Lecture 8 - Connective Tissue & Cartilage (Z. Kalnina 2024) PDF

INTRODUCTION IN CELL BIOLOGY Lecture 8 24/Oct/2024 Dr. Zane Kalnina CONNECTIVE TISSUE (CONTINUED) PART 1 2 CLASSIFICATION OF TISSUES The human body is composed of four basic types of tissues: ❑ Epithelium – lines and covers surfaces ❑ Connective tissue – protect, support,...

INTRODUCTION IN CELL BIOLOGY Lecture 8 24/Oct/2024 Dr. Zane Kalnina CONNECTIVE TISSUE (CONTINUED) PART 1 2 CLASSIFICATION OF TISSUES The human body is composed of four basic types of tissues: ❑ Epithelium – lines and covers surfaces ❑ Connective tissue – protect, support, and bind together; fills the spaces between organs and tissues, and provides structural and metabolic support for other tissues and organs ❑ Muscular tissue – produces movement ❑ Nervous tissue – receive stimuli and conduct impulses 3  CONNECTIVE TISSUE COMPOSITION CONNECTIVE TISSUE CELLS ECM* GROUND FIBERS SUBSTANCE *ECM = extracellular matrix 4  ECM Ground Fibers substance FIBERS Collagen Proteoglycans Reticular Glycoproteins Elastic Glycosaminoglycans 5  COLLAGEN FIBERS AND FIBRILS ❑ The most abundant type of connective tissue fiber ❑ Flexible, have a remarkably high tensile strength ❑ Collagen fibers appear as bundles of fine, threadlike subunits – collagen fibrils ❑ Within an individual fiber, the collagen fibrils are relatively uniform in diameter (ranges from 15-300 nm in diameter in fibers from different tissues) x75,000 x9,500 Electron micrograph of dense irregular connective tissue 6  COLLAGEN FIBRILS HAVE A 68-nm BANDING PATTERN Collagen fibrils exhibit a sequence of closely spaced transverse bands that repeat every 68 nm along the length of the fibril Electron micrograph of dense regular connective tissue Atomic force microscopic image of type I collagen fibrils in the dense irregular connective tissue 7  Fibrillar collagens include types I, II, III, V, and XI collagen molecules with joined sugar groups (collagen is a glycoprotein) The collagen molecule measures about 300 nm long by 1.5 nm thick and has a head and a tail Within each fibril, the collagen molecules align head to tail in overlapping rows with a gap between the molecules in each row and a one quarter – molecule stagger between adjacent rows Every third amino acid of the chain is a glycine 8  RETICULAR FIBERS Reticular fibers in the lymph node x650 ❑ provide a supporting framework for the cellular constituents of various tissues and organs ❑ composed of collagen fibrils (made of type III collagen), exhibiting the same 68 nm banding pattern ❑ the fibrils have a narrow diameter (about 20 nm), exhibit a branching meshlike pattern, and typically do not bundle to form thick fibers ❑ reticular fibers are mainly produced by fibroblasts except for hemopoietic and lymphatic tissues – there RFs are produced by a special cell type, the reticular cell 9  RETICULAR FIBERS IN HUMAN BODY FOUND: ❑ at the boundary of connective tissue and epithelium, as well as surrounding adipocytes, small blood vessels, nerves, and muscle cells In most locations, reticular fibers are produced by fibroblasts, and are composed ❑ in embryonic tissues of type III collagen ❑ prominent in the initial stages of wound healing and scar tissue formation Important exceptions to this general rule As embryonic development or wound healing include the endoneurium of peripheral nerves, progresses, reticular fibers are gradually replaced by where Schwann cells secrete reticular fibers, the stronger type I collagen fibers tunica media of blood vessels, and muscularis of the alimentary canal, where smooth ❑ also function as a supporting stroma in muscle cells secrete reticular and other hemopoietic and lymphatic tissues (but not in the collagen fibers thymus) 10  ELASTIC FIBERS ❑ Elastic fibers allow tissues to respond to stretch and distension ❑ Typically thinner than collagen fibers, arranged in a branching pattern to form a 3D network ❑ The fibers are interwoven with collagen fibers to limit the distensibility of the tissue and prevent tearing from excessive stretching ❑ produced by many of the same cells that produce collagen and reticular fibers, Photomicrograph of a mesentery spread (x200) particularly fibroblasts