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Transcript for the Connective tissue recording Connective Tissue (Title) This topic will focus on connective tissue and wound healing (next lecture), and this recording will represent the connective tissue, part one of this topic. Connective Tissues: We have connective tissues throughout our body,...

Transcript for the Connective tissue recording Connective Tissue (Title) This topic will focus on connective tissue and wound healing (next lecture), and this recording will represent the connective tissue, part one of this topic. Connective Tissues: We have connective tissues throughout our body, and the mechanical strength of connective tissue varies widely from the stiffness and hardness of bone to the the softness of many organs in between types of connective tissue with different mechanical properties. Tendons resists tension and do not stretch, making them ideal for linking muscle to bone and cartilage resists compression. Large blood vessels can withstand, stretch and recoil in response to changes in blood pressure. All of these mechanical properties are mediated by connective tissue. Now, connective tissue is typically classified on the basis that the type of matrix, for instance, the density of fibre and how those fibres are organised, and connective tissue usually will have relatively few cells. The major characteristic of connective tissue is an abundant extracellular matrix containing varying amounts of protein fibres. For instance, blood is considered a special type of connective tissue, which has a fluid matrix called plasma without any fibres, whereas tendon is a fibrous connective tissue and it's predominantly made of fibres with very few cells. Connective Tissue Functions: So connective tissue has various functions, which is dependent on the organisation of that matrix and the role of that matrix. It can act as/have a mechanical support function or has mechanical strength in terms of tensile strength. It can protect and cushion and insulate, or insulate. It can take part in the organisation of the tissue structure, as well as other matrix components, it can offer or assist in metabolic support, and there are a variety of cells that are present within different connective tissues, which may have differing roles. For example, the cells in blood are important for the immune response, as well as the transport of oxygen and carbon dioxide. The function of a connective tissue, therefore, is dependent on the matrix constituents and also the proportion of those matrix constituents; how they're organised and their ability to interact with each other. And we'll have a look at that in the course of this topic. So any alteration in the original quantity or proportion of the individual ECM components, its structure, the type of component and the way it's organised, in terms of assembly together, will change the functional property, and this is particularly important if a connective tissue matrix is wounded or those components that originally were in that connective tissue that ensured that that connective tissue was able to perform its function may be damaged if wounded. So in the wound healing part of this topic, we will look at how those connective tissue components are reassembled and resynthesised in order to bring that tissue back to its original structure and function and to return to its tissue integrity. Connective Tissue (Areolar & Fibrous): So now we'll have a look at different types of connective tissue, so connective tissue proper is represented here and there are two types. There's the areola or loose connective tissue, and then there's the more fibrous, regular connective tissue. So if we look at the areola loose connective tissue, this is what holds organs in place and also can be underlying epithelium. It contains a variety of loose, proteinacious fibres, which may include collagen and elastin. In the case of fibrous or dense connective tissues, examples are those of irregular fibrils or irregular, dense connective tissue in the dermis, or regular dense connective tissue in ligaments and tendons, and here the fibrous connective tissue has its collagen fibrils, densely packed, provide great tensile strength, so tendon is a good example here. In skin, they have both loose and dense connective tissue, obviously you have your epidermis that sits on the basement membrane, which itself contains extracellular matrix components and is important in its role, and we look at that a bit later. But then underlying this, in the upper parts of the skin, we have that loose connective tissue and then we have the more densely packed collagen fibrils in the lower parts of the dermis. Special types of Connective Tissue: If we look at special types of connective tissue, as I ‘ve mentioned earlier, blood is a special type of connective tissue and that it has a matrix not containing protein fibres, but is essentially composed of fluid and those cells that we talked about in both the adult stem cells and the immune lectures, and also in the blood lecture by Malgorzata. So we know that those cells within there have a huge range of functions. If we look at adipose tissue next, adipose tissue is predominantly composed of cells surrounded by a thin matrix, and these cells, as you can see, occupy a large expanse of that connective tissue, and essentially these