Histology Notes I.1 PDF

Histology I 1 SCT: st Epithelial and Connective Tissues *These notes are based on: Ross Histology book 7th edition, lectures slides, and notes fr...

Histology I 1 SCT: st Epithelial and Connective Tissues *These notes are based on: Ross Histology book 7th edition, lectures slides, and notes from class (taught by Professor Antal). Good luck! Lior Onn  Epithelial tissue Overview Epithelium covers body surfaces, lines body cavities, and constitutes glands. Avascular tissue. Form secretory portion (parenchyma) of glands and their ducts. Specialized epithelial cells function as receptors for the special senses (smell, taste, hearing and vision). Three principal characteristics of epithelial cells: 1. Attach closely to one another via specialized cell junctions, cell-to- cell adhesions. Adjacent cells are separated by a very narrow EC space which can’t even be seen in light microscope resolution. 2. Functional and morphological polarity: three surface domains each with specific properties and different functions: a. Free surface, apical domain b. Lateral domain c. Basal domain 3. Basement membrane in the basal surface (noncellular, protein-polysaccharide-rich layer) Epithelioid tissues: epithelial cells without a free surface. Typical of most endocrine glands (interstitial cells of Leydig in the testis, lutein cells of the ovary, islets of Langerhan in pancreas, etc.). also formed by accumulations of CT macrophages in response to injury or infection. Creates a selective barrier between external environment and underlying CT. Classification Based on two factors: 1. number of cell layers: a. Simple: one cell layer b. Stratified: two or more cell layers c. Pseudostratified: appears stratified, but all cells rest on basement membrane. 2. shape of surface cells (in stratified epithelium, the shape of the outer/surface cells is used to classify): a. squamous: width >> height I. Keratinizing – only in the skin (covering epithelium) II. Non-keratinizing (oral cavity) b. cuboidal: width ~ depth ~ height c. columnar: height >> width (low columnar is when height > width) Classification into four main groups: 1. Lining epithelium: covers body surfaces 2. Glandular epithelium: produce and secrete substances 3. Sensory epithelium: associated with senses (taste, vision, olfactory, hearing) 4. Pigment epithelium: found in the retina of the eye, in charge of eye pigmentation. Reflects on structure, not on function. 1  There are also pyramidal-looking cells in some exocrine glands, but they’re classified as cuboidal/columnar. Specialization of the apical domain may also be added to classification: ciliated, keratinized/non keratinized. Transitional epithelium (urothelium): epithelium lining the lowr urinary tract (minor calyces->prox. urethra). Stratified epithelium with special ability to distend. When the bladder is full the epithelium is flattened due to hydrostatic pressure, but when it’s empty the hydrostatic pressure drops and the epithelium becomes columnar and there are even more cells. Endothelium: simple squamous epithelial lining of blood and lymph vessels. Exception: endothelium is cuboidal in high endothelial venules (HEV). Endocardium: epithelial lining of ventricles and atria of the heart. Mesothelium: simple squamous epithelium that lines the walls and covers the contents of the closed cavities of the body (abdominal peritoneum, pericardial, and pleural cavities = SS epithelium of serous membranes). Functions: 1. Secretion (columnar epithelium of the stomach). Glandular epithelium. 2. Absorption (columnar epitheliu of the intestines) 3. Transportation: a. of materials or cells along the surface of an epithelium by motile cilia b. of materials across an epithelium to/from CT 4. protection (epidermis of the skin, SSE). Lining epithelium. 5. receptor function: receive and transduce external stimuli (taste buds, olfactory epithelium, retina). Sensory and pigment epithelium. Cell polarity epithelial cells have three surfaces/domains, each with specific biochemical characteristics and functions. Polarity is needed to create a fully functional barrier between adjacent cells The apical domain and its modifications May contain specific enzymes, ion channels, and carrier proteins. Three structural surface modifications: 1. Microvilli: cytoplasmic processes with core of actin filaments 2. Sterocilia (stereovilli): microvilli of unusual length 3. Cilia: cytoplasmic processes containing microtubule bundles 1. Microvilli (small intestine slide): Fingerlike cytoplasmic projections, 1-3 µm length Number and shape vary, and correlate with the cell’s absorptive capacity: i. Transport fluid and absorb metabolites àmany closely packed tall microvilli ii. Less active trans-epithelial transport àsmaller, irregular shape microvilli (may escape light microscope) Create striated border in the intestinal absorptive cells (highly ordered, uniform), and brush border in kidney tubule cells. 2  Cytoskeletal composition of these cytoplasmic projections is different than that of the cell! Contain core of actin filaments, cross-linked by actin-bundling proteins: fascin, fimbrin, and espin. Villin anchor the plus end of villi (also an actin-bundling protein) Terminal web: horizontal network of actin filaments, with myosin II and tropomyosin, just below the base of the microvilli. Seen in slides. Contractile property à cause an increase in inter-microvillous space. myosin I binds the actin filaments to the plasma membrane of the microvillus. 2. Sterocilia (stereovilli): Microvilli that are unusually long (up to 120 µm) and immotile (can’t move). Found only in: 1. Epididymis (of ductus deferens): absorptive structures. Arise from an apical cell protrusion with thick stem portions that are connected by a- actinin cytoplasmic bridges. Similar to microvilli: internal actin bundles cross-linked by fimbrin. Differences from microvilli: 1. ezrin protein anchors the actin filaments to the plasma membrane. 2. Longer (lateral additions of actin) 3. No villin at the tip (plus end) 4. Larger diameter 2. Sensory (hair) cells of the inner ear: serve as sensory mechanoreceptors. Uniform in diameter, show staircase pattern of increasing height. High density of actin, cross-linked by espin. Lack ezrin and a-actinin. Can be easily damaged à have molecular mechanism of regeneration = treadmilling effect (actin monomers are removed from the base and added to the tip, and everything is pushed down). 3. Cilia (trachea slide): Present on nearly all cells of the body Extensions of the apical plasma membrane (not a cytoplasmic protrusion) Axoneme: internal core of microtubules, which extends from a basal body (a centriole-derived, microtubule organizing center MTOC located in the apical region of a ciliated cell. Dark-staining band at the base of the cilia). The basal bodies appear as a continuous band in light microscope but in electron microscope the basal body of each cilium appears. 3  Ciliogenesis: o the first stage is generation of centrioles, which then assume function of basal bodies. Then there is an elongation stage of motile cilia, polymerization of tubulin molecules, creating 9+2 arrangement. Then axenome grows upward from the basal body, cell membrane is pushes outwardàmature cilium. o Ciliogensis depends on the bidirectional intraflagellar transport mechanism that supplies precursor molecules to the growing cilium. Classification according to functional characteristics: 1. Motile cilia: Move fluid and particles along epithelial surfaces. Trachea, bronchi (sweep mucus and other junk toward oropharynx for elimination), oviducts. Short, fine, hairlike structures, sit on basal bodies. 9+2 axonemal organization, from tip of cilium to its base: 9 pairs/doublets of circularly arranged microtubules, surrounding two center microtubules (which are separate but partially enclosed by a central sheath projection). Microtubules of axenome are highly stable and can resist depolymerization. Ciliary dynein: motor protein, shown on each of the 9 microtubule doublets. Uses energy from ATP. Radial spokes: extend from microtubule doublets toward the central ones, allowing movement. Basal body: 9 short microtubule triplets in a ring. No central microtubules. A and B continue into the axenome as (the) pairs, C microtubules extend into the transitional zone. Transitional zone: transition between basal body and the axenome. Origin of the two central microtubules. Basal body-associated structures: 1. Alar sheet: transitional fiber, from top end of the basal body C microtubule and into the cytoplasmic domain of the plasma membrane. 2. Basal foot: midregion of basal body. Coordinate ciliary movement. Associate with myosin. 3. Striated rootlet: protofilaments that anchor basal body within the apical cytoplasm. Contain rootletin protein. Ciliary movement originates from the sliding of microtubule doublets, which is generated by the ATPase activity of the dynein arms. Effective stroke (a rapid forward movement featured by a cilium doesn’t have enough ATP and remains rigid) and recovery stroke (slower, return movement of the previously rigid cilium, after enough energy is accumulated). 4  Motile cilia beat in a synchronous pattern, creating a wave across the epithelium called metachronal rhythm which allows moving mucus over epithelial surfaces or facilitating flow of fluid through tubules and ducts. Basal feet of basal bodies are responsible. 2. Primary cilia: Non-motile=no active movement (lack microtubule-associated motor proteins needed to generate motile force) àpassively bend due to fluid flow 9+0 pattern of microtubules No central pair of microtubules Its formation is synchronized with the cell cycle and with centrosome duplication Found in a variety of cells called primary cilia/monocilia. Each cell has only one such cilium. Also found in epithelial cells. In many mammalian cells, signaling through the primary cili seems to be essential for controlled cell division and gene expression. Function in secretory organs (kidney, liver, pancreas) as signal receptors, sensing a flow of fluid. Example: mechanoreceptors in glomerulus and tubular cells of the kidney. Fluid flow in renal tubules àcilia bendàCa2+ influx initiated. Polycystic kidney disease, as well as cysts in the pancreas and liver, may be caused by mutations in genes that affect development of primary cilia. 3. Nodal cilia: Found in embryos during early embryonic development 9+0 microtubule pattern (like primary cilia) Motile: contain motor proteins (dynein and kinesin)àmove in full circles, creating a course resembling a full cone (motile cilia did only half cone movement. Shown in table 5.2 on next page). Important for embryonic development: generate left-right asymmetry of internal organs. Without nodal cilia (or if nodal cilia are immotile)ànodal flow doesn’t occuràno flow detection by sensory receptors on the left side of the bodyàrandom placement of internal organsàprimary cilia dyskinesiaàsitus inversus (position of heart/abdominal organs are reversed). 