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Module 1: Scaffolds

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Video 1: Synthetic Polymers
So, today we will talk about Synthetic Polymers.So, this will again be a reasonably brief talk.I will only focus on things which have been studied extensively.So obviously, we can look at any synthetic polymer as long as it is biocompatible; however,we will focus on some things where people have actually demonstrated reasonable successwith respect to using it for tissue engineering applications ok.So, why do you want to be use synthetic polymers yeah, as long as natural polymers do the work,you would not have to look at synthetic polymers right.But we already discussed natural polymers have some serious limitations; like sourcecould be a problem because it can actually cause immunogenicity and contamination issuesare there, batch to batch variations are there, your properties are not really tailorable.There are some problems which has made people look at synthetic polymers.So, the advantages; they are more uniform and they have predictable chemical and mechanicalproperties.however, problem with this would be it is a foreign material right.It could have some problems, but you can actually design it for specific purposes, you can actuallymake it free from toxins and contaminants because you are going to be synthesizing itin a lab.You will have a lot of control over the quality of the material you are producing.So, you have choice over the monomers, the initiators, reaction conditions and any additivesyou want to add to the polymer and so on.So, this way you can actually tailor many of the properties like crystallinity, meltingand glass transition temperatures, molecular weights and side groups.So, these will give you desired properties which will actually be suitable for appropriateapplications, yeah.What is the major difference between bioimplants and scaffold?Ok.So if I am gone a say an implant, an implant is generally not biologically active.In a sense that it is just there to provide some kind of a replacement, it does not helpin regeneration of the natural tissue.A hip and joint replacement is an implant right; so, you basically use a titanium alloyand you put it in the body that is not going to help in your bone growing back whereas,scaffold tissue engineered scaffold is supposed to help the bone to grow back.So, that is why if you were to use a hydroxyapatite ceramic that would help in bone ingrowth.It will help in the cells for attaching and formation of new matrix and it will integrateitself with the bone, with the existing bone.So, that would be a scaffold from a tissue engineering perspective.Sir, most of the times it will be inactive that implant.Implant will usually be inert yes.But there are some degradable bio implants which can be helpful for the bone growth right.So, you have degradable implants yes.So, degradable sutures, all those things are there.So, because it is not permanent does not make it bioactive.I am talking about activity.So, if you are going to load so, if you are going to use something where you are deliveringmolecules then it automatically becomes active that would make it more related to a deliverymechanism.So, it is a signal-based approach so, which you are talking about right.So, here we are looking at the material itself which can help in cell adhesion and so on.So, does that answer your question.Yeah.So what was the material you are talking about when you are saying material degrades.Like magnesium degradable implants are there of course, the reaction it will be made tothe you know the prolong.So, immediate degradation it will leads to that bone growth.Yeah.So, I have not actually read a lot about magnesium and it is bioactivity.I know professor Sampath Kumar does that.So, I do not know exactly the biology behind it, but if magnesium by degradation can actuallystimulate it, then it would actually be a bio active material.So, like a hydroxyapatite.Right, so it just depends on the biology behind it and I am not very confident about the biologyof magnesium.Because nowadays the all the implants are coming in hydroxyapatite coating only.So, that the reaction will be more.Yeah.They are active, bioactive in nature.So, that that is for host integration.So, to make sure that if you have a hydroxyapatite, some ceramic coating it makes sure that thecells can attach and your chances of dislocation and loosening actually come down because itnow integrates with the bone alright.So, that is the idea.So, host integration is another aspect of implantation.So, that is another major concern when it comes to even tissue engineered products right.How you integrate it with your body is going to be a question ok.So, with synthetic polymers you have certain advantages.So, people have tried different synthetic polymers and they always stuck to things whichhave already been proven to be biocompatible.Most of, there are a lot of biocompatible synthetic polymers