Video 1: Cornera
Good morning everyone.So, this lecture is about Corneal Tissue Engineering.We are going to see why Tissue Engineering is necessary for corneal repair.So, there are going to be two videos on this, the first one will cover about the cornealanatomy, the physiology, the tissues, the layers between the cornea’s layer, whatis the function of the cornea and why there is a damage or why there is a necessity forrepair.And we should also know what are the treatment approaches that is there and why do we needa tissue engineering approach for this corneal repair.So, the first video we will be covering about the skin physiology, corneal physiology andanatomy and the next one will cover about the tissue engineering approaches of cornealrepair.So, taking you through the parts of the eye as a first one, we have the cornea to be thefirst one.So, the cornea is the outermost layer of the eye and this is where the light enters andthe transparency is the major region, major reason or the major strength of what the corneallayer or the skin barrier is.And then we have the aqueous humor.So, the aqueous humor is the fluid that is behind the cornea that provides the nourishmentto the eye and then we have the iris and the pupil.So, we all know what is a function of the iris and the pupil, that the light that passesthrough the cornea enters and reaches the iris and the pupil and that is where you getyour vision.And the dilation and the contraction helps you in seeing the near and the far distantobjects.And then we have the lens that is behind the pupil, here this is the lens and then we havethe ciliary muscles surrounding the lens.And then we have the vitreous humor that is the transparent watery gel that supports theeye.And then we have the optic nerve that is where the nerve enters the eye and then it moveson to different ciliary nerves and enters the corneal layer that is through the sclera,ok.So, there is a connection between the cornea and the sclera and this region is called asthe limbus, and is where your nerves enter.So, out of all this we are going to be studying about cornea.Why a tissue engineering approach is necessary for corneal repair.So, why do we need a tissue engineering for corneal repair?So, there as I have already mentioned the corneal is avascular membrane and it givesyou the strength and the transparency that is necessary and it is a major region of whya refraction of the light happens and that is the reason why a vision is possible.So, the corneal blindness is a major cause of vision loss which is affecting over 10million people worldwide and the options for corneal transplantation is there.So, either you can take an autograft or an allograft option.So, in the case of an autograft either from a the, if there is one eye that is affectedthe tissue from the other eye which is fine can also be used, but the corneal layer orthe corneal tissue is very extensive which will we will be covering in the next few slides.So, the transplantation is not as easy as it can be explained.And the another thing is the corneal donors which will have you a tissue rejection, whichis the major criteria.So, to address all these issues, to avoid this, researchers are developing new materialsand strategies to repair, to regenerate or to replace the diseased cornea.So, here in short, the same eye that we are looking at we have the cornea that is theouter transparent region, which is the major refractive zone.So, since it is avascular, but it is innervated, ok.There are millions of nerves that goes through the cornea that is from the optic nerve whenwe see this red and the blue line here.So, these are the nerves.So, these nerves enter and through the limbus enters through the lower regions of the corneagoing up to the epithelial region.So, the primary function of the cornea is to transmit and to refract light and thatis the reason we are able to see and visualize things.And it also, protects the eye from mechanical damage, UV light and infection, and the cornealnerves are very important for maintaining the integrity of the ocular surface and thecorneal sensation.So, let us go in detail about the corneal anatomy and that of the physiology.So, the first one we will look about the dimensions of the cornea.So, this picture A, gives you the position of an anterior position of cornea and thena posterior view.So, when we see here, the anterior position is elliptical in shape with a 11.7 mm herewith a width and then a 10.6 mm of the vertical region and then the posterior one is circularwith 11.7 mm of radius all this is very necessary because the corneal curvature and the cornealshape is very important.Because when there is a repair that is either there is a bulge or a swelling or a decreasein the transparency there is a change in these elliptical anterior and the posterior positions.So, when there is a change in your bulge what happens is people when they use contact lensesthe contact lenses do not sit on their eye properly because the curvature of the eyechanges.So, the radius of the curvature is also controlled by your corneal tissue.So, the anatomy of the cornea is very important and over age though there are still