5 6 Lateral domain and its specializations in cell-to-cell adhesion The lateral domain is characterized by having special proteins called cell adhesion molecules (CAMs) which are part of junctional specialization. Molecular composition of lipids and proteins of the lateral cell membrane is very different from the apicals. Later cell surface membrane of some epithelia may form folds and processes àtongue-and-groove margins between neighboring cells. Junctional complex: specific structural components that make up a barrier to the passage of substances between adjacent epithelial cells and attaches between the cells. There are three types: 1. Occluding junctions=zonula occludens (aka tight junctions): o Impermeableàform the primary intercellular diffusion barrier between adjacent cellsàmaintain physicochemical separation of tissue compartments. o Located at most apical point between adjoining cells àprevent migration of lipids and membrane proteins between apical and lateral surfacesàmaintain integrity of apical and lateral domains (example: Na/K ATPase is restricted to the lateral plasma membrane, below zonula occludens). o Recruit signaling molecules to the cell surface and link them with actin filaments of the cytoskeleton o Features network of anastomosing particle strands in which protein particles from opposing (adjacent) cell membrane surfaces cause complementary grooves in each otheràfunctional seal in intercellular space. o Three major groups of transmembrane proteins of the zonula occludens: 1. Occludin: a. Maintains barrier between adjacent cells AND between lat. And apical domains b. Present in most occluding junctions BUT without it cells may still have fully functional zonula occludentes 2. Claudin: a. family of about 24 proteins that form the backbone of zonula occludens strands b. able to form extracellular aqueous channels for paracellular passage of ions and other small molecules 7  c. mutation in claudin-14 gene à hereditary deafness 3. Junctional adhesion molecule (JAM): a. protein of the immunoglobulin family b. associates with claudins in endothelial cells o These three proteins have amino acid sequences in their cytoplasmic portions which attract PDZ-domain proteins (ZO-1, ZO-2, ZO-3) which they interact with during the formation of the zonula occludens and they also help the transmembrane proteins interact with actin cytoskeleton. o Play an important role in selective passage of substances from one side of an epithelium to the other. Two distinct pathways of transport across epithelium: 1. Transcellular: across the plasma membrane, via active transport and energy-dependent transport proteins and channels. Movement across apical plasma membraneà cytoplasmàacross lateral membrane, below level of occluding junctionà intercellular space 2. Paracellular: across zonula occludens between two epithelial cells. The amount of substances transported is relative to the tightness of the zonula occludens=regulation. o Permeability of the zonula occludens depends not only on the complexity and number of strands but also on the presence of functional aqueous channels formed by various claudin molecules. Combination and mixing ratios of claudins to occludins and other proteins found within individual paired zonula occludens strands determine tightness and selectivity of the seal between cells! o Although it is the job of zonula occludens to restrict free passage across epithelium, the adhesive properties of zonulae and maculae adherents guard against physical disruption of this barrier. 2. Anchoring junctions: o Provide mechanical stability to epithelial cells by linking the cytoskeleton of one cell to the cytoskeleton of an adjacent cell. o Lateral adhesions, found on both lateral cell surface and basal domain as well o Signal transduction capability à important roles in cell-to-cell recognition, morphogenesis, and differentiation. o Two types of anchoring cell-to-cell junctions: 1. Zonula adherens: interacts with actin filament network in the cell 2. Macula adherens or desmosome: interacts with intermediate filaments in the cell o Two types of cell-to-connective tissue matrix anchoring junctions: 1. Focal adhesions 2. Hemidesmosomes 8 o Cell adhesion molecules (CAMs): § Essential part of anchoring junctions on both lateral and basal cell surfaces. § CAMs of neighboring cells interact with one another either in heterotypic binding (binding between two different types of CAMs) or homotypic binding (between two CAMs of same type). § CAMs have selective adhesivenessàcells easily join and dissociate § CAMs bind their cytoplasmic domains to the cytoskeleton of the cellàcontrol and regulate intracellular processes associated with cell adhesion, proliferation, and migration § Four major families, based on molecular structure: 1. Cadherins: -transmembrane Ca2+-dependent CAMs -within zonula adherens (àlinked to actin) -homotypic interactions with similar proteins from neighbor cells -transmit signals that regulate mechanisms of growth and cell differentiation (embryonic cell migration) -E-Cadherin (transmembrane protein) acts as suppressor of epithelial tumor cells, working as E- cadherin-catenin complex bound to Ca2+ (no Ca à dissociation) to interact with actin filaments. 2. Integrins: -two transmembrane glycoproteins -heterotypic interactions -interact with: ECM molecules (collagens, laminin, etc.), actin, intermediate filaments -regulation of cell adhesion, control cell movement and shape, participate in cell growth and differentiation 3. Selectins: -expressed on WBC and endothelial cells -Mediate neutrophil-endothelial cell recognitionàinitiate neutrophil migration through endothelium of blood vessels into ECM -direct lymphocytes (WBC) into accumulations of lymphatic tissue (aka homing) -Heterotypic binding 4. Immunoglobulin superfamily (IgSF): -mediate homotypic cell-to-cell adhesions -play a role in differentiation, cancer and tumor metastasis, angiogensis, inflammation, immune responses, and microbial attachment. o Fascia adherens: broad faced, cell-to-cell attachments (desmosomes + broad adhesion plates that resemble zonula adherens of epithelial cells) between non-epithelial cells, i.e. cardiac muscle cells. Contains zonula occludens ZO-1 protein (found in tight junctions of epithelial cells). 9  o Macula adherens is found in small, localized sites of the later cell surface and is thus different than zonula adherens which is a continuous structure around the cell à a section perpendicular to the cell surface will often not include a macula adherens but will always include zonula adherens. o In the area of the macula adherens, desmogleins and desmocollins provide the linkage between the plasma membranes of adjacent cells. o The intercellular space of the macula adherens is much wider than that of the zonula adherens and is occupied by a dense medial band called the intermediate line. This line represents the extracellular portions of transmembrane glycoproteins of the cadherin family of Ca2+-dependent cell adhesion molecules (called desmogleins and desmocollins). o The desmogleins and desmocollins create a cadherin zipper in the area of the desmosome, as seen in between the cells in the above figure. 3. Communicating junctions (aka gap junctions or nexuses): o Intercellular communication: only cellular structure that allows direct passage (diffusion) of signaling molecules between adjacent cells ( providing specificity to basal lamina of different tissues. -Type XV collagen stabilizes external lamina in skeletal and cardiac muscles cells -Type XVIII collagen in vascular and epithelial basal laminae, angiogenesis -Type VII collagen links basal lamina to underlying reticular lamina. 2. laminins: glycoproteins, 15 different variations. Initiate assembly of basal lamina, involved in many cell-to-ECM interactions, and have binding sites for integrin receptors in the basal domain of the overlaying epithelial cells. 3. glycoproteins: entactin/nidogen (rodlike, sulfated). Link between laminin and the type IV collagen network. Bind calcium, support cell adhesion, and promote neutrophil chemotaxis and phagocytosis. 4. proteoglycans: most of the basal lamina volume. -Protein core to which heparin sulfate, chondroitin sulfate, or dermatan sulfate side chains are attached. -Very anionicà regulate passage of ions across the basal lamina. -Perlecan (400Kd, heparin sulfate). Most common. Binds to all other proteinsà additional cross-linkage. -Agrin: almost exclusively in the glomerular basement membrane, kidney. Major role in renal filtration and cell-to- ECM interaction. The molecular structure of type IV collagen determines its role in the formation of the basal lamina network suprastructure. Structure is 3 polypeptide chains, each with: a. short amino-terminus domain (7S domain) b. long middle collagenous helical domain c. carboxy-terminus globular non-collagenous domain (NC1 domain). There are 6 known chains of type IV collagen (named a1 to a6), and they form three sets of triple helical molecules known as collagen protomers. The different collagen protomers are found in different kinds of 13  basal lamina: 1. [a(IV)]2a2(IV): all basal laminae. 2. a3(IV) a4(IV) a5(IV): mainly kidney and lungs 3. [a5(IV)]2a6(IV): only in skin, esophagus, and Bowman capsule (kidney). Type IV collagen and laminins initiate the process of the self-assembly of the basal lamina. The first step is calcium- dependent polymerization of the laminin molecules, on the basal surface of the epithelial cells. Aided by Integrins. At the same time, type IV collagen associates with the laminin polymers. These two structures are joined together primarily by entactin/nidogen bridges. basement membrane: basal lamina + secondary layer of small-unit fibrils of type III collagen (reticular fibers) forming the reticular lamina (which is not a product of epithelium but belongs to the CT). basal lamina stains positive with both PAS and Ag impregnation. Attachment of basal lamina to the underlying CT is provided by: 1. Type VII collagen – anchoring fibrils. Associate with hemidesmosomes. Extend from basal lamina to anchoring plaques in the CT, or loop back to the basal lamina. The anchoring fibrils connect to type III collagen (reticular) fibers in the CT -> anchor the epithel. Mutations in collagen VII gene can result in detached epithelium in a disease called dystrophic epidermolysis bullosa (inherited blistered skin disease). 14  2. fibrillin microfibrils - Attach lamina densa to elastic fibers. Have elastic properties. Mutation in the fibrillin (FBN1) gene -> Marfan’s syndrome. 