Animal Histology B1100E-AH PDF - Fall 2021/2022
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Lebanese University - Faculty of Sciences
2022
Lebanese University
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This document is a course material on animal histology, details various tissue types and epithelial cells, from the Fall 2021/2022 semester at Lebanese University. It covers the basics of histology and classification.
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Lebanese University Lebanese University Faculty of Sciences Faculty of Sciences B1100 Animal Histology Department of Life and Earth Sciences Fall Semester...
Lebanese University Lebanese University Faculty of Sciences Faculty of Sciences B1100 Animal Histology Department of Life and Earth Sciences Fall Semester 2021/2022 ©opyright Reserved 1 INTRODUCTION Histology and concept of tissues Histology is the science that studies the microscopic structure of tissues. Tissues are sets of differentiated cells that are localized in one place and that contribute to the same biological function. Tissues are made up of cells and of all the elements filling the intercellular or extracellular medium. Classification of tissues There are four types of tissues: - Covering (lining or surface) epithelial tissues and glandular epithelial tissues; - Non-specialized connective tissues and specialized connective tissues: adipose tissue, cartilage tissue, bone tissue and blood tissue; - Skeletal striated muscle tissue, striated cardiac muscle tissue and smooth muscle tissue; - Nervous tissue of the central and peripheral nervous system. Epithelium Covering epithelium Glandular epithelium Loose connective tissue Dense connective tissue Reticular tissue Elastic tissue Connective Adipose tissue Cartilage tissue Bone tissue Blood tissue Skeletal striated muscle tissue Muscle Striated cardiac muscle tissue Smooth muscle tissue Nervous Nervous tissue of the central nervous system Nervous tissue of the peripheral nervous system 2 Histogenesis The tissues differentiate during embryonic development from the three primitive embryonic germ layers: 1- Ectoblast: gives part of the epithelial tissues (the epidermis) and the nervous tissue. 2- Mesoblast: it is at the origin of the non-specialized connective tissues, the cartilage and the bone, the wall of the heart and vessels, the smooth and striated muscles. 3- Endoblast: gives rise to the covering epithelium lining the intestine, the respiratory system and the urogenital tract. Cross section of the 3 embryonic germ layers An organ may contain several varieties of tissues that may have a different embryological origin. 3 Chapter 1 EPITHELIAL TISSUES Epithelial tissues are composed of cells that are tightly packed together with very narrow intercellular spaces, at the limit of visibility under the light microscope. Thus, an epithelium is formed of a set of polarized cells, closely juxtaposed, integral with each other, ensuring their cohesion by different adhesion junctions and involved in one or more common physiological functions; it rests on a basal membrane that separates it from a connective tissue. The epithelia of the body derive from the three embryological germ layers: the ectoderm, the endoderm and the mesoderm. They differentiate morphologically and functionally into two main types: Covering (surface or lining) epithelia that cover other tissues, Glandular epithelia (with secretory function) that are specifically involved in the elaboration of a secretory product. First Part: COVERING EPITHELIA The covering epithelia are tissues formed by one or many layers of epithelial cells closely juxtaposed, with sometimes the presence of non-epithelial cells. These tissues rest on an underlying connective tissue through a basal lamina. They are innervated and non-vascularized (the blood vessels of the underlying connective tissue do not cross the basal lamina), and they have a great capacity for regeneration. Lumen Apical pole Epithelium Basal pole Connective tissue Blood vessel Organization of the covering epithelium 4 The epithelia cover: ▪ the surface of the body: the covering epithelium of the skin is the epidermis, ▪ as well as its natural internal cavities: - Open cavities that extend the inside of the body to the outside (airways, digestive tract, urinary tract, genital tract). The epithelium covering these cavities is called mucosal epithelium. - Closed cavities. Those are : - The cardiovascular cavities: the epithelium covering these cavities is the endothelium, - The serous cavities (pleural, peritoneal and pericardial): the epithelium covering these cavities is the mesothelium. Blood vessels Heart Elastic tissue Smooth muscle Basement membrane Endothelium and mesothelium The epithelial cell Epithelial cells have four principal characteristics: 1) Cohesion: adhesion molecules and specialized junction systems attach the epithelial cells and maintain the cohesion between these cells and with the basal lamina. 5 2) Epithelial morphology: because of the strong interactions that exist between the epithelial cells and the basal lamina and their juxtaposition, the epithelial cells adopt either a squamous or cuboidal or cylindrical (columnar) shape. 3) Presence of intermediate filaments of cytokeratin in their cytoplasm: Cytokeratins are a specificity of epithelial cells. 4) Polarity: the polarity of epithelial cells is manifested by Asymmetrical distribution of the components of the cytoplasm Asymmetrical distribution of the components of the plasma membrane (proteins, enzymes, receptors…). Thus, the epithelial cell has three distinct domains: - The apical domain: this domain is in contact with the external environment or faces the lumen of the cavities of the organism. It is the most specialized domain because it contains most of the proteins necessary for the specific functions of the organ. It also has membrane specializations (microvilli, cilia…). - The lateral domain: it designates the surfaces that are apposed to neighboring cells. It contains most of the proteins required for the fundamental processes common to all cells (polarized and non-polarized). There are anchoring and adhering junctions in this domain. Tight junctions connecting adjacent epithelial cells are closer to the apical pole. - The basal domain: it rests on the basal lamina. Hemi-desmosomes and focal adhesion points are observed. 6 Apical domain Apical domain TJ TJ TJ: Tight junction Basolateral domain Basolateral domain Body surface Apical pole Epithelium Morphological polarity Basal of epithelial cells pole Morphological polarity Connective tissue Molecular polarity of epithelial cells Molecular polarity Basement membrane (or basal lamina) The basement membrane (or basal lamina) participates to the polarization of epithelium and plays a mechanical role by ensuring the cohesion between the latter and the underlying connective tissue. It plays a role in the exchange and filtration processes and in the control of cellular metabolism. Basal laminae also exist around some cells (adipose cells, muscular cells…). Classification of covering epithelia The covering epithelia are classified according to three structural criteria: the number of cell layers, the shape of these cells and the specializations at the apical pole of the cells. Thus the epithelia are classified into: 7 Classification of epithelia according to the number of layers and the shape of cells A- Unistratified epithelium (monostratified or simple): They consist of a single layer of cells having the same shape and size. The apical pole of the cells is in contact with the lumen of the cavity bordered by the epithelium, and the cells rest on the basement membrane. These epithelia are specialized in selective diffusion, filtration, absorption or secretion. They are located in areas where the risks of wear and injuries are low. Among the simple epithelia, we distinguish: 1) Simple squamous epithelia: they are formed by a thin layer of squamous cells ie flattened, adjusted against each other. Their nucleus is ovoid or elongated. These epithelia are often permeable and are involved in several processes: filtration, diffusion and osmosis. They are found in the alveolar sacs of the lungs. Two varieties of simple squamous epithelia are particular: - The endothelium: which is the epithelium of the heart and the vessels - The mesothelium which is the serous epithelium. The endothelium and the mesothelium have a basal lamina that is sometimes discontinuous with weak tight junctions. 8 2) Simple cuboidal epithelia: they consist of a single layer of cuboidal cells, with a spherical central nucleus. The cells are tightly attached against each other. They are epithelia that have a function of secretion and absorption and are localized, for example, at the surface of ovaries and in the excretory ducts of certain exocrine glands (salivary glands and pancreas). 3) Simple columnar (or cylindrical) epithelia: the cells are elongated and cylindrical, their nuclei are all at the same level and located in the basal region (lower third) of the cell. These epithelia are associated with absorption and secretion functions. They line the stomach for example. Depending on their activities and location, the cells of these epithelia have apical differentiations. Thus we describe: - Simple columnar epithelia which apical pole is bordered with microvilli with a striated plateau (like the epithelium of the small intestine in which goblet cells are also observed) or with a brush border; - Simple columnar ciliated epithelia which cells have vibratile cilia and which line the fallopian tubes; a) Cilia Fallopian tube Goblet cell Basement membrane Connective tissue b) Simple columnar ciliated epithelium Simple columnar epithelium 9 4) Pseudo-stratified columnar epithelia: these are simple epithelia composed of cylindrical cells of different sizes: the nuclei are located at various heights. All cells rest on the basal lamina in a single layer, but some of them are short and do not reach the lumen. This organization suggests that the tissue has several layers. These epithelia perform functions of secretion and absorption. A pseudo-stratified columnar ciliated epithelium with goblet cells covers most of the upper respiratory tract. It functions in secretion and transport of mucus. Cilia Ciliated cells Goblet cell Pseudo-stratified columnar ciliated epithelium with goblet cells B- Stratified epithelia: these epithelia consist of several layers (at least two) of superposed cells. The deepest layer (basal or germinative) rests on the basement membrane and the most superficial layer is in direct contact with the lumen of the cavity. These epithelia are found in regions subjected to friction. They are resistant and able to protect the underlying tissues. The cells of the same stratified epithelium have several forms; thus the name of these epithelia is attributed according to the shape of the cells located at the surface. Among the stratified epithelia, there are: 1) Stratified squamous epithelia (or Malpighian): the most abundant of stratified epithelia; they are observed in places that wear out a lot and protect against abrasion. Composed of several layers of cells, these epithelia are thick and well adapted to their protective role. In deep layers, the cells are cuboidal or cylindrical with a rounded nucleus: these basal cells are constantly reproduced by mitosis; as they progress to the 11 superficial layers, they flatten out and become squamous with an elongated nucleus. They degenerate later on: they are then eliminated and replaced. A stratified squamous epithelium lines for example the inner wall of the mouth,... The keratinized stratified squamous epithelium constitutes the superficial layer of the skin (the epidermis). In this epithelium, the superficial cells, very flattened, accumulate keratin and reject their nucleus and organelles. This epithelium is also adapted for dessication: it forms a resistant and protective surface. Keratinized = epidermis Non Keratinized (epidermis) e.g. mouth espphagous esophagus vagina E= epidermis E= epithelium D= dermis C= chorion Stratified squamous epithelia (Malpigians), Keratinized and non-Keratinized 2) Stratified cuboidal epithelia: these epithelia are relatively less abundant. They are generally composed of two layers of cells and play mainly a more solid covering role than a simple epithelium. They are found in the excretory ducts of the sweat glands. 