Department of Natural Sciences University of St.

La Salle

GENERAL CHARACTERISTICS
They have fewer cells that are set apart due to an abundance of intercellular substance or ground substance which contains fibers. The different contents of intercellular substance, CT cells and fibers account for the difference in appearance of the various CT. Except in a few CT types (mucous and cartilage), there are blood vessels, lymphatics and nerves found in the ground substance. The physical properties of CT allow it to bind, fill spaces, and to separate functional units of other tissues and organs, allowing maintenance and coordination of all body functions.

All CT cell types are derived from cells of embryonic mesenchyme originating from the embryonic mesoderm.

 In any type of CT, there are 3 basic elements to consider.  CT types and subtypes are classified according to the amount, types, and proportions of these 3 components: 1.Ground substance- forms the extracellular matrix together with tissue fluid (bound water of solvation) and protein fibers; maybe fluid, gelatinous or solid 2.Fibers- maybe white or collagenous, yellow or elastic, reticular or argyrophyl 3.CT cells- of several types, mobile wandering or relatively fixed elements

GROUND SUBSTANCE
This abundant intercellular substance vary in consistency from a thin fluid to a hard, calcareous substance. The formed elements of CT are embedded in this matrix of amorphous, colorless, transparent material with the properties of a viscous solution or thin gel. It is the medium through which all nutrients and waste products must pass in transit between the blood and parenchymal cells of the organs. It consists mostly of tissue fluids, salts and 2 classes of glycoconjugates:

1.PROTEOGLYCANScomposed of a core protein to which glycosaminoglycans (GAGs) are attached. GAGs are straightchain polymers of repeating sugar heterodimers made up of hexosamine and uronic acid. 2.Five major classes of GAGs exist in CT: hyaluronic acid (does not form proteoglycans, and determines the structural and physiological characteristics of ground substance), chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate. Lysosomal enzyme deficiencies for degradation of GAGs lead to their accumulation, resulting to several human disorders e.g., Hurler syndrome, Hunter syndrome, Morquio syndrome

ECM of mouse endometrium after fixation in the presence of Safranin O. A network of proteoglycans fills the intercellular spaces. Some proteoglycan filaments are in close contact with the cell surface (arrows).

Schematic diagram of cell-surface proteoglycan. The core protein spans the plasma membrane through the cytoplasmic domain. The proteoglycans possess 3 heparan sulfate chains and sometimes chondroitin sulfate.

2.GLYCOPROTEINS- globular proteins to which shorter, branched oligosaccharide chains are covalently bound. These so-called adhesion glycoproteins mediate attachment of cells to their matrix, influence the state of differentiation of cells, and organization of their cytoskeleton. Examples: Fibronectin- long flexible molecule with cell-binding, collagen-binding, GAG-binding, and cytoskeleton-binding domains along their length; cell surface receptors are called integrins; plasma fibronectin synthesized by liver and endothelial cells binds to fibrin during blood clotting reactions

 Laminin- binds to membranes of epithelial and muscle cells, to type-IV collagen, and heparan sulfate; significant in the assembly of the basal lamina, influencing the form and function of epithelial cells  Thrombospondin- activated platelet-product; binds to fibrinogen, plasmalogen and its activator; a participant in blood clotting; function is poorly understood  Chondronectin- a component of cartilage matrix that mediates attachment of chondrocytes to their matrix  Fibrillin- a nonsulfated glycoprotein speculated to be essential for normal development. It is often associated with elastic fibers or with epithelial basal laminae Marfan syndrome is due to a defective fibrillin gene on chromosome 15, characterized by excessively long arms and legs and progressive dilatation and fatal rupture of the ascending aorta (Pres. Abraham Lincoln).

A: The structure of fibronectin. Fibronectin is a dimer bound by SS groups, formed by serially disposed coiled sites, that bind to type I collagen, heparan sulfate, other proteoglycans, and cell membrane receptors. B: The structure of laminin, which is formed by 3 intertwined polypeptides in the shape of a cross. There are sites on the molecule with a high affinity for cell membrane receptors and type IV collagen and heparan sulfate, which are components of basal laminae. Laminin thus promotes adhesion of cells to basal laminae.

