Ehlers-Danlos syndrome is clinically characterized by hyperelasticity of the skin (Figure 4-4) and hypermobility of the joints. The major defect resides in the synthesis, processing, and assembly of collagen. Several clinical subtypes are observed. They are classified by the degree of severity and the mutations in the collagen genes. For example, the vascular type form of Ehlers-Danlos syndrome—caused by a mutation in the COL3A1 gene—is associated with severe vascular alterations leading to the development of varicose veins and spontaneous rupture of major arteries. A deficiency in the synthesis of type III collagen, prevalent in the walls of blood vessels, is the major defect. Arthrochalasia and dermatosparaxsis types of Ehlers-Danlos syndrome display congenital dislocation of the hips and marked joint hypermobility. Mutations in the COL1A1 and COL1A2 genes (Figure 4-5), encoding type I collagen, and procollagen N-peptidase gene disrupt the cleavage site at the N-terminal of the molecule and affect the conversion of procollagen to collagen in some individuals.

Figure 4-4 Ehlers-Danlos syndromes

Figure 4-5 Pathology of collagen: Molecular defects

Elastic fibers: Synthesis, secretion, and assembly

Like collagen, the synthesis of elastic fibers involves both the RER and the Golgi apparatus (Figure 4-6).

Figure 4-6 Synthesis of elastic fibers

Elastic fibers are synthesized by the fibroblast (in skin and tendons), the chondroblast, the chondrocyte (in elastic cartilage of the auricle of the ear, epiglottis, larynx, and auditory tubes), and smooth muscle cells (in large blood vessels like the aorta and in the respiratory tree).

Proelastin, the precursor of elastin, is cleaved and secreted as tropoelastin. In the extracellular space, tropoelastin interacts with fibrillins and fibulin 1 to organize elastic fibers, which aggregate to form bundles of elastic fibers. Tropoelastin contains a characteristic but uncommon amino acid: desmosine. Two lysine residues of tropoelastin are oxidized by lysyl oxidase to form a desmosine ring that cross-links two tropoelastin molecules. Cross-linking enables the stretching and recoil of tropoelastin, like rubber bands. Elastic fibers do not contain collagen. Elastic fibers are produced during embryonic development and in adolescence but not so much in adults. Although elastic fibers are resilient during human life, many tissues decrease elasticity with age, in particular the skin, which develops wrinkles.

Under the light microscope, elastic fibers stain black or dark blue with orcein, a natural dye obtained from lichens.

Under the electron microscope, a cross section of an elastic fiber shows a dense core of elastin surrounded by microfibrils of fibulin 1 and fibrillins.

Clinical significance: Marfan syndrome

Marfan syndrome is an autosomal dominant disorder in which the elastic tissue is weakened. Defects are predominantly observed in three systems: the ocular, skeletal, and cardiovascular systems. The ocular defects include myopia and detached lens (ectopia lentis). The skeletal defects (Figure 4-7) include long and thin arms and legs (dolichostenomelia), hollow chest (pectus excavatum), scoliosis, and elongated fingers (arachnodactyly).

Figure 4-7 Marfan syndrome

Cardiovascular abnormalities are life-threatening. Patients with Marfan syndrome display prolapse of the mitral valve and dilation of the ascending aorta. Dilation of the aorta and peripheral arteries may progress to dissecting aneurysm (Greek aneurysma, widening) and rupture. Medical treatment, such as administration of β-adrenergic blockers to reduce the force of systolic contraction in order to diminish stress on the aorta, and limited heavy exercise increase the survival rate of patients with Marfan syndrome.

Defects observed in Marfan syndrome are caused by poor recoiling of the elastic lamellae dissociated by an increase in proteoglycans (see Figure 4-7). In the skeletal system, the periosteum, a relatively rigid layer covering the bone, is abnormally elastic and does not provide an oppositional force during bone development, resulting in skeletal defects.

A mutation of the fibrillin 1 gene on chromosome 15 is responsible for Marfan syndrome. Fibrillin is present in the aorta, suspensory ligaments of the lens (see Chapter 9, Sensory Organs: Vision and Hearing), and the periosteum (see Bone). A homologous fibrillin 2 gene is present on chromosome 5. Mutations in the fibrillin 2 gene cause a disease called congenital contractural arachnodactyly. This disease affects the skeletal system, but ocular and cardiovascular defects are not observed.

Macrophages

Macrophages have phagocytic properties and derive from monocytes, cells formed in the bone marrow (Figure 4-8).

Figure 4-8 Macrophages

Monocytes circulate in blood and migrate into the connective tissue, where they differentiate into macrophages. Macrophages have specific names in certain organs; for example, they are called Kupffer cells in the liver, osteoclasts in bone, and microglial cells in the central nervous system. Macrophages migrate to the site of inflammation, attracted by certain mediators, particularly C5a (a member of the complement cascade; see Chapter 10, Immune-Lymphatic System). Macrophages in the connective tissue have the following structural features:

1. They contain abundant lysosomes required for the breakdown of phagocytic materials.

2. Active macrophages have numerous phagocytic vesicles (or phagosomes) for the transient storage of ingested materials.

3. The nucleus has an irregular outline.

Macrophages of the connective tissue have three major functions:

1. To turn over senescent fibers and ECM material.

2. The presentation of antigens to lymphocytes as part of inflammatory and immunologic responses (see Chapter 10, Immune-Lymphatic System).