3. discrete projections of the lamina densa. Interact directly with reticular lamina -> additional binding site with type III collagen. Functions of the basal lamina: 1. Structural attachment: epithelial cells are anchored to basal lamina by cell-to-ECM junctions, and basal lamina is attached to underlying CT by anchoring fibrils and fibrillin microfibrils. 2. Compartmentalization: separate CT from epithelium, nerve, and muscle tissue. 3. Filtration, barrier: regulation of movement of substances to and from the CT, by ionic charges and integral spaces. Shown widely in the kidney. 4. Tissue scaffolding: basal lamina guides during regeneration, by maintaining the original tissue architecture. Also, epithelial cells can move easily on the basement membrane to the wounded area, in order to replace damaged cells. If the basement membrane is not intact à the epithelial cells cannot reach the wounded area OR it will take them more time to get there. 5. Regulation and signaling: many molecules in the basal lamina interact with cell surface receptors, influencing epithelial cell behavior. Affect morphogenesis, fetal development, wound healing, proliferation, differentiation, motility and gene expression and apoptosis (has been found to be involved in regulation of tumor angiogenesis in endothelial cells). Cell-to-extracellular matrix junctions Anchoring junctions maintain the morphological integrity of the epithelium-CT interface by two major anchoring junctions: 1. focal adhesions: anchor actin filaments of the cytoskeleton into the basement membrane 2. hemidesmosomes: anchor intermediate filaments of the cytoskeleton into the basement membrane TM proteins in the basal domain of the epithelial cell (integrins) also interact with the basal lamina. Focal adhesions -structural link between actin and ECM proteins, and attach long bundles of actin filaments into the basal lamina -prominent role during dynamic changes of epithelial cells (such as migration of epithelial cells during wound repair) -provide molecule bases for cell migration -also found in non-epithelial cells, such as fibroblasts and smooth muscle cells -integrins are the main family of TM proteins involved in focal adhesions. They interact with actin-binding proteins on the cytoplasmic face of the adhesion (such as alpha-actinin, vinculin, talin, paxillin). They also interact with focal adhesion kinase and tyrosine kinase. On the ECM face the integrins bind glycoproteins. 15 -important sites for signal detection and transduction, such as mechanosensitivity: detect contractile forces in EM and convert them into signals. Integrins transmit these signals into the cell. May also transduct signals from growth factor receptors. Hemidesmosomes -occur in epithelia that require strong, stable adhesion to the CT, epithelia subject to mechanical shearing forces that would tend to separate the epithelium from the underlying CT -variant of anchoring junction, similar to desmosomes (half desmosome) -found in cornea, skin, mucosa of oral cavity, esophagus, vagina, on basal cell surface -exhibits an intracellular attachment plaque on the cytoplasmic side of the basal plasma membrane, with protein composition similar to desmosomal plaque with desmoplakin-like family of proteins such as: plectin, BP 230, and Erbin. -in contrast to desmosomes, though, the majority of TM proteins in hemidesmosomes are integrin class of cell matrix receptors (rather than cadherin). Examples: 1. a4b6 integrin: heterodimer, 2 polypeptide chains. Interacts with type IV collagen suprastructure in basal lamina (containing laminins-5, entactin/nidogen, or perlecan). Laminin-5 form threadlike anchoring filaments that extend from the integrin molecule to the basement membrane (above photo, b). Mutations in laminin-5 gene àfunctional epidermolysis bullosa (hereditary blistering skin disease). **note: anchoring filaments mentiones above are NOT the same as anchoring fibrils! Anchoring filaments: laminin-5 and type XVII collagen, attach basal membrane of epithelial cell to underlying basal lamina. Anchoring fibrils: type VII collagen, attach basal lamina to underlying reticular (collagen III) fibers of the CT. 2. type XVII collagen (BPAG2, BP 180): TM molecule, regulates expression and function of laminin-5. 3. CD 151: glycoprotein. Participates in clustering of integrin receptors. Morphological modifications of the basal cell surface -prominent in cells that participate in active transport of molecules (like in proximal and distal tubules of the kidney) and in ducts of salivary glands 16 -example: infoldings that increase surface area à more transport proteins and channels present -many mitochondria at site to provide energy for active transport -mitochondria + basal membrane infoldings à striated appearance à named striated ducts Glands Two major groups, depending on release of product: 1. Exocrine glands: secrete products to a body surface (inner or outer), either directly or through epithelial ducts that are connected to a surface (lecture says that exocrine glands have secretory unit and duct). Classified as uni- or multi-cellular, according to number of cells in the gland: a. Unicellular: secretory component has only one secretory cell, among non-secretory cells (like goblet cells among columnar cells, where each goblet cell is a unicellular gland). b. Multicellular: more than one cell in various arrangements of parenchyme and presence/absence of branching of the ductile element. Two major groups: i. Sheet: simplest arrangement. Each surface cell is a secretory cell. (example: sheet of mucus- secreting cells lining the stomach and gastric pits) ii. Tube: tubular invaginations from the surface. The end pieces of the gland contain the secretory cells, and the duct portion of the gland connects the secretory cells to the surface. May be straight, branched, or coiled. iii. Alveolar/acinar: secretory portion of the gland is shaped like a flask. May be either single or branched. iv. Tubuloalveolar: if the secretory portion is a tube that ends in a saclike dilation. Simple/compound: depending on if the duct is branched or not. All variations of multicellular glands are shown in the following table: 17 2. Endocrine glands: no duct. Secrete their products (hormones) into the CT from which they enter the bloodstream to reach the target cell. 3. Paracrine: individual epithelial cells secrete a substance that affects other cells within the same epithelium, without entering the bloodstream. Secretory material diffuses into target cell. According to Antal, this is gland is a kind of endocrine gland, where the secretion doesn’t enter the blood system. 18 Cells of exocrine glands have three basic release mechanisms: 1. merocrine secretion: membrane-bound vesicles carry secretion to apical surface of cell à vesicles fuse with membrane à exocytosis, continuously in time. Most common. No loss of cytoplasm during secretion. Examples: pancreatic acinar cells, normal sweat glands, salivary glands, goblet cells). 2. apocrine secretion: exocytosis, at discrete moments in time: secretion released in apical portion of the cell, surrounded by thin layer of cytoplasm with an envelope of plasma membraneàsome loss of cytoplasm. Example: lactating (lipid secreting cells) of mammary gland, and apocrine glands of the skin. 3. holocrine secretion: the cell undergoes programmed cell death=apopotosisà secretion with cell debris discharged into gland lumen. Example: sebaceous glands of skin. Classification of exocrine glands according to chemical nature of secretion: Mucus and serous glands secretory cells of exocrine glands in various body tubes are mucus, serous, or both. The secretory unit is called acinus. mucus secreting cells: o secretions are viscous, slimy, high in carbohydrates o Mucinogen granules in the secretory cells are PAS positive o water solubleàlost during formalin fixationàcytop. of mucus secreting cells appears empty in H&E o nucleus is flattened (squamous) against base of cell by accumulated secretory product o lumen is bigger and more irregular o Examples: 1. goblets cells 2. secretory cells of sublingual gland (salivary) 3. surface cells of stomach 19  serous secreting cells: o secretions are watery and rich in proteins o nonglycosylated or poorly glycosylated secretions o nucleus round/oval and (more) central (?) o lumen is smaller and more regular o apical cytoplasm is intensely stained with eosin o protein synthesizingàmuch rERàbasophilic perinuclear cytoplasm o Examples: parotid gland and pancreas. submandibular gland contains acini of both mucus and serous cells. In routine tissue preparations, the serous acinar cells are shaped like demilunes (half-moons) at the periphery of the mucus acinus. exocrine glands are also classified according to localization: endoepithelial or exoepithelial. Epithelial cell renewal Most epithelial cells have a finite life span Surface epithelia and epithelia of many simple glands are continuously renewing cell populations. The rate of cell turnover is a characteristic of a specific epithelium (for example: cells lining the small intestine are renewed every 4-6 days, and stratified squamous epithelium of the skin is replaced during 28 days). Adult stem cells, located in niches, produce the replacement cells by mitotic activity. In small intestine, niches of adult stem cells are located in the lower parts of the intestinal glands/crypts, then they migrate and differentiate into 4 principal cells: 1. enterocytes (columnar, absorptive) 2. goblet cells (mucus-secreting) 3. enteroendocrine (regulatory and hormone secreting) 4. Paneth cells (migrate down, while the other three migrate up to the tips of the villi where they undergo apoptosis) Transcription factor Math1 in intestinal epithelium determines the fate of the cell (secretory cells such as Paneth, goblet, and enteroendocrine cells have increased expression of Math1). Epithelial cells in more complex glands have stable cell populations in which there is little mitotic activity and cells may live for a long time (like in the liver, though there is stimulated mitotic activity of the healthy liver tissue if there is loss of damaged liver tissue). 20 From class (Prof. Antal): glands Axillary skin slide – H&E Three types of glands are shown: eccrine (merocrine), apocrine, and holocrine (sebaceous). Glands have both a secretory compartment (lighter) and a duct/conducting compartment (darker). We will learn about the ducts next semester… 1. Eccrine glands are merocrine sweat glands. They store their watery secretum into secretory vesicles which are transport to the apical membrane where they wait for a signal. Upon signal the vesicles fuse with the membrane and the secretum is released. The height of the cell doesn’t change. They open up individually and directly to the external surface of the skin. Lumen has regular contour. Three kinds of cells make up the secretory acini: 1. Clear cells: close to basement membrane. Light staining, watery substance inside, pyramidal. 