Histology LC5 Connective Tissue PDF

Defense COURSE OUTLINE ○ Due to content of phagocytic and immunocompetent cells I. CONNECTIVE TISSUE...

Defense COURSE OUTLINE ○ Due to content of phagocytic and immunocompetent cells I. CONNECTIVE TISSUE ○ Cell that produce intermediates of A. Brief Overview inflammation B. Function ○ A physical barrier — prevents dispersion of C. Origin bacteria in the body D. 3 Classes of Components Nutrition II. GROUND SUBSTANCE ○ Due to close relation with blood vessels A. Glycosaminoglycans ○ Serves as a medium for the exchange of B. Proteoglycans wastes and nutrients between blood C. Multiadhesive Glycoproteins vessels and cells III. FIBERS A. Collagen Fibers C. ORIGIN A.1. Categories of Collagen A.2. Structure of Collagen Mesodermal cells migrate, surround and invade A.3. Synthesis of Collagen developing cells to become mesenchymal A.4. Collagen Fibers Histology cells, a tissue developing mainly from the B. Reticular Fibers middle layer of the embryo, mesoderm C. Elastic Fibers o Mesenchymal cells have oval nucleus, IV. CELLS OF CONNECTIVE TISSUE prominent nucleoli, fine chromatin, sparse A. Fibroblasts cytoplasm which extends as thin B. Macrophages processes away from the nucleus C. Mast cells (spindle-shaped) D. Plasma Cells o Point of origin of all connective tissue cells, E. Adipose Cells and blood and blood vessels F. Leukocytes V. TYPES OF CONNECTIVE TISSUE D. 3 CLASSES OF COMPONENTS A. Loose Connective Tissue B. Dense Connective Tissue Ground Substance C. Elastic Tissue Fibers D. Reticular Tissue Cells of Connective Tissue E. Mucous Tissue Technically four, considering tissue fluid. However, the main components are these three. I. CONNECTIVE TISSUE A. BRIEF OVERVIEW Provides and maintains the form of the body Major constituent is extracellular matrix — protein fibers, ground substance, tissue fluid ○ Tissue fluid Similar to blood plasma in ion contents and diffusible substances Contains a small percentage of plasma proteins of low molecular weight Insignificant quantity under normal circumstances Figure 1. Cellular and extracellular components of connective Edema — abundance of tissue fluid tissue due to increased blood flow and vascular permeability II. GROUND SUBSTANCE B. FUNCTION Viscous, colorless, transparent, and Structural homogenous o Encapsulates and provides internal Fills space in between cells and fibers structure to organs Acts as lubricant o Tendons, ligaments, and areolar tissue Barrier to foreign particles around organs (for connection and Difficult to examine in fresh and fixed samples support) due to high water content o Bone and cartilage — specialized types BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 ○ Highly hydrated so when mounted in a Fibronectin slide, it will dessicate and only spaces will ○ Molecular Weight (MW) 235 - 270 kDa, be observed dimeric ○ When asked what structures are in here ○ Synthesized by fibroblasts and epithelial (empty space): none; if asked what cells structure used to be here (empty space): ○ Has binding sites for cells, collagen, and ground substance glycosaminoglycans ○ Helps mediate normal cell adhesions and A. GLYCOSAMINOGLYCANS migration Also known as mucopolysaccharides Laminin Linear polysaccharides ○ Large (200-400 kDa), trimeric, Repeating disaccharide units cross-shaped glycoprotein ○ Hexosamine: glucosamine or ○ Detected in the basal laminae galactosamine ○ Partially responsible for the adhesion of ○ Uronic: glucuronic or iduronic acid epithelial cells to basal lamina Largest, almost unique, most ubiquitous — heparan sulfate (100s-1000s kDa) Integrin Intensely hydrophilic and acts a polyanions ○ Acts as matrix receptors for specific (hydroxyl, carboxyl, and sulfate groups) sequences on laminin, fibronectin, some Binds a great number of cations (sodium), collagens, and certain other extracellular intensely hydrated structures (where salt is, matrix (ECM) proteins water follows) ○ Heterodimers of two transmembrane polypeptides: the α and β chain III. FIBERS Distributed unevenly throughout the body Predominant type determines the property of connective tissue 3 main types of fibers: ○ Collagen Fibers ○ Reticular Fibers ○ Elastic Fibers Figure 2. Glycosaminoglycan named depending on the repeating A. COLLAGEN FIBERS disaccharide unit Tough, thick, fibrous proteins that do not branch B. PROTEOGLYCANS All extremely strong and resistant to normal shearing and tearing forces Composed of a protein core to which various Most abundant protein in the human body (30% numbers and combination of GAGs (linear dry weight) polysaccharides) are covalently attached