Transverse section of mouse endometrium. Immunocytochemical staining shows the distribution of fibronectin in the endometrial stroma.

Transverse section of tongue. Immunocytochemical staining shows the distribution of laminin basement membranes in epithelial layer, capillary blood vessels, nerve fibers, and striated muscle.

 GAGs in CT are highly sulfated which attracts water of hydration. If fluid is injected into CT, it remains localized, walled off by a viscous ground substance. This property acts as barrier to the spread of bacteria that gains access to the tissues. Some bacteria secrete hyaluronidase (Staphylo/ Strepto/ Pneumococci), and collagenase (Clostridium perfringens) that breakdown matrix components. EDEMA is a condition characterized by accumulation of excess tissue fluid. The water in tissue fluid is forced out by hydrostatic pressure from the arterial end. Loss of fluid to the tissues increases the colloid osmotic pressure, and draws most of the fluid back into the blood. Excess fluids remaining in tissues is normally drained away by lymphatic capillaries, so that there is no net change in the amount of blood or tissue fluid.

 Edema accompanies pathological conditions that cause:  Increased hydrostatic pressure in capillaries by obstructing venous blood flow (e.g. congestive heart failure)  Decreased colloid osmotic pressure in the blood caused by lack of blood proteins (e.g. starvation)  Increased hydrostatic pressure in the tissue caused by blockage of lymphatic drainage by parasites or tumor cells  Increased colloid osmotic pressure in the tissue caused by excessive accumulation of GAGs in the matrix.  Hypothyroidism resulting from this condition is referred to as myxedema.

CONNECTIVE TISSUE FIBERS

1.COLLAGEN FIBERS Principal producers are fibroblasts; epithelial and smooth muscle cells also secrete their own type-IV collagen. Most numerous CT matrix, running in all directions in a wavy course; dull and opaque in appearance. Fibers bundled together branch and anastomose; individual fibers do not branch. With the EM, unit fibrils of collagen show periodic cross striations every 67 nm of their length.

Unit fibrils are made up of 3 polypeptide chains (E-chains) held together by hydrogen bonds.

 Collagen occurring in bundles, or as individual fibrils, exhibit acidophilic properties in H & E stained sections. In sections stained with Massonss trichome, they stain green, blue with Mallorys trichome stain. Thin fibers (type III) stain darkly with silver stains, but thicker bundles do not. Collagen that do not form fibers (type IV) cannot be distinguished from the surrounding ground substance except by immunohistochemistry.

Synthesis and Assembly of Collagen

1.INTRACELLULAR free polysomes reading collagen mRNA attach to the rER, and protocollagen or precollagen-chains are deposited in the cisternae. Each chain has about 250 amino acids; every 3rd amino acid is glycine. The signal peptide is clipped off. Proline and lysine residues within the chains are then hydroxylated in the ER to form hydroxyproline and hydroxylysine (unusual amino acids present in large amounts in collagen). Core sugars (galactose and glucose) attach to the hydroxylysine residues in the ER. Each chain is synthesized with an extra length of peptides known as registration peptides, which ensure that the appropriate chains assemble in their correct position in the resulting triple helical molecule called procollagen. Further glycosylation may occur in the Golgi complex, where procollagen is packaged for secretion. Golgi vesicles release procollagen into the extracellular space by exocytosis.

2.EXTRACELLULAR- in the extra cellular space, the enzyme procollagen peptidase cleaves the registration peptides from procollagen, converting it to tropocollagen. Catalyzed by lysyl oxidase, these become aligned in staggered fashion to form collagen fibers, possibly under the control of adjacent fiber-producing cells. The turnover of collagen is slowest in tendons, fastest in loose CT. Macrophages and neutrophils break down old collagen, and replaced by fibroblasts. As humans age, extracellular collagen becomes increasingly cross-linked, & turn-over slows down in CT.

