DAVID C. VAN SICKLE

Connective and supportive tissues connect other tissues, provide a framework, and support the entire body by means of cartilage and bones. These tissues also play an important role in thermoregulation and in defense and repair mechanisms. Connective tissues are also a reservoir for various hormones and cytokines that play an important role in growth and development.

Most connective and supportive tissues are derived from mesoderm, which arises from the somites and lateral layers of the somatic and splanchnic mesoderm. In addition, neural crest cells from surface ectoderm form the head mesenchyme, which subsequently develops into the connective tissue of the rostral head. Connective and supportive tissues are composed of cells, fibers, and amorphous ground substance in varying proportions. Embryonic mesenchyme is a unique connective tissue because it lacks fibers during early development. Based on occurrence, connective and supportive tissues are classified as embryonic or adult with several subgroups.

CONNECTIVE TISSUE CELLS

Mesenchymal cells are irregularly shaped with multiple processes (Fig. 3-1). They are smaller than fibroblasts and have fewer cytoplasmic organelles. The large, oval nucleus has a prominent nucleolus and fine chromatin. The mesenchymal cell population serves as a reservoir of pluripotent cells that can differentiate into other types of connective tissue cells as needed.

Fibrocytes and Fibroblasts

The most common cell of connective tissue is the fibrocyte (Fig. 3-2A). Fibrocytes are generally elongated and spindle-shaped, with processes that contact adjacent cells and fibers. Their heterochromatic nucleus is surrounded by a scant amount of pale cytoplasm. Secretory vesicles in the cytoplasm discharge their contents (e.g., procollagen, proteoglycans, proelastin) into the surrounding microenvironment. At the transmission electron microscopic (TEM) level, the cytoplasm has sparse rough endoplasmic reticulum (rER) and a small Golgi complex. Free ribosomes, mitochondria, lysosomes, and vesicles are also present. Actin filaments occur as bundles in the cell processes. Fibrocytes maintain the connective tissue matrix by forming the fibers and constantly renewing the ground substance.

The fibroblast has a larger, more euchromatic nucleus and more abundant, basophilic cytoplasm than the fibrocyte (Fig. 3-2B). At the EM level, abundant rER and a prominent Golgi complex are present in the cytoplasm. These structural characteristics indicate more active connective tissue matrix production in comparison to the fibrocyte. Fibroblasts may arise directly from undifferentiated mesenchymal cells or are transformed from fibrocytes under the influence of microenvironmental factors (e.g., cytokines). In certain situations, fibroblasts may differentiate into adipose cells, chondroblasts, or osteoblasts.

Myofibroblasts are fibroblasts that contain actin filaments associated with dense bodies; hence, they resemble smooth muscle cells. It is believed that myofibroblasts play a role in contraction of the wound during healing.

FIGURE 3-1 Mesenchyme (rat embryo). Note the stellate mesenchymal cells in an amorphous ground substance. Hematoxylin and eosin (×800).

FIGURE 3-2 A. Fibrocytes (arrows), collagen fibers (C), connective tissue, liver (dog). B. Fibroblasts (arrow), tendon (young puppy). The cells are located between collagen fibers (C). Hematoxylin and eosin (×1520).

Reticular Cells

Reticular cells are similar in appearance to the fibrocyte (Fig. 3-3). They are stellate-shaped cells with a spherical nucleus and basophilic cytoplasm. Reticular cells produce reticular fibers, which form the fine structural network of organs such as the lymph nodes, spleen, and bone marrow. These cells are fixed in the tissue and are capable of phagocytosis. Reticular cells should not be confused with the reticulocyte, an immature erythrocyte.

FIGURE 3-3 Reticular connective tissue, lymph node (sheep). Note the numerous interconnected reticular cells, which form a three-dimensional network. Hematoxylin and eosin (×600).

Adipocytes

Adipocytes are also referred to as fat cells or adipose cells (Fig. 3-4). Individual adipocytes or clusters containing multiple cells are normal components of loose connective tissue, but when the fat cells outnumber other cell types, the tissue is called adipose tissue (see more on adipose tissue later in this chapter). Mature unilocular adipocytes are spherical or polyhedral cells that measure up to 120 µm in diameter. Most of the cell is occupied by a single, large nonmembrane-bounded lipid droplet surrounded by a thin layer of cytoplasm. The cell nucleus is displaced to the periphery by the lipid droplet, which is surrounded by cytoplasm that contains a small Golgi complex, mitochondria, rER, and microfilaments.

