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Video 1: Growth Factor
So, today we will continue our discussion on the Introduction to Tissue Engineering.So, we are looked at the two arms of the tissue engineering triad we looked at what biomaterials,and what are biomaterials and how they can be used.We also looked at cells and what are the different sources and types which we can use.So, today we will talk about signaling molecules; signals basically, not just signaling molecules.So, we will first start with the signaling molecules and I will also briefly introduceother signals.So, our focus here is just an introduction.So, we will go into greater details in the later part of the semester ok.So, signaling molecules themselves can actually be grouped into three major categories, buttheir actually overlapping category some of the molecules will act as both as a mitogenand a growth factor or a morphogen and a growth factor and so on.So, these are mitogens growth factors and morphogens.So, classically it is defined as a mitogens are the ones which will simulate cell division,growth factor were initially identify to be the molecules that help in cell proliferationand it was later identified that it actually can have multiple functions.So, the major challenge with respect to signaling molecules is how you deliver these moleculesright.So, you need to have a control delivery with may be spatiotemporal release; so that therecan actually be proper control signaling which will aid in tissue regeneration.So, usually this is delivered as a, using a biomaterial as the carrier and the moleculescan actually be chemically mobilized or physically encapsulated to provide some kind of a controlledrelease ok.So, this is what is currently being looked at.So, what are growth factors?These are soluble secreted signaling polypeptides that are capable of instructing specific cellularresponses in biological environments.So, can you so, identify some growth factors which you already know?BMP.BMP ok.So, that is a Bone Morphogenetic Protein, that is a growth factor.VEGF.VEGF which is?Vascular Endothelial Growth Factor.Vascular endothelial growth factor, you know what is the role for that?It helps in blood vessel formation.Ok Angiogenesis.It is part of angiogenesis.So, there are many other growth factors we will look at some of the examples which arecommonly used in tissue engineering applications.Actually, growth factors can help in so many different cellular responses from cell survivalto proliferation to migration to differentiation and even with tissue formation.So, it can actually have wide range of applications and it is seen that they do not act in anendocrine fashion, it’s not that the growth factors can circulate in your bloodstreamand actually reach different places that is primarily because they have short half livesand because of this, they only go through diffusion.So, but these are actually proteins right? these are reasonably large molecules.So, they are not going to diffuse very fast; so, they have very short-range diffusion andthis diffusion happens through the extracellular matrix which is present in the tissue, andthey will act locally.So, it will not have a large, like a systemic effect.So, this is a general growth factor signaling mechanism.So, this is not for any specific growth factor.So, what you have is a producer cell which would be producing some growth factors.So, all these growth factors are actually secreted by some cells.So, different cells will actually produce different growth factors.And these will actually then diffuse through the ECM and come in contact with the receptoron a cell surface.From there will be some single transduction cascade which will elicit the response whichyou are looking for.And this is a general phenomena which is commonly observed, what you need to look at carefullyhere is what is zoomed here.So, what you see is the ECM. you actually can have the molecule delivered to the siteof, to the cell in a spatiotemporal fashion, it is not that it, all of them go and reachthe cell directly.So, some of them reach the cell at certain regions some of them actually reach at differenttime points and this actually matters.So, this kind of a spatiotemporal releases the major, was one of the major roles of ECMother than just supporting the cells to grow.So, this helps in providing the proper signal for the cell to behave the way it should ok.So, that is why people have seen that if you use; if you just implant stem cells into atissue, they usually tend to differentiate to that particular cell line, because thereare signaling molecules and ECM micro architectures and niches which actually help in that kindof a differentiation ok.So, ECM actually plays an active role in the signaling.So, this is one major aspect which needs to be accounted for when we actually design scaffolds.So, you can, if you are looking to design bioactive scaffolds, mimicking the ECM becomesvery crucial.So, there are different factors that can govern the cellular response for a growth factor.It is not that you have one growth factor it will always cause the same effect at thesame levels; does not work that way.There are many factors which actually govern what response you observe for a growth factor.So, depending on the receptor type to which the growth factor attaches itself, there canactually be a different response.And depending on the cell type again there can be a different response.Some of these