COLLAGEN TYPES- fibril-forming (I,II,III,V,XI), fibrilassociated (IX, XII), network-forming (IV), anchoring (VII): Type I- most abundant and widespread, forms large fibers and fiber bundles. It is found in tendons, ligaments, bone, dermis, organ capsules, and loose CT. Type II- form only very thin fibers; found in embryonic notochord; in adults, only in the nucleus pulposus of the intervertebral disk, vitreous body of the eye, and in the hyaline and elastic cartilage matrix. Type III- similar to type I, but is more heavily glycosylated and stain with silver. Often found in association with type I, these reticular variety forms networks of thin fibrils that are abundant in loose CT, in soft and compliant organs such as the walls of blood vessels, in the stroma of spleen, kidney and uterus, and compose the reticular laminae underlying epithelial basal laminae.

Collagen types I, II, and III are called interstitial collagens which form microscopically visible fibers. Type IV- does not form fibers or fibrils; major collagen type found in basal laminae. Type V- present in external lamina of muscle fibers, epithelial basal lamina, blood vessels and in small amounts elsewhere Type VI- present where types I and III are found; kidney, uterus, cornea. Type VII- largest of the collagen family; form anchoring fibrils of epithelial basal lamina, serving to stabilize and firmly anchor the epithelium to the dermis.

 Type VIII- secretory product of endothelial cells; a major component of Descemets membrane, the corneal epithelium basal lamina. Type IX- found mainly in cartilage; maintains the 3-D arrangement of type II fibrils by serving as coupler in their sites of intersection Type X- found in the matrix surrounding hypertrophic chondrocytes of degenerating growth plate cartilage in sites of future bone formation. Type XI (cartilage) and XII (tendon fibroblasts)very poorly understood types

Weight for weight, collagen tensile strength is greater than that of steel.

Histophysiology of Collagen
Chemical Characteristics- easily stained by ordinary dyes; yield gelatin when boiled; dissolve easily in boiling dilute acids and alkali; easily digested by artificial gastric juice; resistant to solutions of alkaline pancreatic juice. Cortisol (hydrocortisone) produced by adrenal glands under the influence of ACTH inhibits CT fiber synthesis and retards local inflammatory and immune responses by CT cells. Cortisol reduces local heat, redness and tenderness, but delay and impair wound healing.

 Because collagen synthesis depends on the expression of several genes and on several post-translation events, many human diseases are associated with faulty collagen synthesis. Examples: Progressive systemic sclerosis- excessive accumulation of collagen (fibrosis) in almost all organs; Keloid- local swelling caused by abnormal amounts of collagen that form in scars of skin; Ehlers-Danlos type IV- aortic/ intestinal rupture due to faulty transcription of collagen type III; Ehlers-Danlos type VII- increased articular motility due to decreased procollagen peptidase activity; Scurvy- ulceration of gums, hemorrhages due to lack of Vit. C, a cofactor for proline hydroxylase; Osteogenesis imperfecta- spontaneous fractures & cardiac insufficiency due to mutation in collagen type I

Section of a muscular artery stained with picro-sirius and observed with polarization optics. The upper tunica media (muscular layer) contains reticular fibers consisting mainly of collagen type III. The lower layer (tunica adventitia) contains thick fibers and bundles of collagen type I. Deficiencies of collagen type III may result in rupture of the arterial wall

2. YELLOW or ELASTIC FIBERS Form gentle curves or spirals at their free ends when released from tension Do not form bundles; individual fibers branch and anastomose to form networks They can be stretched to 150% of their length without breaking, but lose their resiliency with advancing age. Appears yellowish, highly refractile, homogenous and are not made up of fibrillar subunits that are visible with the light microscope. Composed of 2 elements: microfibrils, aggregated in bundles which are embedded in abundant and amorphous elastin.