3. Production of cytokines (for example, interleukin-1, an activator of helper T cells, and tumor necrosis factor–α, an inflammatory mediator).

Mast cells

Like macrophages, mast cells originate in the bone marrow from precursor cells lacking cytoplasmic granules. When mast cell precursors migrate into the connective tissue or the lamina propria of mucosae, they proliferate and accumulate cytoplasmic granules. Mast cells and basophils circulating in blood derive from the same progenitor in the bone marrow.

The mast cell is the source of vasoactive mediators contained in cytoplasmic granules (Figure 4-9). These granules contain histamine, heparin, and chemotactic mediators to attract monocytes, neutrophils, and eosinophils circulating in blood to the site of mast cell activation.

Figure 4-9 Mast cell

Leukotrienes are vasoactive products of mast cells. Leukotrienes are not present in granules; instead, they are released from the cell membrane of the mast cells as metabolites of arachidonic acid.

There are two populations of mast cells: mucosal mast cells (found predominantly in the intestine and lungs), and connective tissue mast cells.

Connective tissue mast cells differ from mucosal mast cells in the number and size of metachromatic (see Box 4-D) cytoplasmic granules, which tend to be more abundant in connective tissue mast cells. Although these two cell populations have the same cell precursor, the definitive structural and functional characteristics of mast cells depend on the site of differentiation (mucosa or connective tissue).

Box 4-D Metachromasia

• The granules of the mast cell have a staining property known as metachromasia (Greek meta, beyond; chroma, color).

• After staining with a metachromatic dye, such as toluidine blue, the mast cell granules stain with a color that is different from the color of the dye (purple-red instead of blue).

• This phenomenon is determined by a change in the electronic structure of the dye molecule after binding to the granular material. In addition, mast cell granules are PAS positive because of their glycoprotein nature.

Clinical significance: Mast cells and allergic hypersensitivity reactions

The secretion of specific vasoactive mediators plays an important role in the regulation of vascular permeability and bronchial smooth muscle tone during allergic hypersensitivity reactions (for example, in asthma, hay fever, and eczema).

The surface of mast cells and basophils contains immunoglobulin E (IgE) receptors. Antigens bind to two adjacent IgE receptors and the mast cell becomes IgE-sensitized. An IgE-sensitized mast cell releases Ca²⁺ from intracellular storage sites and the content of the cytoplasmic granules is rapidly discharged by a process known as degranulation.

The release of histamine during asthma (Greek asthma, panting) causes dyspnea (Greek dyspnoia, difficulty with breathing) triggered by the histamine-induced spasmodic contraction of the smooth muscle surrounding the bronchioles and the hypersecretion of goblet cells and mucosal glands of bronchi.

During hay fever, histamine increases vascular permeability leading to edema (excessive accumulation of fluid in intercellular spaces).

Mast cells in the connective tissue of skin release leukotrienes that induce increased vascular permeability associated with urticaria (Latin urtica, stinging nettle), a transient swelling in the dermis of the skin.

Plasma cells

The plasma cell, which derives from the differentiation of B lymphocytes (also called B cells), synthesizes and secretes a single class of immunoglobulin (Figure 4-10). We discuss in Chapter 10, Immune-Lymphatic System, details of the origin of plasma cells.

Figure 4-10 Plasma cell

Immunoglobulins are glycoproteins, and therefore plasma cells have the three structural characteristics of cells active in protein synthesis and secretion:

1. A well-developed rough endoplasmic reticulum

2. An extensive Golgi apparatus

3. A prominent nucleolus

At the light microscopic level, most of the cytoplasm of a plasma cell is basophilic because of the large amount of ribosomes associated with the endoplasmic reticulum. A clear area near the nucleus is slightly acidophilic and represents the Golgi apparatus. The nucleus has a characteristic cartwheel configuration created by the particular distribution of heterochromatin.

Extracellular matrix

The ECM is a combination of collagens, noncollagenous glycoproteins, and proteoglycans surrounding cells and fibers of the connective tissue.

Recall that the basement membrane contains several ECM components such as laminin, fibronectin, various types of collagen, and heparan sulfate proteoglycan. In addition, epithelial and nonepithelial cells have receptors for ECM constituents. An example is the family of integrins with binding affinity for laminin and fibronectin. Integrins interact with the cytoskeleton, strengthening cell interactions with the ECM by establishing focal contacts or modifying cell shape or adhesion.

Several noncollagenous glycoproteins of the ECM mediate interactions with cells and regulate the assembly of ECM components. Noncollagenous glycoproteins have a widespread distribution in several connective tissues, although cartilage and bone contain specific types of noncollagenous glycoproteins. We study them later when we discuss the processes of chondrogenesis (formation of cartilage) and osteogenesis (bone formation).

Proteoglycan aggregates (Figure 4-11) are the major components of the ECM. Each proteoglycan consists of glycosaminoglycans (GAGs), proteins complexed with polysaccharides. GAGs are linear polymers of disaccharides with sulfate residues. GAGs control the biological functions of proteoglycans by establishing links with cell surface components, growth factors, and other ECM constituents.

