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Module 1: Signaling, Bioreactors and Challenges in Tissue Engineering

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Video 1: Growth Factors
Hi everyone.So, today we will be speaking about biomolecule delivery for cell signaling in Tissue Engineering.So, we know that the tissue engineering triad is composed of cells, scaffolds and signals.So, signals will be the aspect we will be focusing on today.So, what are signals?So, signal is a function which conveys information.So, in a cell each and every activity and function is controlled by signals.So, the signaling is used to communicate with other cells and also from the environmentthe cell takes in signals.So, this was essential for multicellular organisms to evolve.So, it was a very crucial aspect especially in tissue engineering thereby.So, ability to control the signals would entirely give us complete control on making a tissuegrow in the way that we want because that is what is naturally being carried out bythe organism itself.So, the three kinds of signals which are present are chemical, mechanical and electrical.So, when it comes to chemical signals there are growth factors, mitogens and morphogens.So, growth factors; so they are small soluble proteins which are produced by the cells itselfand they act as signaling molecules between cells like hormones and cytokines.They help in proliferation, growth and differentiation of cells.So, something to note here would be that growth factor is a term which is frequently confusedwith cytokines.So, what is important to notice that growth factors usually always imply a positive effecttowards cell growth whereas cytokines can have a neutral or a positive or negative effecton cell growth.The reason for both of them to have an over clash in terminologies because cytokines derivedfrom the immunology field, whereas growth factors derived from the developmental biologyfield.So, there were many proteins which are found to be having the same function in both thefields.So, in tissue engineering we would be referring to growth factors to refer to all the proteinswhich affects cell growth, migration, proliferation.So, even mitogens and morphogens we would broadly call them as growth factors, but tospeak on what mitogens and morphogens are.Mitogens are usually proteins that initiates cell division where as morphogens controlgeneration of tissue form; that is the tissues form, the structure that it takes.So, it ends up having a concentration gradient.So, based on the concentration gradient the tissue ends up taking its form.Mitogens and growth factors could be confused to have the same effect too at times becausesometimes growth and like cell division are interlinked.So, in mammalian fibroblast for example, cell growth regulates the cell division.And also at times the growth factors would activate the mitogens So, indirectly initiatingcell division.Sometimes a growth factor can be a mitogen or morphogen like VEGF and BMP.So, there is some level of overlapping between them.So, it all depends on how the cells are wired.So, based on that, the growth and proliferation could be interlinked or to a great extentor not.So, now the question is why do we use growth factors?So, we can always see in tissue engineering that there is an advantage to using biologicalmaterials like a cell for treating a disease.So, having a cell in the scaffold ends up meaning that the cells are able to producethese growth factors and proliferate the cells and improving the healing time.So, there are also constricts in which we do not use cells even here the growth factorsare important because you want the cells to actually migrate to the site of wound andend up healing the area.Now, a question would be.So, if we can use cells, then why use growth factors separately?So, when we are using cells it will be useful to add growth factors because it can enhancethe regeneration and also the differentiation.So, example is TGF-b in bone regeneration and BMPs in osteoinduction.So, now on how these growth factors act.So, like all signaling molecules this growth factors bind to specific receptors on thecell surface and they activate a intracellular signal transduction system and thereby attacks.So, the growth factors do not act in a endocrine fashion because it has a very low half lifeusually.So, its not usually released into the bloodstream to act on far off organs or tissues.So, its usually in a local area.Now, the complexity of this growth factor interaction can vary with the receptor.So, one growth factor might activate multiple receptors at times and each of and also itcan activate multiple receptors on different cells and end up having a different effecton each of these cells.And something which adds to this complexity level is the factor fact that the ECM, thetarget cell location, the concentration of the growth factor, all these play a huge rolein how the growth factors act.Now, this is an image of the growth factors, common growth factor families that shows thereceptor and the function.So, in here you can see that the first growth factor for example, the epidermal growth factorsthere is might, it act as a mitogen at the same time for ectodermal and mesodermal cellsand also promotes proliferation.So, this for epithelial cells.So, this goes to show that how growth factors can actually have a lot in common with mitogensand morphogens and also at the same time have a different effect on different cell types.Now, in the clinical applications of growth factors are not limited to just tissue engineering.So, we will take a look at what the other applications commonly have been used for.So, after chemotherapy when there is a low white blood cell count, the granulocyte colonystimulating factor are given to increase the WBC proliferation and get back the count tonormal.And even before stem cell transplant usually that stem cells are taken from the bone marrow.In that case its a painful process to actually take it from the bone marrow.So, an improved technique is actually to give a growth factors; growth factor which inducesthe stem cells to proliferate and thereby come out of the growth bone marrow sorry bonemarrow into the bloodstream.And from the bloodstream it can be easily collected and transplanted to the same patientor the another patient.Then in alleviating anaemia, low RBC count or and low platelet count all these aspectswe can use growth factors are being used.So, now the hurdles in using growth factors in tissue engineering.So, the most critical aspects in using growth factors in tissue engineering is its deliveryto the right site.So, we can always think of injecting this growth factors to the site that we need asa bolus injection.So, now, the problem there is the half life of these growth factors are very short.So, bFGF has just 3 minutes and VEGF has just 50 minutes.this these are very short actually.So, we would have to actually inject a large dose of this growth factors.So, that we have any considerable amount of growth factors which can act on the tissueand bring about the effect.So, this high dose in turn ends up leading to other side effects like edema or an increasedrisk of cancer.So, this is something which you would like to avoid and also growth factors are quiteexpensive to produce.So, the solution would be to actually use advanced drug delivery systems.

