Connective Tissue PDF

5 VetBooks.ir C H A P T E R Connective Tissue CELLS OF CONNECTIVE TISSUE 96...

5 VetBooks.ir C H A P T E R Connective Tissue CELLS OF CONNECTIVE TISSUE 96 Reticular Fibers 106 Fibroblasts 97 Elastic Fibers 109 Adipocytes 97 GROUND SUBSTANCE 111 Macrophages & the Mononuclear TYPES OF CONNECTIVE TISSUE 114 Phagocyte System 97 Mast Cells 99 Connective Tissue Proper 114 Plasma Cells 101 Reticular Tissue 116 Leukocytes 102 Mucoid Tissue 119 FIBERS 103 SUMMARY OF KEY POINTS 119 Collagen 103 ASSESS YOUR KNOWLEDGE 120 C onnective tissue provides a matrix that supports and physically connects other tissues and cells together to form the organs of the body. The interstitial fluid of connective tissue gives metabolic support to cells as the medium for diffusion of nutrients and waste products. migrate from their site of origin in the embryo, surrounding and penetrating developing organs. In addition to producing all types of connective tissue proper and the specialized connective tissues bone and cartilage, the embryonic mesenchyme includes stem cells for other tissues such as blood, the vascular endo- Unlike the other tissue types (epithelium, muscle, and thelium, and muscle. This chapter describes the features of soft, nerve), which consist mainly of cells, the major constitu- supportive connective tissue proper. ent of connective tissue is the extracellular matrix (ECM). Extracellular matrices consist of different combinations of protein fibers (collagen and elastic fibers) and ground › › MEDICAL APPLICATION substance. Ground substance is a complex of anionic, Some cells in mesenchyme are multipotent stem cells hydrophilic proteoglycans, glycosaminoglycans (GAGs), potentially useful in regenerative medicine after grafting to and multiadhesive glycoproteins (laminin, fibronectin, and replace damaged tissue in certain patients. Mesenchyme-like others). As described briefly in Chapter 4 with the basal cells remain present in some adult connective tissues, includ- lamina, such glycoproteins help stabilize the ECM by bind- ing that of tooth pulp and some adipose tissue, and are being ing to other matrix components and to integrins in cell investigated as possible sources of stem cells for therapeutic membranes. Water within this ground substance allows the repair and organ regeneration. exchange of nutrients and metabolic wastes between cells and the blood supply. The variety of connective tissue types in the body reflects differences in composition and amount of the cells, fibers, and ground substance which together are responsible for the › CELLS OF CONNECTIVE TISSUE remarkable structural, functional, and pathologic diversity of Fibroblasts are the key cells in connective tissue proper connective tissue. (Figure 5–2 and Table 5–1). Fibroblasts originate locally from All connective tissues originate from embryonic mesen- mesenchymal cells and are permanent residents of connective chyme, a tissue developing mainly from the middle layer of the tissue. Other cells found here, such as macrophages, plasma embryo, the mesoderm. Mesenchyme consists largely of vis- cells, and mast cells, originate from hematopoietic stem cells cous ground substance with few collagen fibers (Figure 5–1). in bone marrow, circulate in the blood, and then move into Mesenchymal cells are undifferentiated and have large nuclei, connective tissue where they function. These and other white with prominent nucleoli and fine chromatin. They are often said blood cells (leukocytes) are transient cells of most connec- to be “spindle-shaped,” with their scant cytoplasm extended tive tissues, where they perform various functions for a short as two or more thin cytoplasmic processes. Mesodermal cells period as needed and then die by apoptosis. 96 05_Mescher_ch05_p096-121.indd 96 27/04/18 6:38 pm  Cells of Connective Tissue 97 Fibroblasts are targets of many families of proteins called FIGURE 5–1 Embryonic mesenchyme. growth factors that influence cell growth and differentiation. C H A P T E R VetBooks.ir In adults, connective tissue fibroblasts rarely undergo division. However, stimulated by locally released growth factors, cell cycling and mitotic activity resume when the tissue requires additional fibroblasts, for example, to repair a damaged organ. Fibroblasts involved in wound healing, sometimes called myofibroblasts, have a well-developed contractile function 5 and are enriched with a form of actin also found in smooth Connective Tissue Cells of Connective Tissue muscle cells. › › MEDICAL APPLICATION The regenerative capacity of connective tissue is clearly observed in organs damaged by ischemia, inflammation, or traumatic injury. Spaces left after such injuries, espe- cially in tissues whose cells divide poorly or not at all (eg, cardiac muscle), are filled by connective tissue, forming dense irregular scar tissue. The healing of surgical inci- sions and other wounds depends on the reparative capac- ity of connective tissue, particularly on activity and growth of fibroblasts. Mesenchyme consists of a population of undifferentiated In some rapidly closing wounds, a cell called the myo- cells, generally elongated but with many shapes, having large fibroblast, with features of both fibroblasts and smooth euchromatic nuclei and prominent nucleoli that indicate high levels of synthetic activity. These cells are called mesenchymal muscle cells, is also observed. These cells have most of cells. Mesenchymal cells are surrounded by an ECM that they the morphologic characteristics of fibroblasts but contain produced and that consists largely of a simple ground substance increased amounts of actin microfilaments and myosin rich in hyaluronan (hyaluronic acid), but with very little collagen. and behave much like smooth muscle cells. Their activity (X200; Mallory trichrome) is important for the phase of tissue repair called wound contraction. Fibroblasts Adipocytes Fibroblasts (Figure 5–3), the most common cells in connec- Adipocytes (L. adeps, fat + Gr. kytos, cell), or fat cells, are tive tissue proper, produce and maintain most of the tissue’s found in the connective tissue of many organs. These large, extracellular components. Fibroblasts synthesize and secrete mesenchymally derived cells are specialized for cytoplasmic collagen (the most abundant protein of the body) and elas- storage of lipid as neutral fats, or less commonly for the pro- tin, which both form large fibers, as well as the GAGs, pro- duction of heat. Tissue with a large population of adipocytes, teoglycans, and multiadhesive glycoproteins that comprise called adipose connective tissue, serves to cushion and insu- the ground substance. As described later, most of the secreted late the skin and other organs. Adipocytes have major meta- ECM components undergo further modification outside the bolic significance with considerable medical importance and cell before assembling as a matrix. are described and discussed separately in Chapter 6. Distinct levels of fibroblast activity can be observed histologically (Figure 5–3b). Cells with intense synthetic activity are morphologically different from the quiescent Macrophages & the Mononuclear Phagocyte fibroblasts that are scattered within the matrix they have System already synthesized. Some histologists reserve the term Macrophages have highly developed phagocytic ability and “fibroblast” to denote the active cell and “fibrocyte” to specialize in turnover of protein fibers and removal of apop- denote the quiescent cell. The active fibroblast has more totic cells, tissue debris, or other particulate material, being abundant and irregularly branched cytoplasm, contain- especially abundant at sites of inflammation. Size and shape ing much rough endoplasmic reticulum (RER) and a well- vary considerably, corresponding to their state of functional developed Golgi apparatus, with a large, ovoid, euchromatic activity. A typical macrophage measures between 10 and nucleus and a prominent nucleolus. The quiescent cell is 30 μm in diameter and has an eccentrically located, oval or smaller than the active fibroblast, is usually spindle-shaped kidney-shaped nucleus. Macrophages are present in the con- with fewer processes, much less RER, and a darker, more nective tissue of most organs and are sometimes referred to by heterochromatic nucleus. pathologists as “histiocytes.” 05_Mescher_ch05_p096-121.indd 97 26/04/18 11:15 am  98 CHAPTER 5 Connective Tissue FIGURE 5–2 Cellular and extracellular components of connective tissue. VetBooks.ir Blood vessel Ground substance Extracellular matrix Protein fibers Elastic fiber Collagen fiber Reticular fiber Resident cells Mesenchymal cell Macrophage Adipocyte Fibroblast Connective tissue is composed of fibroblasts and other cells and watery ground substance. In all types of connective tissue the an ECM of various protein fibers, all of which are surrounded by extracellular volume exceeds that of the cells. Functions of cells in connective TABLE 5–1 tissue proper. › › MEDICAL APPLICATION Besides their function in turnover of ECM fibers, macro- Cell Type Major Product or Activity phages are key components of an organism’s innate immune Fibroblasts (fibrocytes) Extracellular fibers and ground defense system, removing cell debris, neoplastic cells, bac- substance teria, and other invaders. Macrophages are also important Plasma cells Antibodies antigen-presenting cells required for the activation and specification of lymphocytes. Lymphocytes (several types) Various immune/defense functions When macrophages are stimulated (by injection of foreign substances or by infection), they change their Eosinophilic leukocytes Modulate allergic/vasoactive morphologic characteristics and properties, becom- reactions and defense against parasites ing activated macrophages. In addition to showing an increase in their capacity for phagocytosis and intracellular Neutrophilic leukocytes Phagocytosis of bacteria digestion, activated macrophages exhibit enhanced meta- Macrophages Phagocytosis of ECM bolic and lysosomal enzyme activity. Macrophages are also components and debris; antigen secretory cells producing an array of substances, including processing and presentation various enzymes for ECM breakdown and various growth to immune cells; secretion of growth factors, cytokines, and factors or cytokines that help regulate immune cells and other agents reparative functions. When adequately stimulated, macrophages may Mast cells and basophilic Pharmacologically active leukocytes molecules (eg, histamine) increase in size and fuse to form multinuclear giant cells, usually found only in pathologic conditions. Adipocytes Storage of neutral fats 05_Mescher_ch05_p096-121.indd 98 26/04/18 11:15 am  Cells of Connective Tissue 99 FIGURE 5–3 Fibroblasts. C H A P T E R VetBooks.ir 5 Connective Tissue Cells of Connective Tissue C a b (a) Fibroblasts typically have large active nuclei and eosinophilic (b) Both active and quiescent fibroblasts may sometimes be distin- cytoplasm that tapers off in both directions along the axis of the guished, as in this section of dermis. Active fibroblasts have large, nucleus, a morphology often referred to as “spindle-shaped.” Nuclei euchromatic nuclei and basophilic cytoplasm, while inactive fibro- (arrows) are clearly seen, but the eosinophilic cytoplasmic pro- blasts (or fibrocytes) are smaller with more heterochromatic nuclei cesses resemble the collagen bundles (C) that fill the ECM and are (arrows). The round, very basophilic round cells are in leukocytes. difficult to distinguish in H&E-stained sections. (Both X400; H&E) In the TEM, macrophages are shown to have a characteris- (see Chapter 13). The transformation from monocytes to tic irregular surface with pleats, protrusions, and indentations, macrophages in connective tissue involves increases in cell features related to their active pinocytotic and phagocytic size, increased protein synthesis, and increases in the num- activities (Figure 5–4). They generally have