 Each fibril is made up of still smaller fibrils united by a small amount of ground substance. These smaller microfibrils have periodic cross bandings.  Ground substance stains with PAS due to its proteinpolysaccharide content: mucopolysaccharides, hyalurinate, chondroitin sulfates.  Chemical characteristics:  Stains poorly with standard ionic dyes since it contains a few charged amino acids; as such it requires the special Verhoeffs (Weigerts resorcin fuschin) stain or Halmis aldehyde-fuchsin method.  It does not yield gelatin on boiling  Does not easily dissolve in boiling dilute acids and alkali; and easily digested by pancreatic juice which contains elastase.

In this side by side comparison of fibrous and elastic tendons cut in cross-section, the relative sizes of fibers is compared. Notice how large and robust the collagen fibers are compared to the smaller elastic types. Nuclei of fibroblasts visible along the edges of fibers are a good indicator these are tendons in cross-section.

Synthesis and Assembly of Elastin: 1.Intracellular- microfibrillar proteins containing mostly hydrophilic amino acids, and proelastin (contains large amounts of the hydrophobic amino acids glycine, proline and valine, thus accounting for elastins insolubility) are synthesized on rER and secreted separately. 2.Extracellular- proelastin molecules polymerize extracellularly to form elastin chains. Lysyl oxidases then catalyze the conversion of certain lysine residues of elastin to aldehydes, 3 of which condense with a 4th unaltered lysine residue to form desmosine and isodesmosine. These very rare amino acids found in elastin cross-link individual chains, which then associate with numerous microfibrils to form a branching and anastomosing network of elastic fibers.

Skin dermis, selectively stained for elastic fibers. Dark elastic fibers are interspersed with pale red collagen fibers. The elastic fibers are responsible for skins elasticity . Elastin molecules are joined by covalent bonds to generate an extensive cross-linked network. Because each elastin molecule in the network can expand and contract like a random coil, the entire network can stretch and recoil like a rubber band.

A: Rat mesentery showing

nonanastomosing bundles of collagen fibers, while the elastic fibers appear as thin, dark anastomosing fibers. Collagen and elastic fibers provide structure and elasticity, respectively, to the mesentery. B: The same preparation observed with polarizing microscopy. Collagen bundles of various thicknesses are observed. In the superimposed regions, the bundles of collagen are a dark color.

3.ARGYROPHYL or RETICULAR FIBERS Fibers are not branched, and are not so wavy as the collagenous fibers when released from tension. They are chemically identical to collagen, hence these fibers are considered as precursors of type I and III collagen; however, they are thinner and form delicate networks instead of thick bundles Chemical characteristics- show affinity to silver (black) stains, hence argyrophyl; do not yield gelatin on boiling; not easily dissolved by dilute acids and alkali; not so easily digested by gastric juice; not so resistant to solutions of alkaline pancreatic juice. Distribution- abundant in regions around blood vessels, muscle fibers, fat cells, basement membrane of epithelia, endoneurium, lymphoid organs and red bone marrow.

Section of an adrenal cortex, silver stained to show reticular fibers. This is a thick section made to emphasize the networks formed by these fibers, which consist of collagen type III. Nuclei are black, and cytoplasm is unstained.

CONNECTIVE TISSUE CELLS

1. FIXED CELLS a. Mesenchymal cells- these cells have multiple developmental potentialities, differentiating along several different lines to produce many different kinds of CT cells, smooth muscle cells, blood cells. They disappear when component cells undergo differentiation. Embryonic mesenchyme consists of a loose network of stellate mesenchymal cells and abundant intercellular fluid. Undifferentiated mesenchymal cells remain in adult CT as reserve cells called adventitial cells, which are difficult to distinguish from some fibroblasts.

b.Fibroblasts (fibrocytes or desmocytes) Flattened cells with a finely granular cytoplasm and a vesicular nucleus. Young fibroblasts are stellate cells with long cytoplasmic processes and a large, ovoid, pale-staining nucleus with nucleoli and sparse chromatin. They have long, slender mitochondria extending into the processes, presence of ER and well-developed Golgi complex, a sign of active protein synthesis. Cytoplasm appears acidophilic but becomes slightly basophilic when actively synthesizing ground substance