Types are based on their molecular or amino Carbohydrate portion constitutes 80-90% of the acid composition, morphology, distribution, and weight function Bind to collagen owing to electrostatic 28 types have been described interaction between their acid groups and the Most common types are I, II, III, IV, V, VII, IX, basic amino acid residues of collagen XII, XIV Synthesis of protein moiety begins in the RER Glycosylation initiated in the RER and completed in the Golgi complex where sulfation also occurs C. MULTIADHESIVE GLYCOPROTEINS Also called structural proteins Have multiple binding sites for cell surface receptors (integrins) and other matrix macromolecules Very large molecules with branched oligosaccharide Important roles for the adhesion of cells to their substrates Figure 3. Collagen fibers viewed under light microscopy Water within this ground substance allows the exchange of nutrients and metabolic wastes between cells and the blood supply BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 Categories of Collagen Structure of Collagen Fibrillar Collagen (Type I, II, III) Tropocollagen ○ Function: to provide three-dimensional ○ Protein unit that polymerizes to form frameworks for tissues and organs collagen fibrils ○ Have polypeptide subunits that aggregate ○ Elongated (280 nm in length; 1.5 nm in to form large fibrils clearly visible in the width) electron or light microscope ○ Consists of 3 unit polypeptide chains ○ Collagen type I - most abundant and intertwined in a triple helix widely distributed; forms large, eosinophilic ○ Difference of chemical structures of these bundles usually called collagen fibers polypeptide chains account for the different ○ Densely fill the connective tissue, forming types of collagen structures such as tendons, organ ○ Aggregate into microfibrillar subunits to capsules, and dermis form ○ Aggregation and packing is achieved by hydrogen and hydrophobic interactions ○ Structure is reinforced by covalent cross-links, catalyzed by lysyl oxidase Fibrils ○ Thin and elongated structures ○ Variable diameter (20 - 90nm) ○ Transverse striations Determined by the overlapping arrangement of subunit tropocollagen molecules Dark band retain more stain due to more free chemical groups ○ In types I & III collagen - fibrils associate to form fibers Type I - fibers can associate to form bundles Type II - can occur as fibrils; does not form fibers Figure 4. Types of Collagen Type IV - does not form fibrils or fibers but occur as unpolymerized or Sheet-forming Collagen (type IV) sparsely polymerized procollagen ○ Subunits produced by epithelial cells and molecules are the major structural proteins of external ○ Type I, II, and III (form fibrils) are called laminae and the basal lamina in all interstitial collagens epithelia ○ Type IV and V do not form fibrils Linking/ Anchoring Collagen (type VII) ○ Short collagens that link fibrillar collagens to one another (forming larger fibers) and to other components of the ECM ○ Widespread among many cells ○ Originally thought to be restricted to fibroblasts, chondroblasts, osteoblasts, and odontoblasts Principal amino acids composing collagen: ○ Glycine (33.5%) Figure 5. Tropocollagen Helix ○ Proline (12%) ○ Hydroxyproline (10%) Amount of collagen in a tissue can be determined by measuring the hydroxyproline content 2 amino acid characteristics of collagen fiber: ○ Hydroxyproline ○ Hydroxylysine Not incorporated in the protein itself but results from the hydroxylation of the proline and lysine in the nascent collagen polypeptide in the RER during synthesis A byproduct that can be measured. Figure 6. Type I collagen. BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 Degradation is achieved by collagenases Collagen Fiber Histology Most abundant fibers in connective tissues Causes tissue to become white Birefringent Inelastic and have a tensile strength greater than steel Figure 7. A cross striated structure of collagen fiber. Dark bands Consist of closely packed thick fibrils (75 nm) coincide in the areas where that have the free ends; these are the ones that would retain more stain because of free chemical ends in Can be organized in parallel array forming the areas. The light band doesn’t have free ends so they stain a lot bundles less. Better studied in spread preparations and appear as elongated and tortuous cylindrical Synthesis of Collagen structures - indefinite length; diameter of 1-20µm 1. Polypeptide alpha chains are assembled on the Acidophilic polyribosomes in RER and injected into the ○ Pink - eosin cisternae ○ Blue - Mallory’s trichrome stain 2. Hydroxylation