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Skin Tissue Engineering

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Video 1: Need for Skin Tissue Engineering
Hello, everybody.Today, we are going to talk about Skin Tissue Engineering.So, before going into that skin tissue engineering or tissue engineered graft, we should needto know about the function of the skin.So, as we all know that skin is the largest organ of the body and mainly it does thatprotection from the environment, especially abrasion and fluid loss.Then we have that gives the containment for the body structure and also the organs presentinside the body.It also involves in the heat regulation like in the form of sweat, dilation, contractionand blood vessels.It also involves in the sensation of nerves like while we touch that conduction of thethings will be passing into and will be sensed and it also involves in the synthesis andstorage of vitamin D. Further it also acts as a blood reservoir.So, it also plays an important role in excreting the unwanted substances through sweat.So, before developing any skin graft or something we need to know about the anatomy of the skin.So, if we see the skin, so, it has two layers: first layer is dermis and the second top layeris epidermis.So, and the bottom layer is not of the part of the skin, but it also plays an role whilewe discuss about the skin.So, that bottom layer is subcutaneous tissue or it is called as hypodermis.So, it has many parts.So, attached with the hair follicles we have a sebaceous gland.So, sebaceous gland produces sebum.So, that sebum is given lubrication for that hair follicle and also this is responsiblefor making that waterproof for the skin.And, it also acts as a primary defense system.Then attached to that sebaceous gland we have arrector pili muscle; that arrector pili musclethat is responsible for that pulling of hair when goosebumps happen that pulling off hairhappens because of that muscle and we have the nerve conducts.So, this is responsible for the sensation, sensation and we have blood vessels for transportof nutrients and other things from subcutaneous part to dermis and further dermis to epidermispart.So, we have epidermis.So, epidermis have various layers.So, if we see the first layer is epithelial cells.So, this so, the this first layer contains epithelial cells and this is called stratumbasale then because of the blood vessels whatever is coming from the dermis part the blood supplyis going into that epidermis and the cells will be keep dividing.So, the division of the cells goes in the top portion.Then, from the basale that goes into that next layer that is called stratum spinosum.In stratum spinosum, the epithelial cells gets keratinized and apart from the keratinocyteswe have melanocytes which are responsible for the color of the skin and the next layerof that stratum spinosum is stratum granulosum.Then here the cells flattening happens and over there we have stratum corneum that isa final layer of the epidermis and where that acts as the shed of the old cells.So, we have in between layer epidermis and dermis is that junctioned epidermal dermaljunction.This is called rete ridges.So, this rete ridges plays a main role in giving mechanical strength to the epidermisand it also gives adherence to the dermis and epidermis because of this epidermal junction.Apart from that the soluble molecules whatever is present in the dermis, the transfer ofsoluble molecules from dermis to epidermis is happening through this junction.So, when there is a wound in a normal skin what happens is by innate mechanism the woundwill get healed.So, it it involves series of steps.The first step is hemostasis.In hemostasis what happens is the blood coagulation will occur.In the blood coagulation during that blood coagulation happen at the wound site epinephrinewill be released, and that epinephrine, once that epinephrine is released that plateletscoagulation will start and like the complete process is called hemostasis.Then the next step next to hemostasis is inflammation.So, once the blood started coagulating, the swelling and other things will happen becauseof the inflammation.During swelling, it also produces heat and fluid flow also will be more and obviously,the pain also will be getting.Then herein we have the defense system also will play an important role.When there is a wound, the foreign particles will try to invade into the body.So, that time that leukocytes and macrophages will start developed into that particularsite.So, the first, first site of mechanism is by leucocytes.So, that leucocytes will be replaced by the macrophages.So, that macrophages what it does is, it will cleans that debris of the wound.Then the next step is proliferation.So, proliferation is mainly of that fibroblast whatever is involved in that dermis.So, if that wound this happening only with the epidermis part, that proliferation willhappen with the help of keratinocytes.So, this involves three different types or like classification.The first one is