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Module 1: Signaling, Bioreactors and Challenges in Tissue Engineering

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Video 1: Scaffold Fabrication
Hello everyone, in today’s class we will talk about few of the Challenges in TissueEngineering and few of the developments done to tackle these challenges.The topics which will be covered are Scaffold fabrication, the challenges associated withscaffold fabrication, in we and we are using, we are discussing about three different tissuesthat is Bone tissue, Skin tissue, and Heart tissue.In the as a second challenge, we will talk about vascularization in tissue engineeringand also we will talk about selection of proper cells.Scaffold fabrication as we know scaffolds are temporary support material which willenable cells to attach, grow and form a 3D structure.Hence, scaffolds must meet the chemical and physical properties of the natural ECM.And also, scaffold must degrade over a period of time so that the tissue regeneration totake place.As we know different tissues have different regeneration time, for example skin tissueregenerates faster than the bone tissue.Hence, scaffold must meet the regeneration time of the tissue of our interestAnd biocompatibility of the scaffolds.It is must that scaffold should not cause any toxicity to the host cells.In the 80s, when pioneers in the field, when they proposed polyanhydrides for biomedicalapplication, the scientific community thought that these synthetic, synthetic materialswould cause toxicity to the host cells.This belief was persistent until 1996 until FDA approves first synthetic polymers forbiomedical application.Hence it is necessary to see the toxic compatibility of these scaffolds to the host cells.Let us begin with the Bone tissue, as we know bone tissue bone is a hard tissue.Hence a scaffold, which we fabricate for bone tissue engineering should have high mechanicalstrength.And it is explained in three different steps, which is known as Biomechanical paradigm ofbone tissue engineering.The mechanical strength of a scaffold should match with the our natural bone tissue.At the same time, it should not have, it should not induce stress shielding effect which isprevalent in metallic implant.What happens in metallic implants is due to their high mechanical strength all the loadwill be taken up by the, will be taken up by the metallic implants.As you see here, this is the metallic implant and this metallic implant due to their highmechanical strength they take up all the load.And the region, and the region which is near to the metallic implant fail to take the loadwhich is necessary which is necessary for it to take.So, as you see the region sorry as you see the region further from the metallic implanthas higher bone mass compared to the region, which is near to the metallic implant.This region does not have sufficient bone mass, which is supposed to possess.Hence eventually, the regenerated bone will fail to take up the load.Hence, it is necessary that the scaffold which we prepare should not induce this stress shieldingeffect.Second, second step is the mechanical property of the scaffold should be in such a way thatit induces the scaffold to scaffold to bone Mechanotransduction.Mechanical stimulus provided by the scaffold induces the tissue differentiation.In the undifferentiated stem cells, the mechanical stimulus causes the differentiation of thecells.Whereas in differentiated cells the, it leads to the matrix production by the differentiatedcells.And in the third steps as the host bone tissue regenerates, it takes up the load as the scaffolddegrades.Recent development is degradable, degradable metallic implant.what the, in this study what they have done is they have used a magnesium alloy and tosee their degradation profile.And this image is the, this image is the degradable polymer and this one is the magnesium alloy.In vivo staining using calcein green; as you see here the region which is near to the magnesiumalloy has sufficient bone growth formation compared to the degradable polymer.And here are the few, here are the compressive strength and elastic modulus of cancellousbone and cortical bone are given.And, second most second most important parameter while designing the scaffold for bone tissueengineering is the porosity.Bone is highly porous in nature.And it is defined in three different terminologies, pore size, pore volume and interconnectivitybetween the pores.While designing the scaffolds for bone tissue, we should consider all these three parameters.And as we know the bone is having high mechanical strength and it at the same time it is highlyporous.So, balancing these two is a real challenge in bone tissue engineering.Let us go to the skin tissue.This is the general structure of skin tissue, the upper epidermal layer followed by thedermal layer.If the skin injuries limited to the epidermal layer what happens is the fibroblasts presentin the dermal layer, they migrate towards the epidermal layer.And, they start differentiating and secreting extracellular matrix and thereby they fillup the gap.If the skin injury is very deep wherein the dermal layer is also lost then the clinicalinterventions are necessary.Here are a few challenges listed for skin tissue engineering.Scarring of the tissue; as I explained in the previous slide, the fibroblast presentin the dermal layer, they migrate towards the skin injured site and they start secretingthe extracellular matrix.And, what happens is they, they secrete a collagen in excess then what it is required.This excessive collagen, it crosslinks and contracts and that leads to the scarring ofthe tissue.And avoiding such a scarring of the tissue is one of the challenges in skin tissue engineering.As we know, skin is the outermost layer of our body preventing from microbial infection.Hence, when we prepare scaffold for bone, skin tissue engineering it is necessary thatit should have antimicrobial property.And also, the scaffold which we prepare for skin tissue engineering should be a hydrogelshould able to keep the environment moist and at the same time should able to absorbthe exudates from the wound.Let us go back to the previous slide, as you see here, the epidermal layer and the dermallayer they are connected with the basement membrane.This basement membrane is is rich with extracellular matrix secreted by the fibroblasts cells.And this basement membrane is involved in several functions of the skin tissue.Reconstruction of this basement membrane is one of the challenges in skin tissue engineering.We will talk in great detail about the vascularization and other complicated challenges such as reconstructionof the skin appendages, thermoregulation, touch and excretions are few of the challengesin skin tissue engineering.In the scaffold, they try to see whether they can able to produce the sweat gland, sweatgland.What they have done was the they have, they have isolated epidermal progenitor cells fromthe dorsal region of the dorsal region of dermis and they have mixed with the planterregion, plantar region dermal ECM, which has cues for the sweat gland formation.As a control, they have used dormal, the dorsal region ECM which has cues for these stratifiedepithelium formation, but not for the sweat gland.And this mixture, they fed to the 3D bioprinter, bioprinter and they try to see whether theycould able to produce this sweat gland or not.And the third tissue which we will be talking about is the Heart tissue.These are the few challenges listed in heart tissue engineering.We will talk about only those, which are which are related to the scaffold fabrication.Electromechanical integration between the between the transplanted engineering myocardialtissue and the host myocardium is one of the challenge in heart tissue engineering.