cells are called adipocytes, which store lipids as droplets that fill most of the cytoplasm. Fat contributes mostly to lipid storage and can serve as insulation from cold temperatures and mechanical injuries, and can be found protecting internal organs. If we move on to bone, this is a hard connective tissue and in fact the hardest of our connective tissues. It provides protection to internal organs and supports the body. So, think about the skull, surrounding your brain, and your ribcage which surrounds your lungs, as well as all those other organs below. So it has a hard, or bone has a hard, rigid matrix of connective tissue fibres embedded in a matrix which is mineralised containing hydroxyapatite, which is essentially calcium phosphate. Both components of the matrix, the organic and inorganic components, contribute to these unusual properties of bone. Without collagen, bone would be brittle and shatter easily. Without the mineral crystals, bone would flex and provide little support. The cells within this tissue we will talk about in a minute, are the osteoblasts. Cartilage is a matrix of polysaccharides with few collagen type II fibres in it, and note in the image you can see what look like round holes. These are called lacunae, which contain the cells which are in this case, are the chondrocytes. It has a distinctive appearance, and this is due to the presence of those polysaccharides, including chondroitin sulphate, which we'll look at later, which bind with the matrix proteins to form Proteoglycans. So note that all these tissues are adapted to the function, and in the case of cartilage, as we'll see later, it's adapted to its function of withstanding compression, which is very important if you think about your knee joints. Connective tissue cells: resident cells: Connective tissues contain either resident or transient cells. In the case of transient cells, these are the cells in the blood and the lymph, and include all those different types of immune cells that we've looked at in out lecture on inflammation, as well as the lecture on blood. So you already know the function of these cells. Additionally, the resident cells include fibroblasts, and there tends to be a type of fibroblast in each different type of matrix, and these are the predominant cell type in connective tissues, which is what CT is here and they're important in the secretion and maintenance of the matrix. So fibroblasts, when they're activated, especially in wound healing situations as we'll see in the second part of this topic, will be converted to myofibroblasts and it’s these cells that are responsible for secreting the collagen, and proteoglycans, as well as the enzymes that are involved in remodelling of the actual matrix after you have that primary synthesis of new products after a wound has been created. So if you think about the cornea, the fibroblast cell type, there is the keratocyte and this, too, will get activated in corneal injury to form a myofibroblast. The osteocytes within bone are osteoblasts which synthesise materials and osteoclasts, which produce those degradative enzymes in the remodelling process. In the case of cartilage, chondroblasts are responsible for the synthesis of new matrix components. So macrophages and mast cells are also present, remember as resident cells within connective tissues, and we've talked about the roles of these in the immune response and the inflammatory process in previous lectures. We've also mentioned the role of adipocytes/adipose cells in the earlier slides in terms of fat and energy storage. So fat cells additionally, as well as being a source of energy, can synthesise hormones. Connective Tissues: Diverse structures & functions: So if we look at this slide, we can see that connective tissues can be assembled into a number of different structures by virtue of their function. So, for instance, the tendons and ligaments are a rope-like structure, which makes them fit for purpose, for connecting different structures within our bodies. In the case of tendons, they can transmit forces in the direction of the orientation of the fibres within them. And ligaments will contain elastin as well as collagen, in order that they can have a bit of give in there as well. Blood vessels are tubular, and gels are formed by the vitreous. In the terms of basal lamina and basement membranes, these have a membrane-like structure, for instance, lining the lens capsule, Bruch's membrane or Descemet’s membrane. Remember, those diverse structures are important for the function of that particular connective tissue, so think about the skin, sclera and the the cornea, they all have a protective function. Cartilage and the intervertebral disc are weight bearing; think of your knee joints. Bone is part of our skeleton and they are incredibly hard to ensure that our body can be upright and move, in combination with tendons and ligaments. The cornea is transparent, so the way that those collagen fibrils are laid down, along with various proteoglycans, ensure that the regular array of those collagen fibrils within the lamellae allow the cornea to be transparent. And then in repair remodelling, we get the formation of scar tissue, which is required in terms of new synthesis of collagen and proteoglycans to ensure that new tissue is laid down, but then this has to be remodelled in order to bring the structures