2. Dark cells: close to lumen, darker (cells are active and make a lot of proteins à lots of RNA à basophilic staining). Inverted pyramids, with base facing the lumen and apex away from lumen. 3. Myoepithelial cells: flat cells with flat nuclei. Surround outer surface of basement membrane. Able to contract à ejection/secretion from secretory acini. 2. Apocrine glands Lumen is large and has an irregular contour due to cells of variable heights (before ejection à tall cells, after ejection à short cells) As the lipid droplets grow, they arrive to the apical membrane (where they can really be seen) à cell volume increasesàonly height of cells increase, not width! When apical membrane opens à lipid substance together with some cytoplasm is lost to lumen à height of cell decreases. Secretum is lipid-rich and is not in vesicles but in lipid droplets (smelly) Open up in adjacent hair shaft and NOT directly to surface (unlike merocrine which do open up directly) Myoepithelial cells are here 3. Sebaceous glands, holocrine secretion These glands develop together with hair follicles, and are seen adjacent to them on slide Cells store lipid-rich secretum in small droplets (unlike apocrine gland cells which store secretum in one big lipid droplet) As we move from the bottom/inside of the gland upwards we see the maturation of the cells: in stratum basale layer we see mitotically active cells à we move up and we see cells with many small (unstained) lipid droplets (we find red cytoplasm in between these droplets). These cells have large nuclei, and as we keep moving up we see the nuclei is becoming smaller à cell death is happening as we go up the layers of the gland As we move up more we see that the cells lack a nucleus and that the cytoplasm is broken à these cells have undergone apoptosis! Cytoplasm and lipid production have been secreted to the conducting compartment of the gland, and what is left are completely disintegrated cells. 21  The secretion is called sebum and is made up of cytoplasm debris and the lipid rich substance that was produced by the cells à holocrine secretion! Secretion to space between hair shaft and secretory acini – not directly to surface! Submandibular gland slide – H&E Salivary glands are shown, with regular lumen, all cells have same height (cuboidal) à merocrine There are three different kinds of secretory acini: 1. Serous secretory unit: Spherical nucleus Strong basophilic staining (large protein synthesis lots of rER and RNA) Secrete watery saliva with many proteins, especially amylase 2. Mucous secretory unit: Flat nuclei, pressed against base Cytoplasm is almost unstained because the mucus was dissolved/lost during histological processing Secrete a viscous secretion, mucus 3. Mixed secretory axini: Both types of cells are present- mucous secreting cells, with half-moon shaped serous cells around them= serous demi lunes àMixed but more serous acini than mucous acini! (sublingual has more mucous than serous) Sublingual gland slide – PAS + Hematoxylin PAS staining is specific for mucus substance – stains it in purple. Mucus was preserved here and it is seen! Lumen with red end product can be seen, and nuclei are pressed against base of cells Serous secretory cells are unstained (and are in small amounts) Hematoxylin stains three things: 1. Nuclei (polyanions) 2. Ca2+ 3. Mucin (mucous substance) Differences between serous and mucous secreting acini: 1. Size: mucous are larger 2. Lumen: mucous have larger lumen, while in serous the lumen is sometimes unseen 3. Nucleus: spherical (serous) vs. flat (mucous) 4. Staining: serous are basophilic and mucous are light (almost unstained) Ureter slide – H&E Urothelium/transitional epithelium is seen here Look at cells outlining the lumen and see three kinds of cells: 1. Cuboidal epithelial cells 22 2. Umbrella cells: most superficial layer, cells are wider than the underlying cells à one umbrella cell covers more than one cuboidal epithelial cell. Have a thick apical cell membrane which is discontinuous à Plaques= small thickened areas of membrane are shown with a unique, rigid protein structure, which enables the cells to create infoldings and thus change the thickness of the whole epithelial layer! When there is urine in the bladder à higher surface area is needed to hold the urine à no infoldings When there is urine conduction/no urine à less surface area is needed àcells cause infoldings 3. Pear-shaped cells: light cells, pear shaped, seen in between the other two kinds of cells 23  Connective tissue General structure and function of connective tissue CT = cells + ECM (intercellular material) which is produced by the cells of the CT. ECM= structural CT fibers (collagen, elastic, reticular) + interfibrillar material (specialized carbs and proteins that constitute the ground substance). Unlike epithelial cells, CT cells are NOT connected to each other! Separated by intercellular space Majority of CT contain blood and lymph vessels, and nerves The function of the CT depends on the cells and fibers present within the tissue and the composition of the ground substance in the ECM. Examples: 1. Loose CT: fibroblasts (produce the extracellular fibers and the ground substance), and many other cells as shown in the figures: 2. Bone tissue: only one cell type – osteocyte (produce the fibers that make up the bulk of the bone tissue). Classification of CT is based on composition and organization of the cellular and extracellular components, and on its function: undifferentiated +wharton’s jelly of umbilical cord (remains undiff.) Cell-rich connective tissue 1 Embryonic connective tissue Mesoderm gives rise to almost all CT of the body, except for the head region (where specific cells are from ectoderm by way of neural crest cells). Mesodermal cells and specific neural crest cells proliferate and migrate to establish mesenchyme (sometimes referred to as ectomesenchyme in the head region) which is the undifferentiated (unspecialized) CT of the early embryo. Mesenchymal cells can differentiate to specialized CT cells, depending on how they proliferate and how they are organized. Example: the embryonic CT that’s present in the early embryo and within the umbilical cord: 1. Mesenchyme: primarily found in the embryo. Has small spindle shaped cells with processes and gap junctions. Cells are undiff., pluripotent. Collagen (reticular) fibers are present, and the extracellular space is with viscous ground substance. 2. Mucous CT: in the umbilical cord. Widely separated spindle-shaped cells. Gelatin-like ECM with “wharton’s jelly” ground substance which is found between thin collagen fibers. Mesenchymal cells produce hyaluronic acid rich ECM Mesenchyme develops from mesoderm of the embryo via epithelo-mesenchymal transformation of cells and ECM production Connective tissue proper Cells + ECM (intercellular material). ECM= CT fibers + ground substance (glycosaminoglycans, proteoglycans, glycoproteins) Two general subtypes: 1. Loose CT (areolar): -small amount of collagen fibers -lots of ground substance (which, due to its high viscosity, plays an important role in diffusion of O2 and nutrients from vessels and CO2 and metabolic waste to the vessels) -primarily located beneath epithelia that cover the body surfaces and line its internal surfaces -associated with epithelium of glands and surrounds smallest blood vessels -pathogens that passed the epithelium are destroyed by the immune system here -lamina propria is the loose CT of the mucous membrane and contains large numbers of immune cells 2. Dense CT: a. Dense regular CT: § contains mostly collagen fibers, little ECM, and the cells that produce and maintain the fibers are packed between the fiber bundles § the fibers are arranged in parallel arrays and are densely packed à maximum strength 2 § main functional component of tendons, ligaments, and aponeurosis: 1. tendons: tendinocytes are the fibroblasts situated between bundles of collagen fibers. In H&E-cross section, Tendinocytes appear stellate. In H&E-longitudinal section, Tendinocytes appear as rows of flattened basophilic nuclei. Epitendineum: thin CT capsule around the tendon, with less orderly fibers. Endotendineum: CT extension of the epitendineum which subdivides the tendon to fascicles. 2. ligaments: fibers are less regularly arranged that in tendons à more elastic. some ligaments need extra elasticity (i.e. in the spinal column, ligament flava)àmore elastic fibers and less collagen fibers=elastic ligaments. 3. aponeuroses: resemble broad, flat tendons. orthogonal array= fibers in multiple layers, with 90 degrees between the fibers of each layer in each layer, the fibers are arranged in parallel à dense regular CT b. Dense irregular CT: § contains mostly collagen fibers, and few cells (typically fibroblasts) à strong § relatively little ground substance § irregular à fibers arranged in bundles in various directions § submucosa=dense irregular CT that many hollow organs possess. Allows these organs to resist excessive stretching and distortion. § reticular/deep layer of the dermis=relatively thick layer of dense irregular CT in the skin. Provides resistance to tearing. 3 Cells of connective tissue proper: 1. Resident cells: three groups: a. mesenchymal cells, fibroblasts, fibrocytes, myofibroblasts (rich in motor proteins à able to contract) b. Plasma cells, macrophages, mast cells c. Adipocytes, smooth muscle cell, adult stem cells, pericytes (perivascular, undiff., can diff. into smooth muscle cells/osteoblasts/adipocytes/fibroblasts/and more). 