well-developed ber of Golgi complexes and lysosomes. In addition to debris Golgi complexes and many lysosomes. removal, macrophages secrete growth factors important for Macrophages derive from precursor cells called mono- tissue repair and also function in the uptake, processing, and cytes circulating in the blood (see Chapter 12). Monocytes presentation of antigens for lymphocyte activation, a role dis- cross the epithelial wall of small venules to enter connective cussed later with the immune system. tissue, where they differentiate, mature, and acquire the mor- phologic features of macrophages. Monocytes formed in the yolk sac during early embryonic development circulate and Mast Cells become resident in developing organs throughout the body, Mast cells are oval or irregularly shaped cells of connective comprising a group of related cells called the mononuclear tissue, between 7 and 20 μm in diameter, filled with basophilic phagocyte system. Many of these macrophage-like cells with secretory granules that often obscure the central nucleus prominent functions in various organs have specialized names (Figure 5–5). These granules are electron dense and of variable (Table 5–2). All are long-living cells, surviving with relative size, ranging from 0.3 to 2.0 μm in diameter. Because of the inactivity in tissues for months or years. During inflamma- high content of acidic radicals in their sulfated GAGs, mast tion and tissue repair which follow organ damage, macro- cell granules display metachromasia, which means that they phages become activated and play a very important role. can change the color of some basic dyes (eg, toluidine blue) Under such conditions these cells increase in number, mainly from blue to purple or red. The granules are poorly preserved in the connective tissue stroma, both by proliferation and by by common fixatives, so mast cells may be difficult to identify recruiting additional monocytes formed in the bone marrow in routinely prepared slides. 05_Mescher_ch05_p096-121.indd 99 26/04/18 11:15 am  100 CHAPTER 5 Connective Tissue FIGURE 5–4 Macrophage ultrastructure. VetBooks.ir L L L N Nu Characteristic features of macrophages seen in this TEM of one phagocytic vacuoles near the protrusions and indentations of the such cell are the prominent nucleus (N) and the nucleolus (Nu) cell surface. (X10,000) and the numerous secondary lysosomes (L). The arrows indicate Mast cells function in the localized release of many bioactive Histamine, which promotes increased vascular perme- substances important in the local inflammatory response, innate ability and smooth muscle contraction immunity, and tissue repair. A partial list of molecules released Serine proteases, which activate various mediators of from these cells’ secretory granules includes the following: inflammation Heparin, a sulfated GAG that acts locally as an Eosinophil and neutrophil chemotactic factors, which attract those leukocytes anticoagulant TABLE 5–2 Distribution and main functions of the cells of the mononuclear phagocyte system. Cell Type Major Location Main Function Monocyte Blood Precursor of macrophages Macrophage Connective tissue, lymphoid organs, Production of cytokines, chemotactic factors, and lungs, bone marrow, pleural and several other molecules that participate in inflammation peritoneal cavities (defense), antigen processing, and presentation Kupffer cell Liver (perisinusoidal) Same as macrophages Microglial cell Central nervous system Same as macrophages Langerhans cell Epidermis of skin Antigen processing and presentation Dendritic cell Lymph nodes, spleen Antigen processing and presentation Osteoclast (from fusion of several Bone Localized digestion of bone matrix macrophages) Multinuclear giant cell (several fused In connective tissue under various Segregation and digestion of foreign bodies macrophages) pathological conditions 05_Mescher_ch05_p096-121.indd 100 26/04/18 11:15 am  Cells of Connective Tissue 101 FIGURE 5–5 Mast cells. C H A P T E R VetBooks.ir E G 5 BV Connective Tissue Cells of Connective Tissue M N C a b Mast cells are components of loose connective tissues, often mitochondria (M). The granule staining in the TEM is heteroge- located near small blood vessels (BV). neous and variable in mast cells from different tissues; at higher (a) They are typically oval shaped, with cytoplasm filled with magnifications some granules may show a characteristic scroll-like strongly basophilic granules. (X400; PT) substructure (inset) that contains preformed mediators such as histamine and proteoglycans. The ECM near this mast cell includes (b) Ultrastructurally mast cells show little else around the nucleus elastic fibers (E) and bundles of collagen fibers (C). (N) besides these cytoplasmic granules (G), except for occasional Cytokines, polypeptides directing activities of leuko- to the antigen, it reacts with the IgE on the mast cells, trig- cytes and other cells of the immune system gering rapid release of histamine, leukotrienes, chemokines, Phospholipid precursors, which are converted to and heparin from the mast cell granules that can produce the prostaglandins, leukotrienes, and other important lipid sudden onset of the allergic reaction. Degranulation of mast mediators of the inflammatory response. cells also occurs as a result of the action of the complement molecules that participate in the immunologic reactions Occurring in connective tissue of many organs, mast cells described in Chapter 14. are especially numerous near small blood vessels in skin and Like macrophages, mast cells originate from progenitor mesenteries (perivascular mast cells) and in the tissue that cells in the bone marrow, which circulate in the blood, cross lines digestive and respiratory tracts (mucosal mast cells); the wall of small vessels called venules, and enter connective the granule content of the two populations differs somewhat. tissues, where they differentiate. Although mast cells are in These major locations suggest that mast