Extracellular Matrix

Extracellular matrix (ground substance+ fibers): The Ground Substance: - The ground substance is mainly water (remember that the connective tissue acts as a transport medium for gases, certain molecules and waste products between blood vessels and epithelium for example). - It’s not only water because according to gravity, this water will pool in the lower part of the body so in fact it’s a gel-like structure. - The ground substance is mainly water, but this water is stabilized by macromolecules (called stabilizing macromolecules), it functions like a jello, it transforms water from liquid state to viscous state. For example, in blood, the ground substance lacks stabilizing macromolecules and lacks fibers, so the extracellular matrix is fluid - With different types of C.T we’ll see different components of ECM (like in blood its ECM is fluid but in bone it’s solid because it has high amount of Ca ions), but here we are talking about ORDANIRY C.T which is located directly under epithelium (for example) not about special types of C.T. 1. 2. 3. 1. ✓ ✓ - 3 types of ground substances (macromolecules): Glycosaminoglycan(GAGs) Proteoglycans Glycoproteins GAGs: Long chains of repeating units of disaccharides (2 sugars), unbranched 2 types: Sulfated: contain sulfate group such as (keratin sulfate, chondroitin sulfate, dermatan sulfate, and heparin sulfate (heparin). - Non-sulfated: No sulfate group, the only example is: hyaluronic acid (hyaluronic acid is a long molecule with very high molecular weight). 2- Proteoglycans: consist of proteins (~5%) and polysaccharide chains (~95%). The polysaccharide chains belong to glycosaminoglycans (a protein core attaches to many GAGs). 3-Glycoproteins: consist of proteins (~95%) and polysaccharide chains (~5%). The sugar chains are branched short polysaccharides (oligosacchardies, oligo= few). - The hyaluronic acid acts as a backbone where the other glycosaminoglycans are going to attach through a protein core, so simply the hyaluronic acid interacts with proteoglycans - Ground substance consists largely of proteoglycans and hyaluronic acid. Proteoglycans are very large macromolecules, consisting of a core protein to which many glycosaminoglycan (GAG) molecules are attached. GAGs are long-chained polysaccharides made up of repeating disaccharide units. GAGs are highly negatively charged (polyanions). The high density of negative charges attracts water, forming a hydrated gel. This gel permits the rapid diffusion of watersoluble molecules but inhibits the movement of large molecules and bacteria. - The large structure formed by hyaluronic acid and proteoglycans makes a spherical space; this space is called a domain. So bacteria need to pass between these domains in order to pass through the connective tissue, so that is why we say that the ground substance has filtering effect and prevents the spread of the bacteria. The pathogenicity of a bacterium is indeed to some extent determined by its ability to find its way through the mesh, and some of the more invasive types produce the enzyme hyaluronidase, this enzyme is able to destruct hyaluronic acid which will destruct this domain so they are able to invade the connective tissue - Each domain looks like a Bottle Brush: the handle is hyaluronic acid and the bristles are the proteoglycans - Functions of Proteoglycans ✓ Resistance of compression. - They are stabilizing macromolecules (little amount of it gives water gel state). ✓ Retardation of movement of microorganisms. ✓ Act as a filter Adhesive glycoproteins: Laminin: connects the integrins of the hemidesmosomes with the other components "other proteins" of extracellular matrix. Chondronectin: it is found in cartilage. "chondro " , "nectin means connection".The connection (adhesion) is between the cells of cartilage and ECM Osteonectin: "osteo means it is in the bone". Fibronectin: it is found in basal lamina, connection between cells and ECM. Fibers of connective tissue: 1- Collagen fibers: the main function is to give strength. 2- Elastic fibers: give elasticity "elasticity means, the ability to restore the original shape after distortion". 