that are known.Many of them have even been, even been approved by FDA for specific applications.It might not be specifically for tissue engineering, but you would usually have it approved forvarious applications.And if it has shown to be; if it is shown to be compatible then you would want to tryto see whether it is actually effective as a tissue engineering scaffold.So, broadly these materials are classified as biodegradable and non-degradable.Biodegradable are the ones which basically degrade in your body in vivo, non-biodegradabledo not degrade inside your body.So, examples for the biodegradable would be PLA, PGA, PCL and so on.These usually degrade based on hydrolysis.So, simple hydrolysis reaction which will just degrade these into products that canbe excreted out; whereas, non-biodegradable are do not usually, again it cannot be hydrolyzed,there are no enzymes which can actually break it down.So, because of this they have to be confined for specific types of implant like if it isgoing to be a permanent implant, then you might want to use a non-biodegradable materialor you would try to chemically modify these non-biodegradable materials to make it biodegradableand you can also use it in blocks of low molecular weight.So, that there can actually be elimination from the body.So, for example, polyethylene glycol is not biodegradable, but below a certain molecularweight it can actually be excreted through your kidney.So, if you use it in that molecular weight then it would not be a problem, it can actuallybe removed from your body.So, PVA, poly acrylic acid, poly HEMA are all some of the examples of non-biodegradablematerials which have been tried out for tissue engineering applications.So, all we will go through some of these in reasonable detail so, this is PGA, poly glycolicacid.So, this is the linear polymer of glycolic acid.So, glycolic acid is actually produced in your body during normal metabolism which meansthe degradation products of poly glycolic acid are not going to have any toxic effectin your body; however, this material itself goes through bulk degradation.It is not just surface degradation; surface etching is not what happened.It just go bulk degradation happens which means it can actually get degraded reasonablequickly.So, in vivo degradation happens only through hydrolysis, this process can also be catalyzedfurther by molecules which have esterase activities.So, the enzymes that are, that have esterase activity can rapidly hydrolyze PGA.So, the in vivo degradations studies have shown that it usually loses it is strengthwithin the first 2 months, 1 to 2 months it loses it is mechanical strength and completedegradation happens within 6 months.So, this has been approved for FDA; it is approved by FDA for biodegradable sutures.So, you would have come across these in surgeries people now use biodegradable sutures ratherthan the regular sutures.So, that you do not have to go back again for removing the suture right.So, these will actually be absorbed by your body.So, this is one of the most commonly used polymers which are bioabsorbable.So, it has been extensively studied.There are commercial products out there which use PGA.Yeah.It is only glycolic acid produced in the body or even PGAs.No glycolic acid is produced in your body, not PGA; poly glycolic acid is not produced.you can actually prepare poly glycolic acid which just gets hydrolyzed to glycolic acid.Tissue engineering studies of PGA have actually been done.People have shown that different types of cells can actually adhere and grow on topof these cells, on these polymer.Another set of polymers which have been studied extensively is PLA or poly lactic acid.So, here these are actually made from two different monomers.So, one is; it is possible to use lactic acid and prepare the polymer.Other option is to use the cyclic di-ester, which is lactide and then prepare this.So, it is the direct condensation of lactic acid monomers to form PLA should be done attemperatures which are milder.So, only then you would prevent the formation of cyclic di-esters.So, if cyclic di-esters form then you go for through the ring opening polymerization ofthe lactide so, which is, can be catalyzed by different metal catalyst.So, using this you actually prepare PLA.And lactic acid itself actually exists in two optically active forms which are D andL. So, lactide which is a cyclic di-ester can exist in three isomers; it could be L-lactide,D-lactide or DL-lactide.And the polymerization can form semi-crystalline polymer or an amorphous powder, dependingon what is used for which stereo isomer is used for preparation of PLA.So, if PLLA is formed then it is semi-crystalline DL-PLA which is the both D and L togetherthat forms an amorphous powder.So, the advantages; you can actually tailor the strength and the degradation rate basedon this.So, PLLA is slow degrading with very high tensile properties because of this it hasactually been studied for many bone applications.Commercially, it