3) Cylindrical (or columnar) stratified epithelia: these epithelia are rare. The basal layers are made of irregular polyhedral cells and only the superficial cells are cylindrical. The stratified columnar epithelium plays a role of protection and secretion and is found in a part of the human urethra. 4) Transitional epithelium: this particular type of epithelium (also called urothelium) lines the organs of the urinary system that are subject to significant variations in internal pressure and to considerable stretching. The thickness of this epithelium and the shape of its cells are variable according to the distension of the organ: bulky and rounded (empty bladder), they become squamous and flattened (full bladder). 11 Transitional epithelium Properties of covering epithelium Renewal of epithelia: Covering epithelia are particularly exposed to wear and aging and have a limited life span. Their renewal is ensured by the proliferation of stem cells or replacement cells. Once activated, the stem cells divide and the rate of their renewal is different according to epithelia. Permeability of epithelia: despite their function as a barrier, the covering epithelia may be more or less permeable. The basal lamina, which separates the epithelium from the chorion, acts as a selective filter. 12 Second part: EPITHELIA WITH SECRETORY FUNCTION (GLANDULAR EPITHELIA) Secretory cells A secretory cell is any cell, whether of epithelial nature or not, that exports out of its cytoplasm (in the extracellular medium) specific molecules that it has synthesized. Glandular cells Glandular cells are epithelial cells specialized in secretion: the secretory products are stored in secretory vesicles, and they are then released on demand. Histogenesis of glands The glandular epithelia result from the differentiation of some embryonic covering epithelia that bud to form a cellular mass. This cell mass enters progressively into the underlying mesenchyme; it can then: a) either maintain the contact with the covering epithelium and the lumen of the organ: it thus differentiates into an exocrine gland, b) or become detached from the covering epithelium: it then isolates itself in the vascularized mesenchymal tissue and organizes itself into an endocrine gland. Histogenesis of glands 13 Classification of glandular epithelium Depending on the nature of their secretory product and their secretion modality, exocrine glandular cells and endocrine glandular cells are distinguished. The glands with both secretion modalities are amphicrine glands. I – EXOCRINE GLANDS These are glands that discharge their secretory product in the external environment (on the surface of the skin) or in a natural cavity in continuity with the external environment. The exocrine glandular cells are polarized: just like the covering epithelial cells, the exocrine glandular cells show a morpho-functional polarity in relation to their secretory activity. We distinguish: - an apical pole that borders the lumen. This apical pole is poor in cell organelles and is the place where the elaborated products are stored before their excretion, - a basal pole that rests on a basement membrane that separates the glandular epithelium from the surrounding connective tissue. Contractile myoepithelial cells are sometimes located between the secretory cells and the basal lamina; their contraction helps in the excretion of the secreted product. Classification of exocrine glands: A- According to the number of cells : They can be: - Unicellular: the goblet cell is an example. The goblet cells are dispersed in the covering epithelium lining the digestive, respiratory, urinary and reproductive tract. They produce mucus that lubricates and protects the surfaces of these systems. 14 Unicellular exocrine gland (goblet cells) - Multicellular: The multicellular exocrine glands comprise a secretory part (or glandular unit) and an excretory canal, enveloped by a stroma of connective tissue (containing blood capillaries and which ensures the nutrition of these glands). Secretory Excretoryducts duct Connective tissue Secretory portion Multicellular exocrine gland B- According to the characteristics of the excretory canal: We distinguish: - Simple glands: they have a single unique excretory duct associated either with a secretory portion (simple exocrine gland) or with several secretory portions (branched or ramified simple exocrine gland). - Compound glands: their excretory duct is branched and each branch joins a secretory portion (several excretory ducts and several secretory portions). 15 Classification of exocrine glands according to the excretory duct and the form of the secretory portion C- Depending on the shape of the secretory portion: We distinguish: - Acinous glands: the acinus is spherical or vial-like; the wall is made of glandular cells in the form of truncated cone (or pyramid); the lumen of the secretory portion as well as that of the excretory duct are reduced (simple acinar glands and compound acinar glands). - Tubular glands having an elongated tube appearance with a wall made of cuboidal or columnar cells (straight simple tubular gland, simple branched tubular gland, simple tubular coiled gland, compound tubular gland). - Alveolar glands have a secretory portion in the form of a bag with a wide lumen relatively to the thickness of the wall; the lumen of the secretory portion and the excretory duct is greater than that of the secretory portion and the excretory duct of an acinar gland (simple alveolar gland: the sebaceous gland). These different forms can be observed within the same compound gland: thus tubulo-acinar glands (submaxillary glands) or tubulo-alveolar glands are observed. 16 D- Depending on the nature of the secretory product: We distinguish: 1- The serous glands whose secretory product is an enzyme of fluid consistency (e.g. acinar cells of the pancreas, parotid cells and main cells of the stomach). Serous cells: Have a rounded nucleus and usually located at the junction of the middle and lower thirds of the cell. The apical cytoplasm is dark with granulations. The cells limit a barely visible lumen. The basal pole is very rich in RER and mitochondria. The Golgi apparatus is very developed. The secretory granules occupy the top of the cell and appear dense to electrons. They are vesicles limited by a membrane and which contain enzymes, either in an active form or in the state of precursors (zymogen). At the apical pole, they merge with the plasma membrane before being excreted by exocytosis. 2- The mucous glands whose secretory product is mucus (mucins). It is a protective viscous product that is rich in glycosaminoglycans or proteoglycans. Mucous cells: They are large. They have a cytoplasm filled with mucigenic grains. The nucleus, flattened, is pushed back to the basal pole of the cell with the rest of the organelles. Usually, the abundance of the mucous secretory vesicles causes the mucous cell in OM to have a "clear" appearance that opposes the "dark" appearance of the serous cells. The cells limit a visible lumen. Section of a serous acinus Section of a mucous acinus Wide Wideand and Narrow Narrow Narrow light irregular light lumen lumen Comparison between serous gland and mucous gland 17 a b Comparison between mucous cell (a) and serous cell (b) 3- In addition to the serous glands and mucous glands, widely distributed in the body, there are a number of glands whose secretory product is neither a protein nor mucus, such as sweat glands (sweat), the sebaceous glands (sebum), the salivary glands (saliva). The presence of mixed glands should also be noted. Ex: sero-mucosa (submaxillary glands). E- According to the mode of excretion: Exocrine glandular cells can excrete the secretory products in three modes: - Merocrine mode: this is the most common case among the exocrine glands. The cells produce secretions and they reject them externally by exocytosis, the cell integrity being respected (ex: the parotids). - Apocrine mode: the secretory product is gradually accumulated at the apical part of the cell. This superficial portion of the cell is detached in the form of a large vacuole which is thus released respecting the integrity of the cell membrane and the cell can resume a new secretory cycle (ex. the mammary glands). - Holocrine mode: the secretory product completely fills the cytoplasm of the cell. The cell then undergoes degeneration, which ultimately results in the rupture of its membrane and the release of the accumulated product (ex: the sebaceous glands). 18 The 3 modes of secretions of the exocrine glands II – ENDOCRINE GLANDS An endocrine glandular cell is a cell capable of synthesizing, storing and secreting into the internal medium, via the bloodstream and without excretory duct, a substance called hormone (chemical messenger) that will act on the receptors of target cells. Classification of endocrine glands 1- According to the morphology We distinguish: - The vesicular glands: the glandular cells are vesicles or spherical follicles between which some (a little) connective tissue and many vessels are observed (the thyroid). - The reticular glands: the glandular cells form cords separated by fine connective tissue that are crossed by vessels (the parathyroid). - The diffuse glands: they are either grouped into islets (interstitial glands of the testis) or scattered in an epithelium (glands of the digestive mucosa). 