Transcribed by Janki Gajera!

January 16, 2014

Connective Tissue 2-3

Slide 1 - Composition of connective tissues Dr. Xin Li - So from the introduction lecture, the first connective tissue lecture, we learned connective tissue in general - the definition, and we also discussed several different types of connective tissue - we classified embryonic connective tissue, connective tissue proper, and each subtypes. We also learned the most unique feature of connective tissue is its composition. Unlike other types of tissue we learned about - like epithelial tissue is made of epithelial cells, there are no other major components in epithelial tissue. Unlike connective tissue, which has three major components: one is cells, the other is fiber. connective tissue always has high fiber. Also, ground substance is in connective tissue. (She repeats this again). We call ground substance and fibers extracellular matrix. Slide 2 - Composition of Connective Tissue Dr. Xin Li - Today we will focus on the composition of connective tissue. So first we will start to learn what type of cells can be found in connective tissue. So cells are fixed and wandering in connective tissue. The fibers, there are several types of fibers. Basically, two categories: collagen-based or elastic-based. Ground substance: these are macromolecules, these glycosaminoglycans, proteoglycans, and adhesion proteins make up ground substance. Some textbooks mention tissue fluid - we dont really consider this a major component of connective tissue, but we will also discuss it a little bit in our last lecture. I want you to keep in mind 3 components of connective tissue: cells, fibers, ground substance. I repeated this a couple of times, so I hope you can appreciate this very important concept you need to know about connective tissue. Very basic. This is the basics, we will learn in detail: cells, fibers, and ground substance. Slide 3 Dr. Xin Li - So lets use this connective tissue as an example: We can see many types of cells different shapes. They look different. Here we can see this is a fat cell - an adipocyte. This is a macrophage. This is a fibroblast - weve already heard of it. It is a very common connective tissue cell. And this is a plasma cell. We will also learn today this is an antibody-producing cell. This is a mast cell, also involved in producing immune response. And this is unique - it is a pericyte. As you can see, this a blood vessel or capillary or small blood vessel. These cells cover endothelial cells - these are called pericytes. Here we can see fibers also. So this thin one is elastic fibers, and this thicker we will later learn is many collagens making a collagen bundle. We will see it is obvious under a light microscope. Slide 4 - Connective tissue components: Cells

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January 16, 2014

Dr. Xin Li - So, we will start with different types of cells found in connective tissue. And all of these cells can be classified into three categories: fixed cells, which include fibroblasts, adipocytes, pericytes which cover endothelial cells under vessels, and mast cells; the second category is something between fixed and transient - only one called macrophage. The third type of cell is mostly blood cells - leukocytes. We will leave the blood system, which is a specialized connective tissue, to another faculty member. She will cover blood. We will only learn about plasma cells under transient cells today. Slide 5 - CONNECTIVE TISSUE CELL LINEAGE 1 Dr. Xin Li - This slide summarizes some of the connective tissue cells. We already know connective tissue originated from mesoderm embryonically. So, the middle layer of the embryo. It is all derived from mesenchymal stem cells. These mesenchymal stem cells can give rise to all types of connective tissue cells, including chondroblasts and chondrocytes, which make cartilage. Mesenchymal stem cells also give rise to adipocytes and fibroblasts, and some endothelial cells. We already learned endothelial cells is a type of epithelial cell making up blood vessel. Mesenchymal cells can also become osteoblasts, which make bone. Later on, osteoblasts can also become osteocytes. So here we will talk about adipocytes and fibroblasts. Pericyte is not shown here [in the picture]. Slide 6 - CONNECTIVE TISSUE CELL LINEAGE 1 We highlighted the only cells we talk about. The only cell not shown is the pericyte. Here is a blood capillary, and the epithelial layer is simple squamous epithelia. The outside is covered by pericytes. Slide 7 - CONNECTIVE TISSUE CELL LINEAGE 2 Dr. Xin Li - So there is another group of cells that arise from hematopoetic cells. The exact origin of hematopoetic cells is not known, but it is believed they share same progenitor with mesenchymal. This is also from mesoderm, the middle layer in the embryo. These types of cells can give rise to all blood cells within our connective tissue such as plasma cells and macrophages, as well as mast cells. These are the three types of cells derived from hematopoetic cells. We will leave the rest to blood system lectures. Slide 8 - Morphology and function Dr. Xin Li - In the next part of this lecture, we will talk about 6 types of connective tissue cells that we mentioned earlier, and I will emphasize what they look like - morphology, and their function - what they do. Slide 9 - A-1: FIBROBLASTS