Video 2: Delivery of Growth Factors
So, what is the advantage of using this system is that they are able to give a temporarilyand spatially controlled release profile.So, they can give a sustained release of these growth factors from this delivery systemsfor a prolonged period.So, conventional delivery systems usually cannot do this.A mass amount of large dose of growth factors is not required when you use a delivery system.And also, it is able to go to the right site and act in the right environment alone, therebyreducing the side effect too.And also, it is not exposing the growth factors usually to the biological environment, therebyreduces the rate of its degradation by the system.So, the approaches to deliver growth factors mainly include immobilization or encapsulationof these growth factors onto scaffolds or matrix, else using nanoparticles for deliveryof this growth factors or else gene-based approaches using nucleic acids.So, this shows an overview of all these three approaches.So, there is delivery of growth factors by using nanoparticles or immobilizing them ona scaffold or a matrix or else gene delivery using nucleic acids into the cells.So, when it comes to immobilization or encapsulation of growth factors on to a matrix, there arethe three approaches again there.So, one is physical encapsulation another one is chemical conjugation or there are approacheswherein its ECM inspired from the extracellular matrix of the body itself.So, in physical approaches what we do is we end up physically encapsulating these growthfactors into a scaffold or a polymer and you put it into the site of the disease and itsthe growth factor slowly diffuses out thereby causing tissue regeneration and healing ofthe wound site.In chemical conjugation, the growth factors are conjugated onto a matrix chemically andthen it is again implanted into the tissue site and it stays there and ends up helpingin proliferation of the tissue and tissue regeneration in the end.So, first we will look at physical encapsulation or immobilization techniques.So, the first one is physical encapsulation of growth factors by mixing the growth factorin a polymer before its gelation.So, that the growth factors are trapped inside the polymer and you can transplant it.So, the advantage of this is, its quite easy to make and also the properties of the scaffoldand also the growth factors are not affected much.But the disadvantage is the loading capacity of growth factors into these polymers wouldbe quiet low.The second physical encapsulation technique is absorption of growth factors onto the surfaceof the matrix.So, here a pre-formed scaffold is taken and the growth factors growth factor is actuallyjust dropped onto the surface.Here it just gets absorbed onto the scaffold and it stays there.Scaffold with the absorbed growth factor is implanted in and then it releases over time.But here, the issue is since its just absorbed on to the surface there is this possibilityof burst release happening which is not preferable.Because it can cause side effects by spiking the concentration of growth factors in thebody to a higher dosage than required, but the advantage here again is that its a easiertechnique.Then the third physical encapsulation technique is layer by layer self assembly, here whatwe use is a polyelectrolyte.So, polyelectrolytes are layered and the growth factors are sandwiched in between.So, polyelectrolytes have different charge they can be polycations or polyanions, positiveor negative.So, the growth factors based on that charge too can be sandwiched in between and we cango for multiple layers as shown in this picture.So, there can be multiple layers of growth factors sandwiched in between this polyelectrolytes.So, here what the advantages are that we can we have a very good control over the deliveryrate.So, there is no issue of a burst release here.Then coming to chemical conjugation, one of the most commonly used technique is carbodiimidecoupling immobilization.So, carbodiimide coupling involves crosslinking the growth factor to the scaffold, usuallyusing a cross linking agent.So, the example here is a covalent immobilization of VEGF and angiopoietin to growth factorson a porous collagen scaffold.So, the advantages is, it is quite simple and you get a very high conjugation ratioand a low cost.But the disadvantage is during this coupling when you crosslink the growth factors ontothe scaffold, it can lose its functionality if its active sites are altered.Now, coming to the second chemical conjugation technique, its mussel-inspired bioconjugation.So, here mussels are these organisms that grow on the beach on the rocks, you wouldfind them attached in the sea.So, they actually use chemical called DOPA, which can very strongly attached to almostall surfaces.So, similar to that, what is been developed is polydopamine using dopamine, which cando similar which can, which has a similar property to DOPA.So, this DOPA can be coated on almost any scaffold surface and the growth factors canbe made to conjugate onto it.So, the advantages that it is a strong adhesion.So, you