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Materials and Fabrication Techniques for Skin Tissue Engineering

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Video 1: Biomaterials for Skin Tissue Engineering
So, from here on we will discuss the major components of the Tissue Engineering Triadthat is the materials, the cells and the signals required for skin tissue engineering.So, biomaterials that are used for skin tissue engineering can be classified as natural,synthetic and the combination of the two.So, we will discuss each one of them in detail.Natural polymers that are used for skin tissue engineering can be polysaccharide-based polymersor protein-based polymers.These are some examples of natural polymers that are used for skin tissue engineering;this is however, not an exhaustive list.So, alginates, chitosan, cellulose, hyaluronic acid, dextran, pullulan, carrageenan, konjacglucomannan, and isabgol are newer polymers that are being explored for skin tissue engineering.Protein-based polymers such as collagen, gelatin, silk fibroin, keratin and elastin are beingexplored for skin tissue engineering, we will look at each one of them in detail.So, in natural polymers, chitosan is a very popular material used for skin tissue engineering;obtained from chitin which is found in the cell wall, fungal cell wall and also the exoskeletonof insects.So, chitosan can be processed into different types of matrices such as films, nanofibersand sponges.So, several studies have been done using chitosan.So, here it shows an in vivo study where chitosan has been shown, it is highly biocompatibleand has been shown to accelerate wound healing and also chitosan has helped improve cellularadhesion and proliferation.Collagen is a protein-based polymer.It is the major portion of the extracellular matrix, the ECM; it collagen also can be processedas nanofibers or 3D printed constructs.So, here you can see that using a collagen skin substitute an acceleration in wound healinghas been achieved; in both freeze-dried skin substitutes and electrospun skin substitutes;reduced wound contraction and also a presence of hallmark of morphogenesis and cohesionwhich is the which shows the formation of the natural skin tissue has been observed.Silk fibroin is another natural polymer; here it has been processed into nanofibers usingelectrospinning.Silk fibroin, electrospun silk fibroin has shown improved pore size and interconnectivity.The improved pore size and interconnectivity helps in good cellular infiltration and proliferation.Next stand-alone natural polymers, hybrid of two or more natural polymers have alsobeen used; such as silk/keratin, scaffolds.So, here the keratin component which contains RGD sequences helps in improved cellular adhesionand proliferation; than compared to having silk fibroin scaffolds alone, and the blendedmaterial showed superior properties.Also, collagen/pullulan hydrogels have been synthesized.Collagen is usually used to make hard biomaterials; to make softer biomaterials it is often blendedwith other natural polymers, to make hydrogel systems.So, pullulan is one such example.So, in this hydrogel they obtained an open porous structure with improved cell viabilityand also achieved improved wound closure and vascularization.So, methacrylated gelatin and methacrylated hyaluronic acid have been blended, both naturalpolymers.Hyaluronic acid is also an ECM component, gelatin is a natural protein-based polymer;but both the polymers have their own disadvantages, such as hyaluronic acid does not help withcell adhesion and gelatin is poor in it is mechanical properties.So, the blend of the two can help us overcome the shortcomings and also physical or chemicalmodification of naturally occurring polymers can help improve their properties.So, here in this example, the blend has been processed into a hydrogel and they have embeddedadipose derived stem cells into the material for improved vascularization.So, konjac glucomannan/keratin.So, here konjac glucomannan is a polysaccharide-based polymer and keratin is a protein-based polymer.So, the two have been blended to form scaffolds.In these scaffolds, they have done in vivo study where it showed enhanced collagen synthesisand deposition in diabetic wounds and also the scaffolds have been shown to be highlybiocompatible.Also, other natural