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January 16, 2014

Dr. Xin Li - First, lets start with fibroblasts. It is very common in connective tissue. It has an oval nucleus shape. This is an example of a fibroblast with H&E staining. Slide 10 - Fibrocyte Quiescent Fibroblast Dr. Xin Li - This is also a fibroblast, but this is quiescent fibroblasts - called fibrocytes. They are more flat, indicating they are not as active as fibroblasts. We will talk later to compare these two types of fibroblasts. Slide 11 - FIBROBLASTS Dr. Xin Li - This is electronic microscopy detailed structure showing fibroblast. You can see partially the nucleus and many mitochondria. This is RER. Being rich in cellular organelles suggest these cells are active and protein-producing. Here is showing collagen. The major function of fibroblasts is to secrete extracellular matrix fibers. Slide 12 - FIBROBLASTS Dr. Xin Li - Another electronic microscopy showing collagen fibers secreted by fibroblasts. Again you see the mitochondria, the ER, the many collagen fibrils. Later on, they make collagen fibers. Slide 13 - FIBROBLASTS Dr. Xin Li - This is another picture showing RER. This ER has ribosomes for protein translation and later on processing. Also some lipid droplet can be seen in the fibroblasts. Again, there are so many mitochondria. Slide 14 - Fibroblast Function Dr. Xin Li - This slide summarizes function of fibroblasts. First one is to synthesize extracellular matrix: this includes collagen, elastin, and ground substance components: It also can synthesize growth factors to influence growth and differentiation of other cells either adjacent to it or itself in an autocrine or pericrine fashion. You need to memorize the function of synthesizing extracellular matrix and growth factors for cell growth and differentiation. Slide 15 - FIBROBLASTS VS FIBROCYTES Dr. Xin Li - Lets compare fibroblasts versus fibrocytes. Fibrocytes are more active, indicated by bigger nucleus and many cellular organelles, like mitochondria and RER. This is based on the section you cut - cross-section is different view. Fibrocyte is more flat, no matter which sectional cut. You will have much less cellular organelles.

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January 16, 2014

Slide 16 - Fibroblast vs Fibrocyte Dr. Xin Li - Fibroblasts are very active to synthesize extracellular matrix and growth factors. Abundant with branched cytoplasm, which are irregular. They have large, ovoid pale-staining nucleus. We often mention pale- or dark-stain nucleus. When a nucleus is dark-staining it is in a less active status chromosomes are condensed all together. We know chromatin needs to spread out for transcription. So if it is condensed, it means the nucleus is less active. Fibroblasts have well-developed RER and Golgi. Slide 17 - A-2: Adipocyte Dr. Xin Li - Next cell we already mentioned is adipocytes. As we know, fat tissue is one of the connective tissues we discussed last time. Fat tissue has very few extracellular matrix mainly made by adipocytes. So the major function is to store lipids, as energy supply later when the body needs it. It is padding protection, and shock absorber. It provides insulation and generates heat. We mentioned brown fat especially brown fat will help to maintain body temperature. Fat tissue has endocrine functions they made adipokines which have the important function to regulate the whole body metabolism. Slide 18 - DEVELOPMENT OF FAT CELLS Dr. Xin Li - Here we already know adipocytes are derived from mesenchymal cells and fibroblasts. When mesenchymal stem cells become adipocytes, it goes thru lipoblasts - a precursor of adipocytes. After lipoblasts the cell can become multilocular adipocyte and unilocular adipocyte. One is brown fat unilocular adipocyte makes white fat tissue. My lab does mesenchymal cell cultures to make bone and adipocytes. On the top right is a picture: cell under microscope in petri dish - live cells in culture. Mesenchymal stem cells differentiated into adipocytes - we use special staining to stain cells - oil red. Oil drops into adipocyte into red. Slide 19 - Lipid Transport [Adipocyte Capillary]

So we know adipocytes store fat as energy supply. So this picture explains from digestion from food intake we take in fat, this will go to our blood through cell cycle and reach the capillaries. And then the fat/lipid will become lipoproteins. This will come out and be processed and stored in the lipid droplets inside the fat cells. Then when the body needs it, there is a fat burning process, so these lipid droplets can be released from the adipocytes and cleaved and processed by the enzyme to make glycerol and free fatty acids. And later on, it also can become glucose to supply energy. You already learned this in BBL. Just remember food goes thru capillaries and fat cells store lipids. There is a cycle back-and-forth of storing lipoproteins to fat cells to store lipids and lipids can also be released from fat cells to supply energy. Slide 20 - Unilocular (White adipose cell)