3- Reticular: give a network. - In any connective tissue we can find the 3 types of fibers, but the proportions of these components differ. - If the tissue is composed mainly of collagen type 1, we call it collagenous connective tissue. - If it's mainly composed of elastic fibers, we call it elastic connective tissue. - If it's mainly composed of reticular, we call it reticular connective tissue. *Collagen fibers: We have many types of collagen fibers, each type has different distribution than other types, we will mainly focus in collagen type one, which is the strongest type of collagen. - Collagen is a protein, 30% of the proteins in our body is collagen" it is the most common protein in our body" - Collagen fibers are very strong, to tear them you need hundreds of kilograms per 1 cm2, leather for example consists of Collagenous Fibers - fibers that originate from the skin of large mammals - Collagen fibers are colorless, but the bulk accumulation of these fibers appears white (for example the tendon is composed of collagen type 1, grossly the tendon appears whitish). Birefringence - With hematoxylin and eosin, collagen appears acidophilic (pink) - If we want to find the connective tissue, we look for eosinophilia, which is the collagen fibers; because the connective tissue is composed of cells and extracellular matrix (ground substance "in the section appears unstained"). - Remember: in the sections, the lightly stained nuclei are fibroblasts "active cells", while the darkly stained are fibrocytes. - We have 28 kinds of collagen; each type has different sequence of amino acids, different chains combinations, different distributions and different modifications - Collagen type 1: the strongest type of collagen, most common. Found in skin, tendons, capsule, and bone. - Collagen type 3: Reticuloendothelial system, reticular lamina of basement membrane. - Collagen type 4: basal lamina. - Collagen type 7: it connects collage type 3 to 4 in basement membrane. How the collagen is synthesized? - Fibroblast makes the extracellular matrix; DNA transcription for collagen mRNA in nucleus------ mRNA is translated in the rough endoplasmic reticulum (a glycosylation step + hydroxylation-the addition of a hydroxyl group-)------Then it gets modified in the Golgi apparatus (addition of sugar-glycosylation), then it gets exported from the cell by exocytosis, the product is called Procollagen (not the final form of collagen, pro means before). - Procollagen is made up of triple alpha helical chains + nonhelical parts on the edges - There is a certain enzyme in the extracellular matrix that cuts these edges, after cutting the edges it's called Tropocollagen. - The Tropocollagens align next to each other and above each other to form Collagen Fibril "smaller than the fiber". - Collagen Fibrils aggregate to form Collagen Fiber. - Collagen Fibers aggregate to form Collagen Bundle. - Only collagen type 1 can form collagen bundles, while collagen type 3 forms fibers (thin fibers), collagen type 2 forms fibrils, collagen type 4 in basal lamina forms sheets. - Note that each molecule of tropocollagen has head and tail, so the arrangement is (head to tail.. head to tail and so on), also there is a regular gap between two tropocollagens, so when looking at a collagen fibril under electron microscope, cross striations will appear; these striations are formed by the regular gaps between tropocollagen molecules because of the accumulation of the stain in these gaps. - The longitudinal areas of completely overlapping tropocollagens are called "overlapping areas". - The longitudinal areas where there are gaps between tropocollagens are called non-overlapping areas. - Note the non-overlapping areas appear darker and overlapping areas appear lighter (striations) - In collagen type one each third amino acid is glycine, also each helix is rich in proline and lysine. - The process of synthesizing collagen is a very long process and has many steps; any defect in any step will result in defective collagen (result in diseases). - For example, Hydroxylation step needs Vitamin C. - Our body does not synthesis vitamin C, the source of this vitamin is the diet. Clinical applications: ✓ At the surgical incision site, the site is filled with collagen fibers synthesized by fibroblasts (the fibrocytes in the connective tissue are activated due to injury (became fibroblasts)). This is called scar formation, and it’s part of the normal healing process (formation of pink line). But in certain people, there will be excessive formation of collagen "the fibroblasts form high amount of collagen”, then the incision site will appear larger in size, elevated from the surface and with very ugly appearance. This is called Keloid. Keloid is common in Africans ✓ Scurvy: deficiency in vitamin C, which is very important in the hydroxylation step. This result in defective collagen, the patient has hemorrhage (bleeding) in a lot of areas. The patient is anemic and the gums are bleeding. ✓ The third type is also caused by defective collagen; the joints are hyperflexible and hypermobile. Ehlers–Danlos syndrome Elastic fibers The main function of elastic fibers is to provide elasticity. In other words these fibers are able to restore the original shape of the tissue after distortion. (Think of an elastic fiber as a rubber band, stretched when a force is applied and relaxed (back to its original shape) when the force is released. ** Elastic tissue can deteriorate (be damaged) by three factors: 1-The elasticity of tissue decreases with age, ex: This effect is easily demonstrated by recruiting two volunteers, one youthful and one elderly. Pinch up a bit of skin on the back of each person's hand and then watch how quickly the skin returns to its original position when released 2-Sun exposure: the elastic fibers (like a rubber band) will lose their resilience and elasticity when exposed to sunlight for a long period of time. 3-Could be digested by pancreatic enzyme elastase. - Elastic fibers consist of individual microfibrils, which are embedded in an amorphous matrix. - Components of elastic fiber (2 proteins): 90% of the fiber is elastin 10% of the fiber mostly is microfibrils of fibrillin protein **These are two main characteristics of elastic fibers: 1-Elasticity: due to presence of elastin protein 2-Stability: due to the presence of fibrillin microfibrils. ** Remember that elastic fibers as well as most of ECM compounds are produced by the fibroblasts (with some exceptions according to the site). ** Elastic fibers are found in all types of connective tissues but in some tissues they are the predominant fibers and the tissue is called elastic tissue examples: 1-certain types of ligaments contain elastic fibers as the predominant fibers like the ligamentum flavum (connects the vertebrae of the vertebral column), where flavum = yellow Note: a ligament connects bone to bone and is usually (not always) made of dense collagenous connective tissue. 2-certain types of cartilages are composed mainly of elastic fibers and so they are called elastic cartilages, example epiglottis at the backof the tongue that closes the respiratory tract so that food can enter theesophagus. 3-another important location is in the wall of large arteries to ensure the recoiling of blood vessels after dilation. ** When elastic fibers are the predominant (large amount is present) they appear yellow in fresh tissue (fresh specimen) **Formation of elastic fibers Elastic fibers are produced by fibroblasts 1-what appears first are the microfibrils 2-elastin protein (characterized by being a coiled protein) deposits and accumulates over these microfibrils. Note: under EM elastin protein appears as amorphous material (with no shape). How do elastic fibers perform the elasticity function? Elastic fibers are cross-linked by a special type of link called desmosine When a certain force is applied to this elastic fiber the fiber is going to get stretched When force is released the fiber will restore to its original state. What prevents elastic fiber in the stretched state from over stretching? Collagen, there is always a certain amount of collagen fibers, even in elastic tissues. There is a minimal amount of collagen fibers which prevents overstretching or provides what is called a mechanical stop. **Can you see elastic fibers under LM? Normally elastic fibers in tissues with small amount of them cannot be visualized using H&E and we need special stains in order to view them under the microscope. H&E stain doesn’t view elastic fibers unless they are present in high concentration. 3- Reticular Fibers: ** Because of their different staining characteristics, reticular fibers were initially thought to be completely different from collagen fibers. Crossstriations with the same periodicity as in collagen fibers type 1 are visible using electron microscopy. We now know that reticular fibers consist of collagen - although the main type of tropocollagen found in reticular fibers, type III, is different from that of the collagen fibers type 1 ** Reticular fibers are very delicate and form fine networks (thin and branching) instead of thick bundles ** Location: 1- They are found mainly in the reticular lamina of the basement membrane. 2- They are found in RES organs (Reticulo- endothelium system), which consists of blood channels supported by connective tissue rich in reticular fibers. These organs (bone marrow, liver, lymph nodes and spleen) have a meshwork of reticular fibers which acts as the framework for these organs ( supportive stroma and supporting individual cells ) . ** Why it was thought to be a different type of collagen fibers? * Because it is stained positively by the "PAS" stain, it has a high amount of carbohydrates. and it is also stained positively by silver nitrate (there is a method of staining called silver impregnation which is adding silver to the tissue, because these fibers have a high amount of carbohydrates, and these carbohydrates are negatively charged, thus, they are going to interact with the silver and cause its reduction, which results in deposition of silver inside the tissue, so