polymers have been blended together to have superior properties as skintissue engineered products; such as isabgol/silk fibroin, collagen/fibrin, bioprinted constructsor collagen/chitosan scaffolds.So, now we look at synthetic polymers which have also been used for skin tissue engineering;polyurethane, PVA that is Polyvinyl Alcohol, PEG Polyethylene Glycol, poly hema, polyhydroxyethyl methacrylate and PLGA Poly Lactic Glycolic Acid and polycaprolactone.Polyurethane is a very popular synthetic polymer used for skin tissue engineering; severalcommercially available wound dressings are there, which are made from polyurethane.So, this is an example of a polyurethane foam, also you can load different active agents,bio active agents into the materials to render better wound healing properties.So, here they have blended, they have added silver and asiaticoside into the polyurethanematerial.And tested effect, wound closure in a porcine model.So, here the material has shown to be biocompatible as in as there is no skin irritation and alsothey have observed faster wound healing in deep dermal wounds.So, PLGA is a copolymer of lactic acid and glycolic acid.So, here in this example they have a synthesized nanoparticle and they have loaded curcumina bioactive agent.So, they have observed reduced inflammation, increased re-epithelialization and increasecollagen deposition which are improve, which are very important for wound healing.Polycaprolactone is another polymer, here they have synthesized them as nanofibers usingelectrospinning.So, they have been embedded with placental derived bioactive molecules for wound healing.And polyhema is another synthetic polymer they have synthesized it as a hydrogel, loadedwith moxifloxacin and they have seen antioxidant activity and absence of inflammation.Also, like natural polymers two or more synthetic polymers have been blended to achieve betterproperties.So, such as PCL and PEG block copolymer that has been synthesized and electrospun.So, here there you can see the free amino groups to which they have attached epidermalgrowth factor, and have done in vivo studies to show improved wound healing with epidermalgrowth factor loading.Also, there have been other synthetic polymers, polymer hybrids PCL and PVA, curcumin loadedPCL and PVA nanofibers that showed improved wound healing.PEG and PLGA hydrogel this was a thermo, synthesized as a thermosensitive hydrogel or environmentallyresponsive hydrogel.This showed enhanced engraftment of muscle derived stem cells that were seeded on tothe grafts.So, the combination of natural and synthetic polymers gives the best, gives better propertiesfor skin tissue engineering.A natural polymers can provide biocompatibility to the graft; while synthetic polymers canprovide the required mechanical strength.And also, the natural polymers help with cell attachment and adhesion and proliferation.So, PVA/CMC that is a blend of Polyvinyl Alcohol and Carboxy Methyl Cellulose, which is a carboxymethylderivative of cellulose; here PVA is the synthetic component and CMC is the natural polymer.So, PVA provides the mechanical properties to the material; PVA is however, highly hydrophilicand it does not help with cell adhesion.So, carboxymethyl cellulose can help with cellular attachment and proliferation.So, the blend has been synthesized as a hydrogel; and in this particular study, they have loadedreduced graphene oxide into the material and have found enhanced angiogenesis which iscrucial for wound healing.Silk fibroin/PVA blend has also been used; silk fibroin here is the natural componentand PVA being the synthetic component.So, this has been fabricated as nanofibers.So, here you can see the silk fibroin-based mat; it helps with cell recruitment and also,they have done In vivo studies to show improved wound healing and remodeling of the extracellularmatrix.These are other combination of synthetic and natural polymers, PCL and collagen nanofibers.So, here they have loaded insulin delivering chitosan nanoparticles, which showed excellentin vivo wound healing.And also, more than two components can be blended together to form tissue engineering,skin tissue engineering materials.PVA, starch, chitosan has been used here to form a hydrogel and incorporated with nanozinc oxide to show excellent antibacterial activity and wound healing.