19 2- According to the type of hormone secreted. Depending on the biochemical nature of the secreted hormone, there are 4 groups of endocrine glandular cells: - Steroid secreting endocrine cells (secretion of androgens, estrogen, etc.): these cells are essentially characterized by abundant smooth endoplasmic reticulum, numerous mitochondria and frequent lipid vacuoles. The lipid nature of these hormones allows them to diffuse freely through the plasma membrane of the cell without the need for the exocytosis process. - Thyroid hormones-secreting cells: these are the follicular cells of the thyroid that synthesize the thyroglobulin and then transform it into liposoluble hormones, tri- and tetra-iodothyronine (T3 and T4) that diffuse freely through the cytoplasmic membrane. - Peptides secreting endocrine cells (from a few amino acids to large proteins): these water-soluble hormones (renin, gastrin, insulin, calcitonin, etc.) are released outside the cell by exocytosis. These cells are essentially characterized by abundant RER. - Amino-acid derivatives secreting endocrine cells (serotonin, adrenaline, norepinephrine, melatonin, etc.): these water-soluble hormones are synthesized from amino acids and they are then rejected out of the cell by exocytosis. 3- According to the mode of action of the receptor. Steroid and thyroid hormones act on an intracytoplasmic or even intranuclear receptor, while peptides and biogenic amines bind to a membrane receptor that will act on the metabolism of the cell via a second messenger. III – AMPHICRINE GLANDS These are glands that are both exocrine for some products and endocrine for others. According to the morphological organization, we distinguish: - Homotypic amphicrine glands that are formed by a single type of both endocrine and exocrine cells. Eg: the liver. - Heterotypic amphicrine glands in which two types of cells are found in the same parenchyma: some are exocrine, the others are endocrine. Ex: serous exocrine acini and endocrine Langerhans cells of the pancreas and testes. 21 CONNECTIVE TISSUES The connective tissues are characterized by the presence of distant cells that are separated from one another by intercellular spaces containing a large amount of extracellular material called the extracellular matrix (ECM). Connective tissues originate from undifferentiated primitive tissue, the embryonic mesenchyme. They are highly vascularized except the cartilage, and are the most abundant tissues of the organism. They provide different functions: protection, exchange, nutrition, defense, energy storage, support, transport and communication…. Connective tissues Connective tissue proper (Non- Specialized connective specialized) tissues: Adipose tissue Cartilage tissue Bone tissue Blood tissue 21 Chapter 2 NON-SPECIALIZED CONNECTIVE TISSUES Non-specialized connective tissues (also called proper, common or classic) are characterized by the presence of cells (fixed and wandering) and a very abundant extracellular matrix (ground substance, fibers and structural glycoproteins). They originate from the embryonic mesenchyme. A-Cells of non-specialized connective tissue The cells of non-specialized connective tissue are non-polarized cells and are divided into two categories: fixed cells (resident) and wandering (migrant) cells. Fixed cells: are non-motile. They consist mainly of fibroblasts (and eventually adipocytes). Fibroblasts: are the principal and the least specialized cells of the connective tissue, compared to other cells of specialized connective tissues. They originate from the mesenchymal stem cells. Fibroblasts are spindle-shaped or stellate cells with long cytoplasmic processes more or less branched and lack a basement membrane. The size of fibroblasts varies according to their activity level. They have a significant mitotic activity. Optical microscopy shows a basophilic cytoplasm. Its nucleus is central, ovoid, large, and elongated with one or two nucleolus. Active fibroblasts contain abundant organelles involved in protein synthesis (rough ER, and Golgi apparatus). Their cytoskeleton is well developed and is essentially formed of actin microfilaments. Fibroblasts control and maintain the extracellular material of connective tissue: They synthesize and secrete macromolecules of connective tissue ECM. They produce the enzymes implicated in the catabolism of these macromolecules. They play also an important role in the process of tissue repair. 22 When fibroblast secretion activity stops, its nucleus condenses; its cytoplasm loses its basophilic characteristics and retracts, reflecting the absence of any genetic activity: it is the fibrocyte. Wandering (or free) cells: are cells that move actively, their presence in the non- specialized connective tissue is variable and occasional. They are cells of the immune system. They reach connective tissue through the bloodstream by crossing capillary barriers via diapedesis (lymphocytes, granulocytes, monocytes...). B- Extracellular matrix of non-specialized connective tissue The extracellular matrix (ECM) of non-specialized connective tissue is very abundant. It is composed of amorphous ground substance, fibrous proteins and structural glycoproteins. 1- Ground substance: it is a highly hydrated gel-like material. It is implicated in the diffusion of molecules (nutrients and of waste) between cells and blood. Ground substance is primarily composed of glycosaminoglycans (GAGs) and proteoglycans. Glycosaminoglycans (GAGs): GAGs are polysaccharide chains, made up of repeating disaccharide units. GAGs are hydrophilic and form a matrix like gel 23 that sucks in lots of water. The main GAGs are: hyaluronic acid, chondroitin sulphate, dermatan sulphate and heparan sulphate. Proteoglycans (PG) (10% proteins and 90% carbohydrates): PG are molecules that consist of a core protein to which several sulphated GAGs are covalently attached. Hyaluronic acid can form non-covalent bonds with proteoglycans. PGs form the framework of the ECM and play an essential role in serving molecular exchanges through the connective tissue. They form gels, fix growth factors and control the activity of certain enzymes. They also serve as support for cell migration. Organization of the extracellular matrix of cartilage 2- Fibers a- Collagen fibers: Collagen is the most abundant protein in the human body. Collagens are very resistant glycoproteins. They are insoluble in cold water but soluble in boiling salted water. In the organism, collagen can be destroyed by collagenase. Collagen is synthesized in cells as alpha polypeptide chains organized in triple helix (procollagen). Its composition in amino acids differs according to the type of collagen. In the extracellular space, the two ends of the procollagen molecules are cleaved forming tropocollagen molecules. The tropocollagen molecules assemble end to end and side by side to form the microfibrils. The microfibrils aggregate into fibrils 24 having a periodic transversal striation of 67 nm. The fibrils are then combined into parallel more or less thick fibers depending on the type of collagen. Then, some types of collagen fibers aggregate into bundles. Currently, there are 27 identified types of collagen. The most important are: Type I collagen is the most abundant. It is present as thick bundles in loose connective tissue, in dense connective tissue, in bone tissue... Type II Collagen is present in the cartilage. Type III collagen is that of reticular fibers. These fibers are thinner than other collagen fibers and do not aggregate into parallel bundles. They form the stroma of the lymphoid and hematopoietic organs and the layer of the basement membrane adjacent to connective tissue. Type IV collagen is found in basement membranes Collagen synthesis and assembly 25 b- Elastin: Elastin is the major component of elastic fibers. The elastic fibers are found in variable number in loose connective tissue and are abundant in elastic ligaments, in large arteries, and in elastic cartilage. They are characterized by their abilities to stretch and to recoil back to their original form after being stretched. The biosynthesis of elastin is comparable to that of collagen: proelastin is produced in cells and is transformed in the ECM into tropoelastin, the tropolelastin then interacts with fibrillin to form the elastic fibers. In most connective tissues, elastin is associated with other macromolecules such as collagen, proteoglycans and structural glycoproteins. 3- Structural glycoproteins (90% proteins and 10% carbohydrates): these are globular proteins that have an important role in connective tissue organization, and specifically in the interaction and the attachment of cells to the ECM. Structural glycoproteins are very numerous. The main ones are: Fibronectin: it provides the link between cells and collagen and GAGs Laminins: sulphated glycoproteins intimately related to the basal lamina, they have several binding sites between cells and the components of the ECM. C- Structure of the basement membrane The electron microscope shows that the basement membrane is composed of three layers, which are from the plasma membrane of the epithelial cell to the ECM as following: 26 Lamina rara or lucida: very clear layer, electron lucent Lamina densa: It is the middle layer, wide and electron-dense, it is the main layer of basal lamina These 2 layers are synthetized by the cells that rest on them and are composed of type IV collagen, proteoglycans, several varieties of glycoproteins (laminin, fibronectin,...) Lamina reticularis or pars fibroreticularis: it is a very clear layer that represents the junction zone between the lamina densa and the underlying connective tissue. It is secreted by cells of the underlying connective tissue. It is composed of type III collagen (reticulin). E Basement membrane E: Epithelium C: Connective tissue LL, LD, LF: 3 basement membrane layers The thickness and composition of the basal lamina vary according to the epithelium location. D- The different types of non-specialized connective tissues There are several types of connective tissues that differ from each other in the organization and the relative proportions and of their components: cells, ground substance, fibers, by their differentiation and function. We can distinguish: 1. Mucous connective tissue: It is a particular connective tissue, close to the mesenchyme but more differentiated. The ground substance is very abundant, viscous, and rich in hyaluronic acid. This tissue is found in the umbilical cord and in the pulp of young teeth. 2. Loose (Areolar) connective tissue: It is the most widespread type of non- specialized connective tissue. It is composed of a balanced proportion of cells and fibers (collagen, elastic, reticular) and an abundant ground substance. Areolar 27 connective tissue is flexible and resistant. It provides several important roles: mechanical (support of various tissues and organs), metabolic (passage of various substances between blood and other tissues), defense of the organism (inflammatory reactions, immune processes, repair). 