cells place themselves many respects similar to basophilic leukocytes, they appear to strategically to function as sentinels detecting invasion by have a different lineage at least in humans. microorganisms. Release of certain chemical mediators stored in mast cells promotes the allergic reactions known as immediate Plasma Cells hypersensitivity reactions because they occur within a Plasma cells are lymphocyte-derived, antibody-producing few minutes after the appearance of an antigen in an indi- cells. These relatively large, ovoid cells have basophilic cyto- vidual previously sensitized to that antigen. There are many plasm rich in RER and a large Golgi apparatus near the examples of immediate hypersensitivity reaction; a dramatic nucleus that may appear pale in routine histologic prepara- one is anaphylactic shock, a potentially fatal condition. tions (Figure 5–7). Anaphylaxis consists of the following sequential events The nucleus of the plasma cell is generally spherical but (Figure 5–6). The first exposure to an antigen (allergen), such eccentrically placed. Many of these nuclei contain compact, as bee venom, causes antibody-producing cells to produce an peripheral regions of heterochromatin alternating with lighter immunoglobulin of the IgE class that binds avidly to recep- areas of euchromatin. At least a few plasma cells are present in tors on the surface of mast cells. Upon a second exposure most connective tissues. Their average life span is only 10-20 days. 05_Mescher_ch05_p096-121.indd 101 26/04/18 11:15 am  102 CHAPTER 5 Connective Tissue FIGURE 5–6 Mast cell secretion. VetBooks.ir Antigens 2 IgE IgE receptor 3 Adenylate cyclase Ca 2 + Fusion of granules ATP Phosphorylated cAMP proteins Active Microfilaments ATP Heparin protein kinase Histamine Inactive 4 protein kinase Proteoglycans 1 ECF-A Exocytosis Membrane 5 Phospholipases phospholipids Leukotrienes IgE receptors Mast cell secretion is triggered by reexposure to certain antigens exocytosis of some granules (4). In addition, phospholipases act and allergens. Molecules of IgE antibody produced in an initial on specific membrane phospholipids, leading to production and response to an allergen such as pollen or bee venom are bound to release of leukotrienes (5). surface receptors for IgE (1), of which 300,000 are present per mast The components released from granules, as well as the leu- cell. kotrienes, are immediately active in the local microenvironment When a second exposure to the allergen occurs, IgE molecules and promote a variety of controlled local reactions that together bind this antigen and a few IgE receptors very rapidly become normally comprise part of the inflammatory process called the cross-linked (2). This activates adenylate cyclase, leading to immediate hypersensitivity reaction. “ECF-A” is the eosinophil phosphorylation of specific proteins (3), entry of Ca2+ and rapid chemotactic factor of anaphylaxis. › › MEDICAL APPLICATION Leukocytes Plasma cells are derived from B lymphocytes and are respon- Other white blood cells, or leukocytes, besides macrophages sible for the synthesis of immunoglobulin antibodies. Each and plasma cells normally comprise a population of wandering antibody is specific for the one antigen that stimulated the cells in connective tissue. Derived from circulating blood cells, clone of B cells and reacts only with that antigen or mol- they leave blood by migrating between the endothelial cells ecules resembling it (see Chapter 14). The results of the of venules to enter connective tissue. This process increases antibody-antigen reaction are variable, but they usually greatly during inflammation, which is a vascular and cellular neutralize harmful effects caused by antigens. An antigen defensive response to injury or foreign substances, including that is a toxin (eg, tetanus, diphtheria) may lose its capacity pathogenic bacteria or irritating chemical substances. to do harm when it is bound by a specific antibody. Bound Inflammation begins with the local release of chemi- antigen-antibody complexes are quickly removed from tis- cal mediators from various cells, the ECM and blood plasma sues by phagocytosis. proteins. These substances act on local blood vessels, mast cells, macrophages, and other cells to induce events characteristic of 05_Mescher_ch05_p096-121.indd 102 26/04/18 11:15 am  Fibers 103 FIGURE 5–7 Plasma cells. C H A P T E R VetBooks.ir 5 Connective Tissue Fibers a b Antibody-secreting plasma cells are present in variable numbers in (b) Plasma are often more abundant in infected tissues, as in the the connective tissue of many organs. inflamed lamina propria shown here. A large pale Golgi apparatus (a) Plasma cells are large, ovoid cells, with basophilic cytoplasm. (arrows) at a juxtanuclear site in each cell is actively involved in the The round nuclei frequently show peripheral clumps of het- terminal glycosylation of the antibodies (glycoproteins). Plasma cells erochromatin, giving the structure a “clock-face” appearance. leave their sites of origin in lymphoid tissues, move to connective tis- (X640; H&E) sue, and produce antibodies that mediate immunity. (X400 PT) inflammation, for example, increased blood flow and vascular permeability, entry and migration of leukocytes, and activa- › FIBERS tion of macrophages for phagocytosis. The fibrous components of connective tissue are elongated Most leukocytes function in connective tissue only for a structures formed from proteins that polymerize after secre- few hours or days and then undergo apoptosis. However, as tion from fibroblasts (Figure 5–2). The three main types of discussed with the immune system, some lymphocytes and fibers include collagen, reticular, and elastic fibers. Col- phagocytic antigen-presenting cells normally leave the inter- lagen and reticular fibers are both formed by proteins of the stitial fluid of connective tissue, enter blood or lymph, and collagen family, and elastic fibers are composed mainly of the move to selected