actually for titanium.So, titanium is one of the most stable materials and even with that you have this kind of aproblem.So, ceramics are basically inorganic compounds that contain metallic and non-metallic elementsfor which you have inter-atomic bonding which can either be ionic or covalent and theseare generally formed at very high temperatures.So, a glass mug, a mug you use would be a ceramic, right.So, these are hard and brittle.So, they are very hard, but they break very easily.They have good thermal and electrical resistance, they are good insulators, they are also resistantto high temperature and severe environments.So, ceramics are used as in biomedical applications.Where do you think ceramics can be used widely?Which tissue would you want to use?Bones.Bones and dental tissues is where you use ceramics because that is where you have ceramicsin your body.So, it has used again in structural components like hip and joint replacements.So, you do not use the ceramic completely for joint replacements because as I said,they are brittle.So, you would not want to place only a ceramic there so, but it is used as a coating or itis used in ways that it can help in cell infiltration and so on.So, spinal fusion devices can also be ceramic materials and dental crowns which are commonlyused.So, if you undergo something like a root canal, you would have to get a dental crown and thatwould you can use ceramics, you can also use metals.That is what people used to use now to make sure it fits with what you already have, peopleuse ceramics.So, other applications would be cochlear implants, coatings on heart valves.So, in tissue engineering can you name any ceramic which is used?Hydroxyapatite.So why hydroxyapatite? because it is part of the bone already.Ok, So, your bone tissue contains hydroxyapatite.So, your bone actually has two components.So, one is hydroxyapatite which is in the nano particle size and this hydroxyapatiteis actually the discontinuous phase.The continuous phase is collagen.So, collagen with hydroxyapatite composite is what your bone is ok.So, that is why it is a nano composite.Bone is actually a nano composite material.So, some of the ceramics which are used are alumina, zirconia, zirconia-alumina complexes.So, these are bio-inert ceramics.So, bio-inert ceramics would not promote bone in growth.So, you can have bio-active ceramics which are the hydroxyapatite and tricalcium phosphateand so on which will promote bone in growth.So, there are different types of calcium phosphate bio-ceramics.Hydroxyapatite is a one which has the calcium to phosphate ratio closest to the one in yourbones.So, that is why hydroxyapatite is extensively studied for bone tissue engineering.So, tricalcium phosphate can also be used.So, you can actually tailor the degradation rates by using different types of tricalciumphosphate.So, that is why people try to use that as well.So, people are using bio-active cements and porous scaffolds and composite scaffolds fortissue engineering.So, people also try to emulate what you they have seen in the body, right.So, people try to use polymers along with ceramics, so that you can actually createbone like tissues.So, carbons are basically just different types of, different allotropes of carbon can beused.Pyrolytic carbon is used as a coating for mechanical heart valves.Diamond, like carbon is also used for coating on heart valves and blood contact devicesbecause diamond like carbon has very good haemocompatibility.So, you use it for coating on blood contact surfaces.So, carbon nano tubes also had a lot of interest in the last couple of decades probably.So, it is being used for bio-sensors, bone regeneration and other gene and drug deliveryand so on.In the recent past, graphene and graphene-based graphene type compounds, type components arealso used.So, graphene, graphene oxide, reduced graphene oxide, all these things are being explored.So, the image you see here is actually a TEM image of reduced graphene oxide.So, this is been used in bio-sensors and also for drug delivery and it has very good antibacterialproperties.So, it is being explored for coating on implants and so on.So, recent studies have also shown that at certain concentrations these graphene oxidesand reduced graphene oxides can help in vascularization, whereas at higher concentrations, they willinhibit angiogenesis.So, you can use it at different concentration.If you use it at higher concentration, you can probably use it for treating cancers whileif you use it in lower concentration, you might be able to use it for promoting angiogenesisin tissue engineering.So, obviously most of these carbons, there are lot of detractors for it because peopleare worried about the long-term effects.So, there are very few studies on the long-term toxicities of such carbons, because see theseare nanoparticles and toxicology of nanoparticles itself is a major question mark which is beingexplored.So, this being a carbon which is not been established for a long time, raises seriousred flags in many cases, however the promise