variationsfrom person to person, but with disease and with wound, there is a drastic differencethat is associated with the corneal tissue damage.So, and then the radius of the curvature is given here.So, here we have the picture of the corneal radius of curvature which is around 7.8 mmand the outer radius of curvature is around 11.5 mm that is including the sclera.The thickness is very important.Because; so, the thickness is different in both the regions that is the central regionof the cornea and the peripheral region of the cornea.So, the central region is around 0.52 mm whereas, the peripheral region is around 0.67 mm andthis region around 4 mm to 5 mm of this is the central corneal portion that is of primeimportance.This is where your light enters, refracts, transmits and this region is of importanceand different layers is what we will be looking at.So, any disease or any wound that we will be incurring in the corneal tissue will havea change in either their thickness, the radius of curvature or their size or shape.And so, now we know that there is a differences in the curvature radius or thickness, butwe need to know what is the underlying process or what causes this to happen.So, here we have the corneal tissue layers.So, the figure here will show you an image of the different layers that are being inthe corneal tissues.So, the corneal tissue has previously or for a long time has been divided into 5 layers,that is the corneal epithelium, the bowman’s membrane, the central stroma, descemet’smembrane and the endothelium.Recently in 2013, scientists have reported that there is a 6th membrane that lies betweenthe central stroma that is here to here that is called as the pre-descemet membrane.So, it is the pre-descemet membrane.So, this membrane is between the corneal stroma and the descemet membrane somewhere here andnow reports and scientists have already been identifying because there is a lot of criticalthinking going on in evaluating whether there is a differentiation in the membrane betweenthe descemet and the pre-descemet membrane.And it has found that the cells between them are different and they also have a differentfunction associated to them.So, now the scientists are classifying that we will have 6 different layers in the cornealayer of epithelial, bowman’s, stroma, pre-descemet, Descemet and the corneal endothelium.And the thicknesses of these respective medium ranges from around the corneal epitheliumto be 50 to 90 microns which is a huge and the 500 micron thickness the central stromaaccounts to around 70 percent of the overall region of your corneal layer and then we havethe descemet membrane and the endothelium which is a single layered cells.So, these are stratified squamous cells, the corneal epithelium and then the endothelium.So, there are avascular, sorry cellularized and decellularized cells.So, the corneal epithelium, this stroma and the corneal endothelium are all cellularizedwhereas, the bowman’s membrane, descemet are acellular or de-cellular membranes.So, now we will be looking into further what is the functionality of these systems andhow they repair and the regeneration process of each of these layers will affect the corneal function.
Video 2: Corneal Epithelium
So, the first one is the outermost corneal epithelium that we are looking at.So, this is an more zoomed version of only the top layer of the stratified squamous epithelialcells.So, we all know what an epithelial cell is.It is the outermost cell layer similar to how our skin cells are.These are the corneal epithelial cells that covers the outer surface.So, similar to its function, so the first thing is that it will give you the mechanicalstrength and then it acts as a barrier for any of the infections of or microbes enteringthe cells.So, this is like the wall that is the first and the foremost region.So, and then environment allows and it also allows the environment and oxygen and nutrientabsorption into the cornea from the tear film.So, here this corneal epithelium is further divided into 3 different layers, the superficialcells, the wing cells and the basal cells.So, we have tight junctions within them, these cells are with tight junctions to have a compactlayer and the topmost the superficial cells have a microvilli.So, this is a zoomed portion.We have a like similar to how a compound wall has a broken horned glasses or something wehave the microvilli which with the glycocalyx.So, this interacts with the tear film and this is the outermost region.So, this tight microvilli and glycocalyx is very important because even when you havea corneal transplantation from a donor, only when you have these microvilli and glycocalyxwill it start having the connection with the tear film and the tissue will start havingthe normal functions to be replaced.When this is damaged, the microvilli and the glycocalyx is damaged we need to have differentcell transplant for forming these microvilli and glycocalyx to be formed.So, and then there are 4 to 6 layers of these cells, the superficial cells, the wing cells,the basal cells and the another important thing is the basal lamina which is the bottommost layer of these cells