 The smaller, mature type (fibrocytes) are less active, spindle-shaped with a dark, elongate nucleus and fewer cytoplasmic organelles. They can revert to the fibroblast stage and participate in tissue repair. Differentiated cells ordinarily do not give rise to other CT cell types. They are suspected to become adipose or bone-forming cells, but it is debatable whether it is actually the fibroblasts or their undifferentiated mesenchymal progenitors that undergo these transformations. In other locations, fibroblasts can take on different shapes and acquire additional functions (e.g., intestinal villi, interstitial cells of renal papillae, periniurium of peripheral nerves)

Fibroblasts actively engaged in synthesis (left) are richer in mitochondria, lipid droplets, Golgi complex, and rER than are quiescent fibroblasts or fibrocytes (right).

c.Reticular Cells These make us a functionally diverse yet morphologically similar group. They produce the reticular fibers that form the netlike stroma of hemapoietic & lymphoid tissues. They can phagocytose antigenic materials and cellular debris. Others collect antigens on their surfaces and help activate immunocompetent cells. They are typically stellate with long, cytoplasmic surfaces, with a central, pale, irregularly rounded nucleus and a prominent nucleolus. The number of mitochondria, rER and Golgi complex is variable; less developed organelles maybe stem cells of various blood types.

d.Fat or Adipose Cells These conspicuous cells are a normal component of areolar tissue occurring singly or in clumps along small blood vessels. If they accumulate in large numbers, the tissue is transformed into adipose tissue. These are fixed cells containing fat globules in the cytoplasm. They accumulate so much lipid that the nucleus is flattened and displaced to one side, the cytoplasm so thinned that it appears as a narrow rim around the edge of the single large lipid droplet giving the cell the characteristic signet ring appearance. Individual cells are surrounded by a fine network of reticular or argyrophil fibers. The biggest CT cell while all the others are small and thus are difficult to demonstrate in ordinary slide preparation.

 In fresh or formalin-fixed tissue, the fat droplet can be stained with osmic acid, but in most histological preparations, the lipid has been extracted, leaving only the plasma membrane. Fat cells are fully differentiated and incapable of mitotic division. The fat cells of the brown fat tissue prominent in hibernating animals do not contain one big fat globule but usually several small fat droplets with some brownish pigment granules. In starvation and malnutrition, the fat content of the fat cells disappears; the cytoplasm becomes more abundant, serous in character with very tiny fat droplets. These cells are termed serous fat cells.

2.FREE, MOBILE or WANDERING CELLS: a.Macrophages (phagocytes, clasmatocytes, histiocytes, resting wandering cells or rhagiocrine cells) These are large, irregularly shaped cells in loose CT with vacuoles and abundant cytoplasm, abundant lysosomes, well-developed Golgi complex, and protruding pseudopods. They are infrequent in CT where blood vessels are few; being abundant in richly vascular areas; life span is about 2 months. Maybe either fixed (resident) or resting, attached to fibers or free in the ground substance.

 Fixed macrophages are inactive and immobile; they are elongated or stellate shaped. The fixed variety becomes the rounded and motile free (elicited) macrophages when mobilized at the site in response to a stimulus; the latter become activated macrophages when presented with phagocytic and antigen-processing activity. These phagocytes help maintain the integrity of CT by removing foreign substances and cellular debris, and participate in the immune response by presenting phagocytosed antigens to lymphocytes and plasma cells.

 They may form closely packed masses forming epithelioid cells. In chronic infection, they may fuse to form multinucleated foreign body giant cells around foreign objects that are too large or resistant to be engulfed and destroyed. The cell enlarges during phagocytosis, the Golgi net becomes prominent and the lysosomes discharge enzymes into the vacuoles. Section of rat skin showing multinuclear giant cells surrounded by macrophages