Figure 4-11 Proteoglycan aggregate

Different types of GAGs are attached to a core protein to form a proteoglycan. The core protein, in turn, is linked to a hyaluronan molecule by a linker protein. The hyaluronan molecule is the axis of a proteoglycan aggregate. Proteoglycans are named according to the prevalent GAG (for example, proteoglycan chondroitin sulfate, proteoglycan dermatan sulfate, proteoglycan heparan sulfate).

The embryonic connective tissue of the umbilical cord (Wharton’s jelly) is predominantly ECM material surrounding the two umbilical arteries and the single umbilical vein. Proteoglycans have extremely high charge density and, therefore, significant osmotic pressure. These attributes enable a connective tissue bed to resist compression because of the very high swelling capacity of these molecules. The umbilical blood vessels, crucial elements for fetal-maternal fluid, gas, and nutritional exchange, are surrounded by a proteoglycan-enriched type of connective tissue to provide resistance to compression.

Degradation of the extracellular matrix

The ECM can be degraded by matrix metalloproteinases, a family of zinc-dependent proteases secreted as latent precursors (zymogens) proteolytically activated in the ECM. The activity of matrix metalloproteinases in the extracellular space can be specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs).

The expression of matrix metalloproteinase genes is regulated by cytokines, growth factors, and cell contact with the ECM.

The degradation of the ECM occurs normally during the development, growth, and repair of tissues. However, excessive degradation of the ECM is observed in several pathologic conditions such as rheumatoid arthritis, osteoarthritis, and diseases of the skin. Tumor invasion, metastasis, and tumor angiogenesis require the participation of matrix metalloproteinases whose expression increases in association with tumorigenesis.

Members of the family of matrix metalloproteinases include:

1. Collagenases. Collagenases 1, 2, and 3 can degrade types I, II, III, and V collagens. Collagenase 1 is synthesized by fibroblasts, chondrocytes (cartilage), keratinocytes (epidermis), monocytes and macrophages, hepatocytes (liver), and tumor cells. Collagenase 2 is stored in cytoplasmic granules of polymorphonuclear leukocytes and released in response to a stimulus. Collagenase 3 can degrade several collagens (types I, II, III, IV, IX, X, and XI), laminin and fibronectin, and other ECM components.

2. Stromelysins (1, 2, and metalloelastase), which degrade basement membrane components (type IV collagen and fibronectin) and elastin.

3. Gelatinases A and B can degrade type I collagen. Gelatinases are produced by alveolar macrophages.

4. Membrane-type matrix metalloproteinases are produced by tumor cells. Matrix metalloproteinases are a target of therapeutic intervention to inhibit tumor invasion and metastasis. We come back to this topic in Chapter 23, Fertilization, Placentation, and Lactation, when we discuss the early stages of embryo implantation in the endometrial stroma or decidua.

Clinical significance: Molecular biology of tumor invasion

Invasion and metastasis are two important events of carcinoma (Greek karkinoma, from karkinos, crab, cancer + oma, tumor), a tumor derived from epithelial tissues. Adenoma is a structurally benign tumor of epithelial cell origin lacking invasive and metastatic properties. Malignant carcinomas may arise from benign adenomas. For example, a small benign adenoma or polyp of the colon can become an invasive carcinoma.

Sarcoma (Greek sarx, flesh + oma) is a tumor derived from the connective tissues (muscle, bone, cartilage) and mesodermal cells. For example, fibrosarcoma derives from fibroblasts and osteosarcoma originates from bone.

Invasion is defined by the breakdown of the basement membrane by tumor cells and implies the transition from precancer to cancer. Metastasis is the spread of tumor cells throughout the body through blood and lymphatic vessels, generally leading to death. Figure 4-12 illustrates and describes the initial events of tumor cell invasion.

Figure 4-12 Tumor invasion and metastasis

Many carcinomas produce members of the matrix metalloproteinase family to digest various types of collagen as we have seen in the preceding section. Normal tissues produce tissue inhibitors of metalloproteinases that are neutralized by carcinoma cells. Tumors that behave aggressively are capable of overpowering the protease inhibitors.

One critical event during metastasis is angiogenesis, the development of blood vessels. Blood vessels supply oxygen and nutrients required for tumor growth. Angiogenesis is stimulated by tumor cells, in particular the proliferation of capillary endothelial cells forming new capillaries in the tumoral growth. In Chapter 12, Cardiovascular System, we discuss the mechanism of action and targets of endostatin and angiostatin, two new proteins that inhibit angiogenesis.

ADIPOSE TISSUE OR FAT

There are two classes of adipose tissue:

1. White fat, the major reserve of long-term energy

2. Brown fat, which serves primarily to dissipate energy instead of storing energy.

Under the influence of insulin—bound to insulin-like growth factor-1 (IGF-1) receptor—preadipocytes synthesize lipoprotein lipase and begin to accumulate fat in small droplets. Small droplets fuse to form a single large lipid-storage droplet, a characteristic of mature unilocular (Latin unus, single; loculus, small place) adipocytes (also called adipose cell) (Figure 4-13). The single lipid-storage droplet pushes the nucleus to an eccentric position and the adipocyte assumes a “signet-ring” appearance. In histologic sections, capillaries appear as single structures that may contain blood cell elements, whereas adipocytes form aggregates.