has been approved for orthopedic fixation devices.So, like bone screws and things like that so, they have actually used PLA for doingthis.And the D and L combination has lower strength and a faster degradation rate.So, we can try to combine these two get desired properties.So, PLA is also used, is the one of the commonly used inks in 3D printing.Most of these 3D printers would use PLA.So, PLGA is basically a copolymer of these two so, this is to try and use both the advantagesok.So, ring opening copolymerization of the cyclic dimers of glycolic acid and lactic acid isdone to get this PLGA.So, what people have shown is there are different forms of PLGA can be formed based on the initialconcentrations, initial molar ratios of lactic acid to glycolic acid.And they are usually written as PLGA x:y where, x is the concentration or percentage of lacticacid and y is the percentage of glycolic acid; 75:25 means 75 percent lactic acid 25 percentglycolic acid.So, the advantage of doing this is the physical properties themselves can be tailored.So, this also degrades by hydrolysis in the presence of water.The rate of degradation actually can be related to the monomer ratio.If you have very high glycolic acid then the rate of degradation is faster because glycolicacid, PGA we saw that had a faster degradation compared to PLA right.So, it would have faster degradation; however, this correlation is actually not linear.So, in some points it does not actually fit that one example is 50:50 PLGA.PLGA 50:50 actually shows much faster degradation which is actually faster than both PGA andPLA.So, I do not know the chemistry behind it, but in general this has been observed.So, PLGA has been approved for many drug delivery applications and has also been extensivelystudied for tissue engineering applications.People have shown many advantages to using this.There are some disadvantages as well because of the acidic nature of this material, thePH in the localized regions could actually become lower which could cause cell deathand it could cause some damage.So, we will have, in some studies have shown that.So, trying to use this has it is advantages and disadvantages.Poly caprolactone is an aliphatic polyester with semi-crystalline properties.So, the repeating units are 1 ester group and 5 methylene groups.So, the structure you can see there, it is a highly water soluble polymer and this isformed by ring opening polymerization to form degradable ester linkages.So, the degradation occurs by surface or bulk hydrolysis of these ester linkages.So, degradation can actually be; degradation is actually very slow and it can actuallybe present in your body for up to 2 years.So, it is usually used for processes where the regeneration is going to be very slow.So, PCL has actually been used in bone tissue engineering applications.So, they have shown that osteoblasts can actually adhered to this PCL and produce ALP.ALP is Alkaline Phosphatase, which is a marker for bio mineralization.So, when ALP production is increased then you know that there is mineralization of bone.So, people have shown that when osteoblasts are cultured with PCL, it shows an increasedALP production.So, to optimize the rate of degradation many studies have tried to use PCL copolymerizedwith other materials like PEG and PVA and so on.So, the once which we looked at till now PGA, PLA, PLGA and PCL are all biodegradable so,the once which we are looking from here on out are nondegradable, non-biodegradable.PEG is a non-biodegradable material; however, it is very highly hydrophilic, it has verygood swelling ability.So, because of this reason it is actually used extensively in formation of hydrogels.We will look at what hydrogels are and we will go into details of hydrogels later, butcan you tell me what you understand with the term hydrogels?Have you come across the term hydrogels?Yes simple definition; it is a matrix which holds water.Ok.The water.So, it can absorb water right ok.So, that would be one, sorry?.Highly hydrophilic.OK, highly hydrophilic; so, if you were to take a sponge right, and you dip it in waterthat also holds a lot of water right.So, does that make it a hydrogel.It should interact with water, should hold water.It should hold water; so, it is not just that it absorbs water, it should also hold water.The sponge if you squeeze the water is going to come out; whereas, the hydrogel if yousqueeze the water will not come out.So, PEG is used because it can actually form hydrogels which are very hydrophilic and itcan actually absorb a lot of water.The advantage of that is, it will have very limited diffusion problems right, if it canswell very nicely, then the network can swell very nicely, nutrient transport is going tobe very effective, but the other side of it is the linear chain actually leads to rapiddiffusion of the material outside of it, which means it just dissolves away and it has verylow mechanical stability.So, for this reason people try to create PEG networks by attaching