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January 16, 2014

Dr. Xin Li - So unilocular adipocytes we already know are white adipose tissue. It is very common and very obvious to observe - it is one big lipid droplet. The overall shape of the unilocular cell is polyhedral shaped - 50-150 um - a pretty big cell. This is because one big droplet can push other cells aside to make a signet ring cell. When we do histology, we dissolve lipids in routine histology. When we do culture and stain, we use oil red stain to make lipid red. In histology, this lipid droplet is showing an empty space. So this lipid droplet is pushed aside the cells. Slide 21 - Multilocular (Brown adipose cell) Dr. Xin Li - Multilocular brown adipose cells have many lipid droplets. Because it produces heat, it also has many mitochondria. We call brown adipocyte tissue brown because blood vessels and mitochondria with cytochromes make this brown color. The overall shape is polygonal - it is smaller than unilocular adipose cells. We already know it produces heat - it maintains newborn body temperature. Slide 22 - MULTILOCULAR ADIPOSE CELL Dr. Xin Li - This is a picture showing brown adipose cell. We can see many mitochondria and small lipid droplets inside the cell. Slide 23 - A-3: Pericyte Dr. Xin Li - Third type of cells we will talk about is pericytes. Pericytes - I already mentioned cover the outside of the capillary right next to endothelial cells. Here is a picture of a capillary and we can see squamous epithelia endothelia. It is a single layer - simple epithelia - to make the inside of the capillary the most open to RBC. E = endothelial cells. The cytoplasm makes the connection. Here is the pericyte (P), outside the capillary. The location of the pericyte is surrounding endothelial cells - it is a rounded cell that wraps around blood vessels. The feature of pericytes is that it has characteristics of both endothelial cells and smooth muscle cells. It is participating in contractile movements - we know from myoepithelia - when we say a cell has muscle cell features - we are mostly talking about the contractile function of muscle cells. Pericytes also have contractile function involved in blood flow. There are many studies on pericytes about its function, there is no doubt that it has very important physiological function. It is not clear though. As far as we know now, we know pericytes participate in blood-brain barrier - it is important; once it is damaged, it brings Alzheimers disease and other neurodegenerative diseases. Memorize the blood-brain barrier role. It has characteristics of endothelium, so it is not surprising that it has a role in angiogenesis. It helps in the survival of endothelia. Itself it is multi potential. It can become other types of cells. It is stem-cell like. It plays an important function in wound healing. When there is a need, pericytes can give rise to other types of connective tissue cells.

Transcribed by Janki Gajera!

January 16, 2014

Slide 24 - Pericyte Dr. Xin Li - Electron microscopy showing epithelial cells. This narrow part is the cytoplasm and this red outline is the pericyte. in the bottom panel, you can see better 3D colored structure showing pericyte covering two capillaries. It kind of looks like myoepithelia that we learned in salivary glands. It is contractile. It covers the capillary. Slide 25 - A-4: MAST CELLS Dr. Xin Li - So far we learned halfway to our goal about the 6 types of connective tissue cells. next is mast cells. This is an illustration of mast cells. It has an oval to round shape, the nucleus itself. The picture shows IgE on the surface - it is an immune cell participating in immune response. The unique characteristic of this cell is that there are many granules called metachromatic secretory granules. Here is a long list that all the products can be released or stored in the granules. Slide 26 - MAST CELLS Dr. Xin Li - So here shows this nucleus and the mast cell has the whole cytoplasm filled with granules. Slide 27 - MAST CELLS Dr. Xin Li - Here is another one showing connective tissue and collagen fibers. Again the mast cell = MC. The bottom panel shows sub-epithelial connective tissue. Here the simple columnar epithelia from intestinal-section digestive tract. There is a basement membrane, which defines epithelia and underneath sub epithelia connective tissue. Here we see mast cells. Slide 28 - MAST CELLS Dr. Xin Li - These are plasma cells. This is the liver, showing you Kupffer cells. Well just skip this. [Revisited from slide 42] KC = Kupffer cell. This is a liver section. In the liver, macrophages are called Kupffer cells. [bottom right picture] is a bigger image showing this. This is N = nuclear because of the phagocytosis. You can see the dye inside of the Kupffer cell. Slide 29 - MAST CELLS Again, this is showing mast cells. So i want emphasize about the single nucleus and of course the very obvious granules. This is how you identify mast cells. This is very unique - the cytoplasm filled with these granules. Slide 30 - MAST CELL