FIGURE 3-4 Two white adipocytes surrounded by capillaries. Hematoxylin and eosin (×630). (Courtesy of A. Hansen.)

In contrast, mature multilocular adipocytes contain a more centrally located nucleus with multiple lipid droplets in the cytoplasm (Fig. 3-5). Both the Golgi complex and rER are rather inconspicuous, but many mitochondria are present. The high concentration of cytochromes in the mitochondria is primarily responsible for the brown color of aggregates of multilocular adipocytes, which are referred to as brown fat (described below).

Unilocular adipocytes produce chemical energy, whereas multilocular adipocytes metabolize lipid to produce heat. Leptin, a protein produced by unilocular fat cells, regulates the amount of adipose tissue in the body. Thermogenin, a transmembrane protein in the mitochondria of brown fat, channels protons away from adenosine 5′-triphosphate (ATP) synthesis and into heat production instead.

Because fat is rapidly dissolved by most of the dehydration and/or clearing agents commonly used for the preparation of histologic sections, the lipid droplets appear as clear spaces surrounded by cytoplasm (Fig. 3-4). When rapidly processed, the lipid can be preserved and stained with certain agents, such as osmium tetroxide or Sudan III stain.

Pericytes

Pericytes, also known as Rouget cells or periendothelial cells, are elongated cells that are located adjacent to the endothelium lining capillaries and postcapillary venules. The cells are surrounded by the basal lamina of the blood vessel and make frequent contact with the underlying endothelial cells by extending processes through the lamina. Pericytes resemble fibrocytes in appearance but have contractile filaments similar to smooth muscle. Proposed functions of pericytes include regulating capillary blood flow; serving as multipotent mesenchymal cells with specific ability to form vascular smooth muscle cells; phagocytosing; and regulating new capillary growth. Pericytes also have the ability to differentiate into adipocytes, osteoblasts, and phagocytes.

FIGURE 3-5 Brown adipose tissue. Note the numerous small lipid inclusions in the multilocular adipocytes. Hematoxylin and eosin (×600).

Mast Cells

Mast cells are common in loose connective tissue, especially around nerve endings and microcirculation. The cells are found in the dermis of the skin and connective tissue of the respiratory tract and gastrointestinal system.

Mast cells are large, polymorphic, spherical, or ovoid cells that contain a prominent, centrally located nucleus. Numerous secretory granules are present in the cytoplasm (Fig. 3-6). These cells can be identified with immunocytochemistry or a metachromatic stain, which has the capacity to stain elements of a cell or matrix a different color from that of the dye solution (e.g., toluidine blue, a blue dye that stains heparin-containing granules red). At the EM level, the mast cell granules are membrane-bounded and have crystalline, lamellar, or fine granular characteristics. The remaining cytoplasm is occupied by an extensive Golgi complex, cisternae of rER, free ribosomes, and mitochondria.

Mast cell granules contain histamine, heparin, and various proteases. Histamine, a biogenic amine, is a vasoconstrictor that causes increased permeability of small venules, thereby permitting leakage of plasma, resulting in tissue edema. This localized inflammatory reaction is designed to dispose of foreign antigens rapidly. Histamine also stimulates smooth muscle contraction in small airways. Heparin, a glycosaminoglycan, acts as an anticoagulant and is believed to stimulate angiogenesis. Mast cells can be activated to release their contents (degranulate) by physical stimuli such as trauma or sunlight; immunogenic stimuli including immunoglobulin (Ig) E, complement, or cytokines; and neurogenic stimuli such as neuropeptides.

Three populations of mast cells based on protease content have been identified. The mucosal mast cell (MC_T) contains tryptase only, while the connective tissue mast cell (MC_TC) contains tryptase, chymase, carboxypeptidase, and cathepsin. A third mast cell type (MC_C) has chymase and carboxypeptidase. The dog and cat have about 70% MC_TC cells. Proteases can destroy nearby cells and tissue matrix and activate complement components. Arachidonic acid products (leukotrienes) and cytokines (e.g., various interleukins, stem cell factor, and tumor necrosis factor alpha [TNF-α]) are also produced by the mast cell and immediately released without storage in the cytoplasmic granules. In the past, the mast cell has been described as a “tissue basophil,” but despite some similarities, the mast cell and basophil are different cells. Both cells develop from the same pluripotent stem cell (CD34+), and both have basophilic cytoplasmic granules that contain similar inflammatory products. However, basophils are terminally differentiated before they enter blood circulation, while mast cells leave the bone marrow, pass through the circulation, and differentiate in tissues outside the bone marrow. The mast cell can undergo mitotic division while the basophil cannot. Also, basophils have a short lifespan of days while most mast cells can survive for weeks to months.