 The MONONUCLEAR PHAGOCYTE SYSTEM (formerly the reticulo-endothelial system) is a group of widely dispersed cells types that are related by their morphology, phagocytic functions and a common origin. The phagocytic cells in all of the tissues and organs are derived from precursors in the bone marrow, thru blood monocytes. They all take up vital dyes, react positively for peroxidase, esterase and other lysosomal enzymes, and all have surface receptors for IgG and complement. This system is composed of free or fixed macrophages of CT (histiocyte), liver (stellate cells of Kupffer), pulmonary alveoli, lymph node, spleen, bone marrow, serous cavity (pleural and peritoneal macrophages aggregated in small patches known as milky spots), bone (osteoclasts), nervous system (microglia of the CNS), skin (histiocyte), synovia (type A cell), tissue macrophages

b.Mast Cells- large, round or ovoid cells resembling the basophils of the blood, with palestaining nuclei and large basophilic granules obscuring the nuclei.

b.These granules are easily soluble in water and seldom seen in the specimen. The granules contain pharmacologically active substances such as: Heparin- anticoagulant and antiagglutinant Histamine- together with leukotrienes and the eosinophilic chemotactic factor (ECF-A), its release is responsible for the allergic response to some foreign proteins or anaphylaxis. Histamine release during massive degranulation of mast cells of anaphylaxis results to increased capillary permeability with leakage of plasma into tissues, dramatic fall in blood pressure, unconsciousness, and even death. Serotonin- a vasoconstrictor that increases the permeability of capillaries and venules, having a marked effect upon blood pressure.

Mast-cell secretion. 1: IgE molecules are bound to the surface receptors. 2: After a 2nd exposure to an antigen (e.g, bee venom), IgE molecules bound to surface receptors are cross-linked by the antigen. This activates adenylate cyclase & results in the phosphorylation of certain proteins. 3: At the same time, Ca+2 enters the cell. 4: These events lead to intracellular fusion of specific granules & exocytosis of their contents. 5: In addition, phospholipases act on membrane phospholipids to produce leukotrienes. The process of extrusion does not damage the cell, which remains viable and synthesizes new granules. ECF-A, eosinophil chemotactic factor of anaphylaxis.

c.Plasma Cells
Spherical cells with an eccentric clock-face nucleus resulting from a large, central nucleolus & several large heterochromatin clumps regularly spaced inside the nuclear envelope. EM show the presence of an extensive ER associated with ribosomes or RNP granules which are involved in the synthesis of antibodies. The secreted proteins do not aggregate into secretory granules. The cytoplasm has well developed Golgi complex and centrioles, finely granular and intensely basophilic. They possess a slight amoeboid motility and are not actively phagocytic.

 This cell type arise by further differentiation of B lymphocytes Has a very important function in resistance to disease and is now known to be the principal producer of antibodies, the immune globulins of the blood that participate in the bodys humoral defenses against bacterial infection. These cells are abundant in the mucous membrane of GI tract. When they disintegrate, their cytoplasmic inclusions (believed to be products of defective antibody synthesis) are set free and become acidophilic in staining, termed the Russell granules or bodies.

Portion of a chronically inflamed intestinal villus. The plasma cells are characterized by their size and abundant basophilic cytoplasm (rER). A large Golgi complex (arrows) is where the terminal glycosylation of the antibodies (glycoproteins) occurs.

d. Leukocytes- wandering cell types that originate in the bone marrow and are carried to CT by the blood and lymph. Lymphocytes production of immunocompetent cells; may originate in CT and remain there. However, they may enter the circulation at any time. Eosinophil- abundant in lactating breast and of the respiratory and alimentary tracts. They accumulate in the blood and in the tissues in certain allergic, parasitic infections and sub-acute inflammatory conditions. They contain a slight amount of histamine. Basophil- contain the anticoagulant heparin, which prevents blood from clotting too quickly; the vasodilator histamine; found in sites of exoparasite infection; and where allergic reactions are occurring Neutrophil- generally escapes into CT from capillaries only in regions of inflammation. Monocytes- are rarely seen; are considered as phagocytes because they may become amoeboid and show inclusions that stain supravitally with neutral red.

Section of an inflamed intestinal lamina propria. Inflammation was caused by nematode parasitism. Aggregated eosinophils and plasma cells function mainly in the connective tissue by modulating the inflammatory process.