Video 1: Anatomy and Physiology
Today, we will talk about an application in tissue engineering; so, this is the VascularTissue Engineering.So, the idea for this is to try to bring together all the basic fundamentals which we have lookedat.Ok So, let us start discuss vascular tissue engineering which is one of the applications.So, I am not going to discuss any particular paper; however, or what I am going to do hereis give an overview of how an application should be approached when we are talking abouttissue engineering.You will have to see and understand the some of the biology and then try to come up witha design for how you would tackle the problem.So, we will talk about vascular tissue engineering because that is one of the popular areas whichpeople are researching.And first when we talk about vascular tissue engineering, we need to understand the anatomyand physiology which is very basic to start with, right.So, there are different parts which are involved in the circulation, circulatory system.So, the functional parts which are involved are the arteries, arterioles, capillaries,venules and veins.So, I have put them in this order because an oxygenated blood first enters into thearteries from where it branches out to enter into the arterioles.And, then it goes into the capillaries where oxygen transfer would happen and the deoxygenatedblood is collected by the venules, which then enters into the veins, and this vein takesit back to the lungs and the heart and so on.So, this is the overall structure.So, the arteries transport blood at a very high pressure; arterioles act as control valvesthrough which the blood is transported to the capillaries, because with that high pressureif it cannot enter into capillaries which actually have very thin walls.So, capillaries job is to ensure there is exchange of fluids, nutrients, electrolytes,hormones and any other substance that might have to be exchanged.And venules collect the blood from these capillaries and veins are the conduits which transportthe blood from these tissues back to the heart and so that it can be pumped back again.So, this is your heart, so, this is the detailed diagram which shows all the heart walls andall the arteries and veins which are coming out of the heart.So, we will not bother too much about the heart anatomy and physiology here becausewe are only looking at the vascular tissue engineering not really about cardiac tissueengineering.So, we are not trying to engineer the heart, but it is important to understand which bloodvessels are actually coming out of this heart and which blood vessels are entry are supplyingblood to the heart.So, that based on that you might have to engineer them appropriately because they will experiencedifferent kinds of pressures and flow patterns, ok.So, if you were to look at the pressure which these blood vessels experience, it is notuniform.It is not just because that you have systolic and diastolic pressures, the pressure is alsovarying based on which blood vessel you are looking at.So, if you have looking at an aorta, the pressure is quite high; you are looking at to haveclose to 120, 80 to 120 mmHg which is experienced by many arteries, but as it comes into arteriolesyou can see that the pressure is dropping and in capillaries the pressure is even lesser.And your venules and large veins and hence the venae cavae all have much much lesserpressures, ok.So, basically your others because the heart is pumping the blood into the aorta and becauseof this force there is going to be a lot of pressure on the blood which is flowing intothe arteries, and as it goes into the arterioles which are smaller, they act as control valvesreducing the pressure.And beyond that there is no thrust which is pushing the bloods, rights so, it is not beingpumped in any way; it is just flowing along.So, because of this, the pressure for these blood vessels is lower.So, if you are talking about vascular tissue engineering, we need to understand what theseblood vessels are, what they are made of.A vascular system which composed as; which is composed of arteries, capillaries and veins.You mainly look at arteries when you are talking about the engineering these vascular tissues.These arterial system conducts oxygenated blood from the heart to the peripheral tissue.It contains three regions.So, the artery basically has the innermost region which is the tunica intima and youhave the middle region which is the tunica media and the outer region which is the tunicaadventitia.So, the tunica intima is what is exposed to the lumen, which means that is what is exposedto the flowing blood.So, whereas your other tissues are not exposed to blood unless there is going to be a damageto the tunica intima, ok.So, aorta is the largest artery so, which is the one are which originates from yourheart.And this aorta then starts bifurcating and it bifurcates repeatedly to form multiplearteries which then form arterioles and so on.So, muscular arteries also contain the same three layers, but they have only one elasticlamina, smaller muscular arteries are called as the arterioles.Muscular, muscular arteries they, do they have intima media?Yeah so, they have all the three layers.So, basically these three layers are present in