functional groups toPEG and try to crosslink it using covalent or other types of crosslinking.So, this is non-biodegradable, but can be degraded by copolymerization, can be madeto be degradable by copolymerization with other degradable polymers.And as I already mentioned, below a molecular weight threshold, it can actually be excretedby your kidneys.So, if you were to use it at that molecular weight then it is usually not a problem.Sir, the activated charcoal would be a hydrogel?No.Why do you think it would be a hydrogel?It is solid absorber.It does not absorb water right.It just absorbs other things it is.ok.So, polyvinyl alcohol is another commonly used material.So, this is the water-soluble polymer with excellent biocompatibility.Lot of studies have worked with PVA and shown that it is very useful for biomedical applicationsand vinyl alcohol itself is not very stable so, it is synthesized using a two-step process.So, it is not just a direct polymerization of vinyl alcohol which is done.So, what people do is, they do the, first step is the free radical polymerization ofvinyl acetate and the second step is hydrolysis of the polyvinyl acetate to form PVA, polyvinylalcohol.So, this hydrolysis actually can be controlled.So, you can have varying degrees of hydrolysis.So, just like how you have varying degrees of deacetylation for chitosan; you can havevarying degrees for hydrolysis for PVA, and based on the degree of hydrolysis the mechanicaland the physical properties can actually be different.So, it is been used in many applications.PVA there are actually review articles which talk about PVA for tissue engineering applicationsyou can go and read it up, it is done for many different things.Only limitation with PVA is it is so hydrophilic that it does not support cell addition veryeffectively.So, in some cases you see, you actually need to maintain your balance when it comes tohydrophilicity and hydrophobicity, if it is going to be very highly hydrophilic it doesnot support the cells as much as it should.So, because of this reason people usually use it along with the other natural polymers.So, various types of natural polymers have been used along with PVA; cellulose and cellulosederivatives, hydroxy sorry, hyaluronic acid, collagen all these have been blended withPVA to improve it is cell addition properties.
Video 2: Scaffold Fabrication Techniques
So, this is just a bunch of other synthetic polymers which have actually been studied.So, you have polypropylene fumarate, polyorthoester, polyanhydrides, polyphophazene, polycarbonateand polyurethanes which all been studied for different applications.polyurethane.So, these are all approved for biomedical use and they have been shown to be biocompatible.So, people have just tried it for tissue engineering applications.You will be able to find papers which talk about other synthetic polymers as well, butthese are some of the more commonly used synthetic polymers.So, another class of polymers, synthetic polymers would be the conducting polymers.So, polymers in general do not conduct electricity right so, they are very poor conductors ofelectricity.So, there are a special class of polymers which are called conducting polymers.So, these were these are, this is actually a Nobel Prize winning discovery.They won the, this discovery actually won the Nobel prize in 2000 I think for chemistry.So, the conducting polymers which are shown here are polyaniline, polypyrrole and PEDOTwhich is poly ethylene dioxy thiophene.So, these are some of the common conducting polymers; so, these as polymers themselveshave conducting properties.However, they have some limitations because they do not have the desired mechanical properties.They actually are brittle when they are fabricated into scaffolds and other materials.So, they are blended with other polymers to prepare conducting polymer composites.So, these are; they have actually been fabricated into multiple things and have been used fordifferent tissue engineering applications where electrical properties of the scaffoldplay a role.So, people do try to prepare these materials in different ways and we will actually lookat how to fabricate these scaffolds.So, fabricating a scaffold is actually a crucial aspect right.So, having a polymer is one thing.From the polymer to actually make it resemble the ECM, is a serious thing which we needto look at.So, the fabrication strategies have to be optimized.There are many different strategies.So, these are just a bunch of them which I have shown here and you can look at many otherthings as well.So, leaching methods where you have solvent casting and salt leaching, ice particle-leaching,gas-foaming and salt leaching.These are all some of the leaching methods.And you have microsphere formation where you can have biodegradable microspheres or macroporousbeads or particle-aggregated scaffold being formed and phase separation methods like freeze-dryingor thermally induced phase separation could also be tried, and