and desmosomes and hemi-desmosomes.So, these are new terminologies.So, the desmosomes and hemi-desmosomes are similar to how tight junctions are, but thehemi-desmosomes help in connecting the basal cells to that of the basal lamina.So, going in detailed about each of its functions and roles, first as I have already told whata tear film that is the outermost region.So, this is the tear film that is outside and this is will have a connection or willhave an interaction with that of the microvilli and the glycocalyx helping it as a barrierand allowing only few things that is entering of only the oxygen and certain things intothe system.And then we have the superficial cells.The superficial cells are 2 to 3 layers of glycocalyx covered with a microvilli becausefrom these the microvillis are starting up and they have proven the tears toxins fromentering the eye.And then we have the wing cells.So, wing cells are polyhedral cells, so these form the second that is the middle layer ofthe endothelial cells which are a connection or they have a bridge between that of thesuperficial cells and the basal cells.So, and then we this communicate between the superficial cells and the basal cells.Then we have the basal cells.So, the basal cells are very important it is a major source for the wing cells and thesuperficial cells.So, this is the one which balances the hemostasis of that of the corneal epithelial layer.And so, these basal cells have tight gap connections which is connected by the desmosomes or thehemi-desmosomes to that of the basal lamina.And the basal lamina this is the one that will connect to your lower region that isyour Bowman’s region.So, this is not cellularized.So, this will have glycoproteins which will help in the adhesion to that of the Bowman’sregion they have the collagen, laminin and then the glycoproteins and help in adhesionand then also maintains the mechanical integrity of the corneal epithelium and avoids the influxof the keratocytes from the Bowman’s region to that of the corneal epithelial.So, now we were talking about rejuvenation.So, anything will have a wound, a repair and it is going to repair on itself.So, similarly the corneal wound or tissue damage will also have a repair on its own.The repair mechanism is known.So, generally it will take 7 to 10 days for the regeneration process to happen, but thereason from where this is happening.So, the earlier studies told that it will happen from the basal membrane, that is thecells which are known to have the wing cells and the superficial cells, the lower mostpart of your epithelial.But further later, people have understood that this is just not the source.So, we have the Bowman’s membrane, the basement and this is called as your basal membrane.So, this basal membrane cells is just not the source, but there is something else thatis called as a limbus region.So, this limbus which I have already mentioned is between the cornea and that of your scleraa connection.So, this is having a limbal stem cells.So, the limbal stem cells are the stem cells which migrate.So, they first have a symmetric asymptotic mitosis happening.So, the limbus corneal epithelial stem cells, they are called as LSCs.So, these LSCs divide to produce transiently amplifying cells.So, what happens is, so the first the stem cells they divide into many and then theyform cells.These cells then migrate go to the Bowman’s membrane and from there, form the bigger layer.So, this is the principle.So, now scientists have shown that both are necessary that is the limbal stem cell regionwhich is the major source of this transiently amplifying cells is necessary, their migrationis an important process, and then the basal cells from which the cells are stratifiedinto the wing cells and the superficial cells.So, this theory is being explained by an XYZ hypothesis.So, this picture will tell us what is happening.So, Thoft R and Friend J, these are the two scientists who first proposed this theoryof the XYZ hypothesis in 1983, which showed that there is a corneal balance between thebasal cells and the limbal stem cells, which helps in the maintaining of the corneal epithelialhomeostasis.So, the first thing is, so please note this red colour small stem cells that we are lookingat.So, the limbal stem cells initiate it and then they migrate.That is they are forming transiently amplifying stem cells which can be differentiated intoany stem cells.They migrate.So, here they migrate, this is by the centripetal force migrate to the basal layer.So, this lowermost region is a basal layer and as I said the corneal layer is this, sothis is how we are looking at the eye and these are the different regions.So, we will have a wing cell here and then we have a superficial cell.So, what happens is, so first it migrates, go to this place and then after the migrationthen in this step they are differentiated into the different cells, either their wingcell which is the second layer or the superficial cells that is the third layer or the topmostlayer and they are squamized.So, this is the entire process.So, what is this XYZs theory?So, the Z is the shedding, that is when the cells are worn out, with the