Video 2: Skin Substitutes and Signals
So, skin substitutes can be classified as acellular and cellular matrices; the cellularmatrices have cells loaded onto the graft.So, there are certain advantages to that, the cultured cells can deliver growth factorsand ECM to the wounds; and also, it has been shown that the cellular skin substitutes canshow enhanced angiogenesis.So, what is the source of the cells that are used for seeding?So, they can be autologous, allogeneic or xenogeneic in it as discussed previously orthey can be differentiated or stem cells.So, different cell types are used in skin grafts, they can be skin derived seed cells;such as keratinocytes which majorly occupy the epidermal layer; the dermal fibroblastswhich lie in the dermis, the epidermal stem cells and the melanocytes which provide pigmentto the skin.Non-cutaneous cell such as embryonic stem cells, IPSC that is induced pluripotent stemcells, mesenchymal stem cells and cells derived from the amnion have also been used.So, this is an image of keratinocytes cultured on electrospun chitosan nanofibers.Also, theseeding of cells can be done as a co-culture of two different type of cells.Fibroblasts and keratinocytes for example, have been co cultured; and it has been observedthat by coculturing them together, the fibroblast can actually help the proliferation of keratinocytes.Also, melanocytes have been cocultured with fibroblast and keratinocytes; this can helprecreate the natural pigmentation process.A Langerhans cells have also been cocultured with fibroblast and keratinocytes.So, this can help monitor skin immunological reactions.And dermal fibroblast have also been cocultured with endothelial cells, so this can promotevascularization.Now, we are going to look at the third component of the tissue engineering triad, which arethe signals.So, these signals can be biochemical or biophysical in nature; these signals provide functionaland instructive matrix to allow skin regeneration.These signals can regulate cell-matrix interactions, cell behavior and function.So, we can develop instructive materials rather than having passive grafts, these instructivematerials can harness the bodies innate ability to self-repair.So, these are the different signals that can be given to the graft.So, some of these are biochemical signals, that are growth factors and derivatives, theECM proteins, small molecules, genetic regulators.And on the right, you can see the biophysical signals such as the topography of the matrix,application of negative pressure, electric or electromagnetic stimulation.So, first we will look at biochemical signals.Biochemical signals can be growth factors; growth factors are potent regulator of cellularactivities, such as cellular migration and proliferation and differentiation.Some growth factors that have been looked at for application in wound healing are; vascularendothelial growth factor and epidermal growth factor and basic fibroblast growth factorand transforming growth factor beta.So, these play a part in potent role in all three phases of wound healing.So, conventionally these growth factors were developed, I mean were delivered using conventionalapproaches.This conventional delivery methods however, cause burst release of the molecules.So, we are developing new techniques to deliver these molecules, which can `release the factorsin sustained manner and also, so that they can be effective at a lower dosage; so onesuch example can be attaching the growth factors to ECM proteins.Other than growth factors, their derivatives and certain peptide sequences.And the best characterized peptide sequence is the RGD sequence consisting of the aminoacids, arginine, glycine, and aspartic acid.So, this sequence is found in several ECM proteins and also this can be incorporatedinto synthetic materials which usually lack cell adhesion sites; this can help in cellularadhesion and migration into the graft.So, other molecules such, small bio active molecules can also be incorporated into thegraft.So, one such molecule is oxygen; oxygen levels have been found to be low in chronic woundtissues.Oxygen level is considered important, because it provides energy for bacterial defense,for cell proliferation, and also for cell migration, it is crucial.Oxygen has been used in hyperbaric oxygen therapy, where it is delivered through systemiccirculation; however, it fails to reach the desired site.So, then they started using oxygen as a topical therapy.Now they have come up with oxygen delivering wound dressings such as oxyzyme; which releasesoxygen through a chemical reaction.Another bioactive molecule that can be incorporated into tissue engineered grafts is nitric oxide.Nitric oxide is synthesized from nitric oxide synthase, this enzyme is present in 3 isoformsand all 3 isoforms have been found in the skin; in skin cells such as dermal fibroblasts,keratinocytes.So, this enzyme is present in 3 isoforms and two of the isoforms are constitutively expressedand another one is expressed by inducible expression.It is involved in various stages of wound healing, especially angiogenesis; this enzymeis very important for VEGF activity.Various genetic regulators have also been incorporated into tissue engineered grafts,such as cDNA, small interfering RNA and micro RNA; cDNA can be delivered using various