Figure 4-13 Adipogenesis

Lipid droplets contain about 95% triglycerides rich in carotene, a lipid-soluble pigment that gives the so-called white fat a yellowish color. Each lipid droplet is in direct contact with the cytosol and is not surrounded by a cytomembrane. Therefore, lipid droplets can be classified as cell inclusions (see Box 4-E).

Box 4-E Fat in histologic sections

• Fat is usually dissolved by solvents (xylene) used during paraffin embedding. Only the nucleus and a narrow cytoplasmic rim, surrounding a central empty space, can be visualized.

• Fat that is fixed and stained with osmium tetroxide appears brown. This reagent is also used for the visualization of lipid-rich myelin in nerves (see Chapter 8, Nervous Tissue).

• Alcoholic solutions of fat-soluble dyes (such as Sudan III or Sudan black) can also be used for the detection of fat in frozen sections.

The main function of white fat is storage of energy. Unlike brown fat, white fat responds slightly to cold and acts as an insulator. The blood supply to white fat, mainly capillaries, is not as extensive as in brown fat. Adipose tissue also insulates the body against heat loss, fills spaces, and cushions certain anatomic parts, behaving as a shock-absorber in the soles of the feet, around the kidneys, and in the orbit around the eye. Most adipose tissues form at sites where loose connective tissue is present, such as the subcutaneous layer—or hypodermis—of the skin.

The storage of lipids by mature adipocytes are regulated by insulin and prostaglandins. The breakdown and release of lipids is regulated by epinephrine, glucagon, and adrenocorticotropic hormone (ACTH) (Figure 4-14). Adipose tissue is innervated by the sympathetic nervous system.

Figure 4-14 Regulation of adipocyte function

Preadipocytes can differentiate into mature multilocular (Latin multus, many; loculus, small place) adipocytes of brown fat in the fetus and newborn. Brown fat is found in the neck, shoulders, back, and the perirenal and paraaortic regions of the body. Brown fat is mostly lost during childhood. Brown fat is supplied by abundant blood vessels and sympathetic adrenergic nerve fibers. Lipochrome pigment and abundant mitochondria, rich in cytochromes, give this type of fat a brownish color.

As stated initially, the main function of brown fat is to dissipate energy in the form of heat (thermogenesis) in cold environments as a protective mechanism in the newborn. Thermogenesis by brown fat cells has two requirements (see Figure 4-13):

1. Mitochondrial biogenesis

2. The expression of the transporter uncoupling protein-1 (UCP-1)

As we briefly mentioned in Chapter 2, Epithelial Glands, in our discussion on UCP transporters in mitochondria, UCP-1 dissipates the proton gradient established across the inner mitochondrial membrane when electrons pass along the electron-transport chain. Thermogenesis takes place because UCP-1 allows the reentry of protons down their concentration gradient into the mitochondrial matrix and uncouples respiration from ATP production.

Clinical significance: Obesity

Obesity is a disorder of energy balance. It occurs when energy intake exceeds energy expenditure. Protection against obesity without consideration of energy intake results in an increase in circulating levels of triglycerides, and excessive accumulation of fat in liver (steatosis). The metabolic activities of adipocytes have very significant clinical consequences. An increase in visceral adiposity is associated with a higher risk of insulin resistance (see Chapter 19, Endocrine System), dyslipidemia (alteration in blood fat levels), and cardiovascular disease.

One of the secreted products of adipocytes is leptin, a 16-kd protein encoded by the ob gene. Leptin is released into the circulation and acts peripherally to regulate body weight. Leptin acts on hypothalamic targets involved in appetite and energy balance. Leptin-deficient mice (ob/ob) are obese and infertile. Both conditions are reversible with leptin administration.

The leptin receptor in hypothalamic target cells shares sequence homology with cytokine receptors. During inflammation, the release of the cytokines interleukin-1 and tumor necrosis factor–α increases leptin in serum, an indication that leptin interacts with cytokines to influence responses to infection and inflammatory reactions. Infections, injury, and inflammation up-regulate leptin gene expression and serum protein levels. As we discuss later, leptin has a role in bone formation.

CARTILAGE

Like the fibroblast and the adipocytes, the chondroblast derives from a mesenchymal cell. Chondroblasts contain lipids and glycogen, a well-developed RER (basophilic cytoplasm), and Golgi apparatus. The proliferation of chondroblasts results in growth of the cartilage.

In contrast to typical connective tissue, the cartilage is avascular and cells receive nutrients by diffusion through the ECM. At all ages, chondrocytes have significant nutritional requirements. Although they rarely divide in the adult cartilage, they continuously synthesize molecules to replace a constantly turned-over ECM, in particular, proteoglycans (Figure 4-15; see Box 4-F).

Figure 4-15 Chondrocytes and the surrounding matrix

Box 4-F Cartilage repair after injury

• Cartilage has a modest repair capacity. Cartilage injuries frequently result in the formation of repair cartilage from the perichondrium.

• This repair cartilage contains undifferentiated cells with a potential to differentiate into chondrocytes that synthesize components of the cartilage matrix.

• The repair cartilage has a matrix composition intermediate between hyaline and fibrous cartilage (for example, it contains both types I and II collagen).