FIGURE 3-6 Mast cell (arrow), lung (rabbit). Note the characteristic granules. Hematoxylin and eosin, plastic section (×1200).

Macrophages

Macrophages are phagocytic cells that are scattered throughout the body and form the mononuclear phagocyte system. They are derived from a bone marrow precursor cell (CFU-GM) that divides and produces monocytes that circulate in the blood. The monocytes then migrate across blood vessel walls into the connective tissue or organs and become macrophages. Mobile macrophages wander through the tissues performing their phagocytic function while fixed macrophages remain in one location. The fixed macrophage of connective tissues is also known as the histiocyte. Other macrophages located in specific tissues include the stellate macrophage of the liver (Kupffer cell), the microglial cell, the intraepidermal macrophage (Langerhans cell), and the osteoclast.

Macrophages are large, ovoid, or spherical cells that contain cytoplasmic vacuoles and are readily distinguishable with the light microscope (Fig. 3-7). At the EM level, they are characterized by numerous lysosomes, phagosomes, phagolysosomes, and pseudopodia (footlike extensions of the cell membrane) (Fig. 3-8). Abundant ribosomes, rER, smooth ER (sER), mitochondria, and a Golgi complex are also present. Histochemical stains for lysosomal enzymes, such as acid phosphatase, facilitate the identification of macrophages. Once activated, the macrophage changes morphology with increased microvilli and lamellipodia, which are sheetlike extensions of cytoplasm. The lamellipodia form transient adhesions with the surrounding substrate, enabling the cell to move.

A variety of chemotactic stimuli (e.g., infectious agents, cytokines) cause macrophages to migrate to locations in the body where foreign material must be removed. Macrophages engulf material such as cell debris, abnormal matrix components, neoplastic cells, bacteria, and inert substances by pinocytosis and phagocytosis. Phagocytosis may be indiscriminate (e.g., dust particles in the lung) or may involve specific interaction with receptors on the macrophage surface (e.g., Fc receptors, IgG, and IgM). Macrophages also act as antigen-presenting cells, which process and display foreign substances so that lymphocytes can recognize and respond more effectively to the challenge.

FIGURE 3-7 Macrophage (arrows), connective tissue. Note the numerous cytoplasmic vacuoles and phagolysosomes. Hematoxylin and eosin (×1200).

Inflammation can be characterized as acute or chronic based on the relative proportion of neutrophils to macrophages. Acute inflammation has more neutrophils than macrophages, whereas chronic inflammation has more macrophages than neutrophils. When stimulated, macrophages may form clusters of epithelioid cells, which resemble epithelial cells in morphology. Multinucleated giant cells or foreign-body cells (Fig. 3-9) arise during chronic inflammation and are a result of the fusion of several macrophages in response to the presence of foreign material.

FIGURE 3-8 Macrophage containing abundant pinocytotic vacuoles (arrows) and phagolysosomes (arrowhead) (×8000). (Courtesy of J. Turek.)

FIGURE 3-9 Multinucleated giant cell, lymph node (sheep). Crossman’s trichrome (×435).

Macrophages synthesize and secrete many substances that are a reflection of their multiple functions. These substances include enzymes such as lysozyme, which lyses the wall of many bacteria; cytokines (interferons and interleukin); complement components (C2, C3, C4, and C5); coagulation factors; and reactive chemical species (hydrogen peroxide, hydroxyl radicals, nitric oxide), which are important components of the bactericidal and cytocidal (e.g., tumor cells) activities of macrophages.

