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Module 1: Signaling, Bioreactors and Challenges in Tissue Engineering

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Video 1: Mechanics of Cell Adhesion
So, until now we have been talking about the different aspects of cell when it comes totissue homeostasis, so we talked about cell growth, death, differentiation.So, there are just two more concepts when it comes to cells, the first is adhesion,and the next one is migration.So we will talk about cell adhesion today.So, understanding cell adhesion is crucial when it comes to tissue engineering because,you need to know how you can design your scaffolds in a way that you will be able to help cellsto adhere.So, you need to identify what type of cell you are going to work with, try to understandhow those cells interact with each other and with the matrix and how they adhere to thesurfaces.Based on that, you can actually functionalize your material to get desirable adhesion properties.So, when you talk about cell adhesion there are the aspects which you need to understandare the concept of the surface receptors.So, each type of cell could have different surface receptors which help in cell adhesion.So, these receptors usually have an extra cellular domain which is the one which interactswith the surface or other cells; and you have a transcellular domain and an intracellulardomain, so through which signals and other cascades can actually be triggered.So, you also have cell to cell adhesion where cells of similar kind or different kind adhereto each other using some of these receptors and form different junctions, or you couldhave cell to matrix adhesions.So, it is important to know the difference between the two and they usually are uniquelydifferent, some types of junctions are more commonly seen in cell to cell interactions,whereas others are more commonly seen in cell to matrix interactions.So, when you are talking about mechanics of cell adhesion itself there are, there canbe nonspecific physical forces such as, electrostatic or steric repulsion or Van der Waals forces,which could play a role when it comes to interaction of cells.So, when we are talking about electrostatic adhesion, electrostatic forces, the cell-cellinteraction can basically have an negative effect.Because, you can have a repulsion because, there would be a negative charge on the cellsurface on both the cells, so which could have a, which should have a repulsive propertiesin when it comes to electrostatic forces.Whereas when you are talking about cell-surface interaction you can try to use this to youradvantage, if you have a negatively charged surface, then you can induce repulsion ofthe cells.Whereas if you have a positively charged surface, you could actually attract the cells towardthe surface, so steric repulsion is basically seen.Sir if naturally two cells, both have negative charges and repel each other and how do theysteric.It depends on how far away they are and which forces actually have the biggest impact, soit is not going to be one force which is acting right.So, you have electrostatic forces, steric repulsion, Van der Waals forces, and thenyou have the specific interaction between, because of the ligand-receptor interactions.So, based on the distance at which they are present and based on the affinity of these,like the dissociation constants or the affinity constants of these forces, you would actuallyhave interactions.So, we will talk about that, so each of them would have their own properties.So, steric repulsion is basically seen when a cell approaches an adhesion site what happensis water is excluded.Therefore, membrane bound proteins becomes concentrated which causes a repulsive osmoticforce.So, the compression of the membrane bound proteins initiate an additional repulsiveforce which is seen as the steric repulsion.So, again all these forces are more active when the distance between the two surfaceswhich are interacting.It could be both cell surfaces or a matrix surface and the cell surface is at differentlevels, at some particular distances you would have electrostatic effect having a biggerrole, whereas at some other distance it will be steric repulsion.So, this the last nonspecific physical forces Van der Waals forces.So, attractive charge interactions between polarizable, but uncharged molecules is calledas Van der Waals interactions and this is significant when the distance is greater than20 nanometers and it is less important when it is very close because the other factorsactually play a bigger role.So, this graph which is seen here, the graph you observe here, the interaction potentialversus separation distance.This graph shows you how the overall effect of all these three are, so this is not individualeffects, this is cumulative effect of these three non specific physical forces.So, electrostatic and steric