that the reticular fibers can be seen as black threads. All the rest of collagen fibers types contain carbohydrates but in minimal amount (about 1% only), thus, collagen fibers are considered glycoproteins, but collagen fibers type three (reticular fibers) contains much higher amount of carbohydrates (10% carbohydrates). ** The cells that are specialized in secreting reticular fibers are called reticular cells, which are modified fibroblasts. ** The function of reticular fibers – they form the stroma of certain organs (like lymph nodes and spleen) and support individual cells. They also allow the movement of cells and molecules. Ex: lymphoid tissue, like in lymph nodes, is filled with cells which are called lymphocytes. So, these fibers form spaces in which these cells “lymphocytes " can move freely within the tissue). The structures that are stained positively with silver are called Argyrophilic, so the reticular fibers are called Argyrophilic (silver loving) Remember the stages of the synthesis of collagen, firstly it consists of procollagen , then it becomes tropocollagen then collagen fibril, after that thick collagen fibers and finally they can aggregate to form bundles of collagen . - Collagen type one, two and three form fibrils. - Type one is able to form fibers and bundles of fibers. - In reticular fibers, usually they are in the form of fibrils and they can aggregate to form thin fibers. **** If we want to compare between the previous three types of fibers: Fibers of connective tissue: 1- Collagen fibers: the main function is to give strength. They appear as pinkish bundles by staining with H&E. 2- Elastic fibers: give elasticity. Usually we use special types of stains like Orcein and resorcin (elastin stain). 3- Reticular: form a network. Delicate network (meshwork) of fibers, and they provide the structure of certain organs. - Its stained positively with PAS stain as well as Silver stain. In any connective tissue we can find the 3 types of fibers, but the proportions of these components differ. If the tissue is composed mainly of collagen type 1, we call it collagenous connective tissue. If it's mainly composed of elastic fibers, we call it elastic connective tissue. If it's mainly composed of reticular, we call it reticular connective tissue. All what have been explained earlier is the components of the connective tissue. Now, we are going to consider the classification of it. *** The classification of the connective tissue, the standard classification scheme is based on composition 1- Connective Tissue Proper o o Loose Connective Tissue Dense Connective Tissue 1) Dense Irregular Connective Tissue 2) Dense Regular Connective Tissue 2- Special Connective Tissue (has similar characteristics of the connective tissue) Blood (cells and extracellular matrix with no fibers and stabilizing macromolecules like gags, that’s why it's fluid), bone (cells and mineralized extracellular matrix, that’s why it's hard), cartilage, reticular tissue, elastic tissue, adipose tissue. Note: when we refer to one type of special connective tissue, we refer to it by its name. 3- Embryonic connective tissue Connective tissue proper This is the type that we talked about in the previous lectures. - It contains the components that were mentioned (fibers, cells, ground substance), with different proportions. - When we say connective tissue we mean connective tissue proper. *if the main component of tissue is collagen fibers then it is called dense collagenous connective tissue *if the main component is cells or ground substance (or equal proportions) then it is called loose connective tissue Loose connective tissue Cells and/or ground substances predominate, but still there are some fibers (thin or delicate fibers) Function: 1. Diffusion (medium for transport) because it has a lot of spaces filled with ground substance, Found around blood vessels, directly under epithelium layer, highly vascular (high amount of capillaries in order for the molecules to pass from the blood vessels to the epithelium and backward. 2. Second line of defense below basement membrane of epithelial cells because it has inflammatory cells (ex macrophages) 3. Fills spaces inside any organ ** Found in lamina propria of mucus membrane below epithelium (special name for the loose connective tissue of mucus membranes=mucosa) ** It is a flexible structure, because it has high amount of ground substance, high amount of water. The extracellular matrix is gel like. ** Not resistant to stress because it has low amount of collagen, flexible but not very strong (the function is diffusion and defense). ** It has more regenerative power than dense connective tissue because it has more cells and more vascularized 2-dense irregular connective tissue (collagenous) *mostly composed of collagen fibers *collagen fibers are arranged haphazardly (randomly) Collagen fibers add strength to the connective tissue so it's very resistant to compressional forces and tensile forces *provides powerful resistance in many directions (according to the orientation of the fibers) *location: 1- Deeper layer of dermis of skin: below the loose connective tissue layer (exposed to many forces in different directions) First layer (superficial) of connective tissue is loose, the next layer (deeper) is dense irregular connective tissue (to give strength for loose and epithelium) Epithelium of the skin is epidermis, and the connective tissue of the skin is dermis 2- Organ capsules, capsules that surround any organ are whitish because they have high amount of collagen but the orientation of the collagen fibers is irregular. 3- submucosa (mucosa=mucous membrane= epithelium and thin layer of loose connective tissue (lamina propria)) Dense regular connective tissue (collagenous) *fibers arranged in bundles (oriented in certain direction) *provides resistance to stress in one direction *fewer cells and ground substance and most of the cells found are fibroblasts not inflammatory cells *ex; Ligaments: connect bone to bone, usually are collagenous (whitish in color) dense regular type of connective tissue except certain types of ligaments (elastic/yellowish in color). Tendons: connect muscle to bone. Aponeurosis: flat tendon (sheet like) instead of cord like structure -connective tissue has variable regeneration power according to the type; loose has a higher regeneration power than dense connective tissue because it has higher amount of cells and capillaries in comparison with dense connective tissue (fibers and little amount of cells), so if a tendon gets injured usually the regeneration is poor because it's less vascularized, and repair of damaged tendons is very slow Reticular tissue • consists of reticular cells (modified fibroblasts) and a network of reticular fibers formed by them • forms the structural framework in which the cells of the organ are suspended • in the liver, bone marrow, lymph nodes and the spleen Embryonic connective tissue: The connective tissue that presents in embryo. ✓ Embryonic connective tissue: It contains star or spindle shaped mesenchymal cell (stem cells), gel- like ground substance with fine fibers (more ground substance than fibers). If you go back to embryo you will see three germinal layers; ectoderm, mesoderm and endoderm ✓ The mesoderm differentiates into a tissue called mesenchyme or embryonic connective tissue ✓ Mesenchyme forms the filling material of the early embryo and it is the common origin of all other connective tissue types ✓ With fetal development, mesenchyme forms the connective tissue between and within the developing tissues and organs -Remember that epithelium is derived from the three germinal layers which is a characteristic for it, while connective tissue is derived from mesenchyme (or mesoderm). -The mesenchymal cells of this tissue (mesenchyme) can form bone, blood or connective tissue proper …….etc. So different types of connective tissue cells are produced by the mesenchymal cells. Mesenchymal tissue or embryonic tissue is a tissue found only in embryo while after birth we can’t find mesenchymal tissue because all mesenchymal tissues have differentiated into different types of connective tissue. EXCEPT in two places; in the pulp of the tooth (be careful we talk now about a tissue not individual stem cells) and the umbilical cord - Mesencymal cells can differentiate into osteoblasts which form the bone or fibroblasts which form the connective tissue or adipoblasts which form the adipose tissue or………etc. So these cells are stem cells which can differentiate into many types of connective tissue cells (pluripotent). - Stem cells mean undifferentiated cells that can become a variety of cells - The extra cellular matrix of mesenchymal c.t contains more ground substance than fibers (gel like structure because it has large amount of hyaluronic acid, chondriotin sulfate (sulfated GAG) and water) - Mesenchymal stem cells are characterized by having a little amount of cytoplasm with fine processes and large oval or rounded nuclei. - The mesenchymal c.t of the umbilical cord is called also mucoid connective tissue (also called Wharton’s jelly ), because its ground substance is mucous like, it’s made up of mucopolysaccharides (hyaluronic acid and chondroitin sulfate) - Mucoid connective tissue forms a compliant cushion around the vessels of the umbilical cord Umbilical cord banking simply involves collecting blood left in newborn's umbilical cord and storing it for future medical use (cord blood and/or tissue) (regenerative medicine).