and smooth muscle elastic fibers (E), collagen fibers (C) - much thicker, cells although they cross one another, they do not branch 11  ELASTIC FIBER STRUCTURE ❑ EFs are composed of two structural components: a central core of ELASTIN and a surrounding network of FIBRILLIN MICROFIBRILS ❑ Central core of ELASTIN – a protein that, like collagen, is rich in proline and glycine – the random distribution of glycines makes the elastin molecule hydrophobic and allows for random coiling of its fibers ❑ Elastin also contains desmosine and isodesmosine, two large 1.5 x amino acids unique to elastin, which are responsible for the covalent bonding of elastin molecules to one another ❑ Elastin forms fibers of variable thicknesses, or lamellar layers (as in elastic arteries) – it is the major extracellular substance in vertebral ligaments, larynx, and elastic arteries 12  FIBRILLIN MICROFIBRILS ❑ Fibrillin-1 (350 kilodaltons) is a glycoprotein that forms fine microfibrils measuring 10 to 12 nm in diameter ❑ During the early stages of elastogenesis, fibrillin-1 microfibrils are used as substrates for the assembly of elastic fibers ❑ The microfibrils are formed first; elastin material is then deposited on the surface of the microfibrils and thus play a major In mature fibers, the fibrillin microfibrils are located role in organizing elastin into fibers within the elastic fiber and at its periphery 13 Scanning electron micrograph of an elastic fiber C – collagen fibrils in a collagen fiber E – elastic fiber Arrows – small fibrillin microfibrils 14  ECM Ground GROUND Fibers substance SUBSTANCE Collagen Proteoglycans Reticular Glycoproteins Elastic Glycosaminoglycans 15  EXTRACELLULAR MATRIX (ECM) A complex structural network that surrounds ECM and supports cells within the connective tissue GROUND SUSBSTANCE is composed from a Ground high water variety of Fibers content! substance ❑ proteoglycans ❑ multiadhesive glycoproteins Collagen Proteoglycans ❑ glycosaminoglycans (GAGs) – most Reticular abundant components of ground Glycoproteins substance, highly negatively charged Elastic Glycosaminoglycans 16  GROUND SUBSTANCE = EXTRAFIBRILLAR MATRIX ❑ amorphous gel-like substance surrounding the cells ❑ traditionally does not include fibers (collagen and elastic fibers), but does include all the other components of the ECM ❑ primarily composed of water, glycosaminoglycans (GAG*), proteoglycans, and glycoproteins, components vary depending on the tissue 17 *GAGs (or mucopolysaccharides) are long unbranched polysaccharides consisting of a repeating disaccharide unit  EXTRACELLULAR MATRIX ❑ All molecules found in the ECM share common domains, and the function of the ECM relies largely on the interactions between these molecules ❑ Each connective tissue cell secretes a different ratio of ECM molecules that contribute to the formation of many different architectural ECM also influences extracellular arrangements – therefore, the ECM possesses communication – ECM form a dynamic specific mechanical and biochemical properties characteristic for the tissue in which it is present and interactive system that informs cells about the biochemical and mechanical ❑ For instance, the properties of the ECM in loose changes in their extracellular connective tissue are different from those of the ECM in cartilage or bone environment 18  GLYCOSAMINOGLYCAN MOLECULES (GAGs) Proteoglycans are composed of GAGs covalently attached to core proteins ❑ GAGs (or mucopolysaccharides) are long unbranched polysaccharides ❑ GAGs are the most abundant components of ground substance responsible for its physical properties ❑ synthesized by connective tissue cells as a covalent, posttranslational modification of proteins called proteoglycans (covalently bound to the proteoglycans) 19 GAGs (continued) ❑ GAGs (or mucopolysaccharides) are long unbranched polysaccharides consisting of a repeating disaccharide unit ❑ GAGs are highly negatively charged – this attracts water, forming a hydrated gel! 20 On the basis of differences in specific sugar residues, the nature of their linkages, and the degree of their sulfation, a family of seven distinct GAGs is recognized 21  HYALURONAN (HYALURONIC ACID) It differs from