things actually have; some of these things actually have effects on multiplecell types, it is not that the growth factor can act only on one type of cells.So, when there are multiple cells which can actually act on the, sorry, multiple cellswhich can be acted upon by the growth factor, it depends on the cell itself.Because the intracellular machinery where the signaling cascade you are going to observe,is going to be different for different cells.so that means, by cellular response can also be significantly different.And the ability of the growth factor to bind to the ECM will also matter.So, if a growth factor can bind to the ECM, then the diffusion is going to be much lower,its going to a get stuck in the ECM for a while.So, that is going to have a role and ECM degradation.So, if you have something where the growth factor is encapsulated in the ECM, then lookingat the ECM degradation will actually control the release profiles and the release patternof the drug, sorry, of the biomolecule and this will actually play a role.And obviously, concentration and cell target location can also play major roles when itcomes to what you observe for a cellular response for a growth factor.Ok.So, this is a bunch of growth factors that have actually been tried out for differenttissue engineering applications.So, I am not sure if you can read it from there, but I can read it from here so, I willread it out for you ok.So, ANG-1 is angiopoietin and ANG-2 is also angiopoietin 2.So, these have been used for treating blood vessels and heart and muscle tissues.ANG-1 has an effect on blood vessel maturation and stability, where as ANG-2 can destabilize,regress and disassociate endothelial cells from the surrounding tissues.And FGF-2 is a blood vessel, is been used in blood vessel, bone, skin, nerve, spineand muscles.So, this helps in migration, proliferation and survival of endothelial cells, it alsoinhibits differentiation of embryonic stem cells.So, FGF has again multiple roles depending on which cell is being targeted.BMP-2 and BMP-7 are bone morphogenetic proteins, see many of these growth factors you wouldalways have some associated number with because there are many variations of these.So, BMP is a class of morphogenetic protein, bone morphogenetic proteins.So, BMP-2 and BMP-7 have been extensively studied because they actually take part indifferentiation and migration of osteoblasts, BMP-7 as also been shown to have an effecton renal development.So, that is why it has been used in kidney tissue engineering as well.EGF is Epidermal Growth Factor, EPO is I think erythropoietin.So, and EGF has been used in skin and nerve, where as EPO has been used in nerve, spineand even wound healing.So, all these molecules actually have significant effect on different cellular responses andbased on understanding what cellular response they have you can use it for the appropriateapplication.So, these are all the bunch of other things which you can look up.So, VEGF is another thing which is commonly studied because it helps in migration, proliferationand survival of endothelial cells.So, this means you would, if we need angiogenesis you can actually use VEGF and hopefully geta vascularized tissue.So, that something which people have been exploring for a while now.So, using this signaling molecules people have actually taken some products to higherlevel.They actually even able to take it even up to clinical studies ok.So, these are the clinical studies and you have VIVA where 178 patients have been testedwith VEGF165, and here they have just done infusions where they gave intravenous andintracoronary injections to deliver this molecule, and this was done to treat cardiovasculardiseases.So; however, the results were not very promising, the results were actually negative in, I thinkin phase 2 probably.So, clinical trials have multiple phases.So, you have phase 0 where you tested in other animals then you have phase 1, phase 2, phase3 and finally, phase 4 is where it is in the market and you get market feedback ok.So, most of these things; so, except for the last 2 BESTT and OP-1 Putty the others oneshad actually not gotten past phase 2.So, these two have actually been commercialized, but even with OP-1 Putty.So, Ramya; I actually was looking up OP-1 Putty the today morning and looks like itis been taken off the market, I am not very sure as to how it was.So, it was initially brought in by striker and it was purchased by Olympus and 4 yearsback it was dumped.But I do not know if somebody is has taken it up or if it has been, because there hasbeen a lot of controversy with respect to the side effects of it.I was just reading up a little bit on that and saw that there was controversy associated.But the other one BESTT, that is basically a product which is currently also availableit is infused by Medtronic, we will talk about that product a little bit later.So, they have actually tried different growth factors and what you see is the first twothings are just a simple infusions, where you just deliver using some kind of an injection,intravenous or intracoronary and they have actually failed, and people also tried alginatemicrocapsules and that did not work either.So, collagen sponges and collagen matrices have actually shown reasonable promise whenit comes to delivering these molecules, and it is understandable, right?collagen is what your ECM is.So, you are going to have a better chance of mimicking the ECM when you use that.