granulation.In the granulation what happens is the cells will start dividing.If we, if we take fibroblasts or dermis as the wound site, so, the fibroblasts will startdoubling and it will be creating multiple cells and after that ones that cells startdividing; obviously, the size of the things will come down and it will contraction willhappen, then epithelialization will happen.The next step is remodeling, in the remodeling what happens is that fibroblasts will developextracellular matrix; that extracellular matrix contains collagen fibril, proteoglycans andother fibronectins.So, fibroblasts initially will produce that procollagen, that procollagen will twist togetherand that form a strong collagen.So, many collagen molecules will come together and they form a very strong network.So, because of this network only, we get the fresh or regenerated tissue.So, if this collagen is not twisted and it is not forming proper network what happenswill be like that skin tissue will not that mechanical strength of the tissue whateveris formed will not be equivalent to the normal skin.So, if we get a wound, we ourselves will be able to see such kind of difference.So, when that tissue starts forming, that strength of the tissue will be very smooth.So, once that new tissue is formed, we will be able to see that strength of the tissuewill be equivalent to the earlier tissue.So, this is what happening.The reason is that collagen remodeling that is what happens at that site.So, this is normal mechanism of a wound healing.So, when this normal mechanism is in few conditions these normal mechanism will not happen.So, the first mechanism is like poor circulation.When there is poor circulation of blood into that wound site that normal wound healingwill not happen and when there is a edema or insufficient nutrients that time also thenormal wound healing process will not occur.Then when there is a repetitive trauma to the wound in that time also the wound willnot get healed by its in own mechanism.When there is a extensive wounding then also that wound will not heal.Then the burn lead to deep wound; if there is a deep wound it is very difficult for thatwound to heal.Then, the next reason could be extensive skin loss due to infection such as necroticizingfascititis.Then we have when there is a surgery so, during surgery also we get skin loss.So, that time also the it is very difficult for that skin to regenerate and wound to heal.So, the probable solution for these like when there is innate mechanism is not happeningthe solution could be induced primary healing.So, induced primary healing will be like consider we have wound here like this.So, in induced primary healing what we do is this portion and this portion will be combinedtogether like this way and they put some stitches over here.So, that will be totally covered.So, when this is happening we will be; we will be able to heal, but when that woundbed size is more it is very difficult to bring that both the sides of the wound and to bringthat down into that portion.Then the second step is a delayed primary healing.So, in the delayed primary healing only we will be talking about that skin graft or tissueengineered based product.So, in delayed primary healing what happens is; so, consider we have wound like this.So, this both the skin cannot be brought together and we cannot stitch it.So, for making that fibroblasts to grow we need to keep something over here.So, that will be achieved by the graft whatever we are developing.So, a classification of skin involves two grafts, like first one is split thicknessgraft and the second one is full thickness graft.Split thickness graft involves only the epidermis regeneration; full thickness graft involvesthe epidermis together with the dermis regeneration.So, nowhere that hypodermis is involved.As I told in that anatomy part, hypodermis is not part of the skin, but it plays a rolewhen we discuss about that engineering thing.We have is skin graft we have autograft.Autograft is from the own patient sample; they will be taking the tissue and they willbe placing it.Isograft genetically equivalent sample will be taken and from the species same species,the graft will be taken.In the xenograft from one species to other species it will be transferred.So, among this autograft is advantageous because of various reason.And, the sources for a split thickness graft could be like stomach or thighs or backsideof the buttocks and like as I told that split thickness graft involves only that epidermispart.In the epidermis part what they do is they use a dermatome took at that splice.So, that splice will be further.So, that splice will be further what they do is they make mesh into it and they willbe placing into that wound site.Then we have sources for the full thickness graft.So, in the full thickness graft what they do is they will cut the graft from the backside of the head and buttocks and also armpits from these portions they can get the fullthickness graft.So, full thickness graft is like excision of that complete