would get a high affinity and also stability.So, there would not be a burst release or uncontrolled release pattern here also.The disadvantages again as previous chemical conjugation technique, the growth factor canlose its functionality during the immobilization technique.Now the ECM inspired immobilization approaches.So, in the ECM we find a lot of proteins or molecules which can actually control the releaseof growth factors.They act as reservoirs by having a very high affinity towards these growth factors andthereby altering how the growth factors are distributed in the environment.So, one such molecule is heparin sulfate.So, it has a very high affinity to growth factors like BMP 2 and VEGF.So, you can use this molecules to actually coat the surface of your scaffolds and ensurethat the growth factors like, the ones which have affinity towards heparin sulfate endup getting immobilized on the surface.So, the advantages it mimics the ECM, so, it is much more natural environment that weare simulating here, and the we get a very good spatial and temporal control.Another ECM based immobilization approach is adhesive protein-based binding.So, just like heparin sulfate there are proteins too like, collagen and fibronectin, whichhave affinity to certain growth factors.So, based on that, we can even coat the surface of your scaffold with these proteins likefibronectin or fibrinogen.So, the example given here is BMP 2, which has a higher affinity to fibronectin fragments.So, we can do that and ensure that the growth factors remain attached stably onto the scaffold.Another approach inspired by ECM is using the structure that the ECM provides.So, biomimetic ECM nanostructures can be achieved using your scaffold itself.This too can improve the growth factor affinity towards the molecules.So, the next approach in delivering growth factors we are going to look at is nanocarrier-basedapproach wherein you use a nanoparticle to deliver this growth factors.So, the advantage to this is nanocarrier-based delivery has already been studied quite wellbecause it is commonly used in drug delivery systems.And mainly the advantage of using this technique is, it can have a very high loading efficiencyand also it can quickly respond to environmental stimuli such as temperature or pH.So, spatially it would be easy to control.So, it can go to the target site and only then release the growth factors, which isa great advantage.Also, they protect the growth factors from the bio-environment, thereby not preventingthe, thereby preventing the growth factors from being degraded easily by the physiologicalsystem.So, the first nanoparticle-based system we will be looking at is synthetic polymers.So, synthetic polymers like PLA, PGA and PLGA these are widely studied in drug delivery.So, PLGA is FDA approved and also it protects the drug inside and its well studied.So, one problem when it comes to PLGA is its poor affinity to the proteins when it comesto physiological conditions.So, the protein retention might be low.So, the proteins can actually leach out in the physiological environment.Protein based nanoparticles.So, albumin is protein which has been widely studied for this application.So, it has multiple drug binding sites.So, you can conjugate the growth factors quite easily and also their surface properties arewell tolerated by charge polymers.They are biodegradable and also, they are easy to fabricate and they are reproducible.So, usually a stabilizer coating is given to these nanoparticles, usually of glutaraldehydeor other polysaccharides; this is done to prevent aggregation.So, an example of this protein-based nanoparticle approach, which has been integrated with themicroarchitecture approach, which is previously mentioned is this study.So, wherein chitosan alginate coated bovine serum albumin nanoparticles was used.So, two different growth factors as you can see has been incorporated into this nanoparticlesand they have been arranged based on the charge in a biomimetic nanostructure.So, it has shown that it has a very sustained release.So, you can see the two growth factors releasing in a very controlled fashion in a very sustainoverall sustained period.So, its around 30 days its releasing for.Also, the micro architecture has been shown to have a synergistic effect along with thisgrowth factors in improving the cell behavior.So, the example shown here is BMP-2 which is been encapsulated in a polysaccharide-basednanoparticle, chitosan base.And also, it is arranged in a polyelectrolyte complexation fashion, which was is similarto the layer by layer approach that we saw.So, chondroitin sulfate is similar to heparin is able to easily attach to BMP-2 becauseof its high affinity.It forms a complex and chitosan ends up complexing with this, due to polyelectrolyte becauseof the charge, charges between them and they form a nanoparticle.Other nanoparticles which are worth mentioning are liposomes which has been widely studiedfor drug delivery applications.So, it is a phospholipid bilayer vesicle actually, which has hydrophobic and a hydrophilic region.So, based on