3. Dense Connective Tissues: They are rich in fibers, contain few cells and non- abundant ground substance. Their role is almost exclusively mechanical (resistance). Connective tissues with a predominance of collagen fibers (dense fibrous connective tissues) contain mostly collagen fibers. We distinguish: Dense fibrous irregular connective tissues: are found in regions where tensions are exerted in different directions. The bundles of fibers are intertwined and do not have a regular orientation, allowing this tissue to resist strong tensions from different directions (dermis of the skin, capsules that surround certain organs,..) Dense fibrous regular connective tissues: are found in regions where tensions are exerted in only one direction. They consist of parallel bundles of collagen fibers (tendons, ligaments). 4. Elastic connective tissues (dense tissues): Elastic connective tissue is very rare. It contains mostly elastic fibers which ramify. Rare fibroblasts or smooth muscle cells are found between the elastic fibers. This tissue is characterized by its ability to stretch and then snap back to its original shape. It is mainly found in the trachea. 5. Reticular connective tissues (loose tissues): These tissues are rich in cells and are composed only of reticular fibers (type III collagen) dispersed within a matrix rich in proteoglycans. Reticular connective tissues form the stroma of hematopoietic and lymphoid organs (spleen, bone marrow, lymph nodes) and liver. 28 Chapter 3 ADIPOSE TISSUE Adipose tissue is a loose connective tissue with a predominance of cells, specialized in fat storage; it is composed mainly of adipose cells (adipocytes) separated by a thin layer of ECM comprising a thin network of reticular fiber and many vessels. Adipocytes Adipocytes or fat cells are cells specialized in the storage of lipids. They are highly differentiated; hence they are unable to divide and their replacement is provided by the differentiation of adipoblasts. Their metabolic activity is important and comprises three steps: The synthesis of lipids (liposynthesis), their storage in the form of triglycerides and their rapid release in the form of fatty acid (lipolysis). There are two types of adipocytes: white adipocytes (white fat) and brown adipocytes (brown fat). White adipocytes: They are round and large cells (100 to 150μm in diameter). When observed with an optical microscope, their small nucleus is flattened and pushed to the periphery of the cell. Their cytoplasm is reduced to a thin rim that surrounds a large lipid droplet (vacuole). In electron microscopy, the cytoplasm contains all the organelles including abundant smooth endoplasmic reticulum, numerous mitochondria and many pinocytosis vacuoles. The cytoskeleton disappears and the accumulated lipids are found in small unbound vacuoles that fuse to form a single central lipid vacuole (unilocular). A thin basement membrane surrounds the plasma membrane. In the non-specialized connective tissues, the white adipocytes are scattered and isolated while in the adipose tissue, they are packed against each other. White adipose tissue represents 15 to 25% of the weight of an adult. Its distribution in the body varies according to age and sex. It is generally found around the kidneys, around the heart and in the hypodermis. 29 White fat plays several roles according to its localization: it constitutes the main energy storage which is immediately released in case of an acute metabolic stress. It also acts as a thermal insulator and a shock absorber. It protects various organs. Brown adipocytes: are round or polygonal cells, of about 50μM of diameter. Their nucleus is eccentric and their cytoplasm is rich in mitochondria, and in small lipid vacuoles which do not fuse together (the brown adipose cell is multilocular). They are involved in thermogenesis: when stimulated, they transform stored energy into heat. Brown fat is abundant in hibernating mammals, but it is also found in humans, mainly at the beginning of life. Development of white and brown adipocytes 31 Chapter 4 CARTILAGE TISSUE Cartilage tissue is a specialized connective tissue. It is part of the skeletal tissues. Skeletal tissues are specialized connective tissues whose ground substance varies, depending on the applied pressure, from a semi solid state to a solid state. There are different types of skeletal tissues: cartilage tissue, bone tissue... The immature cartilage tissue arises from embryonic mesenchyme: in some regions of the mesenchyme, mesenchymal cells gather into clusters, differentiate into cartilage cells, the chondroblasts. These cells show a high mitotic activity and actively synthesize the macromolecules of the ECM. Chondroblasts, while continuing to synthesize ECM, differentiate then acquire, with time, the characteristics of adult or mature cartilage cells and become chondrocytes. I - Components of cartilage tissue Cartilage tissue contains only one type of cell: the chondrocytes and a cartilaginous ECM. It lacks blood, lymphatic vessels and nerve fibers. 1- Chondrocytes Chondrocytes are cells that differentiate from chondroblasts. Chondrocytes are spherical or rounded cells that send very short and thin processes into the ECM. Their nucleus is large, central and spherical and contains one or two nucleoli. Their basophilic cytoplasm contains the usual cell organelles with abundant RER and free ribosomes. The chondrocytes are enclosed in small lacunae without a wall. They synthesize all the macromolecules of the cartilage ECM. Chondrocytes maintain the integrity of the cartilage matrix by ensuring its renewal. 31 2- Cartilage extracellular matrix Cartilage ECM appears homogeneous in OM and consists essentially of water (70- 80%), mineral salts (in particular sodium salts), GAGs (chondroitin sulfate, keratan sulfate, hyaluronic acid), proteoglycans (essentially aggrecan: hyaluronic acid + proteins), collagen fibers (type II essentially) and structural glycoproteins (chondronectin, chondrocalcin, fibronectin, tenascin, cartilage matrix glycoprotein...). This matrix also contains nutrients, metabolites and molecules resulting from the activity of chondrocytes (cytokines, growth factors, etc.). It is highly basophilic. The high water content and the arrangement of collagen fibers give cartilage strength, flexibility and resistance to compression, to tension and to deformation. II - Perichondrium Cartilage, with the exception of articular cartilage and fibrocartilage, is surrounded by a layer of a connective tissue, the perichondrium, which separates it from neighboring connective tissues. It is a vascularized tissue that plays a role in the nutrition, growth, maintenance and repair of cartilage. It consists of two layers: - A chondrogenic layer: deep (inner) layer formed of loose connective tissue. It is poorly vascularized. It makes the connection with the cartilage tissue (fine Sharpey fibers firmly fix the perichondrium to the cartilage). It is rich in cells (cellular perichondrium) and the deepest mesenchymal cells and fibroblasts differentiate into chondroblasts that synthesize the cartilage ECM. - An outer tendiniform layer composed of a fibrous dense irregular connective tissue (containing collagen fibers, elastic fibers and dispersed fibroblasts). It is highly vascularized: this is the fibrous layer of perichondrium. Perichondrium 32 III - Cartilage Classification There are 3 types of cartilage tissue that are distinguished by the composition of their ECM: the nature and proportions of the ground substance and the different types of fibers (collagen and elastin). The cellular density is inversely proportional to the thickness of the cartilage. 1. Hyaline cartilage: This is the most common cartilage in the body. Chondrocytes are few in number and represent 10% of the cartilage mass; they are found in lacunae and are dispersed in an abundant ECM rich in ground substance, but not in fibers (90% of the cartilage mass). Hyaline cartilage constitutes the largest part of the fetal skeleton, is subsequently transformed into bone and persists as growth cartilage in children and adolescents. We distinguish: a- Non- articular hyaline cartilage which persists in the trachea and bronchi. b- Articular hyaline cartilage: it is located selectively in the joints of the long bones. It provides the mobility of the joint. With the synovial fluid, it prevents the friction of the bone surfaces. It is rigid but also deformable to ensure a harmonious distribution of the pressures exerted on the articulation. 2. Elastic cartilage: it is a modification of the hyaline cartilage. It is rich in chondrocytes that contain large lipid inclusions. Numerous bundles of elastic fibers are observed in the ECM, in addition to the few type II collagen fibers. Elastic fibers are organized into a dense network around each chondrocyte. The elastic cartilage is surrounded by perichondrium and grows by apposition and interstitial growth. This cartilage is found in places where flexibility (resistance and flexibility) is needed. It 33 thus supports slight reversible temporary deformations. It is found in the nose and the auricle of the ear. 3. Fibrous cartilage or fibrocartilage: this tissue is devoid of perichondrium and grows by interstitial growth. Unlike elastic cartilage, it is not a modification of hyaline cartilage. The chondrocytes are few in number, more or less fusiform or elongated, devoid of lipid inclusions and surrounded by ground substance without fibers. The ECM fibers are of type I and type II collagen; they are abundant and grouped into bundles and oriented according to the forces of tension: it is therefore a cartilage which is particularly adapted to withstand very high pressures while keeping certain flexibility. Fibrous cartilage forms the intervertebral discs and the articular menisci of the knees. IV - Nutrition of cartilage Cartilages are devoid of blood and lymphatic vessels. They are nourished by the diffusion of small molecules into their ECM from the capillaries of the outer layer of the perichondrium (when it exists). However, articular hyaline cartilage and fibrocartilage are nourished by synovial fluid, and partly by the exchanges with subchondral bone. 34 V - Cartilage growth Cartilage can grow in two ways: a) Interstitial (or endogenous) growth: it is mainly observed in the fetus but also during the post-natal growth of the long bones (growth cartilage). Chondroblasts and chondrocytes are divided by successive mitosis and two or more daughter cells remain for some time in the same lacuna. This multiplication of chondrocytes ensures an expansion of the internal mass of the cartilage and the synthesis of the ECM gets daughter cells apart from each other. If mitoses take place in one direction, the cells will be arranged in line (axial isogenic group) and the cartilage increases in length (this is the case of growth or epiphyseal cartilage). If the mitoses take place in different directions or radially, the cells will be arranged circularly (radial isogenic group) and the cartilage increases in girth (in thickness). This mode of growth is rare in adults. b) Appositional or perichondral (or exogenous) growth: it is mainly observed during fetal development. It is carried out by the proliferation and progressive differentiation of mesenchymal cells and fibroblasts into chondroblasts, then functional chondrocytes that secrete ECM cartilage. Thus, there is apposition of successive layers of cartilage on the surface of the pre-existing cartilage that grows in thickness. 