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Module 1: Hydrogels, Bioceramics and Scaffold Fabrication

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Video 1: Types of Hydrogels
So, today we will start with a small demo.So, we will just have a; so, this swelling itself will take some time, but we will justhave the experiment start, while I start talking about smart Hydrogels.So, these are hydrogels.These are different kinds of hydrogels which we have prepared in our lab.So, the one which is here that is isabgol, one is konjac glucomannan, this is just PVAand this is poly HEMA.So, these are the hydrogels which we have prepared and you can just have a look.So, isabgol is a polysaccharide; so, it is primarily arabinoxylan.So, what we do is, we buy what is called isabgol commercially and we just dissolve it in waterto prepare this hydrogel.KGM is konjac glucomannan which is again a polysaccharide; and PVA and poly HEMA aresynthetic hydrogels, since synthetic polymers which are used for hydrogels.So, we have prepared all of these; some of these are will swell a lot, some might notswell as much.So, we have only a couple of beakers, but we do have a 6-well plate.So, what we will do is, we will cut a small piece of it and put it inside these well,6-well plates and we will pour the buffer.So, she has given me PBS.So, we will pour a little bit of this buffer and we will see how the final state of itis, that will show us how the swelling has actually taken out.And, what we can do is, after it swells, we will take it out and blot it with a tissuepaper and then we can see whether the water actually comes out or not.So, the loosely adhered water can only come out, if it is properly swollen and the wateris closely interacting, it cannot actually come out.So, we will try and do this.So, what we will first do is, I will ask for volunteers who can cut it up into smallerpieces and we will put it in this and we will wait for how it swells, ok.So, after we put up a small piece, you can actually feel the texture as well the dryones, so that when we, after swelling you will be able to see how it feels.So, that is PVA.So, it is quite strong actually.So, one is PVA and I already have poly HEMA.Sir, the other ones.These are sample too.So, these are natural polysaccharides.As you can see it is so much easier to cut, they are such they are weak, mechanicallyweak compared to what you had with PVA.Do not cut too much of that, that will swell a lot.Ok.So, you can show it around to people the; so, the people can take it up and have a look.So, I am not measuring any volume or anything, volume of the buffer because that does notreally matter.We just have to add enough so that it can absorb.So, what buffer you are adding?So, this is PBS.So, let us see how well it swells and just leave it here.Today, we will talk about a special class of hydrogels which are called as a intelligentor smart hydrogels.So, these hydrogels are basically stimuli responsive hydrogels.See when hydrogel swell, they just swell when you put it in PBS right.So, that is what is happening to these.These when you put it there, they are going to absorb a lot of water and starts swelling.Whereas, if you are talking about the stimuli responsive hydrogels, they respond to externalstimuli.The stimuli could be pH, temperature, electrical field, light, magnetic field and so on.In response to these stimuli, they will either shrink or swell or bend or degrade and thiscan be used to tailor it is applications in tissue engineering.So, this is basically prepared using a stimuli response of monomers or pendant groups whichare the side chains which would respond to certain stimuli, right.So, an unswollen hydrogel in the presence of certain stimuli can actually form the swollenhydrogels, right.So, it would, this is the most common thing which we look at like swelling, there is alsoresponse with respect to gelation and so on.So, in some cases the gelation itself is dependent on how well the, it responds to stimuli ok.So, these are some of the common environmental stimuli and the type of hydrogel which is,which can respond to these kinds of stimuli and the mechanism and its applications.So, when you have a hydrogel which can respond to electrical stimuli, it would usually bea poly electrolyte hydrogel.So, when you have a poly electrolyte hydrogel there are ions which can actually respondto the external electrical stimuli.So, when you have a change in charged distribution, it can cause swelling or drug release or biomolecule release in case of tissue engineering.So, this is used in case of actuators, for engineering artificial muscles and also ason-off drug delivery vehicles.So, with respect to thermal things they are called as thermoresponsive hydrogels.So, these thermoresponsive hydrogels actually respond to particular temperature, even