2. Transient cells: (blood born cells) white blood cells: lymphocytes (T/B cells), granulocytes (neutrophils- phagocyte bacteria, eosinophils-release histamine), and monocytes (phagocytic, APC, diff. into macrophages). These cells migrate through the walls of the capillaries (extravasation) and are immunocompetent cells (fulfill immunological functions in the CT). they may return to the blood stream or differntiate into resident cells. More info on cells will come soon… Connective tissue fibers Each fiber is produced by fibroblasts and is composed of proteins with long peptide chains Three types of fibers: 1 Collagen 2 Reticular 3 Elastic Collagen fibers and fibrils Most common CT component; main structural protein in various CT of animals Flexible and strong Stain with eosin and other acidic dyes, aniline blue (Mallory’s stain), and the light green stain in Masson’s. Arranged in subunits called fibrils which are uniform in diameter (in dense reg. CT of tendons they are very large). In each fibril, the collagen molecules align head to tail in overlapping rows (the strength is given by the bonds between the molecules in different rows). Collagen molecule: o right-handed triple helix (~300 nm) of three intertwined polypeptide (a) chains. The helix is stabilized by hydrogen bonds between OH groups (of hydroxylated proline and lysines). The triple helices form supramolecular arrangement à cross striations are shown o Every 3rd amino acid is glycine, with hydroxyproline before it and proline after it. Also rich in alanine. Collagen is a glycoprotein (sugar groups associate with the helix – posttranslational modification) 4  There are 28 kinds of collagen, named collagen I to collagen XXVIII, each with different a chain combinations (homo/heterometric). Examples: o Type I collagen: heterometric- 2*a1 and 1*a2 so: [a1(I)]2a2(I). Found in loose and dense CT. o Type II collagen: homometric- [a1(II)]3. Found in hyaline and elastic cartilage. Most of the collagen molecules polymerize into supramolecular aggregates such as fibrils or networks, and there are several classes of collagen based on the polymerization pattern: o Fibrillar collagens: types I, II, III, V and XI. Glycine-proline-hydroxyproline repeats. o Fibril-associated collagens with interrupted triple helixes (FACITs): interruptions in the helixes à flexibility. Types IX, XII, XIV, XVI, XIX, XX, XXI, XXII. Regulate fibrillogenesis and diameter of fibers, stabilize fibers, etc. o Hexagonal network-forming collagens: types VIII and X. o Transmembrane collagens: types XIII (in focal adhesions), XVII (in hemidesmosomes), XXIII (in metastatic cancer cells), and XXV (a brain-specific collagen). o Multiplexins: multiple triple-helix domains. Types XV and XVIII in the basement membrane. o Basement membrane-forming collagens: type IV (in b.m. of epithelium), types VI and VII. Biosynthesis and degradation of collagen fibers Biosynthesis: Production of fibrillary collagen (types I, II, III, V, and XI) involves several events within the fibroblast (in membrane- bound organelles in the cell) that produce procollagen, precursor of collagen. Production of the fibril occurs outside of the cell, involving enzymatic activity and assembly in the ECM. Collagen a chains are synthesized in the rER as pro-a chains (preprocollagen molecules). The newly synthesized polypeptides are discharged to the cisternae of the rER where posttranslational modifications of the pre-procollagen molecules occur: 1. SS cleaved in N terminus 2. proline and lysine are hydroxylated (ascorbic acid, vitamin C, is required as a cofactor) 3. glycosylation occurs 4. globular structure formed at the C terminus 5. triple helix is formed by three a chains (starting at C terminus) 6. intrachain and interchain hydrogen bonds form 7. chaperone protein hsp47 binds to form procollagen. 8. Procollagen is then packed into secretory vesicles and transported out of the cell, while converting to a mature collagen molecule (by cleavage of the uncoiled ends of the procollagen by procollagen peptidase). 9. Aggregated procollagen molecules form collagen fibrils in fibrillogenesis. 10. The cell creates invaginations on the cell surface called coves where molecules may concentrate where the self-assembly, head to tail alignment, will occur. Covalent bonds between lysine and hydroxylysine aldehyde groups form to create the fibril. 5 Extras from lecture slides: Synthesis: Collagens are synthesized intracellularly as protocollagens Then they are secreted from cell cytoplasm to extracellular space Then they are enzymatically cleaved (C and N terminals are removed): propeptides à tropocollagen Tropocollagen molecules then associate to form collagen fibrils, which then form collagen fibers Three collagens types can associate into fibers: type I (skin, joint capsule, fibers, tendons, bone, fibrous cartilage), type II (hyaline cartilage), and type III (forms reticular fibers, is highly glycosylated, produced by reticular cells in internal organs àforms stroma of internal organs, and is part of basement membranes). Type IV collagen forms a network in the lamina densa (layer of basal lamina) Cells adhere to collagen fibers (and other ECM proteins) via transmembrane receptors such as integrins. Integrins can bind cells to multiadhesive proteins: intracellular domain connects to actin filaments, and extracellular connects to collagen. They regulate cellular functions and connect to each other àextracellular macromolecular networks are formed. All collagens can be stained blue with Azan staining. 6 7  Collagen fibrils usually consist of more than one type of collagen. For example, type I collagen fibrils often contain small amounts of types II, III, V, and XI. Collagen molecules are largely synthesized by CT cells, including fibroblasts. Collagen molecules of basement membranes are produced by epithelial cells. Synthesis is regulated by growth factors, hormones and cytokines: Inhibition: Steroid hormones (glucocorticoids) Stimulation: TGF-b (transforming growth factor) and PDGF (platelet-derived growth factor) Collagen degradation: Initial fragmentation of insoluble collagen occurs through mechanical wear, the action of free radicals, and protease cleavage (collagenase, members of the family of Matrix Metalloproteinases). The resulting collagen fragments are phagocytosed by macrophages and degraded by lysosomal enzymes Excessive degradation is observed in diseases such as rheumatoid arthritis (degradation of cartilage collagen) and osteoporosis (degradation of bone collagen). Two main pathways of degradation: 1. Proteolytic degradation: outside the cells, through MMP enzymes (matrix metalloproteinases). In general, triple-helical un-denatured forms of collagen are resistant to degradation by MMPs, while damaged or denatured collagen (gelatin) is degraded by many MMPs – gelatinase prominent. 2. Phagocytotic degradation: intracellular. Involves macrophages or fibroblasts (work in lysosomes). Turnover of collagen is low in adult tissues (0.1-1% changes daily). Mechanical load can stimulate collagen synthesis in tendons, ligaments, bone or cartilage. Reticular fibers provide a supporting framework for the cellular constituents of various tissues and organs. Both reticular fibers and type I collagen consist of collagen fibrils Composed of type III collagen, with individual fibrils of narrow diameter (20 nm) and branching pattern. Don’t stain in H&E but have a thread-like appearance in light microscopes. Contain more sugar groups than collagen fibers à stained with PAS (and Ag impregnation, as they are argyrophilic, as shown in photo). Found in the boundary between epithelium and loose CT Found surrounding adipocytes, small blood vessels, nerves, and muscle cells. Also in embryonic tissue. Indicators of tissue maturity: prominent in initial stages of wound healing and scar tissue formation, but as embryonic development or wound healing progresses reticular fibers are gradually replaced by stronger type I collagen fibers. Reticular cells in hemopoietic and lymphatic tissues produce the collagen of the reticular fibers. In most other locations, reticular fibers are produced by fibroblasts. 8 Elastic fibers Typically thinner than collagen fibers Arranged in branches to form a 3D network à high expansibility. Fibers are interwoven with collagen fibers to limit the dispensability of the tissue and to prevent tearing from excessive stretching. Can’t always be distinguished from collagen fibers in H&E because they don’t stain well with eosin (collagen-red, elastic-pink). May be stained brown with orcein or black with resorcin-fuchsin. Produced by many of the same cells that produce collagen and reticular fibers, particularly fibroblasts and smooth muscle cells. composed of two proteins (produced intracellularly, then secreted to ECM where fibers are built): 1. Fibrillin: Secreted first. Forms microfibrils. Fibrillin-1 is a glycoprotein that forms fine microfibrils and is used as a substrate for elastic fiber assembly. Abnormal expression of fibrillin gene (FBN1) is linked to Marfan’s syndrome (CT disorderàtall, long limbs, flexible joints, flexible skin). 2. Elastin: makes up the central core of elastic fibers. Globular protein that connects to the microfibrils. -Rich in glycine and proline (like collagen). Unlike collagen it is poor in hydroxyproline and lacks hydroxylysine. -Elastin contains desmosine and isodesmosine (large AAs) which create covalent bonds between each 4 elastin molecules à cross links of microfibrils à expansibility but low strength. -Coded by one of the largest genes in the human genome. -Synthesis parallels collagen production, and both processes may occur simultaneously in a cell (synthesis of both procollagen and proelastin is coordinated by signal sequence in the beginning of the polypeptide chain. -Elastases can degrade elastic fibers Hydrophobic àrandom coiling of the fibers. Elastic material is a major extracellular substance in vertebral ligaments, larynx, and elastic arteries. In elastic ligaments, the elastic material is in thick fibers within collagen fibers (like in ligament flava in the vertebral column). Finer fibers are present in elastic ligaments of the vocal folds in the larynx. 9 Extracellular matrix Complex structural network that surrounds and supports cells within the CT. Contains variety of fibers such as collagen and elastic fibers, as well as the part of the ECM that occupies the spaces between the cells and fibers called ground substance: a variety of proteoglycans, multi-adhesive glycoproteins, and GAGs (glycosaminoglycans) Each CT cell secretes a different ratio of ECM molecules à formation of many different architectural arrangementsà ECM is different in different tissue. The ECM anchors cells within tissues through cell-to-ECM adhesion molecules à cell migration pathways The ECM binds growth factors àmodulates cell growth and affects embryonic development. The ECM influences transmission of information across the plasma membrane of CT cells. Ground substance More than just mechanical and structural support, but also forms direct pericellular macromolecular environment of cells. Transmits mechanical and osmotic changes to cells, influences proliferation, differentiation, and migration. Three types of substances are found: 1. Glycosaminoglycans, aka GAGs: polysaccharides, with disaccharide subunits (hexose-amine+hexouronic acid; mostly sulfated). o Responsible for physical properties of ground substance: highly negatively charged à stain metachromatic (strongly) with basic dyes (such as toluidine blue) + attract water à form hydrated gel à ground substance has a gel-like compositionà rapid diffusion of water-soluble molecules is allowed. o Hyaluronan (hyaluronic acid) is a special GAG, non-sulphated, that doesn’t form side chains of proteoglycans but does form proteoglycan aggregatesàcommon in ground substance of cartilage àallows cartilage to resist compression without inhibiting flexibility, àcartilage is an excellent shock absorber. Hyaluronan also immobilizes certain molecules in desired locations of the ECM. Produced by hyaluronan synthases, degraded by hyaluronidases. o Most GAGs in CT are linked to core proteinsàproteoglycans form (syndecan, aggrecan). See figure 6.17. 