35 Interstitial growth of cartilage 36 Chapter 5 BONE TISSUE Bone tissue is a specialized skeletal connective tissue. It has a mesenchymal origin and is composed of cells and an extracellular matrix impregnated with crystallized mineral salts that make it rigid and impermeable. The bone tissue is hard and resistant but brittle. It is innervated and richly vascularized. It is a dynamic living material, constantly being renewed and throughout the lifetime of the individual. Bone tissue is the largest part of the skeleton of higher vertebrates; it fulfills several basic functions: - A supporting role: it supports the body and the soft tissues. - A mechanical role: it serves as a point of attachment to the muscles to produce a movement (locomotion). - A protection role: it protects the internal organs, including fragile ones. - A metabolic role: it regulates the phospho-calcium metabolism. - A role in hematopoiesis: it hosts the red hematopoietic marrow. A- CELLS OF BONE TISSUE The aspect of bone tissue is the result of a balance between the activities of two cell populations: - The osteoblastic line: The bordering cells, the osteoblasts and the osteocytes are of mesenchymal origin and elaborate and maintain the bone tissue; they are responsible for osteogenesis. - The osteoclastic line: of hematopoietic origin, the osteoclasts destroy the bone tissue (bone resorption). Osteoclast Bordering cells Osteoblasts Bone cells Canaliculi with thin ECM Osteocytes cytoplasmic processes 37 Bordering cells Bordering cells are flat, very thin, elongated cells that cover the surface of bone areas that are not subject to bone formation or resorption in adults. They contain few organelles and are not very active. They communicate with each other by gap junctions. They emit prolongations within the bone matrix. Some histologists believe that bordering cells are osteoblasts '' at rest ''. Others say that if activated, these cells proliferate and differentiate into osteoblasts. Due to their position at the surface of bone, the bordering cells play two roles: - Protective role against the degradation of the ECM - Role in regulation of bone nutrition Osteoblasts Local undifferentiated mesenchymal cells called osteoprogenitor cells are at the origin of osteoblasts. Mature osteoblasts are arranged in a continuous layer, just like the bordering cells, but at the surface of developing or growing bone tissue. They are cuboidal or polyhedral. Their large oval nucleus is usually located in the basal region; it contains a large nucleolus. Their cytoplasm is basophilic and rich in organelles involved in protein synthesis. Mitochondria are numerous. The shape of osteoblasts is irregular because they have long cytoplasmic expansions directed towards the developing bone tissue and the neighboring cells. The contact between the cell body of two osteoblasts or between their expansions is characterized by gap junctions. Osteoblasts are young and active cells. They have an important synthesis activity: they participate in the construction of the bone by synthesizing and secreting the organic component of the extracellular matrix of new bone (known as osteoid) which will then be added to the surface of a pre-existing organic matrix. A few days later, osteoblasts will release tricalcium phosphates and alkaline phosphatase to control the process of mineralization of this formed bone tissue. Osteoblasts also participate to the regulation of bone remodeling: directly, by secreting proteolytic enzymes, and indirectly by releasing factors that will act on osteoclasts. 38 The fate of osteoblasts is defined by 3 ways: - some die by apoptosis, - others rest in the form of bordering cells, - those that lock themselves into the mineralized matrix that they produce (10%) will gradually turn into osteocytes. Diagram showing an osteoblast at the surface of the bone matrix and an osteocyte inside the matrix within an osteoplast Osteocytes These are differentiated cells that can no longer divide (but that may persist for several decades). Star-shaped, they have a cellular body from which fine extensions detach and are embedded in the surrounding matrix. Their nucleus is central and ovoid. In the extensions, there are no organelles. The RER and Golgi apparatus are reduced (their synthesis capacity is thus reduced) whereas mitochondria and lysosomes are poorly developed. Cytoplasmic basophilia is less marked than that of osteoblasts; it varies according to the activity of the cell. Each osteocyte is contained in a lacuna, the osteocyte lacunae or osteoplast. The lacunae are interconnected by canaliculi which contain the fine cytoplasmic prolongations of the osteocytes whose ends connect via gap junctions. These canaliculi allow all osteocytes to access different molecules (nutrients...). Between the osteoplast and the plasma membrane of the osteocyte, there is a thin, non-mineralized periosteocytic space containing collagen fibers and a high concentration of proteoglycans. 39 Osteocytes are metabolically less active than osteoblasts: - They are responsible for the permanent exchange of minerals between bone tissue and blood and they maintain the phospho-calcic balance; - They ensure the maintenance of bone tissue by ensuring the renewal and maintenance of the ECM; - They are involved in the regulation of bone remodeling and presumably in bone resorption at the periphery of the lacunae, by secreting citric acid, acid phosphatase, proteolytic enzymes and peptidases: these are certain localized bone tissue demineralizations without disorganization of the other constituents of the matrix. Osteoclasts a- Formation of osteoclasts: Monocytes migrate through the bloodstream to the bone tissue and differentiate into preosteoclasts. Then several preosteoclasts fuse together to form multinucleated osteoclasts. Osteoclasts are localized on the surface of bone tissue, in the spaces left free by bordering cells and osteoblasts. b- Microscopic aspects: Osteoclasts are large (50 to 100 μm in diameter) multi-nucleated (containing 30 to 50 nuclei) cells. They are polarized cells. The surface adjacent to bone shows a radial striation (a differentiation in the form of a brush border with long irregular microvilli) and is called ruffled border. Osteoclasts are rich in vesicles, phagocytic vacuoles and lysosomes grouped preferentially under the ruffled border. Cytoplasm ranges from basophilia (when synthetic activities predominate) to acidophilia (when resorption activity predominates); this change marks the functional difference between osteoclasts. c- The function of osteoclasts, bone resorption: Osteoclasts are mobile and phagocytic cells, their function is the destruction of bone tissue. Bone resorption is controlled by osteoblasts through multiple growth factors, allowing the coordination of bone tissue synthesis and destruction. Once the osteoclasts are activated, their microvilli bind to the ECM. The osteoclasts then provide, via the proton pumps located in the plasma membrane of the ruffled 41 border, an acidification of the microenvironment located between the ruffled border and the bone and where the pH is 5. This acidification causes the dissolution of the mineral fraction. Lysosomal hydrolases cause the degradation of the organic matrix. Some products of the demineralization and the protein hydrolysis can be resorbed by osteoclastic endocytosis to be fully degraded. The cavity then formed due to the resorption at the ruffled border of the osteoclasts constitutes the Howship lacuna. Howship Lacuna Microvilli Osteoclast Osteoclast B- EXTRACELLULAR MATRIX OF BONE TISSUE The ECM of the bone is formed of two parts intimately related to each other: an organic matrix (30% of the dry weight of the bone) and a mineral matrix (70% of the dry weight of the bone): The organic matrix (osteoid) is acidophilic and is formed of: - collagen fibers (90%): type I (80%), but also types III and V. Their orientation and their disposition determine the morphology of the bone. - a fundamental substance (10%): it is poorly hydrated (water forms only 50% of this fundamental substance). It consists of proteoglycans and sulphated glycosaminoglycans, growth factors, signaling molecules, as well as a very small amount of lipids (cholesterol and triglycerides). Structural glycoproteins are also found in the organic ECM. The mineral matrix: it consists of a complex of ions and mineral salts that are arranged along and inside the collagen fibers and that give the bone its hardness and 41 rigidity. Calcium and phosphorus predominate in this mineral fraction,, in the form of hydrated hydroxyapatite crystals (formula Ca10(PO4)6(OH)2). Other calcium salts (bicarbonate, citrates and calcium fluoride) and magnesium salts are also observed. C- PERIOSTEUM AND ENDOSTEUM The bone tissue is bordered by non-specialized connective tissue, except at the level of the articular cartilages. - The periosteum covers the outer surface of the bone. In adults, it has a thin outer layer of dense connective tissue (tendiniform or fibrous layer) and a predominantly cellular and well vascularized inner layer (osteogenic fibro-elastic layer) of loose connective tissue, containing stem cells. There are bundles of non-oriented collagen penetrating deep into the bone tissue: these are Sharpey's fibers. They attach the periosteum to the bone. - The endosteum is a thin layer of non-specialized connective tissue that lines all the internal cavities of the bones. It is mainly related to the red bone marrow. The endosteum has the same osteogenic potentialities as the periosteum: its cells can be transformed into osteoblasts in case of bone fracture (osteogenic role). The periosteum and the endosteum contribute to the growth in thickness of the bone. They also participate in bone repair. Periosteum Haversian canals Epiphysis Haversian systems Metaphysis Endosteum Diaphysis Periosteum Structure of bones 42 D- VARIETIES OF BONE TISSUES Bone tissues differ in the abundance and especially in the arrangement of their different constituents. Thus we distinguish: 1. Immature bone tissue (woven or non-lamellar or reticular): It is the first to form. It is elaborated from cartilage or connective tissue. It occurs mostly in the fetus