gelationcan actually be dependent on temperature.And the change in polymer-polymer and polymer-water interactions happens with, at specific temperaturesand this causes either swelling or shrinkage, which will lead to drug release or biomoleculerelease.So, this again is used for on-off drug release and also for squeezing devices; in the sensethat when the temperature changes instead of swelling it just shrinks.So, it will just squeeze out whatever is present inside.So, that is kind, that kind of a mechanism has been used.So, one of the common thermoresponsive hydrogels which we will talk about is poly N-isopropylacrylamide,it is also called as PNIPAAm, all right.So, it is just, acronym is that, this is very well studied because of it is, the temperatureat which it goes through these changes.So, we will go into details of that in the next few slides.So, pH plays a crucial role.In your body your pH is not the same, right.So, your pH can actually be different based on which part of the body you are talkingabout and which state of homeostasis your body is in.So, under normal condition your physiological pH is 7.4, around 7.4, but does not mean thatyour, your pH is always 7.4.In case of injury or something your pH will actually go down significantly.And, in certain regions in your body, like your stomach your pH is actually significantlylower.So, using this difference, just leave it here.So, using this difference to actually deliver molecules or cause gelation has been extensivelystudied.So, acidic and basic hydrogels are the most common examples; polyacrylamide and PDEAEMare all molecules which have been shown to have pH responsive properties.So, ionization of the polymer chain.Sir, are these smart hydrogels made from special polymers or can regular hydrogels be modifiedto have smart properties?They have to have some monomer or a side chain which will respond to this stimuli, right.So, if you were to just take PVA, a regular PVA hydrogel cannot behave like that.So, if you can create side chains which can provide stimuli responses, then you can doit.So, which would mean you are modifying PVA into some other polymer before you do it.Will the surface modification help?For a hydrogel a surface modification would not be very helpful because hydrogel is the3D structure, right.So, surface modifications are usually done for polymers which are being implanted.Whereas, in case of polymer which are going to be fabricated into something else, thenyou need a chemical modification of the entire thing.A surface modification alone cannot actually address the required needs.So, what happens in a pH responsive hydrogel is the ionization of the polymer chain occursdue to pH change and this leads to swelling and further release of the molecule whichyou have loaded.So, this is primarily used in pH dependent drug delivery systems.So, magnetic fields have also been explored as external stimuli.So, magnetic particles which are dispersed in a hydrogel matrix can cause this kind ofa magnetic response and the advantage of doing this would be by applying magnetic stresses,you might be able to alter the pores in the gel or cause swelling which will lead to thedrug release.So, this can be used as an on-off drug delivery system, right.So, on demand you can provide a magnetic field to release the drug molecule and otherwiseit will not be delivered.So, this kind of a thing is been extensively studied for cancer therapy and so on.So, ionic strength has also been used.You use an ionic hydrogel.So, depending on ionic strength you will be able to control the release.And this is primarily used as biosensors for glucose and people have tried to use thisto identify glucose, sense glucose and then supply insulin and so on.So, chemical stimuli is basically in the presence of other electron accepting groups so, therecould be other chemical molecules which can trigger the swelling or change in the physicalproperties leading to release of the molecules loaded.Enzyme responsive or bioresponsive hydrogels are the ones where there is an enzyme substratecorrelation.So, you would have hydrogels which are immobilized with the enzymes and the production of thisenzymatic conversion will lead to swelling or release of the molecule.So, it could also be the other way, where the enzyme acts on it for degradation to haveit release.So, it; your hydrogel could be a substrate for the enzyme as well.So, light again is used for delivery.So, using optical or other waves, light waves like UV radiation; see whether that can alterthe crosslinking of the hydrogels thereby you can change how it is; how the gelationhappens and how the release happens and so on.. Thermoresponsive hydrogels are the ones whichhave been extensively studied.Many polymers exhibit thermoresponsive phase transition property.So, these hydrogel; these polymers