[HISTO]LEC_006_CONNECTIVE TISSUES.pdf

(005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 OUTLINE II. 3 CLASSES OF COMPONENTS I. CONNECTIVE TISSUE A. FUNCTION...

(005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 OUTLINE II. 3 CLASSES OF COMPONENTS I. CONNECTIVE TISSUE A. FUNCTION A. Ground Substance B. ORIGIN B. Fibers II. 3 CLASSES OF COMPONENTS C. Cells A. GROUND SUBSTNCE B. FIBERS A. GROUND SUBSTANCE C. CELLS III. TYPES OF CONNECTIVE TISSUES Viscous, colorless, transparent, and homogenous Fills space in between cells and fibers Acts as a lubricant Barrier to foreign particles I. CONNECTIVE TISSUE Difficult to examine in fresh and fixed samples due to high water content Provides and maintains the form of the body - Ground substance is difficult to examine in fresh or Major constituent is extracellular matrix – protein fibers, even in fixed samples because they become ground substance, tissue fluid dehydrated and can only see them as spaces. Tissue fluid - Simmilar to blood plasma in ion contents and diffusible 3 Classes of Components: substances 1. Glycosaminoglycans - Contains a small percentage of plasma proteins of low 2. Proteoglycans molecular weight 3. Structural proteins - Insignificant quantity under normal circumstances Glycosaminoglycans A. FUNCTION - Linear polysaccharides - Repeating disaccharide units (Table 1. see.Appendix) Structural uronic and a hexosamine - Encapsulate and provides internal structure to organs o Hexosamine: glucoseamine or - Tendons, ligaments, and areolar tissue around organs galactosamine - Bone and cartilage – specialized types o Uronic : glucoronic or iduronic acid Defense - largest, almost unique, most ubiquitous heparan - Due to content of phagocytic and immunocompetent sulfate (100s-1000s kDa) cells - Intensely hydrophilic and act as polyanions - Cell that produce intermediates of inflammation (hydoxyl, carboxyl, and sulfate groups) - A physical barrier – prevents dispersion of bacteria in - Binds a great number of cations (sodium), the body intensely hydrated structures Nutrition Proteoglycans - Due to the close relation with blood vessels - composed of a protein core to which various numbers - Serves a medium for the exchange of wastes and and combinations of GAGs are covalently attached nutrients between blood vessels and cells - carbohydrate portion constitutes 80-90% of the weight o One of the characteristics of epithelial tissue - Proteoglycans bind to collagen owing to electrostatic is being avascular unlike connective tissue interaction between their acid groups and the basic which is vascular amino acid residues of collagen o The nutrition of epithelial tissue comes from - Synthesis of protein moiety begins in the RER the underlying connective tissue because it's - Glycosylation initiated in the RER and completed in the closely related to blood vessels Golgi complex where sulfation also occurs B. ORIGIN Multiladhesive Glycoproteins - have multiple binding sites for cell surface receptors Mesoderm (integrins) and other matrix macromolecules Mesodermal cells migrate and surround and invade - very large molecules with branched oligosaccharide developing cells to become mesenchymal cells chains - oval nucleus, prominent nucleoli, fine chromatin, - important roles for the adhesion of cells to their sparse cytolplasm –extends as thin processes substrates away from the nucleus - Fibronectin - Point of origin of all connective tissue cells, and o (MW 235 - 270 kDa), dimeric blood and blood vessels o Synthesized by fibroblasts and epithelial cells o Has binding sites for cells, collagen, and glycosaminoglycans o Helps mediate normal cell adhesion and migration PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 - Laminin o Amount of collagen in a tissue can be o large (200-400 kDa), trimeric, cross-shaped determined by measuring the hydroxyproline glycoprotein content o Detected in the basal laminae ▪ The building blocks of proteins are o Partially responsible for the adhesion of amino acids epithelial cells to basal lamina - Integrin 2 Amino acids characteristic of collagen protein: o act as matrix receptors for specifc sequences - hydroxyproline on laminin, fbronectin, some collagens, and - hydroxylysine certain other ECM proteins o Not incorporated in the protein itself but o heterodimers of two transmembrane results from the hydroxylation of the proline polypeptides: the α and β chain and lysine in the nascent collagen polypeptide in the RER during synthesis B. FIBERS ▪ Hydroxyproline and hydroxylysine in the buy product of hydroxylation of the proline and lysine. To 3 Main types measure it accurately, examine