and it usually has a very short lifetime because it is transient and is replaced in adults by mature lamellar secondary bone tissue. It is mechanically weak and formed of trabeculae. Osteocytes are numerous and arranged without order and without precise orientation. Their shape is irregular and their size is variable, but larger than that of the cells of the secondary bone. The matrix is poorly mineralized. It has randomly arranged interlocking bundles of collagen fibers. The fundamental substance is richer in proteoglycans than that of lamellar bone. 2. Mature bone tissue (lamellar): it derives from the secondary ossification of a primary tissue and develops according to strong mechanical constraints. The lamellar bone tissue constitutes almost all of the adult bone. It is mechanically resistant. It is formed by superposed concentric lamellae. In each lamella, the collagen fibers are fine, oriented homogeneously. But this orientation varies between lamellae. Osteocytes are regularly distributed within the matrix and elongated parallel to the lamellae. Depending on the arrangement of the bone lamellae, we distinguish the Haversian compact lamellar bone tissue, the non-Haversian compact lamellar bone tissue and the spongy or trabecular lamellar bone tissue. a) Haversian compact lamellar bone (cortical or dense): this tissue forms the major part of the diaphysis of long bones. It consists of cylindrical structures: osteons or Haversian systems. An osteon is made of 4 to 20 cylindrical bony lamellae concentrically arranged around the haversian canal. A haversian canal contains blood capillaries and amyelinated nerve fibers embedded in loose connective tissue. In osteons, collagen fibers are arranged parallel to each other in the same lamella but in varying directions 43 from one lamella to another. Between the lamellae are the osteoplasts containing the osteocytes. The osteons are arranged parallel to each other and parallel to the major axis of the diaphysis. They are interconnected with the medullary cavity and with the surface of the bone by transverse canals: the Volkmann canals. Incomplete osteons - remnants of old, partially resorbed osteons, forming the interstitial lamellar bone (or intermediate Haversian system) – are observed between complete osteons. b) The compact lamellar bone (non-Haversian): the diaphysis of the long bones is bordered externally and internally by large bony lamellae concentric to the medullary cavity and which separate the Haversian bone from the endosteum and the periosteum. c) Trabecular or spongy lamellar bone: it is organized in trabeculae separated by large cavities. The trabeculae, of various thicknesses, consist of a few bone lamellae. Collagen fibers have the same orientation in each lamella. There are osteoplasts, containing the osteocytes. The cavities, of irregular size and shape, are communicating with each other and contain red bone marrow. Spongy lamellar bone forms epiphyses and metaphyses of long bones and the diploë of flat and short bones. E- GENERAL ARCHITECTURE OF BONES Long bones Anatomically, a long bone has a middle part, the diaphysis and two generally bulging ends, of variable form, the epiphyses. Diaphysis and epiphyses are connected by metaphyses. - The diaphysis comprises a hollow cylinder with walls of compact bone tissue surrounded by the periosteum on the outside and bordered by the endosteum on the inside. The center of the cylinder is the medullary cavity or canal, which is occupied by a yellow bone marrow (adipose tissue) and vessels. - The epiphyses consist of cancellous trabecular bone tissue. On the periphery, they are covered with dense bone tissue, except at the articular surface, covered with articular 44 cartilage. The cavities of this spongy tissue are filled with hematopoietic red bone marrow and communicate with the medullary diaphyseal cavity. - The metaphyses correspond to a conical segment situated between the epiphysis and the diaphysis. They are made of spongy bone tissue, surrounded by a layer of compact bone that extends the diaphysis bone. Short bones The short bones have a structure comparable to the epiphysis of long bones. They are formed of trabecular cancellous bone surrounded by a thin cortex of periosteal and dense bone tissue. Flat bones The flat bones consist of a central layer of trabecular cancellous bone tissue (the diploë) surrounded by compact bone tissue (the outer and inner tables covered by a periosteum). Long bone Epiphysis Spongy bone Short bone Periosteum Diaphysis Compact bone Medullary cavity Flat bone External plate Metaphysis Epiphysis Diploe Internal plate Articular cartilage 45 F- OSTEOGENESIS AND DYNAMICS OF BONE TISSUE I– Primary ossification 1-Before birth This is the initial formation of bone tissue in the fetus. This primary ossification results in the formation of non-lamellar primary bone from an embryonic connective or cartilaginous model. a. Endomembranous ossification (intramembraneous or endoconnective or ossification of the membrane): it concerns most of the bones of the face and the cranial flat bones. During this ossification, embryonic mesenchymal cells proliferate around the blood vessels and differentiate into osteoblasts that secrete an osteoid substance that will then mineralize rapidly. b. Endochondral (or endocartilaginous) ossification: This is the form of ossification of most skeletal bones (long bones, short bones and some flat bones). Its main consequence is the growth in length of the bone. It continues during childhood until bone growth stops at adulthood. → Ossification of the diaphysis of a long bone: In summary, going from the epiphysis towards the diaphysis, we distinguish the following structures characterizing this stage: - Hyaline growth cartilage (Reserve zone) - Serial cartilage formed by axial isogenic groups (Proliferative zone) - Hypertrophied cartilage with large cells and reduced fundamental substance (Hypertrophic zone) - Calcified cartilage, which fundamental substance is infiltrated with limestone; its cells are in the process of degeneration (Calcification zone) - A line of erosion where the cartilage begins to be destroyed by the vasculo- conjunctive buds. - An osteogenic zone where bone tissue replaces cartilaginous tissue (Ossification or osteogenic zone). 46 → Ossification of the epiphyses: the primary ossification the epiphysis starts late (at birth) at a time where the primary ossification of the diaphysis is already advanced. It is carried out by endochondral ossification. This endochondral ossification, which is carried out according to a process comparable to that described at the level of the diaphysis, respects on the periphery of the epiphysis a layer of cartilage destined to become articular cartilage (on the outside) and the epiphyseal growth plate (on the inside). 2-After birth The long bones continue to grow in length and thickness (width) during childhood and adolescence. a) Growth in bone length: growth in bone length after birth is by endochondral ossification in the same way as before birth. b) Growth in bone width: it is ensured at the level of the diaphysis by successive peripheral apposition of new layers of bone tissue resulting from the activity of the periosteum (osteogenic layer): the osteoblasts add bone lamellae to the external surface of the bone. In the beginning, bone tissue is spongy in nature, but during remodeling, it is transformed into a compact bone. 47 II- Secondary ossification It is the bone maturation that is achieved at the end of puberty. It consists of a bone remodeling, that a replacement of the bone tissue developed during primary ossification by a new bone tissue: the woven (reticular) bone tissue, whether of endoconnective or endochondral origin, wether diaphyseal or epiphyseal, is replaced by lamellar bone tissue. Bone turnover is fast. The lifespan of osteons is a few months, but gets longer in the elderly. However, the mineralization of the matrix is progressive, the younger osteons being slightly mineralized. 48 Chapter 6 BLOOD TISSUE Blood tissue (blood) is a specialized, mesenchymal connective tissue that includes free cells (the formed elements) suspended in an extracellular liquid matrix (the plasma). It is contained in a network of vessels outside of which it coagulates. It ensures the constancy of the internal environment (acid/base balance, water balance, body temperature regulation) and transmits to all tissues the oxygen and nutrients they need. It evacuates the CO2 and the waste products. It transports hormones, allows the defenses of the body to quickly reach the place where they are needed and ensures blood clotting during bleeding. In humans, it represents 8% of the body weight (5 to 6 liters). A- PLASMA It is an aqueous solution that corresponds to the ECM of blood tissue and represents 55% of the total blood volume. The plasma is composed of: - water [91.5%] - plasma proteins [7%]: albumins (55%), globulins (38%), fibrinogen (7%). The plasma proteins confer to blood its viscosity. They also allow the maintenance of an osmotic balance and a slightly alkaline pH (pH 7.35 - 7.45) - and other solutes [1,5%]: organic materials (triglycerides, cholesterol, glucose, urea...), mineral salts and electrolytes (chloride, sodium, potassium…) and dissolved gases. B- FORMED ELEMENTS The formed elements represent 45% of the total blood volume. They form a morphologically and functionally heterogeneous cellular population, continuously being replenished. They are either real cells (leucocytes), or cells lacking nucleus and organelles (red blood cells), or cell fragments that contain certain cytoplasmic structures (platelets). 49 In a normal state, red blood cells (RBCs) and platelets do not leave the blood compartment and exert their physiological function in the circulating blood, whereas leucocytes are the only ones able to cross the capillary wall and are found in the tissues where they exert their functions. The serum contains all plasma elements except fibrin and proteins involved in coagulation. THE HEMOGRAM The blood count: It consists in counting the number of red blood cells, white blood cells and platelets per mm3 of blood. The normal results are as follows : Red blood cells: 4,500,000 to 5,500,000 / mm3 White blood cells: 4,000 to 8,000 / mm3 Platelets: 150,000 to 400,000 / mm3 The blood formula (leukocyte count): it is the counting of the different varieties of leucocytes. Normal results, expressed in percentage of the total number of leukocytes (or in absolute number), are as follows: Neutrophil granulocytes: 50 to 70% Eosinophilic granulocytes: 1 to 3% Basophilic granulocytes: 0 to 1% Lymphocytes: 25 to 40% Monocytes: 2 to 10% Blood vessel Leukocyte Plasma Red blood cell Platelets The blood tissue 51 1. Red blood cells (or erythrocytes): The red blood cells (RBCs) are the most abundant formed elements of the blood. They are highly differentiated, biconcave disc shaped cells, flattened in the center, particularly deformable and with a diameter of 6 to 8 µm. They are anucleated and do not possess cytoplasmic organelles and therefore do not reproduce and do not carry out any significant metabolic activity.The cytoplasm consists mainly of hemoglobin, water (60%) but it also contains a cytoskeleton, ADP, enzymes, ions and glucose (anaerobic glycolysis). Hemoglobin accounts for 33% of the total cell mass; it gives blood its red color and fixes oxygen (oxyhemoglobin) and CO2 (carbaminohemoglobin). The cytoskeleton is closely related to the plasma membrane. It maintains the characteristic shape of the cell and allows its deformability. Glycoproteins called agglutinogens responsible for the determination of blood groups (such as the ABO system) are found at the RBCs plasma membrane level. The lifespan of red blood cells is between 100 and 120 days. They are eliminated by hemolysis by macrophages in the liver and spleen. 