part of that skin.It is not like we do not need to use any dermatome or any other specialized instrument to cutthat splice whereas, that complete skin will be taken.So, they will be excising that portion and they will be placing into that place whereverit is required.So, when they do the surgery like after cutting the portion from the particular part whatthey do is they will give some pressure onto that place and they will be placing a supportlike this way.So, this is called a bolster.So, they will keep the bolster and they will stitch it.So, the reason for doing such thing is if you do not keep something over that portionwherever that graft is placed that will not be in that position for long time.So, for making that sure they are keeping some bolster into that surgery wherever theyhave done and they will be covering it by stitching.So, advantages of this skin grafts is like a it is taken from the own body.So, it is biocompatible and it is non-immunogenic.It also has disadvantages.So, the main disadvantage is like that is taken from the own body, so, obviously, itwill give pain to the patient and then another thing is it will again create secondary wound.So, that secondary wound healing will be again a problem.So, these are all the drawbacks whichever we get from the allograft, autograft-basedskin graft.So, what could be the solution means like have come across from stone age to now weare in the plastic.So, in the plastic age we are using multiple polymers and we are using polymers.We can use polymers for various good applications like these kind of things.So, we can make some prosthetic implant that prosthetic implant can be made with the polymer.So, with that polymer can be used as a skin graft; instead of that graft is taken fromown patient body we can use the polymer as a skin graft because ultimately we are developingthe graft just to cover that wound bed.So, once we covered it and that graft is completely biocompatible, the cells will start infiltratinginto it and it will try to form the normal structure of the skin.So, this is how that will work.So, consider this is our skin graft and that skin graft is placed into that wound bed.So, once it is placed and it will be stitched or they will be doing some dressing, thenonce that dressing is completed; obviously, that wound healing will occur and that willform the normal skin.So, global tissue engineering market involves like highest market is based on the skin.So, once we develop that skin graft, so that can be used for two different applications.The first application could be that skin graft can be used for wound healing.As I told the wound whichever cannot be healed at by wound innate mechanism that type ofthings need some graft.So, for such kind of applications, you can use this skin graft.And, another type of applications is like for cosmetics related things we are developingso many drug and molecules.So, for checking or screening that drug molecule we are using so much of animals and humans.So, that involves ethical considerations and many problems.If we are able to develop a skin graft equivalent to the normal skin so that skin graft itselfcan be used for drug screening implications.

Video 2: Design of Artificial Skin
Hello, everyone.I am Vasudha an MS student in Doctor Vignesh Muthuvijayan’s lab.Today, we are going to discuss various design strategies that have been used to synthesizetissue engineered skin grafts.Artificial skin is designed in two stages – stage 1 and 2.Stage 1 is applicable to short term acute use, while stage 2 is applicable to long termchronic use.Stage 1 deals with physical and mechanical aspects of the design while stage 2 dealswith biochemical aspects.In stage 1 the major critical graft properties that are considered include bending rigidity,surface energy which affects the wetting of the graft, moisture flux rate through thegraft, tear strength and blood compatibility.The critical wound bed properties such as viable tissue are of considerable importanceand the critical properties the graft wound interface include wetting and the peel strength.And, the clinical functions that are most important at this stage are infection andfluid loss control.In the stage 2 the graft properties that are important are biodegradability and non-antigenicityof the membrane is crucial.Also porosity of the membrane, the pore size of the membrane, the thickness and the bloodcompatibility.So, the desired events in the graft lifetime include migration of non-inflammatory cellssuch as fibroblasts and keratinocytes, a synthesis of neo-dermal tissue and also the metabolicdisposal of the graft.So, in this stage other than the infection and fluid loss control, contracture and scarcontrol are considerably important.So, there are different physicochemical and mechanical aspects that are important forachieving effective wound closure.So, in this representation, you can see the formation of air pockets because of ineffectivewound closure.In figure b, you can see that, due to excessive flexural rigidity of the