the 1. Collagen fibers amount of collagen in the tissue 2. Reticular fibers Structure of Collagen 3. Elastic fibers - Tropocollagen - Protein unit that polymerizes to form collagen - Distributed unevenly throughout the body fibrils - Predominant type determine the property of connective tissue - Elongated (280 nm in length and 1.5 nm in Collagen width) - Most abundant protein in the human body (30% dry - Consists of 3 unit polypeptide chains weight) intertwined in a triple helix - 28 types have been described but the most important, - Difference of chemical structures of these best studied, and most common polypepetide chains account for the different - Types are types I, II, III, IV, V, VII, IX, XII, XIV (Table 2 types of collagen. see.Appendix) - Tropocollagen molecules aggregate into o There have been only types described and microfibrillar subunits packed together to form then after a few decades it becomes 13 fibrils. types and now there are 28 types of Aggregation and packing is achieved by hydrogen and collagen. hydrophobic interactions Categories of Collagen Structure is reinforced by covalent cross-links, catalyzed by - Fibrillar Collagen (types I, II, III) lysyl oxidase - have subunits that aggregate to form large fibrils clearly visible in the electron or light microscope Fibrils - Collagen type I, the most abundant and widely - thin and elongated structures distributed collagen, forms large, eosinophilic bundles - Variable diameter (20 to 90 nm) usually called collagen fibers. - Traverse striations (periodicity of 64 nm) - densely fll the connective tissue, forming structures - Determined by the overlapping arrangement of subunit such as tendons, organ capsules, and dermis tropocollagen mollecules. - Dark band retain more stain due to more free chemical - Sheet-forming collagens (type IV) groups - subunits produced by epithelial cells and are the major o Dark bands are the areas that have more structural proteins of external laminae and the basal stained due to more free chemical groups, lamina in all epithelia these are free ends. o Light bands would be less stained because it - Linking/anchoring collagens (type VII) doesn’t have free chemical groups inside the - short collagens that link fbrillar collagens to one polypeptide another (forming larger fbers) and to other components of the ECM In types I and III – fibrils associate to form fibers - Type I – fibers can associate to form bundles Synthesis of collagen - Type II – can occur as fibrils does not form fibers - is very widespread among many cells though originally - Type IV – does not form fibrils or fibers but occur as thought to be restricted to fibroblasts, chondroblasts, unpolymerized or sparsely polymerized procollagen osteoblasts, and odontoblasts molecules Type I, II, and III – (form fibrils) are called interstitial collagens Principal amino acids composing collagen type IV and V – do not form fibrils - Glycine (33.5%) - Proline (12%) - Hydroxyproline (10%) PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 o Different collagen – different amounts of carbohydrate (galactose or glycosylgalactose linked to hydroxylysine) 4. Alpha chain is synthesized with registration peptides (NH- and COOH- terminal ends) assemble to form the procollagen which is transported into the extracellular environment o Registration peptides ensure proper positioning of the alpha chains, makes the procollagen soluble and prevents premature assembly and precipitation in the cell 5. Registration peptides are removed by procollagen peptidases to turn procollagen into tropocollagen 6. Fibrils aggregate to spontaneously form fibers Figure 1. Tropocollagen Helix is a three-polypeptide chain that is made up of different amino acids in different sequences that 7. Fibrilllar structures are reinforced by covalent determine the type of collagen that is form. crosslinks , catalyzed by lysl oxidases Figure 2. Dark bands coincide in the areas where that have the free Degradation is achieved by collagenases ends; these are the ones that would retain more stain because of - “ases” is a suffix used for naming an enzyme free chemical ends in the areas. The light band doesn’t have free (e.g. Collagenases) ends so they stain a lot less. Collagen fibers Synthesis - Most abundant fibers in connective tissues 1. Polypeptide alpha chains are assembled on the - Causes tissue to become white polyribosomesin RER and injected into the - Bifringent cisternae - Inelastic and have a tensile strength greater than steel 2. Hydroxylation of proline and lysine occurs - Consist of closely packed thick fibrils (75 nm thick) o Hydroxylation begins after the