everything.So, the intima, media and adventitia are present in all arteries; however, in aorta and theselarger arteries what happens is this media is much thicker.So, it has multiple layers of smooth muscle cells and it is very thick because that providesthe ability for it to withstand higher pressures as you, as it becomes a movement to musculararteries and arterioles this layer becomes thinner.So, you just have one layer instead of multiple layers of smooth muscle cells in your tunicamedia.So, capillaries are the smallest blood vessels which contain a single layer of endothelialcells, and a subendothelial basal lamina, it does not have anything else.Why do you think this is, this has to be thin?Easy exchange of.Yeah, So, you want the nutrients and other things to be transported.So, you want to make sure that there is no diffusion limitation for this reason it hasto be thin.So, venous system consists of venules as well as small and small, medium and large veins.The veins are basically again composed of tunica intima, tunica media and tunica adventitia;however, the compositions of these regions are different.Intica and adventitia, sorry intima and adventitia are similar to arterial structures; however,the media contains loosely organised elastic fibres instead of tightly packed as you wouldsee in an artery.So, veins also contain a single layer of smooth muscle cells in contrast with the multiplelayers which you see in arteries.So, this is all dependent on what kind of pressures these are facing, right.So, arteries, large arteries especially face very high pressures.So, the structure is designed to be appropriate for that; however, the smaller veins; theveins do not face that kind of pressure or even smaller arterioles do not face that kindof pressure.So obviously, their structure and organisation is going to be slightly different to fit theneeds of that tissue.So, when you are talking about cells, there are three major cells which people look at.So, you have the tunica intima which is the innermost layer which is nothing, but an endothelialcell lining.So, this endothelial lining basically prevents blood from getting activated, platelets fromgetting activated, it maintains blood homeostasis, ok.So, only when there is a rupture in this endothelial cell lining, you will have blood coagulationand wound healing cascades starting, ok.So, smooth muscle cells are the cells which are aligned in the tunica media.So, here these are very tightly packed and these cells constitute the major componentin that region.And finally, you have the fibroblast which are present in your tunica adventitia.So, this tunica adventitia has fibroblast, but it is not as dense as you would see inthe, it is not as dense as a smooth muscle cells in your media, ok.So, these are the three cells which people have to work with and you might have to arrangethem in that particular structure to get the tissue to emulate your, engineered tissueto emulate what the natural tissue is.So, endothelial cells are nothing, but monolayer epithelial cells that line the blood contactingsurfaces of the blood vessels.They express specific surfaces receptors which include von Willebrand factor which is vWFand VEGF receptor 1 and receptor 2 and also factor VIII.So, these are all involved in different aspects of either angiogenesis or blood homeostasis.So, they allow selective transport of plasma substances and molecules, they regulate coagulation,they regulate leukocyte transmigration, contractility, vascular cell proliferation and migration.So, that is the role of the endothelial cell lining in your blood vessels.So, smooth muscle cells are basically fibre like muscle cells which are found in the tunicamedia of arteries and veins.They are aligned in a circumferential direction of the blood vessels.And arteries actually contain multiple layers, as I was saying whereas veins contain a singlelayer.And this multiple layers in the arteries are organised between the elastic laminae.So, that provides the elasticity for the material, for the tissue.Veins contain a single layer which is found right underneath the intimal layer of thevenous wall.So, just below the endothelial cell lining you have another layer of smooth muscle cells.So, this is actually just defined by the location in the tissue, because they are present inthe media they are the smooth muscle cell.All the cells which are localise in this region are classified as smooth muscle cells.So, some of the markers which are used to identify smooth muscle cells are alpha actin,smooth myosin 1 and calponin.So, they allow for vessel contraction, relaxation and regulate vascular cell proliferation andmigration, they are also involved in ECM, secretion and production.Fibroblast are the last set of cells which are found in the tunica adventitia and theseare found as a random organised, randomly organised cells.And the cell density is significantly lower as I was saying.They act to produce either ECM and regulate vascular cell proliferation.They can synthesise type III collagen and other proteoglycans, they also regulate thefunction of vascular cells through growth factor production.So, some of the growth factors which are produced are fibroblast growth factors and epidermal growth factors.