Growth of cartilage (chondrogenesis)

Cartilage grows by two mechanisms (Figures 4-16 and 4-17):

1. By interstitial growth (from chondrocytes within the cartilage; see Figure 4-16).

2. By appositional growth (from undifferentiated cells at the surface of the cartilage or perichondrium; see Figure 4-17).

Figure 4-16 Chondrogenesis: Interstitial growth

Figure 4-17 Chondrogenesis: Appositional growth

During chondrogenesis, chondroblasts produce and deposit type II collagen fibers and ECM (hyaluronic acid and GAGs, mainly chondroitin sulfate and keratan sulfate) until chondroblasts are separated and trapped within spaces in the matrix called lacunae (Latin lacuna, small lake). The cells are then called chondrocytes. The space between the chondrocyte and the wall of the lacuna seen in histologic preparations is an artifact of fixation.

The matrix in close contact with each chondrocyte forms a bluish (with hematoxylin and eosin), metachromatic (see Box 4-D), or PAS-positive basketlike structure called the territorial matrix.

Each cluster of chondrocytes (known as an isogenous group) enveloped by the territorial matrix is separated by a wide but pale interterritorial matrix.

Types of cartilage

There are three major types of cartilage (Figure 4-18):

1. Hyaline cartilage

2. Elastic cartilage

3. Fibrocartilage

Figure 4-18 Types of cartilage

Hyaline cartilage is the most widespread cartilage in humans. Its name derives from the clear appearance of the matrix (Greek hyalos, glass).

In the fetus, hyaline cartilage forms most of the skeleton before it is reabsorbed and replaced by bone by a process known as endochondral ossification.

In adults, hyaline cartilage persists as the nasal, laryngeal, tracheobronchial, and costal cartilage. The articular surface of synovial joints (knees, shoulders) is hyaline cartilage and does not participate in endochondral ossification.

Articular surfaces are not lined by an epithelium. The hyaline cartilage contains:

1. Cells (chondrocytes)

2. Fibers (type II collagen synthesized by chondrocytes)

3. ECM (also synthesized by chondrocytes)

Chondrocytes have the structural characteristics of a protein-secreting cell (well-developed RER and Golgi apparatus, and large nucleolus) and store lipids and glycogen in the cytoplasm. Chondrocytes are coated by a pericellular matrix, surrounded by the territorial and interterritorial matrices, respectively. A lacunar rim separates the cell from the territorial matrix.

The surface of hyaline cartilage is covered by the perichondrium, a fibrocellular layer that is continuous with the periosteal cover of the bone and that blends into the surrounding connective tissue. Articular cartilage lacks a perichondrium.

The perichondrium consists of two layers:

1. An outer fibrous layer, which contains bundles of type I collagen and elastin.

2. An inner layer, called the chondrogenic layer, formed by flat chondrocytes aligned tangentially to the margin of the cartilage.

The ECM contains hyaluronic acid, proteoglycans (rich in the GAGs chondroitin sulfate and keratan sulfate), and a high water content (70% to 80% of its weight). Aggrecan is a large proteoglycan characteristic of cartilage (see Boxes 4-G and 4-H).

Box 4-G Cartilage repair after injury

• The specialized extracellular matrix of hyaline cartilage has a dual role:

1. It acts like a shock absorber, because of its stiffness and elasticity.

2. It provides a lubricated surface for movable joints.

The lubrication fluid (hyaluronic acid, immunoglobulins, lysosomal enzymes, collagenase in particular, and glycoproteins) is produced by the synovial lining of the capsule of the joint.

• The analysis of the synovial fluid is valuable in the diagnosis of joint disease.

Box 4-H How chondrocytes survive

• In cartilage, chondroblasts and chondrocytes are sustained by diffusion of nutrients and metabolites through the aqueous phase of the extracellular matrix.

• In bone, deposits of calcium salts in the matrix prevent the diffusion of soluble solutes, which thus must be transported from blood vessels to osteo-cytes through canaliculi (see Bone).

The transcription factor Sox9 is required for expression of cartilage-specific ECM components such as type II collagen and the proteoglycan aggrecan. Sox9 activates the expression of collagen by the C0L2A1 gene. A lack of Sox9 expression prevents the chondrogenic layer to differentiate into chondrocytes. Mutations in the Sox9 gene cause the rare and severe dwarfism campomelic dysplasia (Figure 4-17).

The structure of the elastic cartilage is similar to that of hyaline cartilage except that the ECM contains abundant elastic fibers synthesized by chondrocytes. Elastic cartilage is found in the auricle of the external ear, a major portion of the epiglottis, and some of the laryngeal cartilages. The specialized matrix of the cartilage has remarkable flexibility and the ability to regain its original shape after deformation.

Unlike hyaline cartilage, fibrocartilage is opaque, the matrix contains type I collagen fibers, the ECM has a low concentration of proteoglycans and water, and it lacks a perichondrium.

Fibrocartilage has great tensile strength and forms part of the intervertebral disk, pubic symphysis, and sites of insertion of tendon and ligament into bone.

The fibrocartilage is sometimes difficult to distinguish from dense regular connective tissue of some regions of tendons and ligaments. Fibrocartilage is distinguished by characteristic chondrocytes within lacunae, forming short columns (in contrast to flattened fibroblasts or fibrocytes lacking lacunae, surrounded by the dense connective tissue and ECM).