repulsions are seen when the cells are farther away and youhave the receptor-ligand binding which can have a significant impact and Van der Waalsbinding which would have a lesser impact ok.So, the specific physical forces, unlike the nonspecific ones are the receptor-ligand adhesions.So, this depends on what surface, and what are the ligands present and what cell andwhat are the receptors present ok.So, the strength is much higher when it is when it is compared to other non-physicalforces, again this is not a chemical bond which is being formed it is a still a physicalinteraction.However, the binding is very strong it overcomes all the other repulsive nonspecific forceswhen this happens.Because it is much much stronger than all the other forces put together.So, the specificity also provides ways to bind specific cells to matrix to the matrix.So, you could have some cells adhere to a surface whereas other cells might not actuallylike this surface as much.So, depending on the receptors which are expressed you can actually tailor your material in away that the specific cell types are adhere to the surface.Sir.Yeah?Sir, these interactions are with respective to tissue engineered cells or like.No in general; even in nature, so if you have an extracellular matrix the interaction isgoing to still be there right.It is like why are we talking about like distance between the cells like, a natural tissue islike, is there a distance between two cells?Yeah, because the cells, it is not that the cells are always present there and they areproliferating right.So, cells could have, would have to migrate from the stem cell niche and come to a specificsite and at that, there will be factors which will play a role.So, it would have an impact even in natural conditions and it will be crucial to understandthat to exploit it in tissue engineering applications ok.So, this receptor-ligand binding actually controls the binding strength and the bindingpersistence because, there are limited number of receptors and ligands on the surface.So, based on that you can actually regulate how strong it is, where it actually bindsand so on.So, when we talk about cell adhesion in general, we usually talking about this ligand-receptorbinding.So, this reversible association of protein receptors in the membrane to the complementaryligands, which could be present on the surface or on another cell is what we talk about,when we talk about cell adhesion.So, this is what you would have studied, when you studied cell biology right.So, I hope you were taught cell adhesion as part of cell biology, so it is actually animportant aspect when it comes to cells.So that, when we talk about that from a biological perspective, this is what we are talking about.So, if we are looking at it from a chemical stand point, it is again similar to the ligand-receptorbinding which we looked at when we talked about growth factors earlier right.So, what would happen is, the ligand and receptor forming a complex.And this would be obviously, a reversible reaction because than specific physical force,but it is a reversible process.So, there would be a dissociation constant, and association constant and if the dissociationconstant is very high, then the affinity it is lesser.If the dissociation constant is low the affinity is much better.So, the interaction would be much stronger ok.So, this shows you the association and dissociation constants for different-ligand receptor complexes.So, you can see that these are classified based on the type of junctions or adhesionwhich would be done, so integrins is one aspect and you would see, so one type.So, integrins you see fibrinogen interacting with alpha 2b beta 3 receptor and you seethat the dissociation constant is really really small right.So, that shows that there is a significant in, significantly strong interaction, therewould be some which are not that strong.So, if you where to take selectins where GlyCAM-1 and L-selectin, it dissociation constant isnot that small it is orders of magnitude higher, so indicating that it is much weaker.So, there are other things which are as close to even covalent bonds like the biotin avidincomplex.So, biotin avidin complex is as strong as a covalent bond, so the dissociation constantis about 10 power minus 15 ok.So, understanding these things can help because specific cell types will actually expressspecific surfaces, sorry surfaces receptor right, we would know.So, fibroblast cell surface will have receptors which will bind with RGD, that is known.So, if you can tailor your material to have RGD, then your fibroblast can be recruited.So, similarly you would have to figure out what would be the suitable ligand for differentcells which you are working with.So, if you have a bone tissue engineering in your mind, we need to figure out what receptorosteoblast would like and try to modify your surfaces