Video 2: Early Studies
So, as you see early studies were focused on treating ischemic diseases that is whyyou saw the first few things where about cardiovascular diseases right.So, you want to treat ischemic diseases using intracoronary or intravenous injections ofangiogenic factors.So, it was simple and straightforward approach.People just thought ok, I know this molecule can trigger angiogenesis, I know this tissueis not getting blood supply because it does not have enough blood vessels.So, you put this growth factor there, you should have new blood vessels formed, butit did not turn out to be as simple.Because the results where promising from the animal studies and phase 1 trials, but phase2 trials did not show expected results.Why do you think it failed?What could be the potential reasons?Think of all possible reasons from a clinical trial stand point has to, why something canfail? and then going to specifics of why this probably failed?Side effects.Ok Side effects; so, why do you think side effects would come?Because it is not targeted enough.Ok, it’s not targeted enough.Ok, You have a question?What is the different between phase 1 and phase 2 trials?Ok, So phase 1 you are supposed to give sub-therapeutic levels of sub-therapeutic dosage and it isgiven for a smaller number of patients and in phase 2 you are supposed to deliver therapeuticlevels to a larger patient sample size.Sub-therapeutic, what is a different between both of them therapeutically sub?Ok So, for any drug you actually have something called the therapeutic index right.So, you have to have a minimum effective concentration and a maximum toxic concentration.So, if the drug is, drug or any molecule is at concentration higher than the toxicitylevels, it is going to have serious side effects, if it is less than the minimum effective concentrationyou are not going to have any effect.So, within this window is called the therapeutic concentration.So, you initially you give something which is sub-therapeutic, which is lower than thetherapeutic concentrations, because you want to know that, see you would have studied andidentify that this level, this concentration, this dosage, will have the desired effect,but you do not know if that dosage will cause negative effects ok.So, what you do is; you start with a very low effect thinks, knowing that it will notdo any good, but you still want to know if there is any harm which is being done ok.So, that is phase 1.In phase 2 what you do is you do both, you do some, you basically group them into grouppatients into control groups and other groups and you put them as one group with sub-therapeuticand one group with therapeutic levels.So, if the sub-therapeutic levels do not show any negative effects then you go to phase2; then you compare sub-therapeutic with therapeutic effects as phase 2 trial and then phase 3you do all therapeutic levels for a much larger sample size.The sample sizes will depend on what type of drug we are dealing with and whether thereis looking at, you are looking at specificity.For example, if you are looking for a cancer drug then you might want groups for differenttypes of cancers and see how the drug would have an effect on different cancers, you mightsay that it works for oral cancer, but it does not work for some of colon cancer right.So, there could always be differences.So, those kinds of things you need to look at; so, that is what the clinical trials are.So, what do you think could be the potential reasons?Side effects is the serious cause, but what do you think causes the side effect?Immune response like the immune system there is slight variation from person to person.Ok.So yeah; so, that is a more of personalized medicine which you were talking about.So, you would not have if you are using one general thing there can always be a problem.But here chances of an immune rejection would be much lesser because you are only lookingat a protein which is already present in your body and you are just supplying it as an intravenousinjection or an intracoronary injection.Then may be the drug delivery aspect of it.How you deliver it, the mode of delivery could be a problem; so, the other, yeah.Because in vaccination like I read that it is better to inject the vaccination in themuscle rather than directly in the blood stream because muscle or skin surfaces would.So, what he is talking about is just a mode of delivery related to whole ideas for vaccination.Yeah.So, see mode of delivery can have a role to play here, but not really about how it isintravenous or intramuscular or intracoronary whatever.So, it could have an effect with respect to what; whether it reaches the site or not.When it comes to vaccine, its different mechanism.Here you are looking for it to reach the site.If I give an injection; intravenous injection, it is probably not going to reach the ischemictissue which is probably close to my heart right.So, intracoronary would probably be better because it delivers it directly to the site.So, that mode, mode of delivery could have a role, but not exactly the way you say, butin a different approach.Sir because it worked in animal studies and did not work with people basically.It I mean you mentioned some factors which determine the response of ECM whether it isencapsulated the ECM and affinity to the ECM and cell type and its receptors, it mightbe something to do with that.Yeah.So, it also depends on like what actually, how it is deliver, not just the mode of deliverywe were looking at whether it is encapsulated in a molecule.Because you are, you need to how it interacts with the with the organism right.So, it would differently, it would interact differently in a smaller species, like a rator a rabbit and when you take it to larger animals or humans it is going to have a slightlydifferent effect, quite possibly can have different effects.So, usually not more than one-third of the animal studies are actually reproducible inhumans.So, there can always be some issues with that, Ok anything else? so.When we say animal studies, do we even consider monkeys in that?So, monkeys are animals so, we would consider.They are very close to human.So, is there.See not really, see no; technically speaking apes are the closest to humans, monkeys arenot; and even amongst apes its actually