lymphoid organs. protein elastin. These fibers are distributed unequally among the different types of connective tissue, with the predominant fiber type conferring most specific tissue properties. › › MEDICAL APPLICATION Increased vascular permeability is caused by the action of Collagen vasoactive substances such as histamine released from mast The collagens constitute a family of proteins selected during cells during inflammation. Classically, the major signs of evolution for their ability to form various extracellular fibers, inflamed tissues include “redness and swelling with heat sheets, and networks, all of which extremely strong and resis- and pain” (rubor et tumor cum calore et dolore). Increased tant to normal shearing and tearing forces. Collagen is a key blood flow and vascular permeability produce local tissue element of all connective tissues, as well as epithelial basement swelling (edema), with increased redness and warmth. membranes and the external laminae of muscle and nerve cells. Pain is due mainly to the action of the chemical mediators Collagen is the most abundant protein in the human on local sensory nerve endings. All these activities help body, representing 30% of its dry weight. A major product protect and repair the inflamed tissue. Chemotaxis (Gr. of fibroblasts, collagens are also secreted by several other cell chemeia, alchemy + taxis, orderly arrangement), the phe- types and are distinguishable by their molecular composi- nomenon by which specific cell types are attracted by spe- tions, morphologic characteristics, distribution, functions, cific molecules, draws much larger numbers of leukocytes and pathologies. A family of 28 collagens exists in vertebrates, into inflamed tissues. numbered in the order they were identified, and the most important are listed in Table 5–3. They can be categorized 05_Mescher_ch05_p096-121.indd 103 26/04/18 11:15 am  104 CHAPTER 5 Connective Tissue according to the structures formed by their interacting Network or sheet-forming collagens such as type IV α-chains subunits: collagen have subunits produced by epithelial cells and VetBooks.ir are major structural proteins of external laminae and all Fibrillar collagens, notably collagen types I, II, and epithelial basal laminae. III, have polypeptide subunits that aggregate to form large fibrils clearly visible in the electron or light micro- Linking/anchoring collagens are short collagens that link fibrillar collagens to one another (forming larger scope (Figure 5–8). Collagen type I, the most abundant fibers) and to other components of the ECM. Type VII and widely distributed collagen, forms large, eosinophilic collagen binds type IV collagen and anchors the basal bundles usually called collagen fibers. These often lamina to the underlying reticular lamina in basement densely fill the connective tissue, forming structures such membranes (see Figure 4–3). as tendons, organ capsules, and dermis. TABLE 5–3 Collagen types. α-Chain Type Composition Structure Optical Microscopy Major Location Main Function Fibril-Forming Collagens I [α1 (I)]2[α2 (I)] 300-nm molecule, Thick, highly picrosirius Skin, tendon, bone, Resistance to tension 67-nm-banded fibrils birefringent, fibers dentin II [α1 (II)]3 300-nm molecule, Loose aggregates of fibrils, Cartilage, vitreous Resistance to pressure 67-nm-banded fibrils birefringent body III [α1 (III)]3 67-nm-banded fibrils Thin, weakly birefringent, Skin, muscle, blood Structural argyrophilic (silver-binding) vessels, frequently maintenance in fibers together with type I expansible organs V [α1 (V)]3 390-nm molecule, Frequently forms fiber Fetal tissues, skin, Participates in type I N-terminal globular together with type I bone, placenta, most collagen function domain interstitial tissues XI [α1 (XI)] [α2 (XI)] [α3 300-nm molecule Small fibers Cartilage Participates in type II (XI)] collagen function Network-Forming Collagens IV [α1 (IV)]2 [α2 (IV)] Two-dimensional Detected by All basal and external Support of epithelial cross-linked network immunocytochemistry laminae cells; filtration X [α1(X)]3 Hexagonal lattices Detected by Hypertrophic Increases density of immunocytochemistry cartilage involved in the matrix endochondral bone formation Linking/Anchoring Collagens VII [α1 (VII)]3 450 nm, globular Detected by Epithelial basement Anchors basal laminae domain at each end immunocytochemistry membranes to underlying reticular lamina IX [α1 (IX)] [α2 (IX)] [α3 200-nm molecule Detected by Cartilage, vitreous Binds various (IX)] immunocytochemistry body proteoglycans; associated with type II collagen XII [α1 (XII)]3 Large N-terminal Detected by Placenta, skin, tendons Interacts with type I domain immunocytochemistry collagen XIV [α1 (XIV)]3 Large N-terminal Detected by Placenta, bone Binds type I collagen domain; cross-shaped immunocytochemistry fibrils, with types V molecule and XII, strengthening fiber formation 05_Mescher_ch05_p096-121.indd 104 26/04/18 11:15 am  Fibers 105 FIGURE 5–8 Type I collagen. C H A P T E R VetBooks.ir 5 Connective Tissue Fibers C a Subunits of type I collagen, the most abundant collagen, assemble to form extremely strong fibrils, which are then bundled together further by other collagens into much larger structures called col- C lagen fibers. C (a) TEM shows fibrils cut longitudinally and transversely. In longitu- b dinal sections fibrils display alternating dark and light bands; in cross section the cut ends of individual collagen molecules appear as dots. Ground substance completely surrounds the fibrils. (X100,000) the extracellular space. Subunits for these fibers were secreted by (b) The large bundles of type I collagen fibrils (C) appear as aci- the fibroblasts (arrows) associated with them. (X400; H&E) dophilic collagen fibers in connective tissues, where they may fill Collagen synthesis occurs in many cell types but is a spe- undergoes exocytosis and is cleaved to a rodlike procollagen cialty of fibroblasts. The initial procollagen α chains are poly- molecule (Figure 5–9) that is the basic subunit from which the peptides made in the RER. Several different α chains of variable fibers or sheets are assembled. These subunits may be homotri- lengths and sequences can be synthesized from the related colla- meric, with all three chains identical, or heterotrimeric, with two gen genes. In the ER three α chains are selected, aligned, and stabi- or all three chains having different sequences. Different combina- lized by disulfide bonds at their carboxyl terminals, and folded as a tions of procollagen α chains produce the various types of colla- triple helix, another defining feature of collagens. The triple helix gen with different structures and functional properties. FIGURE 5–9 The collagen subunit. 