with that.Hopefully that will help you, help in making sure that more bone cells are attached tothe surface.So, as I already mentioned affinity is represented by the dissociation constant, dissociationconstant is basically numerically equal to the ligand concentration required to achieve50 percent receptor binding.So, this is like your Michaelis-Menten constant right.So, that is a 50 percent of your reaction rate is achieved at a particular substrateconcentration that is you km.So, similarly the numerical value at which you can get 50 percent receptor binding isthe ligand concentration is the dissociation constant.So, this varies between 10 power minus 6 to 10 power minus 12 molars, so 10 power minus6 is considered to be low affinity versus 10 power minus 12 is considered to be muchhigher affinity.So, to predict the strength of specific cell adhesion by multiple non covalent bonds, thetensile strength of the receptor ligand bond has to be estimated.So, basically to see how strong this binding is, there are experiments to do this, thereare many studies, so like there, I will give you a reference which you can go through.So, that discusses some of the experiments that can be done to study the receptor-ligandstrength, binding strength.So, the strength of the affinity bond relates to the standard free energy which is deltaG dot G0 and the bond length, so what does this delta, G what is Gibbs free energy.At least the ones who are studying thermodynamics in this semester.The spontaneity of the process.That is the outcome.That is how you, I am, so if your Gibbs free energy is negative then the reaction willhappen spontaneously that is fine.But what is Gibbs free energy?Your quiz 2 would have gotten over just now right?I am pretty sure he would cover Gibbs free energy.Already.What is Gibbs free energy?You studied Gibbs free energy right.Yes.So what is it?Nature of feasibility of a thermodynamic energy or any reaction.So that is one aspect, so in general, it is a thermodynamic potential.Basically, it is a thermodynamic potential and it measures the useful or process initiatingwork obtainable from a thermodynamic system.So, it could be a reaction or any other process ok; like, reaction is what we commonly lookat when you talk about Gibbs free energy.So, we always try to associate it with that, but it is not necessary that it is needs tobe for that.So, Gibbs free energy is the capacity of system to do non-mechanical work and delta G basicallymeasures this non-mechanical work done on the system.Anyways so, the standard free energy can actually be calculated from the binding constants,so you would get delta G0 equals kT ln of Ka by K0 ok.So, Ka here is the binding constant and K0 is the binding constant at a standard conditionusually one mole inverse and k is the Boltzmann’s constant and T is the absolute temperature.So, bond length is assumed to be greater than the size of the individual weak bonds withinthe binding site and less than the size of the binding site itself.So, it will be somewhere in between using this we can calculate the free energy fromwhich you will have an understanding of what would be the strength of the bond affinity.

Video 2: Cell Junctions
So, coming back to the biology aspect of this, so cell junctions are created when we talkabout cell adhesions.The binding of cell surface receptors show complementary ligands is the process in whichthere is association between the cell and external stimuli.External stimuli could be cells, materials, or cells, matrix or foreign objects, so ourscaffold would be technically a foreign object right.So, we would have to figure out how the interaction happens, the stability of these cell-cellor cell-matrix assembly can be enhanced by formation of these cell junctions right.So, instead of just having physical attachments, physical, nonspecific physical forces, ifyou can have these specific junctions which are formed, then you are going to have muchtighter and much stronger interactions.So, there are different classes of the cell junctions, so the first type is called asa tight junction, you also have anchoring junctions and communicating junctions, sowe will quickly go through what these are.So, tight junction is basically something where closely associated areas of two cellswhose membranes are joined together, form a virtually impermeable membrane barrier toa fluid.So, this is a very tight interconnected junction, so the transmembrane proteins interact witheach other to form a very strong protein complex.So, here both the cells are contributing equally, so you would have the receptors and ligandsinteracting from both cells at an equal level.So, this allows molecules to move against concentration gradient, that is the importanceof having these type junctions.It is not just diffusion through which it is going, it can active transport of moleculesis possible through this