peptide chain - Can be organized in parallel array forming bundles has reached a certain minimum length and is - Better studied in spread preparations and appear as still bound to the ribosomes elongated and tortous cylindric structures – indefinite o Enzymes:peptidyl proline hydroxylase and length; diameter of 1-20 µm peptidyl lysine hydroxylase - Acidophilic 3. Glycosylation of hydroxylysine o Pink – eosin o Blue – Mallory’s trichrome stain o Green – Masson’s trichrome stain PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 o Red - Sirrius red Reticular fibers - Extremely thin (0.5 to 2 µm) - Extensive network in some organs - Non visible in H&E preparations - Stained black by silver salts (agyrophilic) - PAS positive o Staining due to high content of glycoproteins - Have 6-12% hexoses compared to 1% in collagen - Composed mainly of type III collagen - Formed by loosely packed, thin (45nm) fibrils bound together by small interfibrillar bridges (proteoglycans and glycoproteins) - Weak bifringency Figure 4. Spleen. Reticular fibers form scaffolding and capsule - Abundant in smooth muscle, endoneurium around the spleen and the framework of hematopoeitic cells - Constitute a network around cells of Elastic fibers parenchymal organs - Fibers branch and unite with one another into an - Abundance of reticular fibers in irregular network embryogenesis, inflammatory processes, - Characteristic yellow color in fresh samples wound healing but is replaced eventually by - Capable of stretching to 150% of their length and regular collagen fibers yields very easily to traction but readily returns - Create a flexible network in organs subjected into original shape when relaxed to increase volume - Methods to demonstrate o Reticular fiber creates a flexible o Resorcin-fuschin: purple Network in organs that are o Aldehyde-fuschin: black subjected to increased volume like o Orcein:dark blue in the urinary bladder for instance. 2 components - Amorphous central region containing elastin - Sheath of 14 nm tubular microfibrils Development: - Microfibrils appear first - Amorphous material fills the space in tube ‘ Elastin - Secreted as proelastin( MW 70,000) which polymerizes to form elastin - Produced by fibroblasts in skin, tendon, smooth muscle - Resistant to boiling, extraction with dilute acids and alkali and not digested by trypsin - Has a tertiary and quaternary structure, stabilized by hydrophobic interactions between the non polar peptide chains o Unlike Collagen fibers that have polar ends, elastin has nonpolar peptide chain so they Figure 3. Right silver salt stain.Reticular fibers are abundant in a are connected to different chains that give hematopoietic organ, like the spleen them a very strong tertiary and quaternary structure Amino acid composition resembles collagen but has greater quantity of valine and alanine Two unusual amino acids: desmosine and isodesmosine, (formed by covalent reactions among 4 lysine residues) thus crosslinks elastin and is thought to give its elastic properties Occurs in a nonfibrillar form as fenestrated membranes in some walls of blood vessels PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 C. Cells Fibroblasts - Most commonly found cell - Synthesizes fibers and amorphous intercellular substrate 2 Different morphologic types: 1. Younger, more actively synthesizing cell (fibroblast) o Abundant and irregularly shaped cytoplasm; nucleus is ovoid, large and pale staining, with fine chromatin, prominent nucleolus; cytoplasm rich in RER, well developed Golgi complex Figure 4. Elastic Connective Tissue. Unlike reticular fibers that 2. Mature, queiscent (fibrocyte) usually form a network elastic fiber is wavy under a microscope o Found in the already formed tissue -Smaller cell, spindle shaped, fewer processes; nucleus is smaller, darker; acidophilic cytoplasm; less well developed RER and Golgi complex ▪ Fibroblast is a very robust and young-looking cell. Found in forming tissue ▪ Fibrocyte is a withered-out cell. Found in mature tissue Figure 5. Schematic diagram of elastic fiber. Microfibrils surround the amourphous elastin. (40-nanometer microtubules). These are the ones that are deposited first to be later filled in with the amorphous material that elastin core. Figure 7. Fibroblast v.s Fibrocyte When actively stimulated, mature cells may revert to younger, active form (i.e. wound healing) Synthesize collagen reticular and elastic fibers, glycosaminoglycans, and glycoproteins Fibroblasts rarely undergo division Figure. 