8.6 nm In the most abundant form of collagen, type I, each procollagen hydrophobic interactions. The length of each molecule (sometimes molecule or subunit has two α1- and one α2-peptide chains, each called tropocollagen) is 300 nm, and its width is 1.5 nm. Each com- with a molecular mass of approximately 100 kDa, intertwined in plete turn of the helix spans a distance of 8.6 nm. a right-handed helix and held together by hydrogen bonds and 05_Mescher_ch05_p096-121.indd 105 26/04/18 11:15 am  106 CHAPTER 5 Connective Tissue › › MEDICAL APPLICATION which the process can be interrupted or changed by defective enzymes or by disease processes (Table 5–4). VetBooks.ir A keloid is a local swelling caused by abnormally large Type I collagen fibrils have diameters ranging from 20 amounts of collagen that form in scars of the skin. Keloids to 90 nm and can be several micrometers in length. Adja- occur most often in individuals of African descent and can cent rodlike collagen subunits of the fibrils are staggered by be a troublesome clinical problem to manage. Not only can 67 nm, with small gaps (lacunar regions) between their ends they be disfiguring, but excision is almost always followed by (Figure 5–11). This structure produces a characteristic feature recurrence. of type I collagen visible by EM: transverse striations with a regular periodicity (Figure 5–11). Type I collagen fibrils assemble further to form large, extremely strong collagen fibers that may be further bundled by linking collagens and proteoglycans. Collagen type II (present in cartilage) occurs An unusually large number of posttranslational process- as fibrils but does not form fibers or bundles. Sheet-forming ing steps are required to prepare collagen for its final assembly collagen type IV subunits assemble as a latticelike network in in the ECM. These steps have been studied most thoroughly epithelial basal laminae. for type I collagen, which accounts for 90% of all the body’s When they fill the ECM (eg, in tendons or the sclera of collagen. The most important parts of this process are sum- the eye), bundles of collagen appear white. The highly regular marized in Figure 5–10 and described briefly here: orientation of subunits makes collagen fibers birefringent with 1. The procollagen α chains are produced on polyribosomes polarizing microscopy (see Figure 1–7). In routine light micros- of the RER and translocated into the cisternae. These typi- copy, collagen fibers are acidophilic, staining pink with eosin, cally have long central domains rich in proline and lysine; blue with Mallory trichrome stain, and red with Sirius red. in type I collagen every third amino acid is glycine. Because collagen bundles are long and tortuous, their length 2. Hydroxylase enzymes in the ER cisternae add hydroxyl and diameter are better studied in spread preparations rather (-OH) groups to some prolines and lysines in reactions than sections, as shown in Figure 1–7a. A very small mesentery that require O2, Fe2+, and ascorbic acid (vitamin C) as is frequently used for this purpose; when spread on a slide, this cofactors. structure is sufficiently thin to let the light pass through; it can be stained and examined directly under the microscope. 3. Glycosylation of some hydroxylysine residues also occurs, Collagen turnover and renewal in normal connective to different degrees in various collagen types. tissue is generally a very slow but ongoing process. In some 4. Both the amino- and carboxyl-terminal sequences of organs, such as tendons and ligaments, the collagen is very sta- α chains have globular structures that lack the Gly-X-Y ble, whereas in others, as in the periodontal ligament surround- repeats. In the RER the C-terminal regions of three ing teeth, the collagen turnover rate is high. To be renewed, selected α chains (α1, α2) are stabilized by cysteine disul- the collagen must first be degraded. Degradation is initiated fide bonds, which align the three polypeptides and facili- by specific enzymes called collagenases, which are mem- tates their central domains folding as the triple helix. bers of an enzyme class called matrix metalloproteinases With its globular terminal sequences intact, the trimeric (MMPs), which clip collagen fibrils or sheets in such a way that procollagen molecule is transported through the Golgi they are then susceptible to further degradation by nonspecific apparatus, packaged in vesicles and secreted. proteases. Various MMPs are secreted by macrophages and 5. Outside the cell, specific proteases called procollagen play an important role in remodeling the ECM during tissue peptidases remove the terminal globular peptides, con- repair. verting the procollagen molecules to collagen molecules. These now self-assemble (an entropy-driven process) into polymeric collagen fibrils, usually in specialized niches › › MEDICAL APPLICATION near the cell surface. Normal collagen function depends on the expression of 6. Certain proteoglycans and other collagens (eg, types V many different genes and adequate execution of several and XII) associate with the new collagen fibrils, stabilize posttranslational events. It is not surprising; therefore, many these assemblies, and promote the formation of larger