the other GAGs in several respects: ❑ It is an exceedingly long, rigid molecule composed of a carbohydrate chain of thousands of sugars rather than the several hundred or fewer sugars found in other GAGs – they are very large (100 to 10,000 kDa) and can displace a large volume of water ❑ They are synthesized by enzymes on the cell surface; therefore, they are not posttranslationally modified like all other GAGs, do not contain any sulphate ❑ Each hyaluronan molecule is always present in the form of a free carbohydrate chain; in other words, it is not covalently bound to protein, so it does not form proteoglycans. ❑ By means of special link proteins, proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called proteoglycan aggregates 22  Chemical structures of HA disaccharide unit (A) and HA tetrasaccharide unit where the hydrophilic functional groups and the hydrophobic moieties are respectively evidenced in blue and yellow, while the hydrogen bonds are represented by green dashed lines (B) Water bridge https://www.semanticscholar.org/paper/Hyaluronic-Acid-in-the-3rd-Millennium-Fallacara- 23 Baldini/496a884f0a03a083ccbcff62137cfede5b6ddb0f/figure/0  BIOLOGICAL IMPORTANCE OF HYALURONAN Chemical nomenclature: Poly{[(2S,3R,4R,5S,6R)-3- acetamido-5-hydroxy-6-(hydroxymethyl)oxane-2,4- diyl]oxy[(2R,3R,4R,5S,6S)-6-carboxy-3,4-dihydroxyoxane-2,5- diyl]oxy} ❑ These molecules are abundant in the ground substance of cartilage – the pressure, or turgor, that occurs in these giant hydrophilic proteoglycan aggregates accounts for the ability of cartilage to resist compression without inhibiting flexibility, making them excellent shock absorbers ❑ To immobilize certain molecules in the desired location of the ECM – i.e., ECM contains binding sites for several growth factors, such as TGF-. The binding of growth factors to proteoglycans may cause either their local aggregation or dispersion, which in turn either inhibits or enhances the movement of migrating macromolecules, microorganisms, or metastatic cancer cells in the ECM ❑ They act as efficient insulators, because other macromolecules have difficulty diffusing through the dense hyaluronan network. With this property, hyaluronan (and other polysaccharides) regulates the distribution and transport of plasma proteins within the connective tissue 24  PROTEOGLYCANS ARE COMPOSED OF GAGs COVALENTLY ATTACHED TO CORE PROTEINS The majority of GAGs in the connective tissue are linked to core proteins, forming proteoglycans The linkage of GAGs to the core protein involves a specific tetrasaccharide composed of a xylulose residue, two galactose residues and a glucuronic acid The tetrasaccharide linker is coupled through an O-glycosidic bond to the protein core that is rich in serine and threonine residues, allowing multiple GAG attachments 25  PROTEOGLYCANS ARE REMARKABLE FOR THEIR DIVERSITY ❑ The number of GAGs attached to the protein core varies from 1 (i.e., decorin) to more than 200 (i.e., aggrecan) ❑ A core protein may have identical GAGs attached to it (as in the case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan) ❑ Proteoglycans are found in the ground substance of all connective tissues and also as membrane-bound molecules on the surface of many cell types – for example, a transmembrane proteoglycans syndecan anchors the plasma cell to the ECM proteins of the connective tissue 26 27  MULTIADHESIVE GLYCOPROTEINS – MULTIDOMAIN AND MULTIFUNCTIONAL PROTEINS IN ECM ❑ Play an important role in stabilizing the ECM and linking it to cell surfaces ❑ possess binding sites for a variety of ECM proteins such as collagens, proteoglycans, and GAGs ❑ also interact with cell-surface receptors such as integrin and laminin receptors ❑ regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation 28 29 CONNECTIVE TISSUE CELL POPULATIONS RESIDENT – typically exhibit little TRANSIENT / WANDERING – cells that movement have migrated into the tissue from the blood in response to specific stimuli fibroblasts and a closely related cell type, the myofibroblast lymphocytes macrophages plasma cells adipocytes neutrophils mast cells eosinophils adult stem cells basophils monocytes 30  Fibroblasts in connective tissue FIBROBLASTS ❑ Principal cell of connective tissue Responsible for the synthesis of ECM fibers and the complex carbohydrates of the ground substance Research suggests that a single fibroblast is capable of producing all of the ECM components! ❑ When ECM material is produced during active growth or in wound repair (in activated fibroblasts), the cytoplasm of the fibroblast is more extensive Fibroblasts in cell culture 31 32  MYOFIBROBLASTS FIBRONEXUS ❑ These cells have elongated spindle-shaped morphology and are characterized by the presence of bundles of actin filament with associated actin motor proteins such as nonmuscle myosin ❑ They are contractile cells resembling smooth muscle cells, but they lack a surrounding basal lamina (smooth muscle cells are surrounded by a basal or external lamina). Also, it usually exists as an isolated Fibronexus resembles focal adhesion found in the epithelial cell cells is of the basis of a mechano-transduction system in which ❑ Expression of the -smooth muscle actin (- force that is generated by the contraction of intracellular actin SMA), actin isoform found in the vascular bundles is transmitted to the ECM smooth muscles) in myofibroblasts is regulated by TGF- 33 34 Myofibroblasts are the mesenchymal cell type responsible for wound healing and tissue repair across all organs and various physiological states, including cancer 35  https://www.youtube.com/ watch?v=D-SzmURNBH0 Tissues, Part 3 - Connective Tissues: Crash Course WATCH THIS! 36 CARTILAGE PART 2 37 Chapter 7 38 CARTILAGE IN HUMAN BODY Cartilage is a key tissue in the development of the fetal skeleton and in most growing bones! 39 39  GENERAL FEATURES OF CARTILAGE ❑ Cartilage is a form of connective tissue composed of: ❑ cells called CHONDROCYTES – produce and maintain matrix ❑ a highly specialized EXTRACELLULAR MATRIX – the functional element (95% of cartilage volume), solid, firm, pliable ❑ No vascular network – ECM takes care of the survival of chondrocytes ❑ high GAG content (weak in shear) and fibers (highly resistible to tension), permissive to diffusion ❑ Regenerates poorly 40  THE THREE TYPES OF CARTILAGE Different in appearance and mechanical properties are distinguished on the basis of characteristics of their matrix: ❑ HYALINE CARTILAGE is characterized by matrix containing predominantly type II collagen fibers, GAGs, proteoglycans, and multiadhesive glycoproteins ❑ ELASTIC CARTILAGE is characterized by elastic fibers and elastic lamellae in addition to the matrix material of hyaline cartilage ❑ FIBROCARTILAGE is characterized by abundant type I collagen fibers as well as the matrix material of hyaline cartilage 41 HYALINE CARTILAGE 42  HYALINE CARTILAGE ❑ Complex living tissue that: provides a low-friction surface participates in lubricating synovial joints distributes applied forces to the underlying bone …and under normal circumstances shows no evidence of abrasive wear over a lifetime, but tends to calcify with ageing ❑ Hyaline cartilage matrix is homogenous, amorphic – highly hydrated to provide resilience and diffusion of small Chondroblasts (perichondrial cells) – metabolites mesenchymal progenitor cells of chondrocytes in the growing cartilage matrix ❑ Throughout the cartilage matrix are spaces called lacunae – located within these lacunae are the chondrocytes Perichondrium – the connective tissue that envelops cartilage (where it is not at a joint) 43  TYPICAL HYALINE CARLITAGE IN MICROSCOPE ❑ Dense connective tissue (DCT ) over the perichondrium (P), from which new cartilage cells are derived ❑ A slightly basophilic layer of growing cartilage (GC) ISOGENOUS underlying the perichondrium contains GROUP chondroblasts and immature chondrocytes ❑ Mature chondrocytes with clearly visible nuclei (N) reside in the lacunae ❑ They produce the cartilage matrix that shows the dark-staining capsule or territorial matrix (TM) immediately surrounding the lacunae ❑ The interterritorial matrix (IM) is less intensely stained 44  MOLECULAR COMPOSITION OF HYALINE CARTILAGE ECM Proteoglycan aggregates containing GAGs 45  COLLAGEN MOLECULES IN HYALINE CARTILAGE Because specific types of collagen molecules (e.g., II, VI, IX, X, and XI) are found in significant amounts only