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January 16, 2014

So this is the electron microscopy of mast cells, and it is showing the nucleus. And again, all these dark stains - these are granules that filled up the cytoplasm of the mast cell. Slide 31 - MAST CELL Dr. Xin Li - So mast cells has an important function - to mediate the immune response. First one, there is the antigen. We already know the mast cells on the outside surface are IgE. So these can recognize the antigen. So once IgE binds with the antigen, sequentially inside the cellular system being activated - antigen binds to the IgE receptor, you activate adenylate cyclase activation, protein kinase, then phosphorylation these are all the cellular signaling languages going on, the releasing of calcium of phosphatase. Eventually, they will secrete luekotrines (we will discuss later), all these mast cells fill up with granules. These granules can fuse. They fuse, then release whatever stored, including histamine outside the mast cell, to call other cells to help to tackle this antigen. So this is antigen-induced mast cell response. Slide 32 - Inflammatory response Dr. Xin Li - Here is showing the inflammatory response of the sequence of events happening in mast cells in response to inflammation signal. So first, mast cells release histamine. Histamine is a vasodilator, it can increase vascular permeability. Then other components will leak out of blood vessels to participate and help. And mast cells then release ECF - eusinophil chemotactic factor to call BC from the vessel, which is already leaky to tackle down parasites. Meanwhile, neutrophil chemotactic factor is also being released so it can take care of any microorganism like bacteria. If there is an infection, neutrophiles will be recruited to the site to care of the microorganism. Mast cells also release leukotrines to further increase vascular permeability. Leukotrines are much more potent - 1000 times more potent - than histamine, and it is also called bronchospasm. Another factor released is prostaglandin D. Some are released from these granules, and some are secreted directly. This causes further bronchospasms and increases secretion of mucous. Mast cells also release this platelet activating factor - it increases vascular permeability. Also, thromboxane A2 also targets platelets. It is called platelet-aggregating [mediator] and vasoconstriction. So this is the end of the inflammatory response. So first, you have histamine-induced vasodilations. The blood vessel leaks - all the BC are called to the site to fight and then further, if needed, the BC cause further permeability and then the platelets start being recruited - then vasoconstriction, so it goes back to normal. And the last - bradykinin - is a powerful vascular dilator and its also responsible for pain. So these are the sequential events that happen when there is an inflammatory response mediated by mast cells. Slide 33 - Mast Cells Factors and Function

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January 16, 2014

Dr. Xin Li - So this is another way to summarize mast cells - the factors being released from mast cells and their function. This is largely overlapped for this seen here [refers to previous slide of list], I know this looks very overwhelming, but I think is important to know, to memorize the major functions, the basic functions of mast cells. And this table will help you to further understand the factors released by mast cells and their actions. Slide 34 - Mast Cells Factors and Function Dr. Xin Li - Also, this tells you whether these factors are from granules stored in the mast cells, or membrane lipids synthesizing and releasing directly. And secondary, so upon other factors releasing, such as this factor with platelets, activating factors are released. Again this table is not required, but it helps with memorizing information on these slides. [scrolls thru 33, 34, 35] These are important slides. Slide 35 - Summary of the Action of Mast Cell Mediators in Immediate (Type I) Hypersensitivity Dr. Xin Li - So another one simplified to summarize mast cells mediator in type 1 sensitivity. So we have histamine, PAF, leukotrienes C4, D4, E4, neutral proteases, and prostaglandin D2 are all vasodilators, they have similar functions, some come first like histamine, some are more potent like leukotrines. but they have the same function - vasodilation - to increase vascular permeability. And this group of factors [smooth muscle spasm] have the function to cause smooth muscle spasm. And this factor - you can see there is overlap again - this has the function of vasodilation and smooth muscle spasm. And the next group of factors - cellular infiltration. So this is the most participating in the direct target - the eosinophil and neutrophil chemotactic factors. Slide 36 - B-1: Macrophage Dr. Xin Li - [break] Lets continue - come back to lecture :) So we will continue lecture on the cells in connective tissue. The one we will talk about next is macrophage. Macrophages are phagocytic cells - so it can engulf and eat. We will explain that process later on. That is the main function of macrophages. It is an antigen-presenting cell once it eats the foreign particles/materials, it will process and then present to other immune cells to respond. So phagocytic and antigen-presenting - these two processes are closely linked. Macrophages have a long life-span. We already said these are the only cells that are in between fixed and transient - it is fixed and wandering, both. It is mononuclear and has this phagocytic system.