pathologic conditions are directly attributable to insufficient fibers from the fibrils. or abnormal collagen synthesis. A few such genetic disorders 7. Fibrillar structure is reinforced and disassembly is pre- or conditions are listed in Table 5–4. vented by the formation of covalent cross-links between the collagen molecules, a process catalyzed by lysyl oxidase. The other fibrillar and sheetlike collagens are formed in Reticular Fibers processes similar to that described for collagen type I and sta- Found in delicate connective tissue of many organs, notably in bilized by linking or anchoring collagens. Because there are so the immune system, reticular fibers consist mainly of colla- many steps in collagen biosynthesis, there are many points at gen type III, which forms an extensive network (reticulum) of 05_Mescher_ch05_p096-121.indd 106 26/04/18 11:15 am  Fibers 107 FIGURE 5–10 Collagen synthesis. C H A P T E R VetBooks.ir Intracellular environment Nucleus Formation of mRNA for each type of α chain. 5 RER Synthesis of procollagen α chains with propeptides Connective Tissue Fibers at both ends. Clipping of signal peptide. OH OH Hydroxylation of specific prolyl and lysyl residues OH OH in the endoplasmic reticulum. Vitamin C dependent. Gal-Glu OH Attachment of soluble galactosyl and glucosyl sugars to specific hydroxylysyl residues. OH Gal-Glu Assembly of procollagen molecules (triple helix). Nonhelical propeptides. Transfer vesicles Transport of soluble procollagen to Golgi complex. Packaging of soluble procollagen in secretory Golgi vesicles. Centrioles Secretory Secretory vesicles assisted by microtubules and vesicles microfilaments transport soluble procollagen molecules to cell surface. Extracellular environment Exocytosis of procollagen molecules to extracellular space. Procollagen peptidases cleave most of the Procollagen Procollagen nonhelical terminal peptides, transforming peptidases peptidases procollagen into insoluble collagen molecules, which aggregate to form collagen fibrils. Collagen molecules Microtubule arrays Fibrillar structure is reinforced by the formation of covalent cross-links between collagen molecules catalyzed by the enzyme lysyl oxidase. Hydroxylation and glycosylation of procollagen α chains and their α chains and collagen production depends on several posttrans- assembly into triple helices occur in the RER, and further assem- lational events involving several other enzymes, many diseases bly into fibrils occurs in the ECM after secretion of procollagen. involving defective collagen synthesis have been described. Because there are many slightly different genes for procollagen 05_Mescher_ch05_p096-121.indd 107 26/04/18 11:15 am  108 CHAPTER 5 Connective Tissue TABLE 5–4 Examples of clinical disorders resulting from defects in collagen synthesis. VetBooks.ir Disorder Defect Symptoms Ehlers–Danlos type IV Faulty transcription or translation of collagen type III Aortic and/or intestinal rupture Ehlers–Danlos type VI Faulty lysine hydroxylation Increased skin elasticity, rupture of eyeball Ehlers–Danlos type VII Decrease in procollagen peptidase activity Increased articular mobility, frequent luxation Scurvy Lack of vitamin C, a required cofactor for prolyl Ulceration of gums, hemorrhages hydroxylase Osteogenesis imperfecta Change of 1 nucleotide in genes for collagen type I Spontaneous fractures, cardiac insufficiency FIGURE 5–11 Assembly of type I collagen. Gap region Overlapping region Procollagen subunit 1 300 nm 2 300 nm 3 Collagen fibril Gap Overlapping region (about 10% Bundle of region of a procollagen subunit’s length) collagen fibers 67 nm 5 Collagen fiber 4 Shown here are the relationships among type I collagen molecules, 4. Fibrils assemble further and are linked together in larger col- fibrils, fibers, and bundles. lagen fibers visible by light microscopy. 1. Rodlike triple-helix collagen molecules, each 300-nm long, 5. Type I fibers often form into still larger aggregates bundled self-assemble in a highly organized, lengthwise arrangement and linked together by other collagens. of overlapping regions. The photo shows an SEM view of type I collagen fibrils closely 2. The regular, overlapping arrangement of subunits continues aggregated as part of a collagen fiber. Striations are visible on the as large collagen fibrils are assembled. surface of the fibrils. 3. This structure causes fibrils to have characteristic cross stria- tions with alternating dark and light bands when observed in the EM. 05_Mescher_ch05_p096-121.indd 108 26/04/18 11:15 am  Fibers 109 FIGURE 5–12 Reticular fibers. C H A P T E R VetBooks.ir 5 Connective Tissue Fibers a b In these silver-stained sections of adrenal cortex (a) and lymph the black argyrophilia. Cell nuclei are also dark, but cytoplasm is node (b), networks of delicate, black reticular fibers are promi- unstained. (X100) Fibroblasts specialized for reticular fiber produc- nent. These fibers serve as a supportive stroma in most lymphoid tion in hematopoietic and lymphoid organs are often called reticu- and hematopoietic organs and many endocrine glands. The fibers lar cells. consist of type III collagen that is heavily glycosylated, producing thin (diameter 0.5-2 μm) fibers for the support of many differ- in many organs, particularly those subject to regular stretching ent cells. Reticular fibers are seldom visible in hematoxylin and or bending. As the name implies, elastic fibers have rubberlike eosin (H&E) preparations but are characteristically stained properties that allow tissue containing these fibers, such as the black after impregnation with silver salts (Figure 5–12) and stroma of the lungs, to be stretched or distended and return are thus termed argyrophilic (Gr. argyros, silver). Reticular to their original shape. In the wall of large blood vessels, espe- fibers are also periodic acid–Schiff (PAS) positive, which, like cially arteries, elastin also occurs as fenestrated sheets called argyrophilia, is due to the high content of sugar