fiber-spinning methodologiesto form nanofibers, microfibers, nonwoven fibers and so on.Injectable gels can also be prepared.3D printing is one of the latest technologies where people are trying to print many of thesethings ok.So, these are some of the scaffold fabrication strategies.We will discuss some of them here and 3D printing I will discuss in greater length in the nextlecture.So, this is solvent casting and salt leaching.So, this is a very simple technique.So, all you do is you take three things you take the solvent, polymer and a salt.So, the salt her is NaCl.So, you could also sugar or whatever right.So, it is something which can form a crystal.So, you basically mix all of these and now what happens is, the salt has actually dissolvedalong with the polymer and you pour it in the mold, whatever the mold could be.So, here they have just shown a disk like mold, you pour it in the mold and once itis dried out.So, you keep it in room temperature or in vacuum to evaporate the, evaporate the solvent.So, now what you have is polymer disk in which the salt is dispersed all over right.So, you put it in water and wash it so, what will happen is you would have dissolved allthe salt away.So, now, the positions in which the salt was present have these pores.So, that is a salt leaching technique and then you can freeze dry it to eliminate allthe remaining solvents and so on.So, this is a SEM image of solvent cast; salt leached material.So, you can see nice cube kind of pores so; this is where the salt crystals were presentright.So, there is, those got washed away and you actually got these pores.So, here what you think could be the advantage of this technique.Any porous material will give you more surface area.So, this specifically has some advantage also.Semi-permeable property.These are all interconnected pores.Ok that is actually not correct.we will get to it in the next thing but.Easy fabrication process becomes.So, it is a very easy process that is one thing.what else?.There is no heating involved.Ok so, milder conditions for processing all that is fine.Regular matrix of pores quite easily because the salt will be dispersed everywhere.Ok so you are able to get regular pores.So, ok I will rephrase it slightly to fit what I want to say.So, you can actually control the porosity.So, based on the salt concentration and the type of salt you use that pore size and poredistribution can actually be controlled ok.So, that is the advantage here.So, as far as your claim of it being interconnected pores it is actually not true.So, if you were to look at these points.So, these are actually deep pores right.So, the ones where you see the dark black are deep pores, whereas if you look at theseparts which are more greyish or even whitish, they are actually not deep.So, those are like, you are saying the polymers surface itself right, but these are actuallypores which have been formed.So, those are places where the salt was present and it has just been dissolved away.So, in some cases you would have gotten interconnected pores in, but in many cases, you would actuallynot have very good interconnectivity in this method ok.So, to get better interconnectivity that is why you do gas foaming and salt leaching.So, what you do here is a polymer gel is prepared and instead of just adding salt, you add asalt which is, which can actually release gas.So, an example would be ammonium bicarbonate.So, you add this and then you actually evaporate the solvent and put it in water or put itin a buffer, what will happen is you will have the carbon dioxide getting released fromthis right.So, this gas is going to get released from the scaffold which you have prepared.So, when the gas comes out it is going to create these pores.So, you will end up with porous scaffold; this after drying and freeze drying you canget a macro porous scaffold.So, the scaffold would look like this.So, if you look at this even the smaller of the pores would actually have some of theseconnections where you would actually see that it is reasonably interconnected.So, you would have very small, those are the places where the gas would have escaped outand created these pores.So, because of this these are; this shows a very good interconnectivity.So, that is the advantage of using gas foaming technique yes so, but these are all very simpletechniques which people have been using where they have shown that you can create porousstructures, but what would be the disadvantage of this compared to the salt leaching.Density would be very less yeah right.Density would be very less for any porous material.If you have increased porosity your mechanical strength is going to come down, ok.As long as you keep increasing your porosity, the mechanical strength will have to comedown right.Because you have pockets of just air.Can you elaborate what do you mean accumulation?It is non-uniform.So, whenever I ask what is the disadvantage of this compared to something else it wouldusually be the advantage of the other