Plasma Cells

Plasma cells are spherical, ovoid, or pear-shaped cells with a spherical, eccentric nucleus. The chromatin is often arranged in peripherally located clumps or in centrally converging strands that give the nucleus a “cartwheel” appearance (Fig. 3-10). The cytoplasm is intensely basophilic, and a negatively stained Golgi region is usually present. At the fine-structural level, in addition to an extensive Golgi complex, the cytoplasm has an abundant rER with dilated cisternae containing slightly granular and moderately electron-dense material as well as spherical inclusions referred to as Russell bodies (Fig. 3-11). Russell bodies give a positive reaction for immunoglobulin. Free ribosomes and mitochondria are also present in the cytoplasm.

FIGURE 3-10 Plasma cells (arrowheads), connective tissue, duodenum. Hematoxylin and eosin (×900).

Plasma cells are most numerous in lymphatic tissue, especially in the center of medullary cords of lymph nodes. They are also particularly abundant in bone marrow, the loose connective tissue underlying the epithelium of the gastrointestinal tract, the respiratory system, and the female reproductive system.

Plasma cells do not originate in loose connective tissue but develop from B lymphocytes that immigrate into the connective tissue from the blood; they produce circulating or humoral antibodies (see Chapter 8).

Pigment Cells

Cells in connective tissue may contain pigments, including melanin in domestic animals or pteridines and purines in fish and amphibians (Fig. 3-12). When present in large numbers, the cells impart color to the connective tissue. They occur in various locations such as the dermis, uterine caruncles of sheep, meninges, choroid, and iris. Their significance is described in connection with these organs.

FIGURE 3-11 Part of a plasma cell with abundant rough endoplasmic reticulum (ER), mitochondria (M), extensive Golgi complex (G), and Russell bodies (R) (×19,500).

FIGURE 3-12 Pigment containing cells in the lamina propria of a uterine caruncle (sheep). Hematoxylin and eosin (×325).

Other Cells of Loose Connective Tissue

Depending on its location and various other factors (infestation by parasites, presence of bacteria, and the like), loose connective tissue may contain a varying number of lymphocytes, monocytes, and granulocytes (especially eosinophils and neutrophils). The structure and function of these immigrant cells are described in the section on blood (see Chapter 4).

Globule leukocytes are mononuclear cells with acidophilic and metachromatic cytoplasmic granules. These cells are found in epithelium and connective tissues of the respiratory, digestive, reproductive, and urinary systems. Globule leukocytes are believed to be mucosal mast cells, but their origin as a subpopulation of T lymphocytes has also been proposed.

CONNECTIVE TISSUE FIBERS

Collagen polypeptide chains are synthesized in the rER as pro-α chains that contain extension peptides (propeptides) at both ends (Fig. 3-13). Multiple α chains have been recognized. Within the rER cisternae, these pro-α chains assemble into triple helices to form procollagen molecules. The molecules are then transferred to the Golgi complex, packaged into secretory vesicles, and released by exocytosis. At this point in synthesis, collagen can be classed as fibril-forming, FACIT ( fibril-associated collagens with interrupted triple helices), sheet-forming, multiplexin, connecting and anchoring, or transmembrane collagen. Twenty-seven collagen types are currently recognized.

Fibril-forming collagen then undergoes extracellular enzymatic cleavage of the propeptides to yield collagen molecules. These molecules in turn assemble in the extracellular matrix (ECM) to form collagen fibrils. The fibrils are only visible with the electron microscope, are up to several micrometers in length, and vary in diameter (10 to 300 nm), with characteristic cross-striations repeated at 67-nm intervals (Figs. 3-13 and 3-14). Bundles of these fibrils form collagen fibers that are visible by light microscopy (Fig. 3-2B). Fibril-forming collagen includes types I, II, III, V, XI, XXIV, and XXVII. Fibers composed of type I collagen account for 90% of the body collagen. Type I collagen is found in skin, tendon, bone, and dentin, while type II collagen is specific for cartilage and vitreous humor. Frequently colocalized with type I collagen, type III collagen is essential for normal collagen I fibrillogenesis. Type V collagen is necessary for extracellular matrix assembly in connective tissues; it is present in fetal tissues and the placenta. Type XI collagen is found in hyaline cartilage and type XXIV is distributed in developing cornea and bone.

Fresh collagen fibers are white, and in histologic preparations, they stain with acid dyes. Thus, they are red to pink in hematoxylin and eosin (H&E)-stained sections, red with van Gieson’s method, and blue in Mallory’s and Masson’s triple stains (green when light green stain is used). The fibers are flexible and can adapt to the movements and changes in size of the organs with which they are associated. Collagen fibers are characterized by a high tensile strength and a poor shear strength, and stretch is limited to approximately 5% of their initial length. Consequently, they are found wherever high tensile strength is required, such as in tendons, ligaments, and organ capsules.