chains bound elastic lamellae. Elastic fibers and lamellae are not strongly to type III collagen α chains. Reticular fibers contain up to 10% acidophilic and stain poorly with H&E; they are stained more carbohydrate as opposed to 1% in most other collagen fibers. darkly than collagen with other stains such as orcein and alde- Reticular fibers produced by fibroblasts occur in the hyde fuchsin (Figure 5–13). reticular lamina of basement membranes and typically also Elastic fibers (and lamellae) are a composite of fibrillin surround adipocytes, smooth muscle and nerve fibers, and (350 kDa), which forms a network of microfibrils, embedded small blood vessels. Delicate reticular networks serve as the in a larger mass of cross-linked elastin (60 kDa). Both pro- supportive stroma for the parenchymal secretory cells and rich teins are secreted from fibroblasts (and smooth muscle cells in microvasculature of the liver and endocrine glands. Abundant vascular walls) and give rise to elastic fibers in a stepwise man- reticular fibers also characterize the stroma of hemopoietic tis- ner are shown in Figure 5–14. Initially, microfibrils with diam- sue (bone marrow), the spleen, and lymph nodes where they eters of 10 nm form from fibrillin and various glycoproteins. support rapidly changing populations of proliferating cells and The microfibrils act as scaffolding upon which elastin is then phagocytic cells. deposited. Elastin accumulates around the microfibrils, even- tually making up most of the elastic fiber, and is responsible for the rubberlike property. Elastic Fibers The elastic properties of these fibers and lamellae result Elastic fibers are also thinner than the type I collagen fibers from the structure of the elastin subunits and the unique cross- and form sparse networks interspersed with collagen bundles links holding them together. Elastin molecules have many 05_Mescher_ch05_p096-121.indd 109 26/04/18 11:15 am  110 CHAPTER 5 Connective Tissue FIGURE 5–13 Elastic fibers. VetBooks.ir a b c Elastic fibers or lamellae (sheets) add resiliency to connective tis- (b) In sectioned tissue at higher magnification, elastic fibers can sue. Such fibers may be difficult to discern in H&E-stained tissue, be seen among the acidophilic collagen bundles of dermis. (X400; but elastin has a distinct, darker-staining appearance with other Aldehyde fuchsin) staining procedures. (c) Elastic lamellae in the wall of the aorta are more darkly stained, (a) The length, diameter, distribution, and density of dark elastic incomplete sheets of elastin between the layers of eosinophilic fibers are easily seen in this spread preparation of nonstretched smooth muscle. (X80; H&E) connective tissue in a mesentery. (X200; Hematoxylin and orcein) FIGURE 5–14 Formation of elastic fibers. a b c Stages in the formation of elastic fibers can be seen by TEM. are also secreted by the fibroblasts and quickly become covalently cross-linked into larger assemblies. (a) Initially, a developing fiber consists of many 10-nm-diameter microfibrils composed of fibrillin subunits secreted by fibroblasts (c) Elastin accumulates and ultimately occupies most of the and smooth muscle cells. electron-dense center of the single elastic fiber shown here. Fibril- lin microfibrils typically remain visible at the fiber surface. Collagen (b) Elastin is deposited on the scaffold of microfibrils, forming fibrils, seen in cross section, are also present surrounding the elas- growing, amorphous composite structures. The elastin molecules tic fiber. (All X50,000) 05_Mescher_ch05_p096-121.indd 110 26/04/18 11:15 am  Ground Substance 111 FIGURE 5–15 Molecular basis of elastic fiber › GROUND SUBSTANCE C H A P T E R VetBooks.ir elasticity. The ground substance of the ECM is a highly hydrated (with much bound water), transparent, complex mixture of three major kinds of macromolecules: glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins. Filling the space between cells and fibers in connective tissue, ground substance allows diffusion of small molecules and, because it is 5 viscous, acts as both a lubricant and a barrier to the penetration Connective Tissue Ground Substance of invaders. Physical properties of ground substance also pro- Relaxed foundly influence various cellular activities. When adequately fixed for histologic analysis, its components aggregate as fine, poorly resolved material that appears in TEM preparations as electron-dense filaments or granules (Figure 5–16a). Single elastin Cross-link GAGs (also called mucopolysaccharides) are long poly- Stretched molecule mers of repeating disaccharide units, usually a hexosamine and uronic acid. The hexosamine can be glucosamine or galactos- amine, and the uronic acid can be glucuronate or iduronate. The largest and most ubiquitous GAG is hyaluronan (also called hyaluronate or hyaluronic acid). With a molecular weight from 100s to 1000s of kDa, hyaluronan is a very long polymer of the The diagram shows a small piece of an elastic fiber, in two con- disaccharide glucosamine-glucuronate. Uniquely among GAGs, formations. Elastin polypeptides, the major components of hyaluronan is synthesized directly into the ECM by an enzyme elastic fibers, have multiple random-coil domains that straighten complex, hyaluronan synthase, located in the cell membrane or stretch under force, and then relax. Most of the cross-links of many cells. Hyaluronan forms a viscous, pericellular network between elastin subunits consist of the covalent, cyclic structure that binds a considerable amount of water, giving it an impor- desmosine, each of which involves four converted lysines in two elastin molecules. This unusual type of protein cross-link holds the tant role in allowing molecular diffusion through connective aggregate together with little steric hindrance to elastin move- tissue and in lubricating various organs and joints. ments. These properties give the entire network its elastic quality. All other GAGs are much smaller (10-40 kDa), sulfated,