actually have something called UCST or LCST.UCST is the upper critical solution temperature and LCST is the lower critical solution temperature.So, when a polymer exhibits a UCST what happens is, it shrinks when you cool it below UCSTand it, it is present us chains when it is above UCST and the other way around happenswhen the polymer has LCST.So, poly N-isopropyl acrylamide or PNIPAAm is one of the most commonly studied thermoresponsivepolymers.Because, it actually has thermally reversible property with an LCST at 32 degree Celsiusin pure water.So, this 32 degree Celsius is very close to your physiological temperature of 37, so thatmakes it an ideal candidate to work with.So, at room temperature you are talking about 25 degree Celsius and at physiological temperatureyou are talking about 37 and this actually comes in between these two temperatures.So, this gives us a nice property which can actually help in using this thermoresponsivebehaviour in physiological conditions.So, above the LCST what happens is, a reversible phase transition occurs and the expanded coilwhich is seen in the hydrophilic environment, which is seen here.So, what you see here, the expanded coils that you see here, when you increase the temperatureabove the LCST it goes into a compact globule formation which causes the shrinkage of thehydrogel.So, whatever molecule is actually loaded inside will get released.Thereby, this squeezing kind of an effect is there for the molecule to be released immediately.So, this is a thermoresponsive hydrogel which has been extensively studied in many differentapplications.So, pH responsive hydrogels display big differences in properties based on the pH itself.They are made from monomers which have ionic groups or using crosslinking with polyelectrolytes.So, even with PVA you can actually prepare it to be a polyelectrolyte, right.So, depending on how you do the crosslinking you can create a polyelectrolyte nature forthe hydrogel and that can help in pH responsive behaviour.So, at appropriate pH and ionic strength the side chains actually ionize to develop fixedcharges.And this can cause either repulsive forces or attractive forces depending on, dependingon whether it is attractive or repulsive forces, there will be swelling or de-swelling of thehydrogel.Even small changes to the pH can actually have significant effects on the mesh size.So, one when the porosity changes obviously, your diffusion properties are going to changeand that will mean your release will also change.Some of the common hydrogels which are used are poly acrylamide, poly acrylic acid, polymethacrylic acid and so on.So, these have actually shown to have pH responsive behaviours.So, this is what happens.So, when you have an anionic; so, you can either have an anionic hydrogel or a cationichydrogel.So, you can start with any ionic hydrogel.So, an anionic hydrogel below it is pKa, basically stays in an unswollen form and above it ispKa, it is starts swelling and it starts releasing whatever molecule would be present at a muchfaster rate.So, even in unswollen state there will be some leakage of the material, it is only diffusionit will, it will be a much slower diffusion because the mesh sizes are very small.So, in case of a cationic hydrogel you will have the reverse, where below pKa you willhave the ions being observed and therefore, the.Drug being.Drug being released, the hydrogel being swollen and the drug being released.So, photo or light-responsive hydrogels are stimulated by light to induce changes in theirphysical and chemical properties.So, this is prepared by functionalization of polymer backbone with photoresponsive groups.So, any polymer chain which has functional groups, you can add these photoresponsivegroups which will respond to different kinds of, different wavelengths of light.The optical signal is first captured by the photochromic molecules which are the sidechains and this converts this photo irradiation to a chemical signal either through isomerisationor cleavage or dimerization which leads to different properties.One of the common parameters which is usually affected by light is the gelation itself.So, when you actually have light source so, this photoresponsive hydrogels will form gelsin the presence of a light source whereas, in the absence of it, it would not form thesegels; it will just be in a liquid form.So, the; this signal which is formed is transferred to the functional part of the hydrogels whichthen controls its properties.So, there are other hydrogels as well like electro-responsive hydrogels.These change their properties in response to changes in electric stimuli.poly electrolyte hydrogels are the ones which can be used for these because they have highconcentration of ionizable groups.So, PVA, acrylic acid, vinyl sulfonic acid and sulfonated polystyrene are all