back to their original structure and function. Remember, all these diverse structures are related to the function of the tissue and are an adaptation of a connective tissue to its function. Connective Tissue Components: So next, we're going to look at the various connective tissue components. These are essentially components of the ECM and include collagen, which is important to impart mechanical strength to a tissue. Elastin within elastic fibres, that are essential for that recoil pressure to allow lungs to return to their original structure and function, and throughout blood vessels, such as they alter to withstand the high pressure of blood as it's pumped into it, from the heart allowing them to expand and then recoil to move that blood on. Proteoglycans and glycosaminoglycans (GAGS) are also essential components, as we will see later in the course of this lecture. They're important in mediating interactions with protein fibres, as well as being important in resisting compression, for example again, in the knee joint in cartilage. Glycoproteins are also very important in the organisation of matrix, and often these are called adhesive glycoproteins, and they can, as well as organising compounds, within the matrix they're important in cell attachment of the matrix. One very important one that we'll talk a little bit about in our wound healing component of this topic is fibronectin, as this can form a temporary matrix on which epithelial cells can migrate across, if the basement membrane has been damaged, in order to reform the skin epidermis or the corneal epithelium if it's wounded. ECM: Collagen: So if we look at collagen first; collagen is the most abundant protein in the body representing about 25 to 30 % of all proteins, and it describes a large family of proteins containing about 29 types of collagen, which are encoded by more than 40 genes, and in fact, I think 42 is the gene number, as of about 2016. There are several different types of collagen, which are all described by their array of supramolecular structure, as you can see in the bottom right hand side of the screen. They range from fibrils to networks, to fibrils with non-fibrillar regions, as well as anchoring fibrils. All collagens, regardless of type are composed of 3 polypeptide chains, which are called alpha chains. Each of these polypeptide chains can be over 1000 amino acids long, such that a collagen molecule has an overall length of about 30 nanometres and a width of 1.3 nanometres. Polypeptides or alpha chains as they are called, wrap around each other and form coiled-coiled interactions. Each alpha chain has a sequence of amino acids in a triplet i.e. a repeat of three amino acids where glycine, which is the smallest of amino acids, is present in every third space, so that it can sit inside the triple helix to ensure that all the amino acids fit in that coiled arrangement. Usually, or frequently, proline will be in the X position and hydroxyproline will be in the Y position. For instance, if you had a sequence of Gly -X- Y. Collagen fibrils can aggregate together to form fibres, and with increasing size they tend to have an increasing mechanical strength. Fibrils can assemble laterally to form 10-300 nm diameter fibrils, and these can assemble further into fibres of about 2.5 to 3 microns in size. Dependent on the type of fibre or determine which supramolecular structure it has, and also its function. So note that 80 to 90 % of total collagen is formed by fibres, which provide the most mechanical strength. We've got a few examples here, but also note that you can have different types of fibrillar collagens within the same tissues, and it's how they're put together within the matrix of that tissue that their function will be determined. Network forming collagen, such as Type IV and Type VIII collagens are found in basement membranes. Remember, the basement membranes tend to sit underneath cells. Type IV collagen is a common component of basement membranes, note BM represents the basement membrane in this image here. Descemet’s membrane, remember, contains type VIII, and it's thought that this type IV and type VIII collagen are important in basement membranes because it gives the membrane some flexibility. Membranes, as well as underlying epithelial cells and endothelial cells, can also surround muscle and fat cells. Anchoring fibrils are another supramolecular structure, and a common one is type VII collagen, and these are essential to act as anchoring fibrils, anchoring cells to membranes such as an epithelial membranes. Type VII collagen is also very rich or enriched within amniotic membrane. FACITS, or fibre associated collagens with interrupted triple helices, as their name suggests, don't actually form fibrils themselves, but they do associate with fibrillar collagens. So it's important to remember that all these macro or supramolecular structures are relevant to the function of the matrix in which these components or these collagens are in, an example of FACITS are Type IX and Type XII collagen. Adaptation to function: Collagen: If we look at how collagen is adapted to its structure and function, orientation and size of fibrils is very important. In tendon and in the retro or postlaminar optic nerve, the collagen is orientated in the direction force. In tendons, as you can see on the right hand side, you have a uniform