Video 2: Blood Vessels and Blood Flow
So, this ECM of the blood vessel is just like many other ECMs you have collagen and otherproteoglycans and, but they also have a lot of elastic fibres to provide the elasticity.And, this ECM provides the structural support, mechanical strength and the elasticity forthe tissue.It plays critical role in development, morphogenesis and pathogenic remodelling.So, basal lamina is the thin membrane which is found underneath the endothelium whichserve as a supportive substrate for the endothelial cells.So, it cannot just be cells right, what we looked at three layers.So, the tunica intima we said had endothelial cells, but these endothelial cells have toadhere to something.So, that is your basal lamina.And basal lamina primarily consists of type IV collagen, fibronectin and laminin and whenthere is an injury or endothelial attachment, the basal lamina initiates platelet and leukocyteadhesion which triggers the blood coagulation cascade.Vascular collagen matrix is composed of primarily type III collagen, it also allows for thesmooth muscle cells and fibroblast to attach, proliferate and create the required patterns.So, the elastic fibres and the laminae which is present provide the elasticity; proteoglycansprovide the structural assembly and organisation of the cells.So, each of these have their own importance and in the way the tissue is developed.So, there is regulation of blood flow in when it comes to these tissues.So, you have to have blood vessel, you have to have blood flowing in one direction.So, there are local signalling molecules which are generated during the metabolic processes,which stimulate relaxation or constriction of the smooth muscle cells.And this also augments to, this provides an resistance to the blood vessel, sorry bloodflow, thereby regulating the blood volumetric flow rate.So, some of these metabolites include carbon dioxide, hydrogen ions, lactic acid, AMP andADP.So, when you exercise you can actually increase these metabolite production, thereby you caninduce dilation and increase blood flow to the heart and other skeletal muscle system.Arteries are also innervated with sympathetic nerves; their main function is to secretenorepinephrine.So, norepinephrine which binds to these, binds to receptors can actually induce smooth musclecell contraction.So, they can, when it binds to some other receptors, the beta receptors, it can actuallyinduce smooth muscle cell relaxation.So, this helps in regulating the blood flow, it helps you make sure that there is a pulsatileeffect which causes the blood to flow.So, exercise can again cause norepinephrine separation, sorry secretion.Therefore, create redistribution of blood flow because you might have more of more pulsatilemoment of your blood vessels.So, the blood flow itself is regulated by, is driven by the arterial blood pressure whichis produced by the beating of the heart; however, this pressure is also highly regulated byother receptors like baroreceptors and chemoreceptors, central nervous system, hormones and the vasopressinsystem and so on.So, baroreceptors are located in the carotid sinus, near the carotid bifurcation and theysense mechanical stretching due to arterial blood pressure.And increase blood pressure can stimulate baroreceptors inducing action potentials thatare transmitted to the central cardiovascular control, which will then reduce the activityof the sympathetic nerve system so, that there is dilation and reduce blood pressure.So, chemoreceptors are similar in location and function, but they sense decrease in oxygenconcentration and increase in carbon dioxide concentration instead of looking at mechanicalstretching.So, the signals which they read are different between chemoreceptors versus baroreceptors.So, why do we need to look at vascular tissue engineering?So, atherosclerosis is actually a very common disease condition, so, this is the progressof development of atheroma which consists of ECM, cholesterol, collagen sorry calcium,smooth muscle cells, macrophages and it is covered by endothelial cells.So, this is a plaque which gets deposited and it is found in the lumen of the largeand medium sized arteries.So, this will basically create a block which prevents blood flow.So, which can cause serious complications right.So, the growing atheroma can partially or completely block this lumen, depending onhow big it is and at what stage you are looking at, this can happen in different parts ofyour body which can cause different kinds of complications.If you are having it in your brain you might end up with the stroke, if you end up youhave it in your heart you end up with the myocardial infarction, if it is in the kidneyyou might have renal infarction and if you have lower limbs it can lead to ischemia,all of these can actually cause serious tissue damage and even loss of life.Does not this happen in small diameter vessels?Yeah it does; so, but see in small diameter vessels if one vessel is blocked it is usuallynot that bigger problem.Because you would have multiple capillaries and even if one of them is blocked, it maynot cause very serious ramifications.However, if your large