Other collagens known as FACIT collagens (fibril-associated collagens with interrupted triple helices) include types IX, XII, XIV, XX, XXI, and XXII. These collagens serve as molecular bridges that are important in the organization and stability of extracellular matrices. Glycosaminoglycans are linked to type II collagen of hyaline cartilage by collagen IX. Type XX collagen is prevalent in corneal epithelium. The ECM of blood vessel walls contains type XXI collagen, while the ECM at tissue junctions contains type XXII collagens. Some FACIT collagens (types XVI and XIX) are unable to bind fibers and are referred to as FACIT-like collagens.

Sheet-forming collagen forms a flexible framework of sheets rather than fibrils. Collagen IV forms the basal lamina of epithelia. In the eye, the posterior limiting lamina (Descemet’s membrane) of the cornea is formed from type VIII collagen. Type X collagen is found in the hypertrophic zone of the physis.

Multiplexins (multiple triple-helix domains and interruptions) are collagens that are associated with basement membranes. This subgroup includes types XV and XVIII.

FIGURE 3-13 Collagen polypeptide chains are synthesized within the cell and released into the extracellular space as procollagen. In fibril-forming collagen, peptides are then cleaved from the procollagen, resulting in a collagen molecule. The collagen molecules are assembled in such a way that hole zones are created, which stain dark, in contrast to overlap zones, which exclude stain. Bundles of light- and dark-banded fibrils form collagen fibers. In a tendon, individual collagen fibers are surrounded by endotendineum and bundles of fibers are bound by peritendineum. In this drawing, the sheath surrounding the tendon is reflected to show the collagen fiber bundles (fascicles) inside. Blood vessels pass through the mesotendineum to supply the tendon.

FIGURE 3-14 Electron micrograph of collagen fibrils with characteristic cross-striations (×88,000).

Connecting and anchoring collagen binds to the surface of fibril-forming collagen and may mediate the interactions of fibrils with one another and with other matrix components. Collagen types VI and VII are believed to perform this function. Type VI collagen forms unique beaded filaments, while collagen VII forms anchoring fibrils that link collagen IV of the basal lamina of the epidermis to the collagen of underlying connective tissue.

Transmembrane collagens include XIII, XVII, XXIII, and XXV. Collagen XVII, a transmembrane collagen of the skin, is associated with hemidesmosomes and mutates in diseases that cause blisters (epidermolysis bullosa). Type XXIII collagen is associated with metastatic tumor cells while type XXV collagen is a neuronal collagen.

Collagen XXVI is unclassified and is found in the testes and ovary.

Reticular Fibers

In routine histologic preparations, reticular fibers cannot be distinguished from other small collagen fibers. These fibers can be identified only with certain silver impregnations (thus the term argyrophilic or argentaffin fibers) or with the periodic acid-Schiff (PAS) reagent (Fig. 3-15). These fibers are actually individual collagen fibrils (type III collagen) coated by proteoglycans and glycoproteins. This coating increases the affinity of the fibers for silver salts. When individual reticular fibers are bundled to form collagen fibers, the coating is supposedly displaced and the argyrophilia decreases.

Reticular fibers form delicate, flexible networks around capillaries, muscle fibers, nerves, adipose cells, and hepatocytes and serve as a scaffolding to support cells or cell groups of endocrine, lymphatic, and blood-forming organs. They are an integral part of basement membranes.

Elastic Fibers

Elastic fibers and/or sheets (laminae) are present in organs in which normal function requires elasticity in addition to tensile strength. Elastic fibers can be stretched as much as two and one-half times their original length, to which they return when released. Found in the pinna of the ear, vocal cords, epiglottis, lungs, ligamentum nuchae, dermis, aorta, and muscular arteries, elastic fibers are one of the most resilient connective tissue fibers, withstanding chemical maceration and autoclaving.

Elastic fibers usually occur as individual, branching, and anastomosing fibers. Their diameters vary within a wide range, from 0.2 to 5.0 µm in loose connective tissue to as large as 12 µm in elastic ligaments, such as the ligamentum nuchae in the neck (Fig. 3-16). In H&E-stained histologic sections, the larger elastic fibers in elastic ligaments are readily distinguished as highly refractile, amorphous, light pink strands; they are stainable by certain selective dyes, such as orcein and resorcin-fuchsin.