technique right.So, the other technique you had control over the pore size and porosity.Here you have reasonable control over the porosity, but not really the pore size andso on.The distribution pore size all those things you are not, it is not very well organized.So, microspheres you guys would have prepared it for different applications, primarily forentrapment of enzymes and things.So, the same thing can actually be used in tissue engineering applications as well, butpeople do not generally use just a microsphere.Although people have earlier studied it.Nowadays people just do not use just a microsphere by itself because that does not resemble whatyour ECM is.So, people will use microspheres along with other materials.so, where you can use the microsphere to load molecules and so on.So, microsphere shown here is quite simple.All you do is dropping it while the crosslinking media is spinning and you create a microsphere.So, this is a simple PLGA microsphere I believe on which human disk cells are actually cultured.So, this shows that cells can actually adhere and it depends on the material you have chosenand you will get nice beads of uniform size.All that you need to control is the viscosity of the material, the rate at which you actuallyrelease the material into the media and also the diameter of the pore which is used forreleasing them again.Freeze-drying is another commonly used technique.This is actually one of the more popular techniques which is currently being used extensively.Even more than salt leaching or gas foaming because it is really really simple.All you do is dissolve it, mold it, freeze it and dry it, that is it, as simple as that.So, you actually create pores because of this freeze drying technique right.So, we in the last class or some class I actually discussed freeze-drying right.So, freeze-drying is lyophilization technique where you actually have a sublimation processhappening.So, can somebody actually draw the phase diagram for water and explain the sublimation process.How would it look?Pressure versus temperature.Pressure versus temperature.Yes.Something like this right.So, which region is which?High pressure, low temperature, phase solid, yeah.High pressure low temperature is.Right, solid.Solid.Then high temperature, low pressure is gas.What?Same.Which is what.Gas that.This one yes.Yeah.Steam.Ok this is liquid.Ok, confident?Yes.Ok.So, you guys are doing thermo now right?Yes.So, now that you have the phase diagram can you actually tell me what is the process oflyophilization from here?Basically, at a low temperature they reduce the pressure.So, all the water coming so, they are actually going from solid to gas, yeah there is noliquid.We just have to go here.So, instead of see, usually solid goes to liquid and then to vapor.So, instead you just so, it depends on which pressure region you are in right.So, if you are in a very low pressure.So, that is why you create vacuum during lyophilization and at vacuum you make sure that you can actuallysublimate it at low temperatures rather than heat it and where you have to have a vaporizationprocess.So, the advantages it is, it creates pores as well as it does not damage the material.It is a very, it is done at low temperatures right, very low temperatures.So, because of this it does not damage the material.So, this is actually, this is a scaffold which we developed in our lab.This was prepared by lyophilization technique.So, you can see very nice pores which are there and like very nicely interconnectedand while it resembles a what an ECM would look like right.So, this is a.What kind of microscope is used here?It is a scanning electron microscope.So, we will discuss characterization extensively.So, there will actually be, there is a lot of things which we can use; so, this is thescanning electron microscope.There are different microscopes which can be used for such things.So, you get these images; so, even the image which I showed, most of these images are actuallySEM images only.These this is also a scanning electron microscope and this is also a scanning electron microscopicimage.So, for observing surface morphology SEM is the most common method used.So, another technique which is commonly studied is electrospinning.So, in this technique what you do is, you take a syringe and you fill it with a polymersolution, and the syringe dispenses the polymer solution which is exposed to a high voltageenvironment.So, and this basically breaks the polymer flow into thin fibers which is collected bya collector which is grounded and the collector can actually be a rotary drum it can be aflat surface and so on.So, depending on the collector surface you can actually get either non-aligned or alignedfibers.So, it will be more like a combination of both, which actually is more close to whatan ECM would be.People are trying different things and these are some of the technologies which are usedfor preparing the scaffolds.So, we will talk about 3D printing in the next class.