some ofthe examples which have been prepared as poly electrolyte hydrogels, ok.Another interesting class of hydrogels is the glucose-responsive hydrogels.So, these are interesting candidates because they can actually be used for insulin deliverysystems.So, what happens is, these act as artificial pancreas and they can basically swell in responseto the presence of glucose.So, which means they will release insulin when there is glucose and they will shrinkagain and not release insulin when there is no glucose, right.So, what happens is you have to immobilize the glucose oxidase and a catalase into apH responsive hydrogel.So, and this is enclosed in a saturated solution of insulin.So, now, insulin is loaded into this, a hydrogel which can sense glucose and respond to pHenvironments, right.So, when glucose concentration is high, the glucose will diffuse into the hydrogel andit is converted to gluconic acid in the presence of glucose oxidase.So, this, because this gluconic acid is formed the pH is going to drop.This decrease in pH will cause swelling of the pH responsive hydrogel.So, insulin is released.And once the glucose concentration reduces because of the insulin which is being released.So, you are obviously, there is lesser glucose present.So, there is no gluconic acid.So, your pH is going to increase leading to the shrinkage of the hydrogel.So, thereby it does not release any more insulin.So, this is an excellent candidate which people are exploring for such demand-based insulinrelease.So, there are some publications on it I do not think it is gone to clinical trial stageyet.So, this is how it is.So, this is from one of the recent papers.So, what they have done is loaded something called Concanavalin A and in a poly GEMA hydrogel.So, when glucose is present, it results in swelling and that releases whatever moleculewill be loaded to it.

Video 2: Hydrogels in Tissue Engineering
So, why hydrogel?Whatever we looked at with respect to hydrogels still now we looked at all the basics of hydrogels,we saw why hydrogels are used and what are the different applications which are usedwhich they have used in.We have not specifically looked at tissue engineering, we just looked at multiple thingswhere hydrogels are used and multiple types of hydrogels.So, what do you think are the advantages of hydrogels?So that is one thing.So, you it is, you can easily engineer it.So, there is enough variability to it that you can engineer it for desired physical propertiesok.ECM, wound healing.Similar to ECM.Wound bandages something like that.Wound bandage would be an application where you can use hydrogels; why do you think itcan be used for wound bandages?Because we can put a drug in it; antibiotic or antibacterial.Yeah.Drug in to prevent infection.That is one thing and moist is another thing.So, providing a moist environment is actually important for wound healing and also it canabsorb the wound exudates.So, the wound exudates many a times can have matrix metal proteases which will actuallydegrade some of the matrix which is being formed.So, it is better to have these absorbed.So, depending on the level of exudates having hydrogels which can swell moderately or significantlycan actually be used, ok.So, it is similar to ECM and that is one thing.Variation and structural properties might it have various structure property.Ok, you can prepare different kinds of; yeah sure.So.Sir, dynamics like swollen states.Ok.So, it can actually be responsive to the environment.So, that is one advantage of using something like this.So, what I have is, some of these things are there.So, the swelling is an important factor which actually helps in transport of nutrients andremoval of toxins, ok.So, easily modified with cell adhesion ligands; so, that is the engineering part of it youhave a lot of option to change it and non-ionic hydrogels are usually non-thrombogenic.So, this is not something we specifically talked about.So, ionic hydrogels are thrombogenic.So, it can actually trigger blood coagulation.So, if you were to use calcium alginate it will trigger blood coagulation.But, if you are using something which does not have ionic properties it is non-thrombogenicwhich means it will not cause blood clotting so, which is a good thing for blood contactapplications.So, it is usually biocompatible and similar to the ECM and so on.So, you also have injectable hydrogels.So, we did not talk about it in detail.So, even the thermoresponsive hydrogels, if I am going to look at it from, from an applicationstandpoint, I can have it as a liquid at room temperature at 25 degree Celsius and if Iinject it into your body, in your body it will form a hydrogel, right.So, it will just be a simple injection for me.So, delivering, site delivery is actually easier.So, if I were to give an injection with a liquid usually what will