in the cartilage matrix, they are referred to as CARTILAGE- SPECIFIC COLLAGEN MOLECULES: ❑ type II collagen constitutes the bulk of the fibrils ❑ type IX collagen facilitates fibril interaction with the matrix proteoglycan molecules ❑ type XI collagen regulates the fibril size ❑ type X collagen organizes the collagen fibrils into a 3D hexagonal lattice that is crucial to its successful mechanical function ❑ type VI collagen is also found in the matrix, mainly at the periphery of the chondrocytes where it helps to attach these cells to the matrix framework 46  GROUND SUSBSTANCE PROTEOGLYCANS The ground substance of hyaline cartilage contains three kinds of glycosaminoglycans (GAGs): ❑ hyaluronan (linear) ❑ chondroitin sulfate joined to a core protein to form a proteoglycan monomer ❑ keratan sulfate The most important proteoglycan monomer in hyaline cartilage is AGGRECAN MULTIADHESIVE GLYCOPROTEINS influence interactions between the chondrocytes and the matrix molecules 47  CHONDROCYTES SPECIALIZED CELLS THAT PRODUCE AND MAINTAIN THE EXTRACELLULAR MATRIX ❑ In hyaline cartilage, chondrocytes are distributed either singularly or in clusters called isogenous groups, meaning cells have recently divided ❑ As the newly divided chondrocytes produce the matrix material that surrounds them, they are dispersed ❑ They also secrete enzymes that degrade cartilage matrix, allowing the cells to expand and reposition themselves within the growing isogenous group ❑ The capsular matrix contains the highest concentration of proteoglycans, hyaluronan and several multiadhesive glycoproteins 48  ELECTRON MICROGRAPH OF A YOUNG, ACTIVE CHONDROCYTE AND SURROUNDING MATRIX ❑ nucleus (N) of the chondrocyte is eccentrically located ❑ the cytoplasm displays numerous and somewhat dilated profiles of rER, Golgi apparatus (G), and mitochondria (M) ❑ large amount of rER and the extensive Golgi apparatus indicate that the cell is actively engaged in the production of cartilage matrix ❑ The numerous dark particles in the matrix contain proteoglycans ❑ The particularly large particles adjacent to the cell are located in the region of the matrix that is identified as the capsular or territorial matrix x 15,000 49 CARTILAGE REMODELLING ❑ Throughout life, cartilage undergoes continuous internal remodelling – the cells replace matrix molecules lost through degradation ❑ Normal matrix turnover depends on the ability of the chondrocytes to detect changes in matrix composition – the chondrocytes then respond by synthesizing appropriate types of new molecules ❑ The matrix also acts as a signal transducer for the embedded chondrocytes – pressure loads applied to the cartilage, as in synovial joints, create mechanical, electrical, and chemical signals that help direct the synthetic activity of the chondrocytes ❑ As the body ages, however, the composition of the matrix changes, and the chondrocytes lose their ability to respond to these stimuli 50  ARTICULAR CARTILAGE Hyaline cartilage that covers the articular surfaces of movable joints ❑ The superficial (tangential) zone contains numerous elongated and flattened chondrocytes surrounded by a condensation of type II collagen fibrils that are arranged in fascicles parallel to the free surface ❑ The intermediate (transitional) zone contains round chondrocytes randomly distributed within the matrix; collagen fibrils are less organized ❑ The deep (radial) zone is characterized by small, round chondrocytes ❑ The calcified zone is characterized by a calcified matrix with the presence of few small chondrocytes ❑ Above tidemark line, proliferation of chondrocytes within the cartilage lacunae provides the new cells for growth 51 ELASTIC CARTILAGE 52  ELASTIC CARTILAGE perichondrium x 180 ❑ Elastic cartilage matrix in addition to those components found in hyaline cartilage contains a dense network of branching elastic fibers (yellow in appearance) and interconnecting sheets of elastic material that gives the cartilage elastic properties ❑ Elastic cartilage is found in the external ear, the walls of the ear canal, the auditory (Eustachian) tube, and the epiglottis of the larynx ❑ Unlike hyaline cartilage, which calcifies with aging, the matrix of elastic