Video 2: Vascularization and Cell Source
Recent, recent development is the usage of conducting polymers.For example, polyaniline is a conducting polymer and the conductivity of this polymers is dependenton the proton based doping process.Wherein what will happen is the imine and amino groups of aniline are protonated inthe presence of protonic acid.And there are few challenges associated with this conducting polymer.When we implant these conducting polymers in vivo, the dopants, the dopants which keepthe imine and amino groups protonated they are lost in the process called dedoping.That leads to the decrease in conductivity of this conducting polymers and it will notlast for longer time.So, another challenge associated with polyaniline is the amine groups of aniline are lost itphysiological conditions.So, in this study what they have done is they have used chitosan.Chitosan is a rich source of amine group and they fabricate it to chitosan film.On top of which they polymerized aniline in the presence of phytic acid and as you aswe knew the chitosan plays a role of plays a role of amine group source and they tryto solve the problem of deprotonation.And another problem is contraction ability of the scaffolds.As we knew the heart is highly contractile in nature.Producing the scaffold which has the contracting ability is one of the challenge in heart tissueengineering.And another challenge is full coaptation.As we knew hearts, heart is made up walls; these walls make sure that the blood is bloodflows in a right direction and these walls are able to contract and relax.Producing such a wall which are which can able to contract and relax is one of the challengein heart tissue engineering.As we all know vascular network mediate gas exchange, they excrete the metabolic wasteand they supply the nutrients.What will happen if we prepare a scaffold without vascular network within it, the diffusionprocess will take place.But I know, but however the diffusion is limited to a 100 to 200 micrometer of distance.Beyond that the cell starve of nutrients and oxygen and they eventually die.Hence, it is necessary to include the vascular network when we design the scaffold.The ideal engineered vessels must be able to withstand physiological pressure withoutany leakage and they should not be thrombogenic, they should not elicit any immunological responseand they must be economically viable.And there are several strategies to introduce vascular network into the scaffold and canbe broadly classified under four categories.That is scaffold design, supplement of angiogenic factors, in vivo prevascularization, and invitro prevascularization.Let us discuss each in detail.The first one is Scaffold design, one of the major prerequisite.For, for introducing vascular network into the scaffold is porosity of the scaffold.The scaffold must be highly porous and and highly interconnect highly interconnected.And there are several strategy through which we can prepare the porous scaffold and fewof the techniques are listed over here.For example: gas foaming, electrospinning, particulate leaching, freeze drying, phaseseparation and microfabrication.The second approach which is supplement of angiogenic factor.Just by supplying the growth factor which are involved in angiogenesis, we can achievethe vascularization in, vascularization in tissue engineering scaffold.Few of the growth factor which are involved in angiogenesis are listed over here.There are two ways through which we can supply these growth factors, one by release by diffusionwhich is a direct method and the second one is released by cell demand this is a indirectmethod.Let us talk about these methods in brief.Release by diffusion, in this method we supply the growth factor which are directly involvedin angiogenesis.For example, VEGF, but we do not know how much exact amount of these growth factorsneed to be supplied for vessel formation.Hence what happens is, that leads to the unhealthy vessel formation as you see in the upper image.The second method is release by cell demand, in this method you supply the growth factorsto the cells and these growth factor in turn stimulate the cells to secrete the angiogenicgrowth factor.Thereby, you can control the release of these angiogenic growth factor, thereby we can achievethe healthy vascular network.As you see in the lower image.The third approach is prevascularization technique, there are two approaches; one is in vitroapproaches and the in vivo approaches.Let us discuss each in detail.First one is in vitro approaches.There are three approaches, cells seeding, generation of spheroids, and cell sheet technology,the first one is cell seeding.In this approach you seed the endothelial cells into the porous scaffold and incubateit over a period of time.Eventually what will happen is there will be vascular network formation within thisporous scaffold and then you implant at the site of interest.The second approach is generation of spheroids; spheroids are structurally three-dimensionalarrangement of cells with intensive cell to cell and cell to matrix interaction.There are several ways through which we can achieve the formation of these spheroids.If we choose endothelial cells and tissue specific cells for the formation of spheroids,what will happen is over a period of time there will be a vascular network formationwithin these spheroids.Once the vascular network forms within these spheroids, then