graft there is formationof air pockets between the at the wound and graft interface.In figure c, due to excessive shear stresses there is formation of air pockets.In figure d, that is, due to peeling excessive peeling forces and then figure e, due to excessivemoisture flux rate through the graft there is dehydration of the membrane at the cornersso, which causes lifting of the membrane.In figure f, the moisture flux rate is insufficient, due to that there is excessive fluid accumulationwhich can cause edema.So, all these should be prevented to achieve effective wound closure.So, what is the long-range design objective for artificial skin?It should you should design a membrane that provides satisfactory wound closure to preventinfection and fluid loss.The graft or the membrane should be solid, degradable and non antigenic and also it shouldprovide early opportunity for cell migration and connective tissue synthesis that is collagendeposition inside the membrane or graft.So, the lifetime of the membrane is a very important aspect.So, here tb is the time constant of biodegradation and tl is the time constant for normal healingof the skin incision.So, the tb that is the biodegradation rate has to be optimized because if the tb is muchlesser than tl, then there is ineffective wound closure that is the membrane is gettingdegraded before the healing process takes place.Also, if it is much higher than tl then it has been observed that there is fibroid tissueformation under the graft.The next aspect is porosity.Porosity is crucial to allow cellular migration into the graft and also important for theexchange of gases and nutrients.Porosity is also important to allow the cells that participate in wound healing processto migrate into the graft; majorly being mesenchymal and epithelial cells.These cells have a size of around approximately 10 micrometers.So, the pore size of the membranes should be larger than this to allow cellular migration,also a different type of cells invade the initially non cellular membrane.This is an image of this is a scanning electron microscope image of a silk fibroin/chitosanblend scaffold.So, you can see the interconnected pores in the image.Cell motility through the graft depends on the availability of gases and nutrients.So, the conditions under which diffusion alone suffices to maintain the supply of nutrientsto the cells that are advancing through the graft is estimated using a dimensionless numberS.So, in this equation gamma is the rate of utilization of a critical nutrient by thecell; l is membrane thickness distance along the membrane thickness along which the cellhas to migrate itself; D is the diffusivity of the nutrient in the membrane; c0 is thenutrient concentration at or near the surface of the wound bed.So, lc is the maximum path length along which cells can migrate without requiring any morenutrient than supplied by diffusion.So, if your membrane thickness l is approximately equal to lc, then the cells can migrate withoutrequiring nutrients other than that supplied by diffusion.However, if the membrane thickness is large much larger then what happens is you needan additional mode of nutrient supply.So, then there is need for migration of endothelial cells and vascularization which is an importantaspect in skin tissue engineering.Endothelial cells have a migration rate of around 0.4 millimeters per day and fibroblastscells around 0.2 millimeters per day.And, this rate is usually insufficient to cover the surface of the graft in a periodcomparable for wound healing.So, an alternative approach that is used is to culture epithelial cells on the membranesurface at an appropriate time following grafting.So, the materials used for skin tissue engineering have to satisfy certain needs.So, major of them include providing a barrier layer of renewable keratinocytes and allowthem to securely attach to the underlying dermis, promote vascularization, and alsoprovide elastic structural support for the skin.So, the biomaterials used for skin tissue engineering can be an epidermal cover whichcan deliver cultured keratinocytes.So, this is the upper thin layer of the skin.this usually is involved in prevention of infection and fluid loss or it can also bea replacement of the dermis which is the underlying thicker layer.So, this layer is mostly composed of fibroblasts.Or it can also be an epidermal and dermal replacement which is usually split skin graft.Also, the tissue engineered skin can contain appendage constructs such as hair folliclesand sweat glands, the presence of these helps us mimic the natural skin more closely.Also, the pre-vascularized constructs can be provided to supply nutrients to the graft,like we discussed if the thickness of the graft is much higher than diffusion aloneis not sufficient to provide gases and nutrients.In the next lecture, we will look at different materials that can be used for skin tissueengineering and we will also look at various fabrication techniques that are used to synthesizetissue engineered skin grafts.