BONE

Bone is a rigid inflexible connective tissue in which the ECM has become impregnated with salts of calcium and phosphate by a process called mineralization. Bone is highly vascularized and metabolically very active.

The functions of bone are:

1. Support and protection for the body and its organs

2. A reservoir for calcium and phosphate ions

Macroscopic structure of mature bone

Based on its gross appearance (Figure 4-19), two forms of bone are distinguished:

1. Compact or dense bone

2. Spongy, trabecular or cancellous bone

Figure 4-19 General architecture of a long bone

Compact bone appears as a solid mass. Spongy bone consists of a network of bony spicules or trabeculae delimiting spaces occupied by the bone marrow.

In long bones, such as the femur, the shaft or diaphysis consists of compact bone forming a hollow cylinder with a central marrow space, called the medullary or marrow cavity.

The ends of the long bones, called epiphyses, consist of spongy bone covered by a thin layer of compact bone. In the growing individual, epiphyses are separated from the diaphysis by a cartilaginous epiphyseal plate, connected to the diaphysis by spongy bone. A tapering transitional region, called the metaphysis, connects the epiphysis and the diaphysis. Both the epiphyseal plate and adjacent spongy bone represent the growth zone, responsible for the increase in length of the growing bone.

The articular surfaces, at the ends of the long bones, are covered by hyaline cartilage, the articular cartilage. Except on the articular surfaces and at the insertion sites of tendons and ligaments, most bones are surrounded by the periosteum, a layer of specialized connective tissue with osteogenic potential.

The marrow cavity of the diaphysis and the spaces within spongy bone are lined by endosteum, also with osteogenic potential.

Microscopic structure of mature bone

Two types of bone are identified on the basis of the microscopic organization of the collagen fibers:

1. Lamellar bone, typical of the mature bone, displays a regular alignment of collagen fibers. This bone is mechanically strong and is formed slowly.

2. Woven bone, observed in the developing bone, is characterized by an irregular alignment of collagen fibers. This bone is mechanically weak, is formed rapidly, and is then replaced by lamellar bone. Woven bone is produced during the repair of a bone fracture.

The lamellar bone consists of lamellae, largely composed of bone matrix, a mineralized substance deposited in layers or lamellae, and osteocytes, each one occupying a cavity or lacuna with radiating and branching canaliculi that penetrate the lamellae of adjacent lacunae. The lamellar bone displays four distinct patterns (Figure 4-20):

1. The osteons or haversian systems, formed by concentrically arranged lamellae around a longitudinal vascular channel.

2. The interstitial lamellae, observed between osteons and separated from them by a thin layer known as the cement line.

3. The outer circumferential lamellae, visualized at the external surface of the compact bone under the periosteum.

4. The inner circumferential lamellae, seen on the internal surface subjacent to the endosteum.

Figure 4-20 Haversian system or osteon

The vascular channels in compact bone have two orientations with respect to the lamellar structures:

1. The longitudinal capillaries and postcapillary venules, running in the center of the osteon within a space known as the haversian canal (Figures 4-20 to 4-22).

2. The haversian canals are connected with one another by transverse or oblique canals known as Volkmann’s canals, containing blood vessels from the marrow and some from the periosteum.

Figure 4-21 Organization of compact bone: Osteon

Figure 4-22 Osteocytes are connected to each other by cell processes

Periosteum and endosteum

During embryonic and postnatal growth, the periosteum consists of an inner layer of bone-forming cells (osteoblasts) in direct contact with the bone. The inner layer is the osteogenic layer. In the adult, the periosteum contains inactive connective tissue cells that retain their osteogenic potential in case of bone injury and repair.

The outer layer is rich in blood vessels, some of them entering Volkmann’s canals, and thick anchoring collagen fibers, called Sharpey’s fibers, that penetrate the outer circumferential lamellae deep in the bone (see Figure 4-20).

The endosteum consists of squamous cells and connective tissue fibers covering the spongy walls housing the bone marrow and extending into all the cavities of the bone, including the haversian canals.

Bone matrix

The bone matrix consists of organic (35%) and inorganic (65%) components. The organic bone matrix contains type I collagen fibers (90%); proteoglycans, enriched in chondroitin sulfate, keratan sulfate, and hyaluronic acid; and noncollagenous proteins. The inorganic component of the bone is represented predominantly by deposits of calcium phosphate with the crystalline characteristics of hydroxyapatite. The crystals are distributed along the length of collagen fibers through an assembly process assisted by noncollagenous proteins.

Type I collagen is the predominant protein of the bone matrix. In mature lamellar bone, collagen fibers have a highly ordered arrangement with changing orientations with respect to the axis of the haversian canal in successive concentric lamellae (see Figure 4-20).

Noncollagenous matrix proteins include osteocalcin, osteopontin, and osteonectin, synthesized by osteoblasts and with unique properties in the mineralization of bone.

Osteocalcin and osteopontin synthesis increases following stimulation with the active vitamin D metabolite, 1α,25-dihydroxycholecalciferol. Osteocalcin inhibits osteoblast function.

Osteonectin is not exclusively an osteoblast product and is present in tissues undergoing remodeling and morphogenesis.

Bone sialoprotein is also a bone matrix component.

As we later discuss in greater detail, osteoprotegerin, RANKL, and macrophage colony-stimulating factor are products of osteoblasts required for regulating the differentiation of osteoclasts.