2. Platelets (or thrombocytes): Platelets are anucleated cell fragments surrounded by a plasma membrane. They are most often grouped into clusters. Their diameter varies from 2 to 4 μm. Their shape varies according to their degree of activity. The cytoplasm of the platelet consists of the inactive peripheral hyalomere and the basophilic central granulomere. The granulomere contains lysosomes, peroxisomes; while the hyalomere contains a cytoskeleton rich in contractile proteins (actin, myosin and thrombosthenin) and microtubules, and an enzymatic system for aerobic and anaerobic metabolism. The cytoplasm also contains glycogen and mitochondria. Platelets play a vital role in stopping bleeding (hemostasis). Their lifespan is 8 to 12 days. The spleen is a big platelets reserve. 51 3. White blood cells (leukocytes): Leukocytes are cells that have a nucleus and organelles. They do not contain hemoglobin and their plasma membrane contains proteins (histocompatibility antigens, MHC or HLA antigens) that serve as a basis for identifying a tissue (transplant rejection). Leukocytes participate in immune and inflammatory reactions developed by the organism to protect itself from pathogenic agents (viruses, bacteria, worms, parasites…). They are also involved in pathological immune reactions (hypersensitivity reactions, autoimmune reactions). Leukocytes are carried by the blood and they can leave it by diapedesis by infiltration between the endothelial cells of the capillary wall to pass into the tissues. The lifespan of leukocytes is a few days (except T lymphocytes and B memory lymphocytes). Leucocytes are grouped in 2 main categories according to the observation or not of specific granules in optic microscopy: - Granulocytes: neutrophils, eosinophils, basophils - Agranulocytes: monocytes and lymphocytes ◄ The granulocytes: they are characterized by a single nucleus that presents several lobes with different shapes and granules in the cytoplasm. According to the dye affinity of these specific granulations, there are 3 groups of granulocytes: neutrophils, eosinophils and basophils. Granulocytes 52 a) Neutrophil granulocytes : the most numerous leukocytes in human blood. They are spherical in the blood. Their nucleus is multi-lobed: it consists of 3 to 5 well- individualized lobes, linked by thin bands of chromatin. Their cytoplasm contains few organelles and two types of granules: - azurophilic, nonspecific granules: the least numerous and the largest. They are considered lysosomes. - secondary specific granules: they have little affinity for dyes, they are the smallest and the most numerous, they are devoid of lysosomal enzymes and peroxidase, but they contain mainly lysosyme, collagenase.... Neutrophils are mainly involved in the non-specific defense processes of the body, including anti-bacterial fight by phagocytosis. Their lifespan is 24 hours in the circulating blood. b) Eosinophilic granulocytes (acidophilic): They are slightly larger than neutrophils, their diameter being between 10 and 14 μm. They have a nucleus usually made of 2 lobes joined by a rather thick chromatin bridge. Their cytoplasm contains the usual organelles of the cell, a small number of azurophilic granules, but especially numerous and large eosinophilic specific granules, rounded, of lysosomal nature. Eosinophils are mainly involved in the defense reactions against parasites. They also have bactericidal and phagocytic properties (but to lesser degrees than neutrophils). They also participate in immediate and delayed hypersensitivity reactions (their plasma membrane has a receptor for IgE). They play a role in the neutralization of histamine by releasing histaminase. After a half-life of 4 to 5 hours in the bloodstream, eosinophils pass into the tissues where they remain for 8 to 12 days. c) Basophilic granulocytes: They are the least abundant granulocytes, and are very difficult to find. Their diameter is between 8 and 10 µm. The nucleus, is indistinctly lobed, irregular and can take a clover aspect. The cytoplasm contains the usual organelles of the cell and especially large basophilic granules, fewer than those of neutrophils and eosinophils, covering the nucleus. These granules are not considered 53 as lysosomes and they contain heparin, histamine... The plasma membrane of basophils has receptors for IgE hence their role in allergic reactions. The lifespan of basophils is of the order of 3 to 4 days. ◄ The agranulocytes: They are characterized by a regular nucleus and a cytoplasm which does not present visible granules in optical microscopy. a) Lymphocytes: are found throughout the body and in particular: the lymphoid organs, the loose connective tissue and the covering epithelia. These are the only cells in the lymph. Their morphological aspect is characterized by: 1) their regular, rounded shape; 2) their size, usually small (7 to 8 μm in diameter); however, alongside these small lymphocytes, medium and large lymphocytes are distinguished, of moderately larger size (10-12 μm); 3) their nucleus with compact chromatin, spherical, dark, without visible nucleolus, occupying almost the whole cell; 4) their cytoplasm, slightly basophilic, reduced to a thin ring around the nucleus. It contains the usual organelles of the cell but in limited quantity and some azurophilic granules (lysosomes). Lymphocytes are involved in the coordination of the body's immune responses. They group three major functional families that can be recognized by different membrane antigens: T lymphocytes, NK lymphocytes and B lymphocytes. - T lymphocytes: these are the most abundant lymphocytes, they are involved in cell-mediated immunity. Their maturation takes place in the thymus. They are characterized by the presence in their plasma membrane of a protein serving as a receptor for antigens (T receptor). CD4 (helper or T4) lymphocytes and CD8 (cytotoxic or T8) lymphocytes are distinguished. - NK lymphocytes: these are non-T non-B cells identified by NK markers. They often have the appearance of granular lymphocytes containing azurophilic granules. - B lymphocytes: their maturation is in the red bone marrow. They are characterized by the presence of a B receptor. They are responsible for humoral- mediated immunity. In lymphoid organs, they differentiate into plasma cells that synthesize antibodies (IgA, IgG, IgE, IgM, IgD) after presentation of the antigen. 54 Lymphocyte b) Monocytes: They are the main professional phagocytes. In blood, they represent the largest normal leukocytes (15 to 18 μm in diameter). Their shape is rounded and their nucleus is bulky, central or peripheral. It is often kidney-shaped. The cytoplasm is characterized by the presence of some azurophilic granules (lysosomes). In electronic microscopy, the cytoplasmic organelles appear poorly developed. The lifetime of the monocyte in the blood is very short (about 24 hours). It then passes into the tissues where it differentiates into a macrophage. The blood monocyte. Diagram showing pinocytotic vesicles (PV), lysosomal granules (G), mitochondria (M), and isolated rough endoplasmic reticulum cisternae (E) 55 C- CELLS OF HEMATOPOIETIC ORIGIN THAT ARE IN THE TISSUES Lymphocytes and different types of granulocytes migrate from the blood compartment to the tissues where the following cells are observed: 1- Mastocytes : The basophilic granulocytes and mast cells are two types of cells that derive from the same determined cell precursor in the red bone marrow. Mast cells are found in almost all connective tissues, in the vicinity of blood vessels and nerves (but they are more abundant in the skin, the respiratory tract and the digestive tract). They are mobile cells, rounded but with irregular contours. Their nucleus is small, rounded and central. Mast cells are easily recognizable by their cytoplasmic content in basophilic granules (heparin and histamine...). The functions of mast cells and the characteristics of their plasma membrane and granulations are similar to those of basophils. Antigen Receptor IgE antibody Degranulation Diagram of a mastocyte (a) at rest, (b) stimulated by an antigen. 2- Plasmocytes: They differentiate from B cells in lymphoid and hematopoietic organs and loose connective tissue. Their characteristics make them very easy to recognize: 1) an oval shape; 2) a rounded nucleus, located in an eccentric position, with a radially shaped chromatin dispersed in large clumps at its periphery (in a wheel radius shape); 3) a basophilic cytoplasm, occupied by a highly developed RER and a 56 voluminous Golgi apparatus. Plasma cells make antibodies; they do not divide and most die in a few days. Plasmocyte, with chromatin in wheel radius 3- Macrophages: After their passage the tissues, monocytes differentiate into macrophages. The size of macrophages is larger than that of monocytes (20 to 40 μm). Their cytoplasm is slightly eosinophilic. It presents vacuoles and lysosomes, cytoplasmic expansions that form true pseudopodia, a highly developed cytoskeleton related to the extreme mobility of this cell. Macrophages are involved in all the body's major defense mechanisms against foreign agents. They can act by phagocytosis, by secretion of toxic substances, and by triggering an immune reaction. A distinction is made between macrophages recruited from a tissue during an injury or a local immunological reaction and macrophages permanently present in certain tissues. Macrophage. A. Fibroblast B. Macrophage C. Plasmocyte D. Mastocyte 57 D- ORIGIN OF BLOOD CELLS - HEMATOPOIESIS Blood cells are continuously destroyed because their life time is short and limited. Hematopoiesis is the process of continuous and regulated formation of the blood cells in order to maintain their constant and stable number. It is carried out in adults in the red hematopoietic bone marrow present in cavities of cancellous bone, from multipotent stem cells (MSCs). Multipotent stem cells (MSCs) or hemocytoblasts are undifferentiated cells. They are small with a reduced basophilic cytoplasm, a nucleus with one or two nucleoli. They can : - either divide to self-renew: they give birth to new multipotent stem cells, - or differentiate themselves: they will be irreversibly engaged in a differentiation pathway. During hematopoiesis, two major differentiation pathways are described: - The lymphoid stem cell which gives 2 types of "determined" cells that lead to T and B lymphocytes. - The myeloid stem cell which gives 5 types of "determined" cells, which will eventually lead to erythrocytes, platelets, neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes and monocytes. 