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January 16, 2014

Slide 37 - MACROPHAGES Dr. Xin Li - Macrophages present in our body in different subtype - these are all macrophages but based on their location, they have a different name. If the macrophage is in blood, we call it monocyte. This is the pre-cursor of macrophages. The macrophage name was given to these types of cells in connective tissue in lymphoid organs and lungs. This summarizes the main function - produce cytokines, chemotactic factors and other factors that participate in inflammation, antigen presentation is a main function of macrophages. When macrophages are located in liver cells, we call them Kupffer cells - their main function is the same as macrophages. We will not discuss neurons, NT, or skin. But you need to know them by name i.e. that it is in skin and the name we give to the macrophage. Osteoclasts - I believe you will learn later in the bone lecture. Another unique type of macrophage is called the multinuclear giant cell - this is several macrophages fused together to make really huge, giant cells, and the function is digestion and segregation of foreign bodies. So macrophages in connective tissue and Kupffer cells, and multinuclear giant cells, we will see in the next slides. Slide 38 - Phagocytosis Dr. Xin Li - So first lets learn about phagocytosis, this process is the main function of macrophages. As I said, when there are foreign bodies - particles that are considered foreign to our body. So first, macrophage will recognize it through this different receptor - if it is a microbe, a bacteria. So for example, this mannose receptor will recognize the particle. Then the phagocyte membrane will zip up around the microbe during the engulfment. So the membrane will start to engulf, and try to cover this microbe. And this cytoplasm will fuse, ingest this microbe inside to the macrophage. Already inside the cell, we know there is a system called the lysosome. This lysosome full of enzymes will recruit to this phagosome and then these two membranes will fuse, and these enzymes from the lysosome will target on the microbes and to kill it. You dont need to know the [ROI/NO box]. You will need to know this: first, recognizing and then engulfment, ingest into the macrophage and the lysosome comes to fuse with the phagosome, and then the enzymes kill the microbe. This process is called phagocytosis. Slide 39 - Opsonization Dr. Xin Li - So there is another term we will learn, its very important to phagocytosis, it is called opsonization. This is a process during phagocytes when the microorganism is covered by antibodies or its antigens bind complement. So these help the macrophage to recognize this microorganism. Slide 40 - Macrophage

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January 16, 2014

Dr. Xin Li - As we know, the basic feature of macrophages is recognizing and ingesting foreign bodies. Here we use India ink to treat [the tissue], and we can see the macrophage. The ink went inside of the macrophage. So this how we can recognize the macrophage - this phagocytes process is going on in the macrophage. Slide 41 - Macrophage Dr. Xin Li - This is another type of dye - toluidine blue can also stain macrophages. Slide 42 - Macrophage: GIANT CELL Dr. Xin Li - This is showing the macrophage fused together to make a giant cell. This is a section from rat skin. Here the indicator shows the giant cells. And this blow up shows it better - high resolution, showing this giant cell. This is a unique macrophage. I mentioned that we should also see Kupffer cells. [Jumps to slide 28] Slide 43 - C-1: Plasma Cell Dr. Xin Li - The last type of cell we will learn today is called the plasma cell. So the plasma cell in the introduction I already mentioned is an antibody-producing cell. It arises from B lymphocytes. It is kind of a B lymphocyte in connective tissue. We know B lymphocytes are in blood, so plasma cells have the same origin as B lymphocytes, except that it is in connective tissue. The shape is ovoid. It is basophilic. It has a well-developed RER - just think: this is an antibody-producing cell. Antibody is majority protein. So that means this type of cell needs to actively produce protein, so no doubt that it has a very well-developed RER. Another unique feature to help you recognize these cells is the juxtanuclear Golgi - next to the Golgi is an apparatus and [the apparatus] is next to the nucleus. The function, we already know, is to secrete antibodies. Slide 44 - Plasma Cell Dr. Xin Li - This is a bigger illustration showing the plasma cell. We can see the Golgi next to the nucleus, the very well developed RER, and this nucleus. Inside of the nucleus, we can see this dark stain. It looks like the nucleus in the middle and this is kind of like a clock-face. This is typical presentation of the plasma cells, under the microscope. We call this clock-face because the clumps of heterochromatin look dark. Slide 45 - Plasma Cell Dr. Xin Li - Here is the nucleus, and this is the chromatin. And here the juxtanuclear Golgi, next to the nucleus. So from Cellular Organelles, or another course, you should know cellular organelles positions - usually, Golgi is not right next to the nucleus. So, usually you will see nucleus next to mitochondria and ER, RER and then Golgi. But in this, you see the Golgi is right