the molecule of interest you can incorporate it in the hydrophobic or thehydrophilic region, which is a advantage when it comes to delivering nanoparticles.But aggregation is one of the issues in liposomes, but this can be overcome just like previouslydiscussed by giving a polymer or a polysaccharide coating.Another nanoparticle worth mentioning is mesoporous silica.It is a silica nanoparticle with a lot of the very porous structure.It has a very high surface area and the particle size can be easily controlled and has a veryhigh good biocompatibility even the liposomes have a good biocompatibility.So, burst release can be eliminated by giving a polymer coating.Now, coming to the third approach that we will be talking about is the gene-based approach.This is a little more complex method, but it offers us a lot more approaches whereinyou can activate or deactivate genes which are already present in the cell, or else ifyou want, you can even introduce new genes which are not present in the cell.So, that is something which this uniquely this approach uniquely offers.So, the steps mainly involved in delivering a gene into the cell involve, first a complexationor condensation of this nucleic acid and a nanoparticle formation and then this nanoparticleis; then this nanoparticle is taken into the cell by a via endocytosis.So, it forms an endosome bilayer around this nanoparticle.So, now the payload has to break out of this bilayer, that is the release of the cargointo the cytosol.So, if the payload is a small interfering RNA, which can go and bind into mRNA and stoptranslation into proteins, it would take its action in the cytoplasm itself.If it is a DNA which needs to be carried into the nucleus, it is further taken into thenucleus wherein it can incorporate with the host DNA or else stay as plasmids.Then degradation of the delivery system that you used inside the cytoplasm, that is veryimportant this it can lead to cytotoxicity.So, that is something which we look at, we will look at avoiding.So, the delivery vectors usually used for delivering nucleic acids are viral or non-viral.So, in the viral vectors the advantage is it has a very high efficiency of deliveringthis genes, but at the same time safety is a concern and also low payload.So, the safety concern is that although the viral in part of this viral-nanoparticlesare deleted out, it can always mutate back into being virulent; that is the disadvantageof viral vectors.So, known viral, when it comes to non-viral vectors they are less immunogenic.So, they are much more safer when compared to viral vectors and also there is a higherpayload, but its efficiency is quite low.So, both of them have a difficulty in delivering genes in the in vivo delivery.So, the solution to this is to actually take the cells out into an ex vivo environmentand then transfect these cells with the gene of interest that you want and then reintroducethem into the body ok.Now, after looking at the chemical signals, we move onto mechanical signals that are usedfor tissue engineering.So, mechanical signals are normally used by the cell to actually grow, proliferate andalso in almost.So, the normal development of most tissues require some sort of mechanical force is tobe involved.So, these are few of the mechanical forces which are in the development process of atissue.So, spring forces as you can see is its similar to a spring wherein if its compressed itstends to relax.So, this is what drives the sperm cells to penetrate the eggs.Osmotic pressure is something which activates egg cells and shear stress which is foundin the heart wherein the blood is pumped by the heart and it experiences a shear stresson it and this also actually affects the endothelial cell differentiation and its quite importantfor the development of these tissues.Something which is interesting to observe is that only about 10 percent of the postnatalchanges in a bone strength is influenced by hormones where as the rest like around 40percent is just by mechanical factors.Now, moving onto electrical signals these are also very vital for the development ofthe tissues.So, it has been shown that endogenous electrical fields are usually formed at wound sites todirect cells towards this wound site there by helping in wound healing.So, when this electrical fields are not present in wound sites, it ends up comprising howthe wound healing takes place.Neurons cultured in an electrical field have been shown to alter its direction and rateof neurite extension based on the parameter of electrical stimulation.High doses of growth factors can pose serious side effects.So, we also need studies to ensure that we can send the growth factors at the only requiredconcentration you know.High doses of growth factors can pose serious side effects.So, we need to study and make sure that the controlled release of these growth factorsend up giving only the concentration that is required for that.So, cells are just one of the aspects in tissue engineering and we need to put the cells,scaffolds and signals together which requires a multidisciplinary approach.Thank you.