direction of that tendon so that it can transmit force in that direction. In the LC, and other tissues, you can also get like a basket-like weave; you have a criss-cross arrangement of the fibres so that it can withstand both tensional and compresses forces. Depending on the force that's been exerted in the tissue, the collagen appears to be designed to withstand those tensions or forces that are applied to it. If you think about the regular array and those small uniform diameter fibres in the cornea, these are laid down in lamellae and it's that's critical laying down or array of those small diameter fibres that ensures the transparency of the cornea. The type of collagen is also important. Different collagen types,dependent on the type of tissue, would determine its function. For example, Type I and III collagen and are present in skin, lungs, heart and blood vessels. Type I collagen is the one that will give the strongest mechanical force, whereas Type III collagen fibrils, or tissues with type III collagen within them, tend to offer resilience to the tissue. ECM: Elastic Fibres: If you look at elastic fibres, these are very important structures within tissues, as the name suggests, they confer elasticity and extensibility to tissues which undergo physical deformation, tension or pressure gradients. Tissue, in which they are rich in, include the skin, lungs, ligaments, blood vessels and the lamina cribrosa. The elastic fibres consist of an elastin core symbolised by the ‘E’ in this electron micrograph in the upper part of the screen. This is said to have no obvious structure when you look at it, it's said to be an amorphous structure, and it's surrounded by microfibrils and these microfibrils are essentially made of fibrillin. Fibrillin is critical here because it helps by forming a template when elastin is first synthesised so that the elastin will form in globules along the template, and gradually merge together to form that elastin core, and then the fibrillin microfibrils will surround it. Fibrillin is also important in its own right, as it can be present in the zonules i.e., the fibres that suspend the lens in place, and remember when we looked to our introduction pathology, we said a mutation in fibrillin could lead to increased likelihood of lens dislocation in Marfan’s syndrome. Fibrillin also has other roles, it can interact with integrins and key growth factors such as TGFB and BMP. TGFB is very important in terms of connective tissue as it can alter cell behaviour, for instance, transforming fibroblasts into those myofibroblasts that secrete connective tissue proteins, and it can also act to stimulate production of components of connective tissue. So you can see that fibrillin, as well as having a role in the biomechanical properties within elastic fibres, it also is a key signalling molecule. ECM adaptation to function: So here we can see tissues where elastin and collagen are present within the same tissue. This is critical because elastin and collagen will provide different properties to tissues. For instance, elastin allows deformation and recovery of the structure after it has been deformed. Collagen provides tensile strength, limiting the deformation to prevent damage. A good example is your skin; if you look at the back of your hand and you just pull on your skin, the reason you can deform that skin is elastin. The reason you can't rip the skin off is that collagen limits that deformation. When you let go, you get recovery of the structure and that's elastin’s recoil properties that brings that structure back to its original structure and function, so that it can redo the same thing again. So elastin and collagen are present in tissues that need these recoil and deformation properties, whilst strength is maintained. This includes skin, lungs, hearts, blood vessels as examples. Here we have in the upper picture, we've got the pink is second harmonic generation signal, which can be derived from collagen fibrils, due to what we call their non-centrosymmetric centre, but you don't need to worry about that. All you need to know is that second harmonic generation signals can be emitted from collagen fibrils. We can see then next to it, we have MPEF, which is essentially multiphoton excited fluorescence, which is the same as the two photon excited fluorescence in the central retinal artery below. This shows you where the elastic fibres are, as elastin has an endogenous signal which can be emitted using two photon microscopy. If you look at the distribution of the collagen and elastin in the merged image of the alveoli, in the lung, they appear to be distributed in the similar pattern as each other, which is important for lung inflation, and also bringing it back to its original structure after you've breathed in and then breathed out. Below is a magnified image of the central retinal artery and vein. We know that this is the artery because it has a ring of elastin in its wall. This overlay emphasises the differences and also overlap of elastin and collagen in tissues. In the central retinal artery, you can see that distinct ring of elastin around the circle, and if you looked at any blood vessel, you would be able to see the elastin. This obviously is important in blood vessels, as I mentioned, especially arteries, where you have fibres that need to ‘give’ or deform to allow the blood