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next to the nucleus. So this clock-face and this junta nuclear Golgi are two features that can help you recognize plasma cells. Slide 46 - Plasma Cell Dr. Xin Li - So this light microscope sees - again, this is not perfect, you are missing some here in the middle you have a dark stain and it makes a circle with a clock-face. Slide 47 - FUNCTIONS OF CONNECTIVE TISSUE CELLS Dr. Xin Li - So far we already discussed all six types of connective tissue with cells. This table summarizes the basic functions of these cells. I highlighted the cells we discussed here fibroblasts, plasma cells, macrophage, mast cells, adipocytes, and pericytes. So where they are located, presentation/activity, and their main function {know this} Slide 48 - CELLS IN AREOLAR CT 1 Dr. Xin Li - Another table to summarize the function of these cells - I highlighted the cells we discussed here. How the cells look like - the shape - cellular organelles like the Golgi and RER, and cytoplasm, and their main function. Fibroblasts, adipocytes, macrophage, mast cells, and plasma cells. Slide 49 - CELLS IN AREOLAR CT 2

Slide 50 - Composition of Connective Tissue Dr. Xin Li -[clicker: You cut into your hand and in the inflammatory response neutrophils arrive and function in killing bacteria. (Which cell?) _____ then arrive and function in producing collagen and ground substances. A fibrocytes B mast cells C fibroblasts D macrophages] The neutrophils indicate there could be bacteria. The answer is fibroblasts because that is the major function of fibroblasts - to produce extracellular matrix. So fibers and ground substance are mainly made by fibroblasts. It is not fibrocytes because those are inactive cells of fibroblasts. Mast cells call neutrophils in the earlier response. Macrophages we know are antigen-presenting and phagocytic cells to ingest antigens and later present to other immune cells. So fibroblasts made the collagen fiber and ground substance to repair.

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January 16, 2014

So we already covered one of the three main components of connective tissue cells. We discussed in detail six types of cells. Next, we discussed extracellular matrix. We will start with fibers. Slide 51 Dr. Xin Li - Again, I show this slide which shows thicker collagen fibers and elastic fibers in loose connective tissue. Slide 52 - CT Fibers Dr. Xin Li - So fibers, connective tissue fibers, normally we say are collagen-based or elastic fiber-based. But still sometimes you can see reticular fiber. We already learned about connective tissue proper, we called it connective tissue because their fibers are reticular. Reticular fibers are also a type of collagen, so you can say there are three types of fibers: collagen, reticular, elastic. Or you can say there are two types of fibers: collagen and elastic. Both are correct. Slide 53 - Over 25 types of Collagens Dr. Xin Li - So collagen fibers - we have more than 25 types. So we will only discuss these 7 types - the most important to connective tissue. Type 1 is very common type of collagen fund in many organs - dermis, bone, capsules of organs, fibrocartilage, dentin, and cementum. I hope you remember dentin and cementum from the first connective tissue lecture, the structure of the tooth section. We showed dentin and cementum. They are a specialized type of connective tissue and are majorly made of type 1 collagens. Many Type 2 collagens are found in elastic cartilage. And Type 3 - reticular fibers. We dont call Type 3 collagen fibers, we call it reticular fibers, these are the main components of reticular connective tissue. Type 4, you should already have an impression of this because we first mentioned this in our epithelial tissue lecture. This is the main component of basal lamina. Lamina densa of the basal lamina is Type 4. So Type 4 collagen network is incorporation of basal lamina. It is also important in components of basal lamina. Type 5 is found in the placenta. Type 7, I hope you remember, is anchoring fibers. Later we will talk about a mutation Type 7 collagen, causing a severe disease, a very miserable disease caused by Type 7 collagen. Slide 54 - COLLAGEN Dr. Xin Li - The structure of collagen, you heard me say many times about collagen bundles they are obvious under light microscope, and I mentioned they are obviously thicker and look different compared to elastic fibers. collagen bundles are made of smaller fibrils - smaller fibrils are aggregates of tropocollagen molecules. When these collagen molecules assemble, they have overlap. Overlap means there is always connection. As illustrated here, each line