cartilage does not calcify during the aging process Orcein stain reveals the elastic fibers (brown), which are of various sizes and constitute a significant part of the cartilage 53 https://www.youtube.com/watch?v=ycXqvk2Svhw 54 FIBROCARTILAGE 55  FIBROCARTILAGE – shock absorber (resist compression & shearing) 60x ❑ Fibrocartilage is a combination of dense regular connective tissue and hyaline cartilage in various ratios ❑ The chondrocytes are dispersed among the collagen fibers – they appear similar to the chondrocytes of hyaline cartilage, but they have considerably less cartilage matrix material ❑ There is no surrounding perichondrium 700x ❑ Fibrocartilage is typically present in intervertebral discs, the symphysis pubis, articular discs of the sternoclavicular and temporomandibular joints, menisci of the knee joint, the triangular fibrocartilage complex of the wrist, and certain places where tendons attach to bones Fibrocartilage from an intervertebral disc. Collagen fibers are stained green; fibroblasts with elongated nuclei (arrows), chondrocytes with dark round nuclei 56  Small collagen fiber bundles that ECM IN FIBROCARTILAGE run in different directions. Obviously spongey, shock absorbing type of tissue ❑ The cells in fibrocartilage synthesize a wide variety of extracellular matrix molecules during its mature state allowing the fibrocartilage to respond to changes in the external environment ❑ The extracellular matrix of fibrocartilage contains significant quantities of both type I collagen (characteristic of connective tissue matrix) and type II collagen (characteristic of hyaline cartilage) ❑ The relative proportions of these can vary depending on location and it changes with age – in older individuals, there is more type II collagen because of the metabolic activity of chondrocyte ❑ ECM of fibrocartilage contains larger amounts of versican (a proteoglycan monomer secreted by fibroblasts) than aggrecan (produced by chondrocytes) – it can also bind hyaluronan to form highly hydrated proteoglycan aggregates 57  CHONDROGENESIS Most cartilage arises from mesenchyme Chondrogenesis, the process of cartilage development, begins with the aggregation of chondroprogenitor mesenchymal cells to form a mass of rounded, closely apposed cells – chondrogenic nodule Chondrogenesis is Within hyaline cartilage: regulated by many 1. Chondroprogenitor cells are triggered to differentiate into molecules, including chondroblasts via the expression of transcription factor SOX-9 extracellular ligands, nuclear receptors, 2. Chondroblasts secrete cartilage matrix and progressively move transcription factors, apart as they deposit matrix adhesion molecules, matrix 3. When they are completely surrounded by matrix material, the cells proteins, and are called chondrocytes biomechanical forces 4. The mesenchymal tissue immediately surrounding the chondrogenic nodule gives rise to the perichondrium 58 CARTILAGE GROWTH Cartilage is capable of two kinds of growth: appositional and interstitial ❑ Appositional growth, the process that forms new cartilage at the surface of an existing cartilage – new cartilage cells produced are derived from the inner portion of the surrounding perichondrium ❑ Interstitial growth arise from the division of chondrocytes within their lacunae since the chondrocytes retain the ability to divide In its mature state, hyaline cartilage has limited ability for repair due to: avascularity of cartilage the immobility of the chondrocytes and the limited ability of mature chondrocytes to proliferate 59  HYALINE CARTILAGE CALCIFICATION Calcification – a process in which calcium phosphate crystals become embedded in the cartilage matrix – always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during an individual’s growth period The matrix of hyaline cartilage undergoes calcification in three well-defined situations: 1. The portion of articular cartilage that is in contact with bone tissue in growing and adult bones, but not the surface portion, is calcified 2. Calcification always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during an individual’s growth period 3. Hyaline cartilage in the adult calcifies with time as part of the aging process 60