2. Proteoglycans (PG): fibrillary core protein o GAG side chains account for the physiochemical properties of the molecules o Some PGs bind to hyaluronanàaggregates o Syndecans: integrant plasma membrane HS-PGs with receptor functions 10 3. Glycoproteins stabilize the ECM and link it to the cell surface. o Fibronectin is the most common multi-adhesive glycoprotein in CT. o Other multi-adhesive glycoproteins such as laminin, tenascin, and osteopontin and osteonectin are present. See figure 6.18: o Fibronectin and laminin fibers can connect to integrin molecules on cell membrane à signal transduction through either cytoskeleton-mediated signals or soluble signalsàinfluence of organization of sytoskeleton and influence of gene expression àimpact on cell diff., proliferation, protein synthesis, locomotion and cell shape. See figure below: 11 Connective tissue cells Connective tissue cells are either of the resident cell population or the wandering cell population. Resident cell population are stable, little movement, permanent residents of the tissue. Examples: 1. Fibroblasts (and myofibroblasts): a. Differentiated mesenchymal cells. b. Principal cell of the CT. c. Responsible for production of all components of ECM in CT proper: synthesize collagen, reticular and elastic fibers and the complex carbohydrates of the ground substance. (A single fibroblast is capable of producing all of the ECM components) d. Elongated/disc-like cells with basophilic staining (especially during active growth or during tissue repair, the active fibroblasts are seen with more extensive cytoplasm and may show basophilia due to more rER); only nucleus (euchromatic) is shown in H&E e. Once the CT is ready, the fibroblast becomes a Fibrocyte, which is the less active form with eosinophilic cytoplasm and heterochromatic nucleus. f. Myofibroblasts: elongated, spindly CT cells. Are not readily identified in H&E. Contain a-smooth muscle actin filaments which attach to the plasma membrane forming a cell-to-ECM anchoring junction called fibronexus. This arrangement enables a mechano-transduction system: force generated by contraction of intracellular actin is transmitted to the ECM. Photomicrograph and electron micrograph of a 2. Macrophages (histiocytes): macrophage (BV=blood vessel, N=neutrophil): a. Phagocytotic cells, derived from monocytes. b. Members of mononuclear phagocytotic system (MPS) c. Difficult to identify in light microscope or with regular stains unless they are mid-phagocytosis, but have characteristics such as indented/kidney shaped nucleus and many lysosomesà eosinophilic cytoplasm. d. Large golgi apparatus, rER and sER, mitochondria, and secretory vesicles. e. Important role in immune responses- they are APCs that have MHC II on their surface for displaying proteins (antigens) that CD4 T lymphocytes interact with and may trigger a response. f. If a large foreign body is found, the macrophages may fuse to form a large, 100 nucleus-wide cell called foreign body giant cells (Langhans cells). 3. Adipocytes: CT cell differentiated from mesenchymal stem cells, stores neutral fat, produces hormones, inflammatory mediators, and growth factors. Located in loose CT. If accumulate in large numbers à adipose tissue. 12  4. Mast cells: a. Develop in bone marrow from HSC (hemopoietic stem cells); travel via blood to fully differentiate in CT. (Before differentiating in the CT, the cells circulate in the blood as a-granular cells). b. Spherical nucleus and cytoplasm full of intensely basophilic granules (with basic dyes such as toluidine blue) due to the proteoglycan heparin that is found in them. (These granules are also present in basophils). c. Major cellular component of allergic reactions such as inflammation. d. The granules contain mediators of inflammation called preformed mediators (such as histamine, heparin, serine proteases), and upon activation of the mast cells they produce and secrete newly synthesized mediators (mostly lipids and cytokines, such as Leukotriene C, LTC4, tumor necrosis factor a, TNF-a, and interleukins). e. Have IgE receptors f. Triggering of mast cells through Fc receptors (IgE binding to them) and/or through IgE independent mechanisms à granule exocytosis (degranulation) of inflammatory mediators (i.e. TNF-a) into the ECM. g. Especially numerous in the CT of skin and mucous membranes and in the meninges. h. Are not found in the CT around small blood vessels within the brain and spinal cord. 5. Adult stem cells: cannot differentiate into multiple lineage, reside in niches of stem cells in tissues and organs (excluding bone marrow) that are called tissue stem cells. Bone marrow contains hemopoeiteic stem cells, multipotent adult progenitor cells, and bone marrow stromal cells. Niches of adult stem cells are called mesenchymal stem cells and are found in loose CT in adults. -Pericytes are found around capillaries and venules and they are mesenchymal stem cells. During development of new vessels, cells with pericyte characteristics may diff. into smooth muscle. The fibroblasts and blood vessels within healing wounds develop from mesenchymal stem cells associated with the tunica adventitia of venules. Wandering cells population (transient) are cells that have migrated into the tissue from the blood in response to specific stimuli. Examples: 1. Lymphocytes: smallest of wandering cells, hetergoneous population of at least three major functional cell types: T, B, and NK (natural killer) cells. 2. Plasma cells: antibody producing cells derived from B lymphocytes. Common in loose CT. Large, ovoid, basophilic cytoplasm fur to an extensive rER. Spherical, heterochromatic nucleus (chromatin is found 13  in two varieties: euchromatin and heterochromatin. Distinguished cytologically by how intensely they stain – euchromatin is less intense, while heterochromatin stains intensely, indicating tighter packing). 3. Neutrophils 4. Basophils: develop and differentiate in bone marrow, released to circulation as mature WBC. They share many features with mast cells (granulocytes, secrete similar mediators, Fc receptors for IgE, participation in allergic reactions). 5. Eosinophils: function in allergic reactions and parasitic infections, and may be observed in the lamina propria of the intestine. 6. Monocytes More from lecture slides: Basal lamina: special pecicellular ECM of epithelial, muscle, adipose and Schwann cells. During locomotion of cells, dynamic reconstruction and degredation of cell-matrix connection occurs Mucopolysaccharidoses are inherited malfunctions in degradation of glycosaminoglycans (GAGs) due to mutations in lysosomal enzymesà intracellular accumulation of GAGsàdisturbed embryonic development: 14  Histology I 2 SCT: nd Adipose Tissue. Cartilage. Bone: Development and Growth. Muscular Tissue. The Histology of Blood Vessels, Blood and Bone Marrow. Production of Blood Cells. *These notes are based on: Ross Histology book 7th edition, lectures slides, and notes from class (taught by Professor Antal). Good luck! Lior Onn  Adipose Tissue Overview Specialized connective tissue for energy homeostasis. Specialized cells: Adipocytes (found in loose connective tissue) 1. Specialized for storage of lipid 2. Generate ATP 3. Produce hormones 4. Produce cytokines 5. “Sense” energy balance in body In order to meet the body’s energy demands when nutrient supplies are low, adipose tissue stores excess energy within lipid droplets in the form of triglycerides (the body has limited capacity to store carbs and protein). How? Takes fatty acids from the blood and converts them into triglyceride within the adipocytes. This energy can be rapidly released for use at other sites in the body when needed. In the event of food deprivation, triglycerides are an essential source of both water and energy. Adipocytes secrete paracrine and endocrine substances to regulate energy metabolism àadipose tissue is considered a major endocrine organ. Active contribution in the regulation of energy balance. There are both white adipose tissue and brown adipose tissue, based on color in living state. White is predominant in adult humans, while brown is present in fetus and diminishes during first decade. Each adipocyte is surrounded by basal lamina which connects the adipocyte to stromal elements of adipose tissue. Stroma of adipose tissue contains undifferentiated mesenchymal cells, blood vessels, reticular fibers, reticular cells, mast cells, macrophages, and more. The stroma cells are: reticulocytes, macrophages, mesenchymal cells. Generate ATP and/or heat, metabolic water (hump of camel!). Forms cushions around joints and internal organs (mechanical and structural function). Located primarily in skin as subcutaneous fat, and in the peritoneal tissue of the abdominal cavity as visceral fat. Adipocytes develop from mesenchymal cells during embryonic life, and can be generated from adult mesenchymal stem cells (MSC) residing in the adipose tissue stroma during extra-uterine life. Staining: oil red O (lipid staining dye) or heavy metal salts (OsO4) osmium: 1 White Adipose Tissue (unilocular)(univacuolar) Function of White Adipose Tissue Predominant type in adult humans. Forms “panniculus adiposus” or “hypodermis” layer in connective tissue under the skin. Thermal conductivity of adipose tissue is only half that of skeletal muscleàthermal insulation against cold, by reducing the rate of heat loss. Provides cushioning of vital organs, such as: GI, heart, eyeballs, kidneys. Both sexes have a mammary fat pad. In females it is important for the lactating breast function. Leptin, a hormone involved in the regulation of energy homeostasis, is exclusively secreted by adipocytes. It’s a satiety factor, active in endocrine signaling between brain and adipose. Adipocytes synthesize and secrete other hormones as well: 1. Angiotensinogen (AGE) (vasoconstrictor) 2. Adiponectin (increases insulin sensitivity, fatty acid oxidation) 3. Resistin (obesity, diabetes; insulin resistance) 4. Steroid hormones. Obesity increases the secretion of growth factors and cytokines. 