happen?It will get dispersed all over your body which is not really a good thing.Here it will form a gel and it will stay there.So, if you are talking about drug delivery to a specific site or if you are looking at,forming a scaffold that have specific site where cells can migrate.So, this is not going to get leached out.It is going to form the, form a hydrogel there.So, those are some interesting applications for injectable hydrogels.What you think would be the disadvantage?Mechanical properties.Yeah, mechanical properties is one serious problem.You saw some of these hydrogels which are really soft right.So, they are like a sponge.So, mechanical properties can be an issue.What else?For the injectable one you cannot really control this scaffold geometry.Ok, yeah; so, it will depend on the site of injection.It will just form a mould kind of thing, but yeah, you would not have proper control overit.Sir, I cannot really imagine this injectable hydrogel, I mean where do you inject it?To the site where you want to inject it; so, it does, see not all injections are just overyour skin right.So, you might have injections which are much, will reach much deeper in your body.So, if I want to create an injectable hydrogel and use it for prepare, making a liver; Iwould inject it to the portal vein of your body.So, I probably use a bigger gauge needle and inject it to the site where I want.So, injection just means minimal penetration, instead of cutting a person open, I can justuse an incision, small incision kind of thing.So, that is what it is.It is not always mean the small needle which we are talking about ok.Sir, we just if the smart hydrogels which are there, they respond to a particular condition,but is there is any cap over how much, like an upper limit kind of thing over this limitthey were not respond over.They would not respond to a particular concentration of local application range.Yeah, obviously, there will be some effect if you are going to have, up to a certainlevel they will swell, beyond a point they cannot swell right.So, it will stop.If you keep increasing glucose concentration it is not going to continuously keep swelling.It will have to stop at some point.Like for, glucose as an example.Even if you have other things there will always be some range within which they will workand that will depend on the material itself, ok.Can we degrade it in the body?Yeah degrading; yeah it can get degraded, but degrading is not a very bad thing if aslong as you can control the degradation it is not a bad thing.If hydrophilic, will cell adhesion be problem?If it is very hydrophilic cell adhesion can be a problem yes.But people, what people do is they try to attach ligands to it and use proteins whichhave these cell adhesion ligands along with the material and so on, those are ways toovercome it.So, couple of things are, they are actually difficult to handle and sterilization canactually affect its properties.So, it is; so, these hydrogels are actually quite fragile, right.So if, and they need to be sterilized properly and you cannot really put it in 121 degreeCelsius for 45 minutes and hope it will survive.But, there are ways to hydrolyze it, sorry sterilize it and when you are sterilizingit one of the things would be UV sterilization, but UV sterilization you might not be ableto get to the pores because it is a 3D structure and sterilization might not be the most effective,UV sterilization might not be the most effective.There are other things like ETO sterilization where a gas is pumped through this.So, there are other ways to do it, but it gets tedious and you do not know how thateffects the chemical and mechanical properties of the hydrogel.So, that can be a challenge with respect to hydrogels.So, when you are talking about hydrogels in tissue engineering, they are used primarilyas scaffolds because they mimic ECM.Cells can adhere to the matrix or they can also be suspended within the gel.So, that is why you can actually have injectable gels in which cells are suspended.So, for example, if I am going to inject a cartilage, so, I can actually have an injectablehydrogel which is in liquid and cells are suspended in this liquid.And, when I inject it this hydrogel will form into a gel upon injection in your body, whenthe temperature is higher than your LCST in your body.So, this will now have cells seeded to it.So, I can have it suspended within the gel as well.So, which one would you prefer, if I were to give you the option which do you thinkyou would prefer and why?Suspension will have uniform gel distribution of system; cells specially for proliferationinvolved.So, suspension would probably have more uniform cell distribution fine.So, what would be the advantage of cells adhering to the matrix?Better cell proliferation.Ok.Why do you think better cell proliferation?Because