Cellular components of bone

Actively growing bone contains cells of two different lineages:

1. The osteoblast lineage, which includes the osteoprogenitor cells and derived osteoblasts and osteocytes

2. The osteoclast lineage

Osteoprogenitor cells are of mesenchymal origin and have the properties of stem cells: the potential for proliferation and a capacity to differentiate. Osteoprogenitor cells give rise to osteoblasts by a regulatory mechanism involving growth and transcription factors and are present in the inner layer of the periosteum and the endosteum. Osteoprogenitor cells persist throughout postnatal life as bone-lining cells; they are reactivated in the adult during the repair of bone fractures and other injuries.

Osteoblasts differentiate into osteocytes after they are trapped inside lacunae within the mineralized matrix they produce. Their differentiation involves the participation of two transcription factors: Cbfa1/Runx2 and osterix (see Box 4-I).

Box 4-I How osteocytes differentiate

• The osteoblast to osteocyte differentiation process requires the activation of two transcription factors: Cbfa1/Runx2 (for core binding factor a1/runt homeodomain protein 2) and osterix.

• We have already seen that chondrogenesis involves the transcription factor Sox9 (see Figure 4-17). We discuss in Chapter 5, Ossification, that Cbfa1/Runx2 controls the conversion of proliferating chondrocytes to hypertrophie chondrocytes, an event that is prevented by Sox9.

• The transcription factors Sox9, Cbfa1/Runx2, and osterix (the latter specific for osteoblast to osteocyte differentiation) play critical roles in the development of the skeleton.

• Mutations in genes encoding these transcription factors are the genetic basis of skeletal diseases. For example, a total lack of expression of the Cbfa1/Runx2 gene determines that the entire skeleton consists only of cartilage.

The osteoclast lineage derives from the monocyte-macrophage lineage in the bone marrow.

Osteoblasts and osteocytes

Osteoblasts are epithelial-like cells with cuboidal or columnar shapes, forming a monolayer covering all sites of active bone formation. Osteoblasts are highly polarized cells: they deposit osteoid, the nonmineralized organic matrix of the bone, along the osteoblast-bone interface. Osteoblasts initiate and control the subsequent mineralization of the osteoid.

In electron micrographs, osteoblasts display the typical features of cells actively engaged in protein synthesis, glycosylation, and secretion. Their specific products are type I collagen, osteocalcin, osteopontin, and bone sialoprotein (Figure 4-23). Osteoblasts give a strong cytochemical reaction for alkaline phosphatase that disappears when the cells become embedded in the matrix as osteocytes. In addition, osteoblasts produce growth factors, in particular members of the bone morphogenetic protein family, with bone-inductive activities.

Figure 4-23 Function of the osteoblast

When bone formation is completed, osteoblasts flatten out and transform into osteocytes. Osteocytes are highly branched cells with their body occupying small spaces between lamellae, called lacunae. Small channels, the canaliculi, course through the lamellae and interconnect neighboring lacunae. Adjacent cell processes, found within canaliculi, are connected by gap junctions (see Figure 4-22). Nutrient materials diffuse from a neighboring blood vessel, within the haversian canal, through the canaliculi into the lacunae. As you can see, the dense network of osteocytes depends not only on intracellular communication across gap junctions but also on the mobilization of nutrients and signaling molecules along the extracellular environment facilitated by canaliculi running from lacuna to lacuna.

The life of an osteocyte depends on this nutrient diffusion process and the life of the bone matrix depends on the osteocyte. Osteocytes can remain alive for years provided vascularization is continuous.

In compact bone, 4 to 20 lamellae are concentrically arranged around the haversian canal; they contain a blood vessel, either a capillary or a postcapillary venule.

Clinical significance: Osteoblast to osteocyte differentiation

A pluripotent mesenchymal cell is the precursor of osteblasts as well as muscle cells, adipocytes, fibroblasts, and chondroblasts.

The differentiation of the osteoblast is controlled by growth and transcription factors. Several members of the bone morphogenetic protein (BMP) family and transforming growth factor—β can regulate the embryonic development and differentiation of the osteoblast.

Osteoblast-specific genes modulate the differentiation of the osteoblast progeny (Figure 4-24): Cbfa1/Runx2 (a member of the core-binding factor family) encodes a transcription factor that induces the differentiation of osteoblasts and controls the expression of osteocalcin. Cbfa1/Runx2 is the earliest and most specific indicator of osteogenesis and its expression is induced by BMP7, followed by the expression of osteocalcin and osteopontin. Osteocalcin is a specific secretory protein expressed only in terminally differentiated osteoblasts under the control of Cbfa1/Runx2 (see Box 4-I).

Figure 4-24 Osteoblast differentiation

Cbfa1/Runx2-deficient mice develop to term and have a skeleton consisting of cartilage. There is no indication of osteoblast differentiation or bone formation in these mice. In addition, Cbfa1/Runx2-deficient mice lack osteoclasts. As we will discuss soon, osteoblasts produce proteins that regulate the formation of osteoclasts.

Consistent with the skeletal observations in the Cbfa1/Runx2-deficient mice is a condition in humans known as cleidocranial dysplasia (CCD). CCD is characterized by hypoplastic clavicles, delayed ossification of sutures of certain skull bones, and mutations in the Cbfa1/Runx2 gene.