we can transfer these spheroidsinto the scaffold and then thereby we can achieve the vascular network into the scaffold.The third approach is cell sheet technology, this technique is devoid of any scaffolds.In this technique, what we use is thermosensitive polymer.For example, poly isopropylacrylamide, at 37 degree Celsius these thermosensitive polymersthey allow the cell adhesion.So, at 37 degree Celsius you seed endothelial cells and tissue specific cell on to thesethermosensitive polymer and incubate it over a period of time until it reaches the monolayerformation.Once the monolayer is formed, you decrease the temperature to 20 degree Celsius, whathappens is at 20 degree Celsius this polymers they swell.And the cell adhesion property of this polymer is lost, that leads to the peeling off ofthese monolayer from the polymer surface.And these monolayer are stocked and then transfer to the site of interest.Thereby we can achieve the vascular network formation at the site of interest.The next approach is In vivo approaches, in this there is a technique called Arteriovenousloop formation.As you see here, the synthetic, synthetic vessel is connected to the artery and venoussurgically.And this after surgically connected to the artery and venous, this scaffold, this setupis implanted in vivo.As you see here synthetic, synthetic vessel is looped inside this scaffold once we implantinside.Over a period of time there will be a sprouting of capillaries into this scaffold as we seeover here.Once, once there is a sprouting of capillaries into the scaffold, you surgically remove fromthat site and implant it to the site of your interests, that is injured site whatever.Thereby, we can achieve the vascularization into the scaffold.Which is an efficient strategy? as you know all these techniques come with certain advantagesand disadvantages.For example, in, in case of scaffold design it is easy to fabricate, but again the disadvantagesis it relies on the in growth by host vasculature.In the second method in vitro prevascularization, it does not rely on the In vitro in growthby host vasculature, but again the perfusion rate is low compared to the in vivo prevascularization.But in case of in vivo prevascularization, though it does not rely on in growth by hostvasculature and it involves a very rapid perfusion once after implantation.But again, this method involves the surgery.The fourth method is angiogenic factor delivery, this approach has given a promising result.But again, it depends on in growth by host vasculature.So, it is difficult to say which strategy is an efficient one.Again, there are few other challenges in vascularization; for example, it is difficult to constructa tissue engineering scaffold with smaller capillaries whose diameter is less than 1mm.And, perfusion and vascularization is a significant problem in metabolically highly active organslike heart and liver.For example, in heart the inter capillary distances is around 20 meter, achieving suchare highly vascularized tissue is a challenge.We next move on to the third challenge that is Selecting Proper Cell Source.In this section, we will talk about different types of stem cells which we have exploredin tissue engineering applications.As we know stem cells, stem cells have the potency to differentiate into many numberof cells and we will talk about few of these stem cells which have been exploited in tissueengineering.The first one is mesenchymal stem cells.Mesenchymal stem cells are multipotent adult stem cells.These stem cells are able to differentiate in to several cell types of the body but notall.These mesenchymal stem cells can be isolated from different parts of the body, for examplebone marrow, adipose tissue and dental tissues.But the problem with mesenchymal stem cells, they are multi potent but not pluripotent.The second stem cells which we are talking about is human embryonic stem cells.These embryonic stem cells are isolated from inner mass of the blastocysts.And, these stem cells are pluripotent in nature, that means they are able to give rise to anycell types of the body.But the problem is associated with the embryonic stem cells is the ethical concerns as we areextracting this stem cell from the embryo.And the recent development is induced pluripotent stem cells.What they have done is they induce pluripotency in the normal somatic cells.What they have done is they have introduced four genes into the mouse fibroblasts cells.And thereby they could able to reprogram these somatic, somatic cells into these stem cells,stem cells.But the problem with stem cells is their proliferation capacity is very low in two-dimensional cellculture plate.So, it is difficult to it harvest required number of cells with that is required forour tissue engineering application.There are again certain advancements in conventional cell culturing techniques.For example, exposing stem cells to the hypoxic condition led to the improvement in theirsurvival.So, such advancements have been done quite recently.The another problem is maturity of these cells, these stem cells they have, they will largelydifferentiate in to immature cells with variability in structure and function.And, another problem which we face in cells is producing clinically relevant number ofcells.For example, human myocardium consists of 10 to the power 9 cells generating such ahuge number of cells is again a challenge in tissue engineering.Thank you.