non-viralvectors, such as cationic polymers or liposomes or naked plasmids can be used.cDNA encoding various peptides and growth factors have been incorporated.One such protein is the sonic hedgehog protein, a study showed that the sonic hedgehog genevector has been shown to improve diabetic wound healing with micro vascular remodeling.Several small interfering RNA have been used.So, small interfering RNA can cause silencing of disease-causing genes, by binding to thecomplementary sequences of the target mRNA.So, several diseases such as a skin fibrosis have been treated using small interferingRNA.Another set of gene regulators are micro RNA, these are endogenously found repressors; thatbind to the three prime and translated regions of the mRNA.These micro RNA have also been used to regulate angiogenesis and also involved in re-epithelializationand tissue remodeling.Now, we will look at biophysical signals; one such is the topography of the matrix.The topography of the matrix can guide cell migration through the graft.So, this can be achieved by using micropatterned surfaces.So, here you can see there is a PDMS membrane, which has micro fabricated grooves on it.So, which has been synthesized using the mold; and human dermal fibroblasts have been culturedon the membrane; and mechanical wounding is done on the surface and you place your patchon the membrane and observe the cellular in-growth.So, here you can see a video.So, in this video you can observe the difference in cellular dynamics between grafts that haveperpendicular gratings and other grafts blank grafts that do not.So, here at 12 hours, there is considerable in growth of cells into the graft where theperpendicular gratings are present; while in the blank graft, even after 24 hours ofwounding, the graft shows lower confluence compared to the one which has the gratings.Another biophysical signal that has been used is the application of negative pressure.So, this helps in increase in blood flow to the area and also helps in promotion of angiogenesisand granulation tissue formation.The mechanism that has been proposed here is, when you apply the negative pressure thereis a slight deformation of the ECM which generates micro-strain on the cells, this helps in cellularmigration and proliferation.So, several vacuum assisted closure devices have been commercially made available.So, these devices are mainly vary in their filler material that is used.So, either foam or antimicrobial gauze dressing have been used and they also vary in the suctioncatheter that have been used, they also vary in the intensity of the negative pressurethat has been applied, so which varies from 50 to 125 millimeters of mercury.So, in this figure you can see that on application of negative pressure, there is extruded removal,edema reduction and blood supply increase that has been shown.Another biophysical signal used is electric stimulation.So, this is a biomimicry tool that has been used, it mimics the endogenous electric fieldsthat arise after wounding and cause keratinocyte migration.And procellera is one such bio electric dressing, it produces electric currents similar to thephysiological electric fields present in the body; it produces micro currents of the rangeof 2 to 10 microamperes.Electromagnetic therapy has also been used as the biophysical signal, it resulted inup regulation of multiple growth factors; and including nitric oxide which is very importantfor angiogenesis and also this therapy has also been shown to regulate cellular migration.


Video 3: Fabrication Techniques
In the next few lectures, we will look at different fabrication technique; that havebeen used to synthesis tissue engineered skin graft.So, as Vasudha discussed about the design and other characteristics for the for makingskin graft that can be cumulatively made into three categories; the first one is freeze-drying,and the second one is electrospinning, and the third one is injectable hydrogel.So, in freeze-drying what they do is, they make the polymer solution and they will keepit in the frozen condition; based on the melting temperature of that solvent it will get frozenand it will form the crystals.So, based on whatever temperature we keep it that crystal size will be formed, and basedon that the pores will be formed into that scaffold.So, in electrospinning there will be passing that polymer solution under a electric field.So, when it is spinned and that will be collected into that substrate.So, the fibers whatever is collected that will be forming a mesh like thing and thatcan also be used as a skin graft.So, in the injectable gel, in outside of the physiological environment that will be inthe sol form, when it is injected into that physiological environment that will form thegel.So, the advantages is with the injectable gel over that freeze-dried scaffolds and electrospinningwaste scaffolds are; like that automatically fills that wound bed, we do not need any specificmold or something and we do not need any other preprocessing.So, what we need is, we just need the polymer are with or without cells and that can bedirectly put into or injected into that place wherever that wound is and that will fillthat wound and it will form the gel.So, that wound bed will be completely covered.So, another advanced technique which is involved