58 Origin and stages of differentiation of blood cells 1. Erythropoiesis Erythropoiesis is the process of differentiation, proliferation and maturation that lead a pluripotent stem cell to become a circulating red blood cell. It is done by the main action of a growth factor, the erythropoietin. It lasts 5 to 7 days and during this process the size of the cells decreases. The myeloid stem cell, by successive divisions and transformations, gives the proerythroblast, the basophilic erythroblast, the polychromatophilic erythroblast (beginning of hemoglobin synthesis (Hb), observed color change, nucleus is even 59 denser), acidophilic orthochromatophilic erythroblast (hemoglobin synthesis is at its peak, at this stage the nucleus is highly condensed, it is expelled with the organelles outside the cell). The orthochromatophilic erythroblast differentiates into reticulocyte (cell with some ribosomes and some mitochondria and nuclear debris). The reticulocyte leaves the bone marrow by amoeboid movement and passes into a blood capillary. In 48 hours, it achieves its maturation in blood, loses all active mobility and finally becomes a red blood cell or erythrocyte. Erythropoiesis 2. Thrombopoiesis This is the formation of blood platelets. It is regulated by a stimulating factor, thrombopoietin. The myeloid stem cell gives rise to a megakaryoblast, with that has a very basophilic cytoplasm, rich in ribosomes. The DNA replicates several times without any cell division and the megakaryoblast becomes a promegakaryocyte then a basophilic megakaryocyte (50 µm) with a giant lobed nucleus (polyploidy). The cytoplasm is enriched in granulations and the cell becomes a thrombocytogenic megakaryocyte: it is a large cell with a huge nucleus, a network of invaginations of the plasma membrane 61 and whose cytoplasm is distributed in 5 to 8 '' pro platelets territories '', each of these territories getting fragmented later on to release in a sinusoid (blood capillary) of the red bone marrow between 1000 and 1500 platelets. Thrombopoiesis 3. Granulopoiesis The three granulocyte lineages derive from the myeloblast (determined precursor cell) which gives the promyelocyte (appearance of nonspecific "azurophilic" granules) and then the myelocyte. At this stage, specific granules appear (neutrophilic, acidophilic, basophilic). The myelocytes give then the metamyelocytes where the number of specific granules increases. During granulopoiesis, the cell size gradually decreases. The nucleus becomes more and more condensed. Growth factors, called G-CSF, are involved in the regulation of granulopoiesis. Granulopoiesis 61 4. Monopoiesis The monoblast gives a promonocyte that differentiates into monocyte in the blood. The regulating factor is M-CSF. 5. Lymphopoiesis Two determined cells or T and B lymphoblasts give either a T prelymphocyte that will give a T lymphocyte, or a B prelymphocyte that will give a B lymphocyte. 62 Chapter 7 MUSCLE TISSUES Muscle tissues are specialized in the production of mechanical work or muscle contraction. They derive from the embryonic mesoderm. They are made of muscle cells (myocytes or muscle fibers) that contain in their cytoplasm contractile proteins forming myofilaments grouped into myofibrils. There are 3 types of muscle tissues that differ in the structure of their cells, their location in the body, their function, and the triggering mode of their contractions: - Skeletal striated muscle tissue is usually associated with the skeleton. Its fibers are the longest muscle fibers and throughout their length there are regular transverse bands called striations. The contraction of these cells is voluntary and allows voluntary movements and posture maintenance under the control of the nervous system. - Cardiac striated muscle tissue exists only in the wall of the heart. It is a highly differentiated form of striated muscle which cells perform involuntary rhythmic contraction. - Smooth muscle tissue is involved in the processes related to the maintenance of the internal environment. The cells are unstriated and involuntary. These tissues are found in the walls of vessels and hollow visceral organs, and in the respiratory tract organs. A – SKELETAL STRIATED MUSCLE TISSUE 1. The skeletal striated muscle cell (rhabdomyocyte) It is the functional unit of a skeletal muscle. The skeletal striated muscle cell or fiber (SSMC, rhabdomyocyte) is a long cylindrical, regularly contoured cell with transverse striation. 63 Most of the cytoplasm is occupied by myofibrils, which together constitute the myoplasm. In the remaining cytoplasm or sarcoplasm, and between the myofibrils, the various common organelles are observed, notably: - several hundreds of ovoid nuclei, aligned at the periphery of the cell, against the plasma membrane and oriented in the longitudinal direction of the cell, - numerous and large mitochondria grouped in the vicinity of the nuclei and aligned in line between the myofibrils, - highly developed sarcoplasmic reticulum (SER) that forms a network participating in the so-called sarcotubular system or T system. - a cytoskeleton formed mainly of microtubules and intermediate filaments of desmin. We also observe in the sarcoplasm: - Lipid vacuoles and important reserves of glycogen near the mitochondria. - Myoglobin red pigments which fix the oxygen to deliver it to the mitochondria. The plasma membrane, called sarcolemma, is doubled externally by a basal lamina. The SSMC is unable to divide; it constitutes a syncytium formed in the embryonic state by the fusion of mononuclear precursor cells (the myoblasts). Satellite Myoblasts cells Fusion of myoblasts Satellite Differentiation cells Muscle fibers Differentiation of a rhabdomyocyte 64 2. Organization of rhabdomyocytes The skeletal striated muscle cells associate to form together skeletal striated muscles. These are surrounded by a conjunctive envelope. The dense fibrous connective tissue that surrounds the skeletal striated muscle and individualizes it, is called epimysium. The epimysium enters the muscle and forms the perimysium that surrounds groups of muscle cells, leading to partition of the muscle into fascicles. The invaginations of the perimysium, called endomysium, penetrate inside the fascicles to surround and separate neighboring rhabdomyocytes. 3. Ultrastructure, composition and molecular organization of myofibrils: The myofibrils are formed by an assembly of protein myofilaments and are differentiated for contraction. In the myoplasm of an SSMC, several hundreds or thousands of myofibrils are observed, which constitutes the majority of the cell volume. The myofibrils are cylindrical, parallel, elongated in the cell direction, clamped against each other, and extend from one end of the cell to the other. In longitudinal section, they present a transverse striation due to the alternation of bands (disks): - dark, anisotropic (A) in polarized light microscopy i.e. of non-homogeneous appearance, - light (clear), isotropic (I) and homogeneous, divided in their middle by the Z line. The portion of the myofibril between two successive Z lines is called a sarcomere. 65 The sarcomere results from the orderly and parallel arrangement of fine and thick myofilaments along the myofibrillar axis. It comprises the A band in the middle region and two I half-bands on either side: I/2 + A + I/2. It is the smallest structural and functional unit of the muscle cell (contraction unit). The myofibril is thus a sequence of sarcomeres and in a cell, the sarcomeres of all myofibrils are located at the same level, which explains the transverse striation of the myocyte. The dark band A is formed by thick myosin filaments and thin actin filaments arranged in a parallel manner, while the I band is only formed by thin actin filaments. The band A comprises in its middle a clearer transverse zone, called the H zone, only formed of myosin, and divided in its middle by a dark transverse line 66 called the M line. The myosin molecule comprises a cylindrical rod or axis (or tail), which terminates at one of its ends by 2 spherical heads that interact with actin through actin binding sites, and have the ATPase activity necessary for the hydrolysis of ATP. Each half-band A comprises the heads and the tails of the thick filaments, whereas the H zone consists solely of tails. The M line includes myomesin which connects the thick filaments to each other. Each light I band contains fine (or thin) actin myofilaments with troponin and tropomyosin molecules. It is divided transversely into two parts by the very dense Z line. The fine filaments extend on both sides of the Z line over the entire length of the I band and the Z line composed of alpha-actinin is thus the point of attachment of these fine filaments. The fine filaments consist of globular monomers (G- actin) forming the F-actin filaments composed of two helically entwined (double helix) polypeptide chains. G- actin is characterized by binding sites on which the myosin heads attach during contraction. Tropomyosin consists of two α-helical polypeptide chains located in the groove of the F- actin filament. Troponin is composed of 3 globular subunits forming a complex: Troponin T (Tnt): binds the complex to tropomyosin. Troponin I (Tni): inhibits the attachment of myosin heads to actin at rest. Troponin C (Tnc): has a binding site for calcium. 67 A very thin elastic filament formed of the large protein titin (connectin), the third most abundant protein (after actin and myosin) in skeletal muscle, links the extremities of the thick filaments of the A band to the Z line, contributes to the stabilization of the Z line position, and plays a role in restoring the sarcomere resting length during relaxation. 68 4. Smooth sarcoplasmic reticulum and T-System The plasma membrane has transversal invaginations (T tubules), arranged around the junction zone between the A and I bands of each myofibril. The T tubule network is called the transverse system or T system. The smooth sarcoplasmic reticulum is located around each myofibril and runs parallel to it. It consists of tubules and sacs that periodically meet to form terminal cisternae at each A-I bands junction. The SER contains high concentrations of Ca ++ which release in the cytosol is responsible for the muscle contraction. SER cisternae are adjacent to either side of a T tubul