of proline and lysine occurs ○ Green - Masson’s trichrome stain - Begins after the peptide chain has reached ○ Red - Sirius red a certain minimum length and is still bound to the ribosomes A. RETICULAR FIBERS - Enzymes: peptidyl proline hydroxylase and peptidyl lysine hydroxylase Extremely thin (0.5 to 2µm) 3. Glycosylation of hydroxylysine Extensive network in some organs - Different collagen - different amounts of Non-visible in H&E preparations carbohydrate (galactose or Stained black by silver salts (argyrophilic) glycosylgalactose linked to hydroxylysine) PAS (Periodic Acid-Schiff) positive 4. Alpha chain is synthesized with registration ○ Staining due to high content of peptides (NH- and COOH- terminal ends) glycoproteins assemble to form procollagen which is Have 6-12% hexoses compared to 1% in the transported into the extracellular environment collagen - Registration peptides ensure proper Composed mainly of type III collagen positioning of the alpha chains, makes the Formed by loosely packed, thin (45nm) fibrils procollagen soluble and prevents bound together by small interfibrillar bridges premature assembly and precipitation in (proteoglycans and glycoproteins) the cell Weak birefringence 5. Registration peptides are removed by Abundant in smooth muscle, endoneurium and procollagen peptidases to turn procollagen intro the framework of hematopoietic cells tropocollagen Constitute a network around cells of 6. Fibrils aggregate to spontaneously form fibers parenchymal organs 7. Fibrillar structures are reinforced by covalent Abundance of reticular fibers in crosslinks, catalyzed by lysyl oxidases embryogenesis, inflammatory processes, wound healing but is replaced eventually by regular collagen fibers Create a flexible networks in organs subjected to increase volume B. ELASTIC FIBERS Fibers are thin, small, branching and unite with one another into an irregular network Characteristic yellow color in fresh samples Capable of stretching to 150% of their length and yields very easily to traction but readily returns into original shape (recoil) when relaxed without deformation Methods to demonstrate: ○ Resorcin-fuchsin: purple ○ Aldehyde-fuchsin: black ○ Orcein: dark blue 2 components of elastic fibers: ○ Amorphous central region containing elastin ○ Sheath of 14nm tubular microfibrils Figure 8. Collagen synthesis BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 Development: ○ Microfibrils appear first ○ Amorphous material fills the space in the 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 Figure 11. Elastic fiber where microfibrils surround the amorphous ○ Amino acid composition resembles elastin. collagen but has greater quantity of valine and alanine IV. CELLS OF CONNECTIVE TISSUE ○ Two unusual amino acids: desmosine and isodesmosine, (formed by covalent reactions among 4 lysine residues) thus A. FIBROBLASTS crosslinks elastin and is thought to give its elastic properties Most commonly found cell ○ Occurs in a non fibrillar form as Synthesizes collagen, reticular and elastic fenestrated membranes in some walls of fibers, amorphous intercellular substrate, blood vessels glycosaminoglycans, and glycoproteins TWO MORPHOLOGICAL TYPES Fibroblast — younger, more actively synthesizing cell ○ 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 Fibrocyte — mature, quiescent ○ 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 Figure 9. Prominence of networks of delicate, black reticular fibers in the spleen. A loose connective tissue made up of a network of reticular fibers that provides a supportive framework for soft organs. The fibers consist of type III collagen. Cell nuclei are also dark but cytoplasm is unstained. Figure 12. Large fibroblasts in a section of umbilical cord. Figure 13. Fibroblast vs. Fibrocyte Figure 10. Sheets of elastic fibers found in the stroma of the lungs and in the walls of the large arteries. BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 Fibroblasts are targets of growth factors that E. ADIPOSE CELLS influence cell growth and differentiation ○ Fibroblasts rarely undergo division. Specialized for storage of neutral fats or heat However, when actively stimulated by production growth factors, mature cells may revert to Serves to cushion and insulate the skin and younger, active form (i.e. wound healing) other organs ○ Myofibroblasts are fibroblasts involved in wound healing; have well-developed F. LEUKOCYTES (WHITE BLOOD CELL) contractile function and are enriched with a form of actin also found in smooth muscles Leave the blood and migrate to the tissues, Mitoses are only observed when the