with making skin graft is, 3D printing.So, in 3D printing it involves various steps.So, first what we need is, we will be scanning that particular tissue.So, if you take skin.So, that skin will be imaged under CT scan or MRI scan.So, CT scan involves X-ray and that image will be taken at all the 360 degree angle.In MRI, under the magnetic resonance field, under 360 degree the image will be taken.Once that image is taken that will be converted into printable form like CAD format.So, because then only you will be able to print that particular structure whatever weare interested in.Then the next step is material selection.So, in the material selection whatever criteria Vasudha discussed, we all need to go throughall those things and we need to decide what material we need and for what purpose we need.So, all these things need to be decided.Then in case of cell selection, we have epidermis; that epidermis contains keratinocytes anddermis contain fibroblast.So, apart from that we also can go for some stem cells as she discussed.So, like based on our interest we need to choose keratinocytes or fibroblast or togetherkeratinocyte and fibroblast; it is up to us like what we are going to make.If you want to make only epidermal graft, then we need to choose only keratinocytes.If you want to make dermal graft, we need to choose only fibroblast; or like if youwant to make that skin to be in that particular colour, we also need to choose melanocytesand that needs to be co cultured with the keratinocytes.So, it is all up to us that what we are going to make, then the next step is bio printing.So, bio printing can be of, can be printed three ways; like we can use inject printeror like we can go for micro extrusion-based printer or like laser printing.So, we have already discussed about that 3D printing techniques and how all these thingsworks.So, we do not need to go in detail about those things.So, like we, as we know that the hydrogels whatever we are going to make, that hydrogelshould need to be biocompatible; only that biocompatibility and gelling nature will nothelp to use for the bio fabrication.So, there are certain criteria’s that needs to be considered.So, consider like that forms gel, but when you apply some stress or something, the cellswill not be viable; then it is it is not of no use, like when we print it is making gel,then also it will not be useful.So, the printing fidelity and the speed when the gel, gelation occurs all these thingsplay a very important role.So, all these things are mainly controlled by two main parameters; one is viscosity orthe rheology of the polymer whatever we use for bio fabrication, and the second thingis crosslinking mechanism.So, in viscosity what happens is, like viscosity mainly, mainly played by two parameters; firstone is concentration of the polymer, and the second one is molecular weight of the polymer.These are the two parameters decides the viscosity of the any polymer solution.So, if we take high viscous polymer with the low molecular weight, what happens?That nozzle will not be able to print that particular thing.So, if we use that micro extrusion based technique, so we use the needle; and that over that needlewe will be applying the pressure into that needle.So, when we apply the pressure, that will be pushing that needle and that will be printing.If that solution is highly viscous what happens, instead of making solution it will form thedroplet.So, to avoid that what we can go for is, we can mix it with some other polymer or we canuse less concentration of polymer with high molecular weight.So, we can go for this way or that way.So, in the left side we see one fine example.So, in the example what they have done was; in the first image A, so they used gelatinmethacrylate which is the known polymer.So, when they used only that gelatin methacrylate, it was not able to print, whereas, it formedthe droplet; but when they mix that with hyaluronic acid what happens, it formed the clear solution.And once that solution is formed, they were able to get the structure of their interest.So, viscosity plays a very important role in controlling this.So, another thing is that shear stress.So, in the shear stress, like if we apply more shear to the polymer solution; obviously,the cells will get died.So, there are reports that even if we apply 1 Pascal of shear into that hydrogel containingcells, the endothelial cells get detached from the substrate.So, like consider even 1 Pascal of pressure is applied, that cells are getting detached;it means, when we apply that shear into that needle or whatever printing technique we use,the cells will not survive.So, we will get this hydrogel and we will get the resolution of our interest; but thecells will not survive.In that case there is no point of doing 3D printing.So, in the same 1 Pascal of shear stress, like they have also found that cartilage functionis also lost.So, to avoid that what we can do is, we can increase the speed of the printing and reducethe shear stress.Say for example, we have the needle and that needle we are applying some pressure intoit.So, that time of applying that pressure will be less and the printing speed will be faster.So, the shear whatever is given into that particular polymer solution will become reduced.So, the number of cells what we get will be