quite difficult to do very large scale studies withthese kinds of apes.So, if I would want to perform studies with maybe hundred chimpanzees, it is not reallygoing to happen.So, there are other statistical issues associated with how the data is interpreted and you cannotcompletely take this forward.In some cases, it will actually be easy to extrapolate, it depends on how the complexityof the tissue itself and it also depends on how that particular organ functions comparedto human organ.So, for example, if you were to take pancreas, a pig’s pancreas is closer to a human pancreasthan an ape’s pancreas.Because the insulin response, the sugar response and insulin release are quite similar.So, it just depends on all that; so there is a, you cannot just say doing it in apewill be the best thing.So, for that particular tissue what would be the best thing.And again, for thrombotic effects if you can work with lambs or pigs they would have amuch more aggressive thrombus formation compared to what you would see in humans.So, then you are now going to have a very different set of results.So, there is always going to be variations which you have to account for ok.So, what I had here was basically some of the things which you already said, side effectis primarily because of the dose used.Ok.So, when you use sub-therapeutic levels you are basically using very low concentrations.Very low concentrations of, see if you take low enough of a concentration of anythingit is not going to cause any effects, even if cyanide could not kill you, if you takeit in a low enough concentration right.So, when it is in a sub-therapeutic level, it is not going to cause any effect and thatis ok, but once you take it to therapeutic levels, that is when you are actually doingit in a; at level which actually has any meaning and if that causes problems, it basicallythrows away your molecule.And route of administration or mode of administration; so, I called it route of administration andmode of delivery and basically talking about whether it is encapsulated or whether it isin solution and so, on.So, we already; I already said that ECM plays a role and in presenting the growth factorto the cell, it is not just about the growth factor reaching the cell right.The ECM has a way to present it in a spatiotemporal fashion, that actually controls the preciseway the cellular responses happen.So, there are factors and obviously, there can always be inappropriate clinical trialdesigns so, which could also lead to such results, which do not really give you conclusiveproofs.So, basically what was done in the early studies was large doses of potent growth factors whichwere formulated in the solution were directly injected into the body.The bad thing about this is, this can lead to severe side effects, because you have togive a very large quantity of the molecule for it to have any therapeutic effect becauseI already said they have very short half life right.So, they can actually degrade very quickly and they will also get, and if you are goingto give it in intravenous injection, it is not going to a reach the site of action fora long time.So, it will not survive for that.So, for it, for you to make sure that at least the therapeutic level reaches the site ofaction, you have to load a lot of the growth factors into the body which will be a verybad thing to do.So, and again the other side of it is when you the delivery using intravenous thing,even if you load very high concentration it still not going to reach the target tissuein the necessary time frame within which it degrades right.So, then what happens is you are only observing side effects and you are desired effects arenot even see.These are serious problems when you try to give an intravenous injection or an, justsupply it as a solution.So, one example would be VEGF.VEGF has a physiological half life of about 30 minutes when infused intravenously.See as I already said all these things do not; none these growth factors operate inendocrine mechanism right.So, they do not actually flow through the bloodstream.So, when you put it in a intravenous thing, they are actually going to degrade very fastand 30 minutes is probably a very short time for VEGF to reach the site of action and whatwould happen is to ensure something at least reach as a site of action, you are going tohave a huge concentration of VEGF loaded to the with the injection.And when you do that, you are going to have pathological blood vessel formation like whatyou would see in cancer right.In cancer VEGF is actually over expressed.So, there are therapies cancer drugs which basically just block VEGF.So, when you have very high concentrations of VEGF, you are going to have some kind ofa dormant tumor which is going to be formed and that is not really something you wantto do ok.So, there are people need to have better understanding of it how to deliver it that was the majorlacuna which had to be address eventually.And;
Video 3: Commercial Products and Other Signals
So, what people did was to put it in some kind of a matrix and then deliver it.So, one thing which was reasonably successful is the commercial product infuse.So, this was, this is being marketed by Medtronic.So, this is used in anterior lumbar interbody fusion surgery.So, basically spinal fusion surgeries and here what they use is recombinant human bonemorphogenetic protein.So, this is just produced using fundamental molecular biology techniques, BMP is overexpressedin an organism and produces a recombinant protein.So, these bone morphogenetic proteins have the ability to initiate bone growth.So, because of this the material infuse is highly osteoinductive and with this helpsin regeneration of the bone tissues.So, this is used along with a cage and an absorbable cottons collagen sponge carrier.So, they call it ACS carriers ok.So, this can potentially eliminate the need for autogenous bone grafts.So, that is what they claim to do, claim to make, but again there are still limitationsabout the size of it and how you can replace like for in some cases it can actually bea substitute for autogenous bone grafts.So, this is how it works.So, what you have is the first step is implantation.So, you basically implant this combination of BMP-2 with the collagen sponge, and thenyou would see