tissue damageor by age they are, they become dead cells.So, that is desquamation.So, the rate at which the cells are worn out or when the process is being damaged and thenwe have the centripetal migration.So, this migration is termed to be Y and then we have the cell division or the stratificationthat is Z; sorry that is X.So, this all these 3 are combined, that is for this to happen; so, X plus Y is equalto Z.So, this homeostasis is maintained by this theory, that the limbal stem cells along withthe basal stem cells is the differentiation, the mitosis process and the centripetal movementall plays a road, role in maintaining this process.So, this happens upon a normal tissue repair and then when there is a wound that is created.So, this is what an injury happens, limbal epithelial stem cells differentiate and migrateto repair the injury.But what happens when there is a severe injury to your epithelial cell?So, when the damage to the limbus, so when there is a differences in your layers; so,in your corneal layer when you have damage to any one of them we can have the repairprocess happening or when there is a damage to that of your limbus systems itself whatwill happen; in that case a corneal transplantation will not be helpful.So, in that case what they do is a keratoplasty is a normal term for corneal transplantation.So, when any one of these tissues are damaged, you can either take it from your own eye asan autograft taken from the other eye or from an external donor that can be used for anyof the corneal tissues.But when there is a damage to the limbal stem cells, a keratoplasty will not work becauseonly when these cells migrate, there will be no tissue rejection that will come as aproblem.So, for this what we do is, in this case there is a limbal stem cell transplant that is tobe done.So, here we will have a limbal stem cell transplant or placement of the small limbal stem cellitself into the recipient place for a tissue engineer.So, for these, this is the normal approaches.So, for this we need a tissue engineering approach to have the use of the epitheliallayer of mass.
Video 3: Bowman’s Layer and Stroma
And now we are moving into the second layer of the epithelium that is the Bowman’s layer.So, the Bowman’s layer is around 8 to 14 micrometer thick and it is; so, this is theregion that lies between that of your epithelial and that to the stroma.So, here they are not, they do not have cells, they just have proteins, glycoproteins andcollagen.So, they get infused to the stromal layer.The stroma consists completely of collagen fibers.So, this is the border between that of your stroma and that of the corneal epithelium.So, here it is resistant to both mechanical and infective legion.And so, we know that the corneal region upon damage can repair on its own.Only when there is a severe damage it cannot repair.But whereas, the Bowman’s the thickness of this will decrease over age or with anywound or damage, so this keeps decreasing.So, what happens is the, but this will not affect your corneal epithelial layer becauseonly above this is the entire process that is happening and the Bowman’s will not havea region, but there is something else that the Bowman is responsible to that is the influxof nerve impulses.So, we have I have already mentioned that within this Bowman region is where from youroptic nerve your nerves enter the eye.So, from this, the nerve enters and then gets into smaller nerves and goes up to the epithelium.So, when there is a damage to the Bowman, the eye the corneal epithelial repair is notstopped, but there would be a problem in the nerve plexus that can have a major role tobe played.So, this will have pores for the influx of nerves.So, this is the Bowman’s layer.And then we have the stroma.So, we have already spoken about what these layers are, the corneal layer and then wehave the Bowman’s layer and now we move down to the major region or the major cornealfunction that is called as the stromal region.So, the stromal region is the middle layer, accounts to 80 to 90 percent of this entirethickness.So, they have collagen fibers associated.So, the collagen fibers are well aggregated and they are arranged in a parallel fashioncalled as a lamellae with a few keratocytes So, there are two things here primarily maintained,that is the collagen and the keratocytes.So, the arrangement of the lamella is heterogeneous that is; so, how a lamellae is formed is,these fibers are aligned in a parallel fashion in bundles and these bundles are then furtheroriented.So, here they form a nice good lamellae and they are differentiated to the anterior positionand then to the posterior position and in between them we have the keratocytes thatoccupy them to giving them the proper periodic arrangement and the patterning of the system.So, looking at this yellow substance is where these are the small bundles of your fiberslooking them at a lateral view.These are the fibers of your collagen fibers as bundles arrange them and they are arrangedinto a specific diameter of 25 to 35 nanometer.So, what is the functions of this big stromal layer?So, this stromal layer is known for to associate the transparency of