which these materials are showing has madeit an interesting material to work on.There is a lot of research which is happening in this domain.So, moving to the polymers.So, this is where I work.So, most of my lab students work on polymers.We try to develop different types of polymers and polymer composites for various applications,either it could be for an implant application or drug delivery application or tissue engineeringapplication.So, here which type of material, which polymer you use would be driven by the tissue youare trying to regenerate.So, you try to design polymers in a way that it will be similar to the natural tissue itself.So, these things I am assuming you already know.Poly is many, mer is the unit.Polymer is basically a repeating unit.So, these are non-metallic components which form macro molecules.It could be chains, branched chains or cross-linked networks.They have poor thermal and electrical conductivity, however there are some conducting polymersas well, which have been used for nerve tissue engineering and so on.So, polymers are one of the most widely used biomaterials because there are just too manytypes of polymers.There are each polymer has its own property, physical, chemical and biological properties.They are so vast.So, it has been used in all possible applications.So, valves, ducts, catheters, joint replacements, extrusion coatings, encapsulants, tissue engineeringscaffolds, lenses from anything from blood bag to tissue engineered scaffold, you arelooking at using a polymer right.So, there are just a wide array of applications that you can look at and depending on theapplication, you would be looking for very different properties.See if you are going to use it as a lens, what would be the property lens for your eye?What would be the property would focus on?Has be transparent; transparent.Transparent.So, what about if it is going to be used as a blood bag?It should not affect the blood inside.Ok.So, it should not trigger coagulation cascades, right.So, which means it should be haemocompatible, it needs to be blood compatible.So, blood should not, the platelet pathway should not get activated.Platelet should not start adhering and clotting.If that happens, the blood will be unusable.So, each material has to be uniquely different and people try to engineer these materials,we try to modify surfaces to provide desirable properties.So, PMMA is used there and mostly people do not use polymers by themselves.It will again be used with, right now people use it with other combinations, with ceramicsand so on.So, polymers in tissue engineering.Again, collagen is the gold standard because that is what is present in your body.So, you have collagen as the major component of your extracellular matrix.So, people try to use collagen, however collagen can be expensive if you are going to get itas some highly purified form.You could try to extract collagen from other sources.So, we can get it from any source right, any tissue would contain collagen.So, if you were to take just chicken skin somewhere and you start treating and performthe proper procedure, you can actually extract collagen.However, collagen is not the only material you want to use.So, people are trying to look at different things because collagen, see when you talkabout natural materials, you can always have batch to batch variations and there is alsorisk of contamination.So, considering all those reasons, people try to use other sources.So, polysaccharides are again natural sources.You also have PLA, PLGA or PEG and so many other materials which you can use which PVA,all these things that used which are synthetic materials where you have very good controlover the molecular weight which means the physical properties can also be controlledvery well, right.So, we will try to use so many different materials and these materials are also fabricated differently.So, what you see here are different fabrications.The first one you see is probably just a lyophilization and freeze drying, freeze thawing techniquewhich creates a lot of pores.And the next one you see is just a solvent casting technique where you have somethinglike a hydrogel, smooth surface hydrogel and the one you see after that is actually theelectrospinning where you get nice fibers which could be of nano diameters.And the other the next one is more like a printing where you get or molding where youget specific structures.So, there are just many fabrication techniques.We will go into the details of the fabrication techniques when we talk about the individualmaterials.So, composites can be anything.Composites are basically polymers and ceramics put together would be a composite.Collagen hydroxyapatite is a composite in bones and you can have polymers and carbonscombining to form conducting composites.So, people have looked at putting a polymer like PCU along with graphene to show whetherit can improve the electrical properties.So, polymer can also be blended with metals like metal nanoparticles to provide antibacterialproperty or to deliver drugs and so on.