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indicates tropocollagen molecules. Here you can see there is overlap with the other one. A lot of overlap. Some are missing - there is a gap. This is how collagen fibers are assembled. Because of this, it presents a unique feature under staining. I will let you think about what dark and light indicates. Hydroxyproline residues of ! chains are important to maintain stability of tropocollagen molecules. This is very important, keep in mind. Slide 55 - COLLAGEN Dr. Xin Li - This is another way to see from the beginning - of tropocollagen molecules and how they are assembled with the gap and there is overlap. Collagen ends up having some regions that are darker, some are lighter. Slide 56 - Collagen fibrils Dr. Xin Li - This staining shows collagen fibrils staining - you can see some areas are darker, some are lighter. So can you tell me whether its the gap area thats darker or overlap? Gap is darker because the dye can fill in the gap area. So gap area is where you have less fiber, less tropocollagen molecules, but under staining you see it darker because the dye can fill in that space. Light staining in the region with no gap. Slide 57 - RETICULAR FIBERS Dr. Xin Li - Type 3 collagen is reticular fibers. it is collagen but it is thinner and has more carbohydrates than Type 1. Usually, reticular fibers are not visible under normal histology preparation or under the light microscope, so you need to use a special stain, such as a silver stain to visualize reticular fibers. These reticular fibers are produced by reticular cells - it is easy to understand this. But what is a reticular cell? It is actually a fibroblast, but it is producing reticular fibers. Again - fibers are produced by fibroblasts, and reticular fibers are produced by reticular cells, which is also a type of fibroblast. A function of reticular fibers is not like Type 1 collagen where it is assembled very regularly. Many reticular fibers produce a scaffolding network. Slide 58 - RETICULAR FIBERS Dr. Xin Li - We will see from here these dark-staining fibers. These are reticular fibers. This looks like a network if you look in 3D, like a scaffolding. This is reticular tissue commonly seen in the adrenal cortex. Slide 59 - RETICULAR CT Dr. Xin Li - This is another example showing reticular connective tissue in lymphatic tissue. The staining is silver - these are reticular fibers. Slide 60 - Reticular fibers- lymphoid tissue

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Dr. Xin Li - Another lymphoid tissue presents reticular fibers. Slide 61 - Lymph node- medullary sinus Dr. Xin Li - You may remember reticular connective tissue found in the lymphoid system, the lymph node. As I mentioned before, reticular fibers are not visible without special staining so we can see reticular fibers here. But we can see reticular cells, a fibroblast. Slide 62 Dr. Xin Li - This is not required, because from slide 61, you don't know what the lymph node looks like, so this section [slide 61] is from inside the medullary sinus. Slide 63 - Spleen- Reticular Fibers Dr. Xin Li - The spleen is also showing reticular fibers. It has reticular connective tissue, and its major constituent is reticular fiber in splenic cords in the red pulp of the spleen. This is showing blood vessels, WP = white pulp and RP = red pulp. [Left picture] is not showing reticular fibers, but they can be seen in the right picture of the red pulp of the spleen. Slide 64 Dr. Xin Li - Again, this is not required. It is just because I mentioned red/white pulp of the spleen. This will just help you to locate where this structure is. Slide 65 - collagen fibers in a muscular artery Dr. Xin Li - This is a muscular artery with collagen fibers. We can see the collagen fibers show two different colors. So reticular, which is Type 3, showing purple. Tunica media is a very muscular layer. This tunica adventitia is mainly Type 1 collagen, it is a red color. It is stained with picrosirius and observed with polarization optics. It is a very pretty-staining and it shows both types of collagens. It is a unique, different color. underneath is adipose tissue. Slide 66 - Elastic Fibers Dr. Xin Li - We already talked about collagen fibers and Type 3 fibers which are reticular fibers. The last fibers are elastic fibers. Elastic - as indicated by the name - can stretch up to 150 times its length. The fiber is composed of an inner part and outer part. The inner is core of elastin, the main protein, is the core component of the elastin fiber. This is not collagen, it is a protein called elastin. The outer layer is covered by a glycoprotein: microfibrils of fibrillin - this is the composition of elastic fibers. Looks very different from collagen. Slide 67 - Collagen and Elastic Fibers Dr. Xin Li - This is one section, you can see elastic fiber - very dark stain but thin fibers. And collagen fibers - pink-stained and thicker.