tight junction ok.So, permeability of tight junction decreases logarithmically with protein density in athe junction.So, I, because it is like dependent on the concentration of the proteins it will be firstorder mechanism, so it will, you will have exponential increase and logarithmic decreaseand so on right.So, you would have to see that, you would see that as the number of tight junctions,number of proteins or the protein density at the junction increases you are going tohave significant reduction in the permeability.But it will become much stronger interaction thereby helping in crossing the barrier like,helping in transfer from lower concentration to a higher concentration.So, anchoring junctions are the ones where the protein complex is formed where the cellsare actually mechanically attached to another cell or matrix.So, this usually consists of two portions; the first one is the intracellular attachmentof proteins, which connect the cytoskeleton to the membrane, and then there is transmembranelinker proteins which tethers the membranes between the cells.So, the attachment occurs when these transmembrane protein binds to the complementary proteinin the adjacent cell or the matrix.So, this basically acts as a mechanical link, like a hook which has been connected right.So, now you, so this is now connected to the cell, intracell transmembrane protein andthen there is a intracellular attachment proteins which connects it to the cytoskeleton.So, it was now a kind of hook to the surface.So, these are some of the anchoring junctions.So, as you can see your identify there are some of these is shown here, you have thedesmosomes, you have adherins and you have the hemidesmosomes.So, these are the different anchoring junctions which can actually be formed.So, this is the overview of the anchoring junctions, so if you are talking about adherens,their transmembrane protein which is involved is cadherin and the extracellular ligand isalso a cadherin.So, the intracellular linkage which is the intracellular protein, accessory protein whichis present would be actin fibers.In case of desmosomes you would have cadherin-cadherin interaction; however, the intracellular linkages,intermediary filaments which are present in the cell.Hemidesmosomes are not cadherin dependent they form interactions based on the integrinsand the ECM protein.And so, what you can identify from that is adherens and desmosome are usually used incell-cell interactions, whereas hemidesmosome is the type of anchoring junctions which isformed between a cell and a matrix ok.So, again intermediate filaments are involved in the intracellular linkage.So, focal contact is basically an integrin-ECM protein junction which is an actin, actinfilament acts as the intracellular linkages.So, integrins are and cadherins are usually involved in these types of anchoring junctions.So, adherens are basically, adherens junctions are the types of junctions which connect actinfilaments to either the matrix or the cell.And the junctions usually are composed of cadherins, which are transmembrane proteinsthat form homodimers in a calcium dependent manner.So, cadherins require calcium to form these to be active.And p120 binds the membrane region of the cadherin.And beta catenin binds the catenin binding region of the cadherin, binds to the cadherinbinding region of the cadherin.And alpha cadherin binds the cadherin indirectly via the beta catenin or to the cytoskeletonor to the plakoglobin which should be the intermediary filaments and links it to theactin with the cadherin ok.So, these are just the structures, so the mechanism like, the understanding of thisdetails of this is probably not very crucial for tissue engineering applications.But what is more important is to understand how these are formed or like what are thereceptors and ligands which are involved in these things which can be tried to apply totissue engineering applications.So, desmosomes and hemidesmosomes are also anchoring junctions.So, in case of desmosomes what you see is, a connection between the intermediate filamentsto the adjacent cells; the intermediate filaments are usually made of a fibrous protein thatform a much of the structural framework of the cells.So, some of these would be keratin, or vimentin, or desmin, so these are just fibrous proteinswhich are present to create this structural framework.So, the connections here again are mediated by cadherins ok.So, the two these two things, the adherens and desmosomes are cadherin-dependent anchoringjunctions; the hemidesmosomes are integrin dependent anchoring junctions.So, here what happens is there is a connection of the basal surface of the cells by meansof intermediary filaments.So, these interact with the extracellular matrix to form the junctions ok, so hemidesmosomes;these are some of the representations of how this interactions actually happened.