Video 3: Cell Delivery
So, that covers the aspects related to materials.So, you also have cells which can be delivered, right.So, that is the next part of this work.So, how do you deliver cells?So, what cells do you choose to deliver?So, again it depends on the cell source and cell types.Cells can come from different sources.It can also be of different types.So, we need to identify what cell source we want to use for it for a specific applicationand what would be the cell type you would want to use for the specific applications.So, the sources I have classified as autologous, allogeneic, xenogeneic and types; I have classifiedthem as differentiated and stem cells and just put other types of cells where you canuse a cell type which is not directly related to the tissue you are looking at, or you canlook at co-culturing and so on.So, let’s look at these cell sources first.What do you think could be the advantage of using an autologous cell?Less immune response.No, immune response it is from your own body.So, there is no chance of any immune response.Ok.What would be the disadvantage of using an autologous cell?Invasive procedure.Ok.Invasive.sorry?Limited amount.Limited amount ok.It cause problems in the tissues like the location you are extracting.So, that is called Donor Site Morbidity.So, basically you are damaging another tissue to harvest the cells and if a patient is alreadysuffering, creating another damage to the tissue would, can actually be a problem.So, what about allogeneic transplant, allogeneic cells?What would be the advantage of allogeneic cells?Allogeneic is from same species, some other person?So, what would be the advantage?Abundance and higher availability.Abundance Ok, sorry higher?Higher availability.So, it is more easily available.Disadvantages should be?Immune responses.Potential immune responses and rejections would be a limitation.What about xenogeneic; from another species?Readily available.So, much easier availability, right.So, if you are going to get tissues from an animal, you are probably going to get moreof it and it is much easier to get animal tissues compared to human tissues, right.So, and ethically also it is a little easier to justify doing something like that.So, what about; what about the limitation itself?Rejection.Rejection would be the major problem, right.So, that is what I have here.So, basically you have limitations with all these techniques.So, you need to look at how you would optimize it, right.So, people try to work on these, people try to develop technologies, so that they canexpand cells.So, you can only harvest a certain number of cells, right.So, you cannot harvest all the cells you need for creating a tissue.So, you harvest some number of cells and then you grow the cells.So, you provide the right environment, right nutrients, so that the cells grow and thenyou can use this cell population for regeneration.So, that is what people try to do and because of this people have developed different technologiesfor different types of cell, cells themselves and people are working on next improving thisfurther.So, what about cell types?So, I said differentiated cells and stem cells, what do you think would be an advantage ofusing a differentiated cell?If it is the functional tissue which we are making tissue, then we will know that, thatis the tissue which is going to; that is the cell which will be there.So, you know that the functionality is already there ok.So, that is one advantage.Just a tissue is already a functional tissue.What would be the disadvantage?Differentiated cells do not divide.So, difficult in growing.So, they will actually be very slow in growing.So, you would not be able to get enough numbers.So, that can actually be a major limitation.So, what about stem cells?So, stem cells actually, can be from different sources.It can be from adults, from fetuses or they can be embryonic and you also have IPSCs now.Ok.So, there are different types of stem cells which are available.So, we will go into details of each of them later, but right now what would be an advantageof using stem cells?You can differentiate it into any type of cells.Ok.So, you can provide the functionality you want ok.It is abundant.Much more abundant and easy to harvest, and they will divide faster, they will grow ata much faster rate making it easier to culture them.What would be the disadvantage of using a stem cell?You have to provide the right factors for it to differentiate.Ok.So, controlling the differentiation of stem cells can be a challenge especially in vivo.So, in vitro differentiation is one thing, but if you are going to use a stem cell andyou are going to a use it in vivo, if you are going to implant it as a stem cell, howdo you control the differentiation of the stem cells in vivo?So, that becomes a different kind of a challenge, ok.So, what else?Cancer.Can you explain what you are saying is correct but?Like unregulated growth rate.Uncontrolled growth can lead to teratoma formations especially with respect to embryonic stemcells.That is a problem.So, you can actually have that issue where it forms into a teratoma and that can be awhole new complication you have developed now ok.So, that is what I have here.You have required functionalities with differentiated cells, but stem cells can give you differentfunctionalities, can actually be differentiated to specific functionalities, but how you differentiatethem in vivo is a challenge.So, you have to take into consideration all these things while you are developing yourproduct or the problem statement..So, because these are a growth factors or proteins which need to be delivered to thesite, so they are usually loaded to some material and delivered.So, we will look at what are commercially available and what is currently being donein that domain and with that, we will come to the conclusion of the basic introductionfor tissue engineering ok.Thank you.