The main component of elastic fibers is elastin, which contains a network of fibrillin microfibrils that form a supporting scaffold for the elastin. An amorphous protein rich in proline and glycine, elastin contains little hydroxyproline. One theory of elastin structure is that the molecules are randomly coiled and joined by stable, covalent cross-links containing desmosine, an identifying component of elastic fibers. The coils of elastin can stretch and then recoil as needed. Elastin is synthesized by fibroblasts and smooth muscle cells.

FIGURE 3-15 Reticular fibers. 1. Liver (pig). Achucarro silver impregnation (×435). 2. Lymph node (dog). Palmgren silver impregnation (×230).

FIGURE 3-16 Ligamentum nuchae, large ruminant. 1. Longitudinal section. 2. Cross section. Note that the large elastic fibers (arrowheads) are surrounded by networks of collagen fibers. Crossman’s trichrome (×600).

The secondary component of elastic fibers is 10-nm microfibrils that are embedded within and surround the elastic core (Fig. 3-17). The microfibrillar material is composed of a glycoprotein, fibrillin, which is necessary for elastic fiber integrity.

During development of the elastic fiber, fibrillin microfibrils are secreted before elastin and provide a scaffolding on which elastin is deposited. At this stage, the fiber is referred to as oxytalan. In the second stage of development, elastin is deposited between the microfibrils, forming elaunin. As more elastin accumulates within the developing fiber, mature elastic fibers are formed. Developmentally, the elastic fiber is the last fiber to appear in organs (e.g., lung) or connective tissue.

Fibrous Adhesive Proteins

The extracellular matrix contains noncollagenous fibrous proteins that play a role in organizing the matrix and help cells to adhere to it. Fibronectin, a major product of mesenchymal cells, is a fibril-forming protein that binds to various structures, including the cell membrane, collagen, elastin, and proteoglycans, and probably mediates the connection between the cytoskeleton and the ECM. Fibronectin plays a role in a variety of processes, such as cell adhesion, cell differentiation, cell growth, and phagocytosis.

Laminins are large glycoproteins. They are a major constituent of the basement membrane and are synthesized by the cells that are in contact with it (e.g., epithelial cells, smooth muscle cells, neurolemmocytes) (Fig. 16-6). Laminins are present in the lamina lucida (lamina rara) and lamina densa. They are connected to type IV collagen by the adhesive glycoprotein nidogen (entactin). Laminins form a structural network in the basement membrane to which other glycoproteins and proteoglycans attach. Also, they are signaling molecules that stabilize cell surface receptors.

FIGURE 3-17 Electron micrograph of an elastic lamina between collagen fibrils (longitudinal and cross sections). The electron-dense thin lines in the elastic lamina are microfibrils (×21,000).

Other adhesive glycoproteins include fibrinogen (blood-clotting mechanism), link protein (linking of cartilage matrix components), mucins, tenascin (embryonic tissues, uncertain function), and thrombospondin (platelet aggregation).

GROUND SUBSTANCE

Glycosaminoglycans are comprised of unbranched polysaccharides of alternating uronic acid and hexosamine residues. Seven major types of GAGs can be distinguished. Hyaluronan (hyaluronic acid) is a nonsulfated GAG. It is a large, long molecule that forms networks with spaces that are filled with tissue fluid. The resulting gel is particularly abundant in the vitreous humor of the eye and in synovial fluid; it is also found in the umbilical cord, loose connective tissue, skin, and cartilage. Hyaluronan binds proteoglycans into a larger molecule called an aggrecan (Fig. 3-18). Chondroitin-4-sulfate and chondroitin-6-sulfate are abundant in cartilage, arteries, skin, and cornea. A smaller amount is found in bone. Dermatan sulfate is found in skin, tendon, ligamentum nuchae, sclera, and lung. Keratan sulfate is present in cartilage, bone, and cornea. Heparan sulfate is found in arteries and the lung, whereas heparin is found in mast cells, the lung, the liver, and skin. The latter six GAGs are of the sulfated variety.