6. When the elastin is in a relaxed state the chain is tortuous Mitoses are only observed when the organism requires but when stretch it will become one single elastin molecule or chain additional fibroblasts (tissue damage) which is cross-linked with several other chains so that makes it stronger and have more ability to stretch PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 ▪ Dense regular tissue – 3D network and Macrophages provide resistance to stress from all - Phagocytic capacity directions (tendons) - Derive mainly from precursor cells that produce monocytes Dense regular tissue has a ---transported where they mature into macrophages specific and organized o Macrophages are transported as monocytes orientation and commonly see from the hematopoietic organs and when they in the tendons. reach their target organs, they mature become macrophages. - Can proliferate locally to produce more cells - Ingestion of particles and their digestion by the lysosome o Macrophage mast cells and plasma cells these would give Connective tissue its defensive or immune capabilities Mast Cells - Oval to round connective tissue cell (20-30 µm) - Cytoplasm filled with basophilic granules - Nucleus is small spherical and centrally situated - Metachromatic - Release leukotrienes - 2 populations: o connective tissue mast cells: proteoglycan is heparin o mucosal mast cells: proteoglycan is chondroitin sulphate - Contain specific receptor for IgE, Plasma cells - Large ovoid cells, with a basophilic cytoplasm, rich Figure 8. Dense Connective tissue RER - Nucleus is spherical and eccentrically placed with compact coarse hetrochromatin - Responsible for the synthesis of antibodies Adipose Cells - Specialized for storage of neutral fats or heat production o Adipose keeps the body at a constant steady temperature o The combination of the types of cells type of fiber amount of ground substance would determine the type or characteristic of connective tissue. Leukocytes III. TYPES OF CONNECTIVE TISSUES Connective tissue proper: - Loose connective tissue o Areolar tissue o More abundant type o Fills space between fibers and muscle sheaths o Supports epithelial tissue - Dense connective tissue Figure 9. Dense Irregular Connective Tissue o Same component as loose but with a clear predominance of collagen fibers Elastic tissue o Fewer cells – fibroblasts most common - Composed of thick, parallel elastic fibers o Less flexible and resistant to stress - With a surrounding small amount of loose connective o Can be : tissue, flattened fibrocytes ▪ Dense irregular – without definite - Occurs in the yellow ligament of the vertebral column and in orientation the suspensory ligament of the penis PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 Figure 10. Elastic fibers Reticular tissue Figure 11. Reticular Fibers. Reticular cells - Specialized loose connective tissue variation, provides the architechtural framework of the myeloid and lymphoid Mucous tissue hemopoietic cells - Abundance of amourphous ground substance composed - Reticular cells – fibroblasts specialized in the production of chiefly of hyaluronic acid reticular fiber constituents - Jelly like tissue containing collagen fibers and few elastic or reticular fibers - Mainly fibroblasts - Principal component of the umbilical cord, also found in the pulp of young teeth o Mucus tissue is a lot less of fibers and cells and more of the ground substance and it's made of chiefly of hyaluronic acid Figure 12. The ground substance under the microscope is just space because it is intensely hydrated and when preparing them, even in fresh preparation will eventually desiccate the ground substance and removing them. PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 APPENDIX Glycosaminoglycans Repeating dissacharide Distribution Electrostatic interaction with Hexuronic acid Hexosamine collagen Hyaluronic acid D-Glucoronic D-Glucosamine Umbilical cord, synovial acid fluid, vitreous humor, cartilage Chondroitin 4-sulfate D-glucoronic D-Galactosamine Cartilage, bone, High levels of acid cornea, skin, interaction, mainly with notochord, aorta collagen type II Chondroitin 6- sulfate D-Glucoronic D-Galactosamine Cartilage, umbilical High levels of acid cord, skin, aorta interaction, mainly with (media) collagen type II Dermatan Sulfate L-iduronic acid D-Galactosamine Skin, tendon, aorta Low levels of or D- (adventitia) interaction, mainly with Glucoronic acid collagen type I Heparan Sulfate D-Glucoronic D- Galactosamine Aorta, lung, liver, basal Intermediate levels of Acid or laminae interaction, mainly with L-iduronic acid collagen types III and IV Keratan Sulfate (cornea) D-Galactose D-Galactosamine Cornea Keratan Sulfate D-Galactose D-Glucosamine Cartilage, nucleus (skeleton) pulposus, annulu