Video 3: Cell Adhesion Receptors
So, this these are the receptors, so if you look at the major receptors, cell adhesionreceptors which are exposed they can be categorized as into four major groups; integrins Ig-likeCAM, or cadherins or selectins right.So, these have different structures and depending on which cell type you are working with oneor more of them can be over expressed, so you can design your scaffolds appropriately.So, basically these four are can be again classified, grouped as two major things; oneis the calcium dependent and other is the calcium independent receptors.The cell adhesion molecules which come under calcium dependent would be the integrins,cadherins and selectins.Immunoglobulin like receptors or the Ig-CAM receptors are calcium independent.So, integrins are calcium dependent proteins which are involved in cell-matrix adhesion,but some can actually be involved in cell-cell adhesion as well.So, what happens here is there is a heterodimeric membrane protein, which consists of non covalentlyassociated alpha and beta subunits.So, this is the structure of the integrin and the binding is actually very specific.It binds to collagen and laminin through the alpha 1, beta 1 integrin and through whereasto fibronectin through alpha 5 beta 1 integrin ok.So, depending on the two subunits, you can actually have different specificity for attachment.So, if you are talking about this transmembrane protein, this connects the cell to the externalenvironment, and the extracellular domain basically binds to the ECM protein.And the cytoplasmic domain binds to the cytoskeleton in the cell, ok.So, this way you actually have proper interaction within the cell, with the matrix.RGD is one of the ligands which interacts with the integrin.So, many ECM molecules contain RGD domains.So, these are, this is very popular fibronectin actually contains a lot of RGDs, so does collagenand laminin and even keratins and so on.So, cell binding through this RGD domain is often through, mediated through integrins.And so, people have actually, this was one of the early peptides which were used forimproving cell adhesion.So, when you are looking at modifying cells like material surfaces, people are tryingto attach RGD domains or incorporate RGD domains as part of this matrix.Or try to use molecules which would have these RGD domains usually proteins should have RGDdomains to prepare this scaffolds, so that way were trying to improve cell adhesion.So, it can also be used to reduce cell adhesion, if you were to use RGD as a soluble RGD inthe media.So, if you want to just move it away from the scaffold, but that is also, that mightbe required if you are looking for like some kind of separation using this like cell separation.So, where you say that cells which do not express receptors for RGD ligands should beadhered where as the cells which actually express these receptors should be in suspension.So, if you want to do something like that, then you would use RGD in solution therebypreventing adhesion of the cells.Cadherins are again calcium dependent binding, so these.Would you give an example of a why that would be required using RGD to prevent.So, you remember it we talked about cell isolation, so during that time I was saying one of thethings which can be done is to selective adhesion to surfaces or selective separation.So, this would be one of those applications.Sir, I have heard that RGD mutagens is widely used in used in bone tissue engineering.Is it how is it actually.So, basically fibroblasts, all types of fibroblast actually have like receptors which will interactwith RGD, osteoblast are can be, can have similar like surfaces like they are more likefibroblast present in a bone tissue right.So, because of that reason they actually adhere in nicely to RGD.So, this can be seen with chondrocytes, many of these types of cells have integrins whichcan bind to RGD, so that is why people try to use that.And so, these materials can, these cells are advantageous because they can also secretetheir own matrix; like, fibroblasts, and osteoblasts, or chondrocytes they secrete their own matrix.So, if you have adhered them, so as your matrix, as the scaffold you used gets degraded overa period of time you would have new matrix formed because these cells are depositingtheir matrix.So, that is why they try to optimize it that way ok.So, if you are talking about cadherins these are in mostly involved in cell-cell bindingand unlike integrins; integrins are mainly involved in cell matrix bindings.So, these are transmembrane proteins which consists of about 700 amino acids, they contain5 extracellular repeat domains which are called as the CAD domains.And the 3 calcium binding regions, this may, they contain a membrane spanning region whichis the transmembrane region.And then a cytoplasmic region which typically binds to the actin filaments in your cell,the CAD domain consists of histidine, alanine, valine domain which regulates