Proteoglycans are formed by covalently linking GAGs to a protein core and range in size from small molecules (decorin, m.w. 40,000) to large aggregates (aggrecan, m.w. 210,000) (Fig. 3-18). In addition to filling space in the connective tissue matrix and imparting its unique biomechanical properties, proteoglycans may regulate the passage of molecules and cells in the intercellular space. They are also believed to play a major role in chemical signaling between cells and may bind and regulate the activities of other secreted proteins.

Proteoglycans in low concentrations are not detected in H&E-stained sections, but when present in higher concentrations, as in hyaline cartilage, they stain with basophilic dyes. When stained with toluidine blue or crystal violet, a metachromatic change to pink or magenta occurs.

FIGURE 3-18 The matrix of cartilage contains aggrecans, which are composed of proteoglycans bound to a hyaluronic acid chain by link proteins. The proteoglycans are composed of glycosaminoglycans, such as chondroitin and keratan sulfate, bound to a protein core.

EMBRYONIC CONNECTIVE TISSUES

Mesenchyme, the connective tissue of the developing embryo, is composed of irregularly shaped mesenchymal cells and amorphous ground substance (Fig. 3-1). The cell processes contact adjacent cells and thus form a three-dimensional network. Mesenchymal cells undergo numerous mitotic cell divisions and continuously change their shape and location to adapt to the transformations that occur during embryonic growth. During early development, mesenchyme does not contain fibers, and the abundant amorphous ground substance fills the wide intercellular spaces. Mesenchyme gives rise to various types of adult connective tissues, as well as blood and blood vessels.

Mucous Connective Tissue

Mucous or gelatinous connective tissue is found primarily in the embryonic hypodermis and umbilical cord (Fig. 3-19). It is characterized by stellate fibroblasts that form a network. The large intercellular spaces are occupied by a viscous, gel-like amorphous ground substance that has a positive reaction for glycosaminoglycans or proteoglycans. Collagen fibers (types I and III) are also present. In the adult organism, gelatinous connective tissue occurs in the papillae of omasal laminae and reticular crests, the bovine glans penis, and the core of the rooster comb.

FIGURE 3-19 Gelatinous connective tissue, umbilical cord (pig). Fine collagen fibers (arrows) are present. Hematoxylin and eosin (×800).

ADULT CONNECTIVE TISSUES

Loose, or areolar, connective tissue is the most widely distributed type of connective tissue in the adult animal (Fig. 3-20). A The cells and fibers of loose connective tissue are widely separated by spaces filled with ground substance. Compared to other types of connective tissue, the cells in loose connective tissue are more abundant and include both fixed and mobile populations. All three fiber types (reticular, collagen, and elastic) are present. The relative abundance and orientation of fibers vary widely and depend primarily on the location and specific function of the tissue. In cases of injury, the stage of healing also causes variation in fiber arrangement. Early connective tissue is highly cellular with fine reticular fibers; later connective tissue has predominantly thick collagen fibers.

The amorphous ground substance of loose connective tissue is composed of proteoglycans that bind a significant quantity of tissue fluid. Substances dissolved in the tissue fluid can diffuse through the amorphous ground substance and thus have ready access to connective tissue cells. Circulating tissue fluid is formed at the arterial end of capillaries and absorbed by either venous or lymphatic capillaries.

Loose connective tissue is present beneath many epithelia, where it provides support and a vascular supply. This tissue forms the interstitial tissue in most organs, thereby allowing easy movements and shifting of organs. Loose connective tissue is present around nerve and skeletal muscle bundles as named tissue layers (e.g., epineurium) and is found between the layers of smooth musculature of hollow organs. The pia mater and arachnoid of the brain and spinal cord are also composed of loose connective tissue.

The functions of loose connective tissue range from the purely mechanical, such as support and dampening biomechanical effects in various locations (e.g., hypodermis), to more sophisticated functions, such as participation in tissue repair and defense activities (inflammation).

Dense Connective Tissue

The fibers in dense connective tissue are more abundant than cells and amorphous ground substance. Dense connective tissue is commonly classified as either dense irregular connective tissue, with a random orientation of the fiber bundles, or dense regular connective tissue, in which fibers are oriented in a regular pattern.

FIGURE 3-20 A. Loose connective tissue with blood vessels (arrows), liver (dog). B. Dense irregular connective tissue (arrow), liver (dog). C. Dense regular connective tissue with fibrocytes (arrows), tendon. Hematoxylin and eosin (×600).

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