2 Differentiation of Adipocytes Differentiate from mesenchymal stem cells under the control of PPARγ/RXR (transcription factors). Specific cell type derived from undifferentiated mesenchymal stem cells associated with the adventitia of small venules. Initially develop from stromal-vascular cell along the small blood vessels in the fetus and are then free of lipids. Primitive fat organs: collections of proliferating early lipoblasts and proliferating capillaries. Early lipoblasts look like fibroblasts (elongated configuration, multiple cytoplasmic processes, abundant endoplasmic reticulum and Golgi apparatus). During differentiation, small lipid inclusions appear at one pole of cytoplasm and an external lamina. Mid-stage lipoblast: oval configuration, excessive concentration of vesicles and small lipid droplets around nucleus, glycogen particles in periphery. Late lipoblasts: large, spherical, much sER and little rER, small lipid droplets have come together to form a single large lipid dropletàunilocular adipocytes or mature lipocytes. This large lipid droplet pushes the nucleus to the side of the cell. Structure of Adipocytes and Adipose Tissue Nucleus is flattened and displaced to one side of the lipid mass, with cytoplasm ring around the lipid (the thin strands that separate adjacent adipocytes represent the cytoplasm of both cells, as well as the ECM). During histological sectioning, lipid is lost through extraction by organic solvents such as xylene. Adipose tissue is richly supplied with blood vesselsàcapillaries are found at the angles of the meshwork where adjacent adipocytes meet Adipocytes are surrounded by reticular fibers (type III collagen), secreted by the adipocytes themselves. Shown in silver stains. Lipid mass in adipocytes is not membrane bounded, but has a layer of vimentin filament around itàseparation of the hydrophobic content of the lipid droplet from the hydrophilic cytoplasm matrix 3 Regulation of Adipose Tissue Brain-gut-adipose axis: interconnected hormonal and neural signals coming from adipose tissue, alimentary tract, and CNS. Regulates appetite, hunger, satiety, and energy homeostasis. Amount of an individual’s adipose tissue is determined by two physiological systems: short-term weight regulation and long- term weight regulation: 1. Short-term weight regulation: controls appetite and metabolism daily. Associated peptide hormones: a. Ghrelin (appetite stimulant, acts on hypothalamus). Produced by gastric epithelium à makes anterior lobe of pituitary gland release growth hormones. b. Peptide YY (PYY) (appetite suppressant, acts on hypothalamus). Produced by the small intestine. 2. Long-term weight regulation: controls appetite and metabolism on a continual basis (months or years). Associated hormones: a. Leptin: levels of leptin mRNA are elevated in obese humans àtheir adipocytes are resistant to leptinàleptin doesn’t reduce amount of adipose tissue in obese. Protects body against weight loss during food deprivation. Appetite regulation. b. Insulin: regulates blood glucose levels; enhances conversion of glucose into triglyceride (of the lipid droplets) by adipocytesàaccumulation of adipose tissue. Increases insulin sensitivity. Stimulates lipid synthesis enzymes and inhibits hormone- sensitive lipaseàblocks release of fatty acids. c. Thyroid hormone, glucocorticoids, hormones of pituitary gland. Mobilization: stimulation by neural or hormonal mechanismsàbreak down of triglycerides into glycerol and fatty acids. Fatty acids pass through the adipocyte cell membrane to enter capillary (carrier protein: albumin). The FAs then pass to other cells through the blood and are used as metabolic fuel. Neural mobilization: important during fasting and severe cold. Norepinephrine leads the activation of lipases that split triglycerides, which is an early step of mobilization of lipids. Hormonal mobilization involves hormones and enzymes that control fatty-acids release from adipocytes. Examples: o Insulin, thyroid hormones, adrenal steroids, glucagon, and growth hormone. o Elevated levels of TNF-alpha have been shown as a cause in insulin resistance associated with obesity and diabetes. 4 Brown Adipose Tissue = BAT (multilocular)(multivacuolar) Smaller cells. Contain numerous fat droplets. Nucleus is in an unusual position within the cell, not flattened (as it is in white adipose) In H&E the cytoplasm is shown as empty vacuoles cause the lipid is lost during preparation à resemble epithelial cells rather than CT cells Contain: numerous mitochondria with large amounts of cytochrome oxidase à brown color of the cells. Abundant in newborns (important in inhibiting heat loss + avoiding lethal hypothermia - a major risk for premature babies). The amount decreases as body grows but remains widely distributed along the first decade of life (in cervical, axillary, paravertebral, mediastinal, sternal, and abdominal regions). Disappears in adults from most sites, except for regions around the kidney, adrenal glands, large vessels (aorta), regions of the neck, back, and thorax. Subdivided into lobules by CT partitions Differentiate from mesenchymal stem cells under PRDM16/PGC-1 (transcription factors) in the presence of catecholamines. They activate UCP-1 gene à controls brown fat differentiation and encodes a mitochondrial protein called uncoupling protein (UCP-1) or Thermogenin. UCP-1 (found in inner mitochondrial membrane): used to uncouple the mitochondrial respiratory chain = uncouples the oxidation of fatty acids from the production of ATP à generating heat rather than ATP. Thermogenesis: Metabolism of lipid in brown adipose tissue generates heat. Non-shivering thermogenesis: when oxidized, adipose tissue produces heat to warm the blood flowing through the brown fat on arousal from hibernation. Maintenance of body temperature in the cold. In non-hibernating animals and humans: lipid is mobilized, and heat is generated by brown adipocytes when they are stimulated by sympathetic nervous system. Hibernating animals: have large amounts of brown adipose tissue. Metabolic activity is regulated by norepinephrine: increased blood levels of norepi à brown adipose tissue expands. Transdifferentiation of adipose tissue Transdifferentiation: white-to-brown and brown-to-white adipocyte transformations, in response to the thermogenic needs of an organism. Browning phenomenon: 3-5 days of exposure to chronic cold temperatures à increased thermogenic needs of an organism à mature white adipocytes can transform into brown (UCP-1 positive) adipocytes (to generate body heat) Energy balance is positive à the body requires an increase of triglyceride storage capacity à brown adipocytes are able to transform into white adipocytes. Mice with abundant brown adipose (either natural or induced via browning) are resistant to obesity and type 2 diabetes. Beige is the transitional kind of adipose tissue, between brown and white (prof. antal). 5  Exercisingà muscle cells produce irisine àstimulation of browning: Other triggers of transdifferentiation include reprograming of adipose tissue genes by activating specific transcription factors (“master regulators”) and growth factors, such as fibroblast growth factor-21 (FGF-21). 6 Extras from the lecture slides: There is also “Beige” adipose tissue: Obesity leads to inflammation in adipose tissue à increase the probability of various diseases, including cancer. Inflamed adipose tissue à insulin resistance + malfunction of skeletal muscle fibers in obesity. Both white and brown have basal lamina Slides 1. Hairy skin, H&E: Adipose layer is seen underneath the CT (white adipose). Light microscope resolution is 200 nm à plasma membrane cannot be seen (~8 nm thick) à the circle around each adipocyte is NOT membrane à the circles are cytoplasm of adipocytes, pushed to the sides by the large lipid droplet occupying the cell. Narrow ring, pressed against (unseen) cell membrane. Lipid droplet is stored in the cytoplasm without a vesicle à no surrounding structure. Sits in cytoplasm. Peripheral nuclei, pushed by lipid droplet. 2. Adrenal gland (suprarenal), H&E: The gland has an adipose capsule surrounding it à adipose tissue is shown in the slide! In the midst of the white adipose tissue we can see an area of brown adipose tissue: o Small, several lipid droplets, randomly distributed o Nuclei are in variable positions in the cytoplasm o Eosinophilic cytoplasm is seen in between the lipid droplets 7  Cartilage Overview Form of connective tissue Mesenchymal origin: mesenchymal cells à osteochondroprogenitors à chondroblasts à chondrocytes. Two main components of cartilage tissue: 1. Specialized cells, chondrocytes (3-5% of tissue). Chondrocytes are the ONLY cells of the cartilage! 2. Highly specialized extracellular matrix (secreted by chondroblasts and chondrocytes): (95% of tissue) a. fibers b. ground substance à attract water à highly hydrated (water makes up 60-80% of total mass)! Chondrocytes and ECM are adapted to the mechanical demands of the localization of cartilage. Cartilage is avascular (except for some parts of fibrous cartilage in menisci) à nutrients and oxygen received via diffusion. Large ratio of glycosaminoglycans (GAGS) to type II collagen fiber à diffusion of substances between blood vessels in the surrounding CT and chondrocytes. Presence of specific cartilage matrix is essential to keep chondrocytes differentiated. Extracellular matrix is solid and firm, but also somewhat pliable (flexible). Main tissue in the development of fetal skeleton and most growing bone. Low capacity for regeneration. Three main types, distinguished by characteristics of their matrix: 1. Hyaline cartilage: matrix containing type II collagen fibers, GAGs, proteoglycans, and multi-adhesive glycoproteins. 2. Elastic cartilage: whatever there is in hyaline cartilage + elastic fibers and elastic lamellae. 3. Fibrous cartilage: whatever there is in hyaline cartilage + abundant type I collagen fibers. Perichondrium: outer surrounding of most collagen tissue (there are exceptions). Has two layers: 1. Stratum fibroelasticum: outer, fibrous 2. Stratum chondroblasticum: inner, cellular Cartilage is NOT innervated. Perichondrium is innervated. Characteristics of chondrocytes: 1. Only cell type in cartilage. 2. Rich in small proteoglycans 3. Secretes the ECM 4. Anaerobic metabolism 5. Do not proliferate in adults, but have a long-life span 6. Stores glycogen and lipids. No chondroblasts in cartilage, only chondrocytes à create collagen fibrils only for maintenance à unable to create collagen in large amounts (as would be needed in healing) à little to no cartilage regeneration. 1 Hyaline Cartilage Rigid, resistant to compression à shock absorber (found in articular cartilage). Also found in organs of airways (trachea slide), nasal septum. Homogeneous, amorphous (shapeless) matrix. Appears glossy. In the cartilage matrix are spaces called lacunae, and within these lacunae there are: 1. Chondrocytes. 