the binding.So, just like how RGD is one of the ligands, here the domain itself contains this histidine,alanine, valine combination domain ok.So, there are different classes you have E-cadherins which are the epithelial cadherins; P-cadherinswhich are placental and epidermal cadherins, and you have N-cadherins which are seen innerve cells, lenses and the heart cells ok.So, the dominant mechanism, this is the most dominant mechanism for cell-cell interactions.Selectins are again calcium-dependent bindings, these are usually expressed in a transientway, these are not very involved in regular cell adhesion processes.So, these are typically expressed on the surface of endothelial cells that line the arterywalls.And they contain a lectin domain which recognizes the specific oligosaccharides expressed onthe surface of the neutrophils.So, thereby they can actually attract neutrophils when there is an inflammation, so this isover expressed only at that point.So, the neutrophils will bind, migrate and eventually transmigrate through the vesselwall to participate in the inflammatory response.So, that is where selectins play a crucial role.The Ig-CAMs or the immunoglobulin like receptors contain one or more domains that have structuralsimilarity to the immunoglobulin molecules.And this again mediate cell-cell interaction, these are calcium independent binding.There are two major classes, there is a N-CAMS and I-CAMS.N-CAMS is where involved in neural cell adhesion, where as I-CAMS are involved in other intercellularadhesion molecules.So, N-CAMS are present in many cells; however, they are primarily seen in nerve cells.These bind cells by using a homophilic interactions of N-CAMS.Whereas I-CAMs are expressed in activated endothelial sites, endothelial cells and thesebind integrins on the leukocytes by heterophilic mechanisms, so they provide very fine controlof cell adhesion.So, depending on the structure of the immunoglobulin which is present, they can actually selectivelyattach cells and so on.So, we looked at what the cells do, so the other side of it is what are the ligands whichare present on the matrices, you try to understand that to emulate cell adhesion in tissue engineering.So, we have already looked at what ECM is, ECM is made up of different things proteins,proteoglycans, and glycosaminoglycans.So, glycosaminoglycans could be Hyaluronan, Chondroitin sulfate, dermatan sulfate Heparansulfate, Keratan sulfate and so on.And you could have proteoglycans are and proteins such as Collagen, Fibronectin, Laminin, andElastin, and Tenascin, Vitronectin, and Thrombospondin and many other things ok.So, depending on what is present, each of them would have different ligands which willhelp in attachment of cells.The glycosaminoglycans are usually high molecular weight polysaccharides which typically formABn, it is a poly disaccharide, right.So, your hyaluronic acid is a disaccharides, poly disaccharides and so on.So, one sugar residue is always an amino sugar, and the second residue is typically an uronicsugar, usually an uronic acids such as glucuronic acid and so on.So, these are unbranched, inflexible and highly water soluble, they are very hydrophilic andthey are highly sulfated as well therefore, they have a lot of negative charges.So, this induces migration of positive charges into the matrix creating osmotic forces thatensures that the tissue is hydrated.And they can, these are the molecules that is used to form hydrogels even at very lowconcentrations.So, hyaluronic acid is one example for that, it has been shown to help in embryogenesis,tissue repair and it also facilitates cell migration.So, proteoglycans are proteins that possess glycosaminoglycans linked, using some kindof a linker protein.So, they possess an extremely high sugar content which could be about 95 percent of their weightbecause the glycosaminoglycans are very large polymers which can be attached to these proteins.So, they are very highly heterogeneous structures and take part in many functions; they canact as reservoirs for signaling molecules, or mediators for adhesion of cell membranes,or size or charge filters for the molecules which are crossing.So, proteins as I have mentioned collagen which is the most abundant protein in ECM,there are different types of collagen and they are present in different regions.You have laminin; laminin has nonspecific binding; laminin is one of the proteins whichhas very high amount of RGD domain, fibronectin also has RGD domains which help in integrinmediated cell adhesion.So, other things are elastin, tenascin, vitronectin, and thrombospondin.All of these are involved in different aspects and many of them have RGD binding sites.So, as you see RGD is a very ubiquitous ligand seen in the many of the matrix components,so that is why it has been extensively studied.So, if you were to look up strategies to improve cell adhesion are attaching RGD peptide wouldbe one of the most common strategies.