vessels to expand as blood is pumped into them and fill, but then also to recoil as they return to their structure (original shape) to allow the blood to be pumped further through the system. So the dermis of skin also contains elastic fibres and collagen and this is important for the skin to resist tension in different directions, but also to be able to recoil back. If we look at the overlay of the central retinal artery. and remember this central retinal artery is actually in the centre of the LC and this is where this tissue section was taken. Image was taken by the optic nerve group, which we have in the department. You can see the distinct but also, similar regions, where you have both contributing collagen, which is green here and elastic fibres, which are red here. ECM: Proteoglycans (PGNs) NB: Glycosaminoglycans: GAGS We move on to proteoglycans. PGns contain a core protein with GAG side chains of repeating disaccharide units. There are different types of glycosaminoglycans that can be attached to the core protein to form the PGn, and this includes chondroitin sulphate, heparin, heparan sulphate, dermatan sulphate and keratan sulphate. The high sulphation on these GAGs provides a huge negative charge, which is hydrophilic form, which enables hydrated gels to form, which are able to withstand compression. An example of this is obviously cartilage in the knee joint, which we've mentioned several times. PGns are structurally diverse; they can have different core proteins, different GAGs chains and also different lengths of GAGs chains. PGns can attach to basement membranes, they can be within matrix. PGns have a number of functions: they can have a biomechanical function due to those hydrated gel properties, enabling them to resist compression. But they can also regulate collagen fibril diameter, which in itself can have a biomechanical function as small collagen fibrils have more resilience compared to the wider diameter collagen fibrils which have large tensile strength. So dependent on the type of tissue, would be dependent on what type of PGN is present within the tissue. Also, important PGNs can have a role in cell signalling. Adaptation to function: Collagen and GAGs: So here we can see where collagen and PGns are within the same tissue in the case of cartilage. Here we have a large aggregating PGN called aggrecan where the GAGs content represents 15% of the dry weight of the tissue. It's very hydrophilic in nature because of that high negative charge on the GAGs, allowing it to trap water, having a cushioning effect when we walk in the knee joint. Here we have cartilage or a tissue made up of collagen and PGns with a large component of PGns compared to collagen fibrils in the form of Type II collagan fibrils. Those large PGn aggregates of aggrecan can act as the cushion, trapping the water, enabling the tissue to withstand compressive forces, while those collagen type II fibrils impart tensile strength to the tissue, limiting the space in which the deformation of the tissue can occur. ECM: Glycoproteins Another component of the ECM are glycoproteins, one that you will have heard of from me is fibronectin. These are particularly important in cell migration, cell attachment in wound healing - see epithelial wound healing. Fibronectin has two subunits which form a ‘V shape’ in the diagram on the left. It functions as a connection of cells in ECM containing collagen fibrils, and it can bind to collagen, heparin sulphate, hyaluronic acid (HA) and integrins. -what I didn't mention is that HA was also a GAG but it is distinct from the other GAGs as it is not bound to a core protein, but it enables PGN aggregates to form. So going back to glycoproteins, they can also be called adhesive or adhesion glycoproteins because they ensure specific interactions between cells and molecules of the ECM. They are important, as I've mentioned, in cell matrix attachment, cell adhesion and the regulation of cell migration, cell attachment and matrix organisation, as well as maintaining the shape. Examples are laminin, vitronectin and thrombospondin you'll learn more about the role of fibronectin in wound healing in the next part of this subject (i.e. the Wound healing lecture!). Connective Tissue: Extracellular Matrix (ECM) components So we've talked about ECM components that are within connective tissues in the form of collagen, which conform form those fibrils, anchoring fibrils, as well as network forming collagen. We've looked at elastic fibres with their recoil properties and that they contain elastin as that amorphous core protein with the more regular microfibrils that can form around the core protein. We've also looked at PGns and GAGs, glycoproteins, as well as the cells within the tissue. The makeup of the ECM products and the cells within a tissue will be adapted to the function of that tissue, and if any of these are disturbed, if they're wounded, it's important that they are reproduced in the wound healing process in terms and reorganised to bring the tissue back to its original structure and function In the next part of the topic, we'll look at the process involved in healing of wounds, using skin and bone as examples of soft and hard tissues, respectively, and see how our body attempts to restore the tissue integrity after it's been wounded so that it returns to its original structure and function.