2. Newly produced ground substance Provides low-friction surface, lubrication of synovial joints, and distributes applied forces to the underlying bone. Limited repair capacity, but shows no abrasive (grinding, rubbing) wear over a lifetime. Exception: articular cartilage, which may break down with age. ECM is produced by chondrocytes, and contains three major classes of molecules: 1. Collagen molecules: major protein of the matrix. Four types, aka cartilage-specific collagen molecules: a. Type II constitute the bulk of the fibrils b. type IX facilitates fibril interaction with the PGs. c. type XI regulates fibril size. d. type X organizes fibrils into hexagonal lattice which is crucial for the mechanical function of the cartilage. Type VI collagen is also found. 2. Proteoglycans: contains 3 GAGs: a. Hyaluronan b. chondroitin sulfate à PG monomer c. keratin sulfate à PG monomer Most important proteoglycan monomer is aggrecan à many aggrecans associate with one hyaluronan à PG aggregate. Because of the presence of the sulfate groups, aggrecan molecules have a large negative charge with an affinity for water molecules. Other PGs are found as well, but don’t form aggregates. 3. Multiadhesive glycoproteins: AKA- “noncollagenous” and “nonproteoglycan-linked glycoproteins”. Influence interactions between chondrocytes and matrix molecules. Anchorin CII, tenascin, and fibronectin. Highly hydrated with intracellular water (60%-80%). Huge volume of water bound to aggrecan-hyaluronan aggregates à extreme resistance to compressive forces. Changes in water content occur during joint movement or due to pressure. Cartilage undergoes continuous internal remodeling, as the cells replace matrix molecules lost through degradation. Chondrocytes can be distributed either singularly or in clusters called Isogenous groups (chondron) (representing a cell that has recently 2  divided). Chondrons of hyaline cartilage contain 3-4 cells in average (prof. Antal) and they are the cellular unit of the cartilage. Secrete metalloproteinases, enzymes that degrade cartilage matrix, allowing the cells to expand and reposition themselves within the growing isognenous groups. Cytoplasm appearance varies according to chondrocyte activity: active in matrix production à protein synthesis à basophilia. Because proteoglycans of hyaline cartilage contain high concentration of sulfate groups, ground substances stain basophilic (metachromasia). The matrix doesn’t stain homogenouslyà 3 different regions are described based on the staining property of the matrix (seen in the figure of the chondron, shown above): 1. Capsular (pericellular) matrix: a. Ring of densely (basophilic) stained matrix immediately around each chondrocyte. (ECM molecules > collagen molecules à strong basophilic staining). b. Contains highest concentration of sulfated proteoglycans, hyaluronan, biglycans, and multiadhesive proteins. c. Contains almost exclusively type VI collagen, with type XI collagen as well. 2. Territorial matrix: (TM) a. Surrounds the isogenous group. b. Randomly arranged network of type II collagen, with some type XI as well. c. Less sulfated proteoglycans à stains less intensely than capsular matrix. d. More collagen than in pericellular matrix à another reason for lighter staining than pericellular (still more ECM molecules than collagen!). 3. Interterritorial matrix: (IM) a. Surrounding the territorial matrix b. Occupies space between the isogenous groups. c. Collagen molecules > ECM molecules à even lighter staining than territorial matrix! (many GAGs but proportionally they’re less). Precursor of bones that develop via endochondral ossification (lecture slide): 1. Process in which much of cartilage is replaced by bone. 2. Remaining cartilage serves as growth site, epiphyseal growth plate (epiphyseal disc). Epiphyseal growth plate is made of hyaline cartilage and it increases Epiphysial plate is shown in the following imageà Perichondrium: a dense connective tissue (DCT) composed of cells that are indistinguishable from fibroblasts. Surrounds hyaline cartilage. 1. Contains: chondroblasts (immature chondrocytes). 2. Surrounds cartilage of internal organs, ear and nose, and glands. 3. Allows growing and certain regeneration of cartilage. Serves as a source of new cartilage cells. 4. Has two layers: a. Stratum fibroelasticum, outer fibrous layer: dense regular CT. Extremely thick and long collagen fibers + low amounts of fibrocytes. b. Stratum chondroblasticum, inner cell-rich (chondroblastic) layer. Source of young chondrocytes. 5. Not present in fibrocartilage and not in articular cartilage (prof. Antal). Hyaline cartilage of articular joint surfaces do not possess perichondrium. 3  The articular surfaces of movable joints is called articular cartilage, and is a remnant of the original hyaline cartilage template of developing bone. It’s actually a specially layered hyaline cartilage. Articular cartilage is 2 to 5 mm thick and is divided into four zones: 1. Superficial (tangential) zone: pressure-resistant. Elongated and flattened chondrocytes. Not fully diffrentiated. Type II collagen. Parallel fibers. 2. Intermediate (transitional) zone: thickest. round chondrocytes, randomly distributed. oblique fibers. 3. Deep (radial) zone: small, round chondrocytes, in short columns. Parallael. 4. Calcified zone: calcified matrix with small chondrocytes. Separated from deep zone by tidemark. Above tidemark, proliferation of chondrocytes provide new cells for interstitial growth *these four layers can be seen in the articular cartilage around the bones in the knee joint slides! Elastic Cartilage Normal components of hyaline cartilage matrix plus elastic cartilage matrix. Flexibility and resilience (high ration of elastic fibers) à low mechanical resistance. Highly cellular: most cell-rich type of cartilage Smaller chondrons (than in hyaline cartilage), with only 2 cells in average in each chondron. Best stained with resorcin-fuchin and orcein (stain elastic fibers). Found in external ear, walls of external acoustic meatus, auditory tube, and epiglottis (our slide, stained with orcein which stains the elastic fibers between the lacunae/chondrocytes/chondrons in brown, and hematoxylin which stains the nuclei in blue. Seen in the imageà). Surrounded by perichondrium (2 layers). Matrix does not calcify and does not ossify. Doesn’t allow to correct the shape of an ear without plastic surgery. Major differences between elastic and hyaline cartilage: 1. Average number of chondrocytes in each chondron (elastic – 2, hyaline – 3/4). 2. Fibular constituents of ECM (elastic – elastic fibers, hyaline – collagen fibers). 4 trong onenote Fibrocartilage Combination of dense regular connective tissue and hyaline cartilage (the chondrocytes have less cartilage matrix material than hyaline cartilage). No perichondrium! Resistant to torsion and twisting, compression, and shearing. Shock absorber. Chondrons contain 1-2 cells (prof. Antal says only one chondrocyte in chondron of fibrocartilage). EC space made up mostly of collagen fibers, mainly made of type I, but type II is also present (prof. Antal). Cells with rounded nuclei and small amorphous matrix material is typically seen. Within fibrous areas there are fibroblasts, with nuclei that are flattened or elongated. Present in intervertebral discs (equal I and II), symphysis pubis, articular discs of sternoclavicular and temporomandibular joints, menisci of knee joint (less II more I) (shown in slide of knee jointà), triangular fibrocartilage complex of wrist, and certain places where tendons attach to bones. Serves as a resistor to compression and shearing forces. Shock absorber. Responds to changes in external environment. Can be partly vascularized. ECM contains versican (proteoglycan monomer secreted by fibroblasts). Knee joint slide is also shown in toluidine blue staining, displaying metachromasia. The fibrocartilage (as well as the articular cartilage) have many GAGs à purple stain. 5 6 Chondrogenesis and Cartilage Growth Chondrogenesis: process of cartilage development. o begins with aggregation of chondroprogenital mesenchymal cells o site of hyaline cartilage formation is an aggregate of mesenchymal or ectomesenchymal cells known as chondrogenic nodule. o SOX-9 transcription factor triggers the differentiation of these cells into chondroblasts. Secrete cartilage matrix (type II collagen). o When they are completely surrounded by matrix material they’re called chondrocytes. o Mesenchymal tissue surrounding the chondrogenic nodule gives rise to perichondrium. Cartilage can grow in two ways: o Appositional growth: new cartilage at the surface of an existing cartilage. derived from the inner portion of perichondrium. Cells undergo differentiation process with TF SOX-9. o Interstitial growth: new cartilage within an existing cartilage mass. Arise from division of chondrocytes within their lacunae. *with the onset of matrix secretion, cartilage growth continues via a compbination of the above two processes. Repair of Hyaline Cartilage Limited ability for repair due to the avascularity of cartilage, immobility of chondrocytes, and the limited ability of mature chondrocytes to proliferate. Some can be repaired when it involves the perichondrium, thanks to the pluripotential progenitor cells located in it. New blood vessels commonly develop at a healing wound site and stimulates the growth of bone rather than cartilage matrix. When hyaline cartilage calcifies, it is replaced by bone. o Process where calcium phosphate crystals are embedded in cartilage matrix. Calcification can occur in three situations: o The portion of articular cartilage that is in contact with bone tissue in growing and adult bones, but not the surface portion, is calcified. o Always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during growth period. o Hyaline cartilage is calcified in adults as part of aging process. Chondroclast are cells that are involved in removal of cartilage. 7 Extra info from the lecture slides: Chondrocytes The only cell type of cartilage Round shape, adapted to compressive load Embedded into ECM Found in lacunae, surrounded by pericellular fluid and a pericellular matrix rich in small proteoglycans. Secrete ECM components, with low metabolic rate Anaerobic metabolism Do not proliferate in adults BUT live for decades! Store glycogen and lipids ECM of cartilage Macromolecular components are secreted by chondrocytes Two main components: 1. Fibers: a. Collagen type II, III, VI, IX, XI b. Collagen type I (fibrous cartilage) c. Elastin, fibrillin 2. Ground substance: a. Free GAGs: hyaluronan b. PGs: mostly aggrecan, also versican decorin, fibromodulin, biglycan c. GPs: link protein, fibronectin, COMP, thrombospondin, matrilin. The PGs and GAGs bind huge amount