the corneal tissue.So, whenever corneal tissue comes into your mind, the transparency is one of the majorfunctions because that is the reason you are able to transmit and refract light and seeor have a vision.So, these give you that property and then the elasticity and also the strength; refractivepower the elastic modulus and the transparency.So, the fibers are very consistent, consistently arranged as I have said and the spacing betweenthe fibers is tightly controlled to give you a periodicity of around 67 nanometer.These numbers are very important because these numbers give you the periodic arrangementsimilar to how a crystal structure lattice has periodicity in arrangement of these lattices.And then the keratocytes occupy is responsible for the synthesis and maintaining of the collagen,giving you the extracellular matrix.So, this transparency as I have said is the major function of what the stroma is, butwhy will a collagen fiber arranged in a lamellae position give you a transparent membrane,what is the functionality.So, for this two people have developed their own theories.The first theory was by Maurice, the known as the Maurice Theory in 1957.So, the transparency of the stroma is here that he has mentioned is because of the periodiclattice arrangement.As I have mentioned, when you know the crystal lattice the periodicity is a major role.So, when light comes and passes through, so because of the lattice regularity of the arrangementthere is the separation of the backscattered light by a destructive interference.So, there is the vision, there is proper refraction that is coming up and this arrangement isthe one that is the reason.It is what his claim is.There is one more reason that is the interfibrillar distance.The interfibrillar distance as I have said are all cut completely arranged.The bundles will be around 25 to 35 nanometer and the interfibrillar distance is around67 nanometer.So, these numbers are less than that of the incident light.So, the incident light is the light that we are seeing which is around 400 to 700 nanometer.So, whenever there is a lesser than the wavelength of that light there is going to be no interferencethat is being done.So, two factors are responsible one is the arrangement and the arrangement has a specificpattern packing which is helping in responsibility for the transparency.Later, after the Maurice theory in 1957, Goldman based on the diffraction theory proposed anotherstatement that, so this picture sorry I am not explained.So, here we have a picture of the normal cornea and then we have a edematous cornea that iswhen there is edema swelling or something what happens there is a bulge in your corneallayer and your transparency is reduced.So, in the normal cornea there is a periodic arrangement that is happening whereas, inthis there is no periodicity that is seen.So, this shows that the periodicity plays a major role in the transparency that is bulgingor the clouding of your vision.Goldman later said that, the lattice arrangement is not the only requirement.He has told that the fibrillars do not interfere when the transmission of light to the transmissionof light until they are larger than one third of the incident light.So, the fibrillar diameters are very small.So, similarly the transmission of light, that is the light entering through is around 400to 700 nanometer.Based on the diffraction principle he says that since the fibrillars length dimensionsare very small, they would not interfere with the transmission of light, they would interfereonly when they are more than one-third of the wavelength of the incident light.So, periodicity is not a matter, but the fibrillar as such will give you the transparency.These are two theories that are ongoing, but both would say that the stromal layer is madeup of the collagen fibers and the fibril arrangement, their fibril type and the bundle packing andthe lamellar packing is important in giving you the transparency, elasticity, and themechanical strength.So, what happens when there is a stromal injury or a repair?So, as we know there are two major components the keratocytes and the collagen fibers.So, the keratocytes help in giving your increasing the collagen source.So, when they are injured they activate and synthesize the new extracellular matrix.So, this is upon the normal injury and the repair process.Upon severe disease or injury, the cell death occurs and then we have a scar tissue thatis being formed which is reflected as a scarring of your cornea.So, here we have an entire keratoplasty that is to be done.So, keratoplasty as such, as I mentioned is corneal transplantation.So, either it is penetrating keratoplasty or anterior lamellar keratoplasty.So, penetrating keratoplasty is when the scar is deep.Penetrating keratoplasty means when the entire corneal region is to be changed, that is tobe replaced.Like, the stromal region as such or the entire epithelial along with a stromal.But when we have an anterior lamellar keratoplasty only when few of the regions are damaged youcan have a partial replacement of the corneal tissue.So, these are the ongoing current treatment methods for the corneal transplantation.