more and that will be viable.So, that is a ultimate idea in this.And the next property with the shear stress is that shear thinning.So, in the shear thinning what happens is, in the when that is, when the polymer solutionis in the needle or particular dispenser.So, that will be in one structure, and when we apply some shear into it that thinning,shear thinning will happen and the structure will be reformed or reorganized that willbe able to see in the second part of that image; then once it is printed that formsparticular hydrogel or the structure of our interest.So, again that reformed into that shape whatever we are interested in.So, if we apply more shear into that particular thing that reorganization will be a difficultthing.So, we will not be able to achieve that reorganized polymer structure in the substrate.So, if we are not able to achieve that reorganized structure, the gelation and other things alsowill get vary.So, these all the main parameters we need to discuss or we need to know whenever weare going for 3D bio printing based graft development.Then the next step is that gelation that as we all know that gelation can be of physicaland chemical.So, in physical we can use some ionic crosslinkers or that could be of electrostatic based interactionor we may pass even UV or something and we get gelation hydrogels.And chemicals like we can also use some crosslinkers, because of that cross linkers that forms gel.So, these are the main parameter we should know before going for bio printing.So, in case of 3D bioprinted construct, so we, as we discussed in the design criteria,we need to have the scaffolds.So, that graft can be of acellular and cellular.So, if we want to make acellular based scaffold or a graft what we need is, we just need onlypolymer and that structure whatever we need to bring that will be printed.If it is of cell based therapy, then along with the scaffold or hydrogel we need to culturethe keratinocytes and fibroblast or only keratinocyte or only fibroblast according to our interestand they will be grown in vitro and once they mature they will be placed into that patientbody and that will be used as a regenerated tissue, skin.So, far with the various techniques they were able to achieve that particular thickness.So, laser-based technique they were able to achieve 600 microns around thickness of theskin.So, in case of micro wall-based thing, they achieved about 150 micron.And like they have used various materials like collagen, hyaluronic acid, chitosan andvarious type of biopolymers; especially they are all water-soluble polymers.So, far very limited papers are available with hydrophobic polymers and that to be usedfor skin graft applications.


Video 4: Commercial Products
So, far with the design, design criteria and other things, they have developed some commercialproducts.So, herein we are not just going to discuss about the skin graft based products; evenlike if there is a wound, they will be doing some dressings.So, even that also will be going, we are going to discuss here.So, the first one is that Hydrocolloid dressing.So, that will promote that debridement.So, in the debridement one of the finest products from that is comfeel.So, that was developed by Coloplast Denmark Company.So, what it does is, that will promote debridement and it will avoid that moisture to go outside.Then the next second thing is alginate dressing.So, that dressing is made of alginate material and again this also will give, this will actas absorbent and one fine product from that alginate dressing is Sorbsan that was developedby Maersk Denmark based company.So, the next product is non-alginate dressing.So, in the non-alginate dressings we have a Nu-Gel; Nu-Gel developed by Johnson andJohnson.So, this will again provide that, promote debridement and it also gives that absorptionto the wound site.So, now that Nu-Gel comes with alginate also just to promote that hydrophilic nature andabsorption.So, we have a semi-permeable films.So, here we have Bioclusive developed by Johnson and Johnson.So, herein we have a polyurethane film.So, that polyurethane film is coated with acrylic adhesive.So, what it does is, when we place into the wound, that will automatically adhere intothe wound site and it will pass that air to pass and that will be keep maintaining thatmoisture condition.So, if it is dried, it will be difficult for that wound to heal.So, to maintain that they use semi permeable films.So, they have foam dressings also, as we all know that foams mainly made of that polyurethanes.So, this is just to protect from the thermal environment.So, from various temperature it can be protected.So, that is made of that polyurethane.So, we have antimicrobial dressings.So, as we as that name indicates that dressing is to avoid that infection, whatever microorganisminvading into that body that can be avoided by these types of dressings.So, Acticoat is developed by Smith and Nephew Healthcare and that is one of the productwhich is available.So, these are all the products whichever is available for simply wound dressing, not forthe graft.So, when we come to the graft, as we told like the graft can be of split thickness graftor full thickness graft.So, also it can be used for screening various drugs that could be used for various cosmeticapplications.Thank you.