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January 16, 2014

Slide 68 - COLLAGENOUS & ELASTIC FIBERS Dr. Xin Li - Higher magnification - you can see darker stain elastic fiber and lighter-stained collagen fibers. Slide 69 - Medium Artery: Elastic Fibers Dr. Xin Li - Medium artery showing elastic fibers. Sometimes vessels need to dilate and come back to its original size, so elastic fibers required to help function of stretching and returning to normal size. It is kind of like a spring, this curvy elastic fiber. Elastic is always dark and fine stained under microscope.

[clicker: This is oral mucosa showing the junction between normal and dysplesia. The yellow arrow points to: A. macrophage B. Type 4 collagen C. Type 1 collagen D. fibroblast] blood vessel You can see there is a transition - a junction - between normal and dysplesia. But thats not the key part. The key part is this is oral mucosa. Stratified squamous epithelia. You should know now beneath is connective tissue. The [yellow arrow] is indicating not macrophage because the arrow is not pointing to a cell. It is a type of collagen. It is connecting to many epithelia, so it is Type 1 collagen. If I put the arrow at the basal lamina [white arrow], what collagen is here? Type 4 collagen. Slide 70 - Ground Substance Dr. Xin Li - We are done with fibers - lets move on to the last components we will discuss today about connective tissue: ground substance. It is a complex of macromolecules, fills space between fibers and cells, and organizes tissue topography, supports cell migration, orients cells, induces cell behavior, binds growth factors and participates in cellular signaling. Also serves as cytokines reservoir - cytokines stay there. When needed, cytokines are released. There are 3 types of compounds in ground substance: glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins - adhesion proteins. Slide 71 - Glycosaminoglycan (GAGs) (Acid mucopolysaccharides)

Transcribed by Janki Gajera!

January 16, 2014

Dr. Xin Li - GAG has other names, called acid mucopolysaccharides. I dont know how much you learned from BBL, but this is a repeating structure of hexozamine and uronic acid. We will see on the next slide. It can bind cations. It is a part of proteoglycan. Osmotic pressure leads to increased water content - causes firmness and flexibility. Hyaluronic acid is the most ubiquitous GAG - it is the most common one. Slide 72 Dr. Xin Li - This is the structure of the GAG - the top shows the repeating disaccharide unit. Bottom shows GAG together with core protein to make proteoglycan - overall a brush-like structure. Slide 73 - PROTEOGLYCANS Simply, really like a brush used in a lab. Protein core with GAG together is a proteoglycan. Slide 74 - Multiadhesive glycoproteins Dr. Xin Li - Third type is multi-adhesive glycoproteins - it has a structure different from proteoglycan - it is a globular protein with carbohydrates. It is two types: cell-cell and cellsubstrate. Majorly made up of fibronectin, which is synthesized by fibroblasts and epithelial cells. Remember fibroblasts synthesize extracellular matrix, and fibronectin is a protein secreted by fibroblasts. Laminin protein participates in adhesion of epithelium cells to basal lamina. It is also a glycoprotein component. Another component is integrins, which are matrix receptors integrins play very important role in cellular recognition and downstream cellular signaling. Slide 75 - Fibronectin- Laminin Dr. Xin Li - This shows in the laminin structure in the fibronectin. This is a laminin trimer. These have three major binding domains that are required of adhesive proteins. These are adhesive proteins so they need to have binding domains to bind with cells, collagens, fibers, and ground substance components like GAGs. Here we can use fibronectin as an example and see that fibronectin has all three binding proteins that can bind with these three components. connective tissue needs to adhere with cells, collagens, and other ground substance components. Thats the link between structure and function. [Clicker: Connective tissue has a 3D structure and is composed of fibers, fluid, ground substance, cells, etc. Is the following statement correct? Connective tissue has little impact on the biological functioning of biological molecules such as hormones, growth factors, and cytokines, etc. since connective tissue doesnt produce these molecules to influence cell/tissue behavior. A. True B. False]

Transcribed by Janki Gajera!

January 16, 2014

Its false because we know connective tissue has important function in biological functioning. We know connective tissue produces growth factors at least. Fibroblasts can produce growth factors and also cytokines. Ground substance is cytokine reservoir. So all of these definitely impact biological behavior or biological molecules.