Video 4: Descemet’s Membrane and Corneal Endothelium
And now we move on to the pre- Descemet’s and the Descemet’s membrane.As I have already told, the decimate is the 4th membrane, but since from its inceptionin 2013 the pre-decimate is the layer that is before the decimate membrane.So, both these membranes are acellularized membranes, so they contain of collagens, laminin,lectins and different types of collagen.So, like before this, we will have the pre-Descemet membrane and it is made up of type I collagenthat is the pre-Descemet, which assembles into the lamellae and overall thickness isaround 10 to 15 micrometer.After that, we will have the Descemet membrane and in this we have the type IV collagen,laminin, nidogens, vitronectin, and the fibronectin.And the basement, so, this is the basement membrane of your endothelium.So, this membrane will form the basis for the endothelial cells to attach and have thetight junctions.So, and this membrane can regenerate and is assistant to chemical agents and infections.So, we have looked about Descemet and Bowman’s, both are acellularized membrane this willhave, this cannot regenerate whereas, a Descemet membrane can regenerate and they are resistantto chemical agents, infections and pathology.And the other difference is over age the Bowman’s membrane reduces in thickness whereas, theDescemet membranes increases in its thickness over the age.And then we have the endothelium.So, the corneal endothelium is a bottom most cells that we are looking at.So, this is the layer.It is a single layers, one to two layers of cell thickness is what forms your cornealendothelium, the innermost cells of the corneal tissue.And it is a simple squamous cell layer and the cells density is a major role.So, the cell density of these endothelial cells decreases over age.Say for example, this picture will tell you that.So, a new born baby would have around 3000 to 4000 cells per millimeter square whereas,as we all grow the adult would have a 2500 centimeter square, whereas here with age itfurther reduces to 2000 cells per meter square.So, what happens?When these cells are reducing in number there, we are going to have gaps in between them.So, to occupy that space what happens is there is an increase in the size and shape.So, compensated by increase in cell size and cell shape that is polymegathism and pleomorphism.So, this is what it is.So, in early life it is 18 to 20 micron, over age it becomes 40 or more.This is the characteristic of what an endothelial cells is.So, we all know what; since as we know an epithelial cells will have act as a barrier,the endothelial cells will perform the major functions for the cells.So, they will be metabolically very active for the passage of the flows.So, similar in that, the corneal endothelium is also like that.So, it is metabolically active, it has a mitochondria, but it is mitotically inactive.So, that is the reason they will not regenerate.Once it is formed, it just degenerates over age, but it is not going to be replenished.And so, the major functions are they are metabolically active, so they control the sources, thatis the nutrient inflow and outflow from the cells within the rears what goes into thestroma and what is being pumped out of the stroma.And the stroma is an hydrated layer.So, why it gives you a transparency?It also has a hydrated layer.So, this endothelium is acting as the pump, it has the efflux pumps pumping out the excesswater and the fluids outside maintaining the osmotic pressure.So, it maintains the osmotic pressure and then the bicarbonate ions.So, based on that if tissue engineering approaches are used in the form of scaffolds, cells orby stem cells that is the limbal stem cells or corneal stem cells that can be grown outsideand then be infused again, if these are all done then the avoiding of a corneal transplantationscan be done where tissue engineering approaches can be overcome.Thank you.
Nous enverrons les instructions pour reinitialiser votre mot de passe à votre adresse e-mail associée. Veuillez marquer votre adresse e-mail actuelle.