organism where they become functional and perform requires additional fibroblasts (tissue damage) various activities related to the immunity All are rather spherical white suspended in B. MACROPHAGES blood plasma, but they become amoeboid and Also called histiocytes motile after leaving the blood vessels and Phagocytic capacity invading the tissues Derive mainly from precursor cells that produce monocytes — transported where they mature into macrophages V. TYPES OF CONNECTIVE TISSUE ○ Monocytes formed in the yolk sac during early development circulate and become A. LOOSE CONNECTIVE TISSUE residents in developing organs throughout the embryo, comprising a group of related Areolar tissue cells called the mononuclear phagocyte More abundant type system. Fills space between fibers and muscle sheaths Can proliferate locally to produce more cells Support epithelial tissue Ingestion of particles and their digestion by the Composed of all main components of lysosomes connective tissue proper, most abundant cell is the fibroblast and macrophages C. MAST CELLS B. DENSE CONNECTIVE TISSUE Oval to round connective tissues cell (20-30μm) Same component as loose but with a clear Cytoplasm filled with basophilic granules predominance of collagen fibers Nucleus is small, spherical and centrally Fewer cells — fibroblasts most common situated Less flexible and resistant to stress Metachromatic — they can change color of Can be: some basic dye from blue to purple or red ○ Dense irregular — without definite Release leukotrienes orientation; fibers are going haywire Function in the localized release of many ○ Dense regular — 3D network and bioactive substances important in the local provide resistance to stress from all inflammatory response, innate immunity and directions (tendons); has one single tissue repair direction ○ Some bioactive substances include: heparin, histamine, serine proteases, eosinophil and neutrophil chemotactic factor, cytokine, and phospholipid precursors 2 populations: ○ Connective tissue mast cells: proteoglycan is heparin ○ Mucosal mast cells: proteoglycan is chondroitin Contains specific receptor for IgE as part of immediate hypersensitivity reactions Figure 14. Dense regular connective tissue. Long, parallel bundles D. PLASMA CELLS of collagen fibers fill the spaces between the elongated nuclei of fibrocytes (formed mature tissue). Large ovoid cells, with a basophilic cytoplasm, rich RER Nucleus is spherical and eccentrically placed with compact coarse heterochromatin Responsible for the synthesis of antibodies Derived from B lymphocytes that enter the connective tissue from blood vessels, then differentiate into plasma cells when exposed to antigens BATCH 2028 1C ANATOMY LC5: CONNECTIVE TISSUE Dr. Lacuesta, N. B. 09/16/2024 E. MUCOUS TISSUE Abundance of amorphous ground substance composed chiefly of hyaluronic acid Jelly-like tissue containing collagen fibers and few elastic or reticular fibers Mainly fibroblasts Principal component of the umbilical cord (also called Wharton’s jelly) found in the pulp of young teeth Figure 15. Dense irregular connective tissue showing the randomly arranged large collagen bundles. C. ELASTIC TISSUE Composed of thick, parallel elastic fibers With a surrounding small amount of loose connective tissue, flattened fibrocytes Figure 18. Mucous connective tissue of the umbilical cord. Section Occurs in the yellow ligament of the vertebral of umbilical cord showing large fibroblasts surrounded by a large column and in the suspensory ligament of the amount of very loose ECM containing mainly ground substances penis very rich in hyaluronan, with wisps of collagen. References: Mescher, A. L. (2021). Junqueira's basic histology: Text and atlas (17th ed.). McGraw Hill. Eroschenko V.P. diFiore’s Atlas of Histology with Functional Correlation. 13th ed, 2017. Dr. Lacuesta’s PowerPoint Presentation. S.Y. 2024-2025 - Epithelial tissue. University of Northern Philippines. College of Medicine. Figure 16. Elastic tissue. Note the thick, parallel elastic fibers. D. RETICULAR TISSUE Specialized loose connective tissue varistion, provides the architectural framework of the myeloid and lymphoid hematopoietic cells Reticular cells — fibroblasts specialized in the production of reticular fiber constituents. Figure 17. Reticular tissue. Diagram shows the fibers and reticular cells. Silver-stained section of lymph node in which reticular fibers are seen as irregular black lines. Reticular cells are also heavily stained and dark. Most of the smaller, more lightly stained cells are lymphocytes passing through the lymph node BATCH 2028 1C