of better cell adhesion.Better cell proliferation is possible, but not because of better cell adhesion, but primarilybecause cells are to; when you are seeding a cell on top of it, the cells can actuallyget the nutrients more easily.And, they are also nicely adhered and spread on the surface.Whereas, when you are suspending it within the gel, there is no guarantee that they willhave the nutrients and they might not have get the morphology which they need for theproliferation and migration, ok.What would be the disadvantage of seeding it on the matrix other than non-uniform?It can get crowded.What do you mean it can get?So, cell proliferates so much there in one place the nutrients will not be enough forit to be proliferate more.Ok.So, you mean over crowded in that region, it because of proliferation.Ok, usually cells do not divide that rapidly.And especially, in vivo the cells will not divide that rapidly when you have other factorscoming in.So, but the problem usually with adhered thing is cells can actually leach out; cells canmigrate out of the matrix ok.So, when you are placing it in vivo, the cells can migrate out of the matrix whereas, insuspended it is not going to leach out very easily.But, yeah the disadvantage would be it will not get the, it may not get the nutrientsdepending on the size of the hydrogel and how the hydrogel properties are, yeah.So, you can have hydrogels as both biodegradable and non biodegradable hydrogels.So, depending on how these hydrogels are fabricated you can have them both ways.And, they are also used as barriers where it is used for preventing post operative restenosisand thrombosis.So, when there is blood coagulation it can this, these hydrogels; non-ionic hydrogelscan actually prevent this blood coagulation and this prevents the platelets and coagulationfactors from contacting the vascular wall.The ruptured vascular wall will trigger the coagulation cascade.So, by using these as barriers you can prevent the platelets from contacting the rupturedwall and these being non-thrombogenic will not triggered any blood coagulation cascades.So, that is one way it can be used.Is the stent itself made up hydrogel or just by adding the hydrogel with the stent.Restenosis is not only for stent, ok so, even with post operative restenosis can happenwith any other.Surgery.Yeah.So, any surgery.Ok, in tissue engineering it is also used for biomolecule delivery.So, drug delivery is one aspect, but concepts of drug delivery are used in tissue engineeringwith respect to delivery of biomolecules.So, different biomolecules which have different specific applications like growth factorsand other proteins can be loaded to these hydrogels so that it can be released to providedesired signals while the hydrogel is being prepared.So, the rate at which it is delivered can or when it gets delivered or the environmentin which it gets delivered can all be controlled when you use the, you design the hydrogelappropriately.Cell encapsulation is something which I talked about where you can actually suspend the cellsand you can actually encapsulate the cells.So, the advantage here is, it will provide immunoisolation while diffusion can stillhappen.So, one application where this is used, this would be useful is artificial pancreas.People have tried simple microencapsulation using calcium alginate beads where they havejust suspended Islets in calcium alginate beads and shown that it can be reasonablyeffective; however, it still not completely successful.That is one of things which people are working on.These are a bunch of other biomedical applications in which hydrogels are used.So, disposable diapers where they capture the urine, so is one of the common thing.So, you would see a used diaper quite thick compared to the original diaper that is becauseit can actually hold a lot of the water.A contact lenses are made of hydrogels and medical electrodes, you have other coatingsof for lubricating the surfaces and your breast implants, wound healing materials, these areall hydrogels and they are also used as reservoirs for tropical drug delivery and so on.So, currently there are some hydrogels which are commercially available for biomedicalapplications.With respect to wound dressing materials there are quite a few which are hydrogels that arecommercially available Vigilon, Hydron and gel Gelperm are some of the more popular ones.And, people have been exploring these hydrogels for many many applications like kidney membranes,artificial skin, reconstruction of plastic surgery kind of things and even for vocalcord replacements and just like how you can have breast implants other implants and injectionsthese are all different types of hydrogels which have been tried out, ok.So, even these Botox injections, all these things are kinds of hydrogels which have beentried out, ok.