Table 1. Glycosaminoglycans are named depending on the repeating disaccharide unit. PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 Table 2. Types of Collagen PREPARED AND EDITED BY: GROUP 1  (005) CONNECTIVE TISSUES DR. NICANOR B. LACUESTA JR., MD, DPBO-HNS | 10/05/2020 TEST YOUR KNOWLEDGE 8. Research scientists at a small biotech firm are investigating new methods of controlling the growth and metastasis of malignant cells in patients diagnosed with breast cancer. They have developed a 1. Which of the following connective tissue components is located in novel peptide-based drug, potentially deliverable therapeutically, that the ECM but not in the ground substance? disrupts the tumor cells’ ability to adhere to the ECM, which in turn triggers apoptosis. Which of the following is a most likely target of a. Collagen bundles such drugs? b. Fibronectin c. GAGs a. Cadherins d. Hyaluronan b. Adhesins e. Proteoglycans c. Integrins d. Glycolipids of the cell membrane e. Fibrillin 2. What cells numerous in loose connective tissue are filled with secretory granules and stain with metachromasia? 9. A 36-year-old man is referred by his family physician to the pulmonary clinic. He complains of shortness of breath following a. Macrophages physical activity and decreased capacity for exercise. He says that b. Mast cells strenuous exercise including yard work is impossible without sitting c. Fibrocytes down and resting every few minutes. After taking several deep d. Active fibroblasts breaths during the physical examination, he begins to wheeze. He is e. Leukocytes not a smoker and works in an office not exposed to dust, fumes, or other irritants. He appears slightly jaundiced. Serum alpha-1- 3. What is the first step of collagen production that occurs after the antitrypsin (AAT) concentration analysis is below normal and is protein undergoes exocytosis? followed up with AAT phenotype and DNA testing which indicates one copy of S and one of Z mutations with 40% abnormal AAT a. Cross-linking of collagen fibrils with a short linking collagen production. Urinalysis shows elevated levels of desmosine and b. Removal of the terminal nonhelical domains by peptidases isodesmosine. These excreted compounds normally contribute to c. Hydroxylation of lysine and proline efficient lung function by which of the following mechanisms? d. Assembly of subunits to form a larger structure e. Disulfide bond formation a. Post-translational modification of fibrillin b. Cross-linking elastin c. Activating elastase 4. What is an important part of the role played by macrophages d. Activating AAT during maintenance and renewal of strong extracellular fibers in e. Binding type IV collagen to elastin connective tissue? a. Storage for a major energy source needed for ECM maintenance 10. A 33-year-old homeless woman has been living in an abandoned b. Production of specific collagen subunits building eating dried meat and bread from the dumpster behind a c. A sentinel function against invaders entering the ECM delicatessen. She smokes cigarettes “bummed” from others. She d. Secretion of matrix metalloproteinases presents at a free clinic with bleeding under the skin, particularly e. Presentation of antigens important for assembly of collagen around hair follicles, and bruises on her arms and legs. She is bundles irritable, clinically depressed, and fatigued with general muscle weakness. Her gums are bleeding, swollen, purple, and spongy, with 6. Sulfated GAGs are important constituents of what extracellular several loose teeth. She has an infected toe, which may be broken. structures? She is afebrile, a glucose finger-stick is normal, and the urine dipstick a. Hyaluronan shows no sugar, protein, or ketones. You suspect a vitamin b. Elastic fibers deficiency. What might be the underlying mechanism for this patient’s c. Type I collagen symptoms? d. Proteoglycans e. Multiadhesive glycoproteins a. Decreased degradation of collagen b. Stimulation of prolyl hydroxylase c. Formation of unstable collagen helices 7. Dense regular connective tissue typically involves which of the d. Excessive callus formation in healing fractures following features? e. Organ fibrosis a. Contains mostly synthetically active fibroblasts Answers: 1a, 2b, 3b, 4d, 5d, 6b, 7e, 8c, 9b, 10c b. Contains much ground substance c. Contains a similar cell population as areolar connective tissue d. Predominant tissue type in the stroma of most organs REFERENCE e. Predominantly located in tendons and ligaments Mesher, A (2016). Junqueira’s Basic Histology Text and Atlas.McGraw-Hill Education. 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