blood vessels are blocked, larger arteries are blocked, it will be aserious problem because supply to many regions will get affected, ok.So, atherosclerosis has been identified to have a lot of correlation with endothelialdysfunction, and even there is an endothelial damage, what happens is endothelial permeabilitychanges and cellular adhesion also changes.So, you have more fibroblasts and smooth muscle cells adhering there causing this kind ofa plaque deposition.So, that is why a lot of interest has been growing with respect to understanding endothelialdysfunction and atherosclerosis and looking at how diabetes affects endothelial dysfunctionwhich can in-turn cause heart disease.So, all these correlations are being studied to try and understand what is actually thecause; so, that is the basic biology side of it. .So, our goal is to look at treating this right.So, conventionally the treatments are using anti hyperlipidemia agents, which controlthe lipid levels; you just take tablets. or anti proliferative agents, which will preventsmooth muscle cell proliferation.You can take vasodilators which will improve blood flow to the heart and reduce ischemia.You can also take beta adrenergic antagonist which will reduce the hearts work by blockingthese receptors.You can end up having angioplasty, where you mechanically open the arteries using ballooninflation, but restenosis is usually a concerns so, people nowadays do not do just balloonangioplasty they go and place stents after placing, doing a balloon angioplasty.So, stents basically mechanically opens the arteries with the metal frame and keeps itopen as long as the stent does not collapse, it will stay opened.Again, here restenosis a concern.You have arterial reconstruction which is possible where vascular grafts from autologoussources can actually be reconstructed, you can also use polymers and other materials.So, you people have even tried using biodegradable polymers as for temporary fix, while this,while the actual plaque is being treated.So, there have been many strategies so, with angioplasty people had initially also lookedat taking a fan blade kind of a mechanism and actually cleaning out this plaque.But, what people realised was when they do that there is actually aggravation with plaquedeposition.So, they have stopped doing that now, they primarily do only stent placements.So, these are some of the conventional treatmentsThere are also molecular therapies which are done, which attempt to treat atherosclerosisby preventing cell adhesion, thrombogenesis or vascular cell proliferation, migrationor intimal hyperplasia, ok.So, there are different factors which, molecule which have been tried.So, people try to use mitogenic factors or cell cycle inhibitors and nitric oxide propromoters which have actually been used to try to prevent this formation of atherosclerosisor even reduce this problem, ok.So,, our goal for today is to propose a treatment model that will use a tissue engineering approach.So, basically we need to engineer a vascular tissue which would be useful in treating thiscondition.So, if that is the goal, we need to identify what would be the cells that are required,what would be the materials that are required, what would be the signals like?I am just putting it as a bioreactor, but what would be the signals you would want toprovide to this tissue which you are creating to make sure that it is conditioned for theapplication you are looking at.So, this is basically what I had, so, I will just quickly go through because we are goingrunning out of time, so epithelial cells smooth muscle cells endothelial cells.Scaffolds; obviously, biocompatible, flexible and should be able to withstand contractionsand at the same time it should not collapse because it needs to be stable enough to collapse.So, if you are going to use something natural, then using a template of existing vasculaturewhich should be your decellularized matrix or if you are going to use synthetic PET andteflon would be the alternate which are commercially looked at.So, must be rigid and flexible enough for mechanical strength to train the cells, becausethe cells which you are seeding may not have experience the same kind of mechanical pressure.So, you need to condition them.So, the reactor should actually have a pulsatile flow system which can have a flow rate ofabout 5 litres of blood per minute and it should be able to withstand pressures in therange of 1 to 5 kilo Pascal.So, it should also have media exchange for long term growth because you are going tohave multiple layers.The other aspect is people usually work with drug eluting stents, so, you could also lookat loading drugs, drug molecules to these scaffolds, right.So, which could help in anti inflammatory properties and ensure there is better hostintegration.So that is also an aspect which people can look at.But there will be challenges with respect to this being weaker than the natural muscle,there can be immune response and there can be further damage to the tissue while it isbeing placed.So, these are some of the challenges which have to be accounted for.
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