chemotaxis where there is migration of mesenchymal stem cells and other bone formingcells to the site of implantation.So, because you have the bone morphogenetic protein, there is going to be signals sentto the nearby bone cells and bone forming cells to come to this site right.So, they start migrating towards it and then there will be differentiation sorry, thenthere will be proliferation where this particular material provides the environment where stemcells can multiply before differentiation, because you have a collagen matrix on whichthey can attach and start multiplying.And the differentiation happens because you have the BMP-2, it binds to the receptorson the stem cell surface and then this helps in the differentiation of stem cells intoosteoblasts, and then you have bone formation and angiogenesis.So, the osteoblast will respond to the local mechanical forces to produce a new mineralizedtissue which replaces the collagen matrix which they had used, and new blood vesselformation is also observed at the same time.So, this is, this is not truly triggered by this molecule itself, but there have beenstudies which say that, BMP and VEGF can actually have an dialogue basically.So, they can actually interact and create vascularization.So, there are some studies which suggest that.And there is; so, finally, you have remodeling where the body continues to remodel the bonein response to the environment and the mechanical forces.So, this will finally, form the trabecular bone.So, this is the mechanism of action so, this is directly from their website and this iswhat they claim to have been the mechanism of action for infuse, ok.So, the important thing which we need to look at is the importance of the carrier.So, whenever we are looking at signaling molecule delivery, again the material we use as a carriercomes into play.So, that is why biomaterial is actually a very crucial component when comes to tissueengineering; it depends on, it is not just about providing a scaffold and support.It is also about delivering the molecules and presenting the molecules in the rightway.So, the protein should actually be retained at the site of implantation for a certainperiod of time, only then it can have an effect.If it is going to get diffused away right, in a very short time, you are not going tohave enough time for the cells to migrate to the towards the site or to differentiateor proliferate and do whatever.So, people have shown that retention and bone regeneration have a positive correlation.So, if you can retain this molecule for a longer period of time, you are going to havebetter bone regeneration ok.So, those; so, that is why people try to look at controlled release of these molecules ok.So, people wanted to come out in a very slow and sustained fashion.So, there are many other signals as well, which I will not go in to great detail, whichcan actually be used for stimulating cellular responses.Mechanical stimulation has been explored extensively recently and there are other things like electrical,optical, magnetic and ultrasound signals as well, which can be used for stimulating cellseither in vitro or in some cases even in vivo ok.So, mechanical simulation is basically deformation which is transduced by a biophysical mechanismto create a biochemical response, and this will result in either the cell migration orgene expression and so, on.So, people have shown that the mechanical property of a material can such as elasticityor strength of the material and all those things can actually affect how the stem celldifferentiates, cells which are cultured on these can differentiates, even without thepresence of other growth factors.So, these are also factors which need to be considered while you are taking; while youare developing any tissue engineered product.So, if you are going to develop a reactor, you would want to provide these kinds of signalsas well.So, the idea is reactors for tissue engineering are slightly different from the reactors youwould have studied as part of your bioprocess training.So, these reactors are here to provide signals as well as maintain the controlled environment.In case of a bioprocess you want a controlled environment, and maybe do large scale production,but here this can also provide signals and usually it is the reactors are designed ina way that you can deliver desired signals either it could be mechanical or electricalor whether its perfusion and so on.So, there are so many factors which should be looked at.So, we will talk about how reactors can be designed in later in the semester ok.So, now that we have looked at the basic introduction.So, this is the summary of the issues in tissue engineering; when we talk about a tissue engineering,the first question we need to have an answer to is, do we want an in vitro tissue to beengineered or and in vivo tissue regeneration right.So, there are two terms tissue engineering and regenerative medicine right.So, these are used interchangeably, but technically speaking they are slightly different.So, tissue engineering is engineering tissue in vitro and then implanting it in human body,where as regenerative medicine is providing the support environment which can actuallyhelp in regeneration of the tissue in vivo.So, that is actually the difference between the two; however, it is very commonly usedin an interchangeable fashion there are journals and association which are just called tissueengineering and regenerative medicine because they kind of come together when it is discussedfor research ok.So, once we know what we want to do there, then we need to identify how we take the threequestions; the three arms of the triad like what scaffold to use, what cells to use andwhat signals do you want to deliver.So, what scaffolds?So, it is not just about what material you use, it is also about how you fabricate it,what should be the pore characteristics, what should be the surface characteristics, whatshould be the physical and mechanical properties the chemical properties of it.So, you would have to address that and as you work on the field ok.So, this is what the state of tissue engineering overall can be given as and we need to focuson different applications you see where exactly we stand, how exactly it can actually be takenforward ok.Thank you, guys.