Leptin, a peptide synthesized by adipocytes with binding affinity to its receptor in the hypothalamus, regulates bone formation by a central mechanism. Although details of the leptin-hypothalamic control mechanism are unknown, mice deficient in leptin or its receptor have a considerably higher bone mass than wild-type mice. In fact, patients with generalized lipodystrophy (absence of adipocytes and white fat) exhibit osteosclerosis (increased bone hardening) and accelerated bone growth.

Osteoclasts

Osteoclasts do not belong to the osteoprogenitor cell lineage. Instead, osteoclasts derive from the monocyte-macrophage progenitor cell lineage in the bone marrow, which diverges into the osteoclast progenitor pathway.

The osteoclast precursor cells are monocytes, which reach the bone through the blood circulation and fuse into multinucleated cells with as many as 30 nuclei to form osteoclasts by a process regulated by osteoblasts and stromal cells of the bone marrow.

After attachment to the target bone matrix, osteoclasts generate a secluded acidic environment required for bone resorption. Bone resorption involves first the dissolution of the inorganic components of the bone (bone demineralization) mediated by H⁺-ATPase (adenosine triphosphatase) within an acidic environment, followed by enzymatic degradation of the organic matrix (type I collagen and noncollagenous proteins) by the protease cathepsin K.

Osteoclasts play an essential role in bone remodeling and renewal. This process involves removal of bone matrix at several sites, followed by its replacement with new bone by osteoblasts.

The osteoclast is a large (up to 100 μm in diameter) and highly polarized cell that occupies a shallow concavity called Howship’s lacuna or the subosteoclastic compartment (Figures 4-25 and 4-26).

Figure 4-25 Function of the osteoclast

Figure 4-26 Osteoclast differentiation

The cytoplasm of the osteoclast is very rich in mitochondria and acidified vesicles. The membrane of the acidified vesicles contains H⁺-ATPase; mitochondria are the source of adenosine triphosphate (ATP) to drive the H⁺-ATPase pumps required for the acidification of the subosteoclastic compartment for the subsequent activation of the enzyme cathepsin K. Cathepsin K breaks down the bone organic matrix following removal of the mineral component of bone. Figure 4-26 provides a step-by-step sequence of the activation of an osteoclast. We discuss in Chapter 15, Upper Digestive Segment, that the mechanism of production of HCl in the stomach is very similar to the acidification of Howship’s lacuna.

The cell domain facing the lacuna has deep infoldings of the cell membrane, the ruffled border. When the cell is not active, the ruffled border disappears and the osteoclast enters into a resting phase. Around the circumference of the ruffled border—at the point where the cell membrane is closely applied to the bone just at the margins of the lacuna—actin filaments accumulate and participate, together with α_vβ₃ integrin, to form a sealing zone. The sealing zone seals off the bone resorption lacuna.

Osteoclasts are transiently active in response to a metabolic demand for the mobilization of calcium from bone into blood. Osteoclast activity is directly regulated by calcitonin (synthesized by neural crest derived parafollicular or C cells of the thyroid follicle), vitamin D₃, and regulatory molecules produced by osteoblasts and stromal cells of the bone marrow (see Osteoclastogenesis).

Osteoclastogenesis (osteoclast differentiation)

Osteoclastogenesis is triggered by two relevant molecules produced by the osteoblast: (1) macrophage colony-stimulating factor (M-CSF), and (2) nuclear factor kappa B (NF-κB) ligand (RANKL).

The osteoclast precursor, the monocyte, responds to M-CSF, a secretory product of osteoblasts. M-CSF is required for the survival and proliferation of the osteoclast precursor (Figure 4-27). Its role was established by studies of the op/op mouse, which does not express M-CSF, lacks osteoclasts, and has an increase in bone mass (osteopetrosis; Greek osteon, bone; petra, stone; osis, condition). In humans, osteopetrosis is characterized by high-density bone due to absent osteoclastic activity. In long bones, this condition leads to the occlusion of marrow spaces and to anemia.

Figure 4-27 Osteoblasts regulate osteoclastogenesis

Both osteoblasts and stromal cells of the bone marrow produce RANKL, a member of the tumor necrosis factor (TNF) superfamily. RANKL binds to RANK receptor present on the surface of differentiating osteoclasts. RANKL binding leads to RANK trimerization and the recruitment of an adaptor molecule called TRAF6 (for TNF receptor-associated factor 6).

TRAF6 stimulates a downstream signaling cascade, including the nuclear relocation of two transcription factors: NF-κB and NFATc1 (for nuclear factor—activated T cells c1). In the nucleus, these two transcription factors activate genes leading to osteoclast differentiation. We discuss in Chapter 3, Cell Signaling (see Figure 3-8), that NF-κB is a critical transcription factor heterodimer activated in response to inflammatory or immunologic signaling.

TRAF6 also interacts with c-Src to stimulate a pathway leading to cytoskeletal reorganization and prevention of apoptosis. Figure 4-28 summarizes the relevant signaling steps following RANKL binding to RANK.

Figure 4-28 RANK-RANKL signaling

The interaction of the RANK receptor on osteoclast precursor cells with RANKL, exposed on the surface of osteoblasts, determines cell-cell contact required for further maturation of the osteoclast precursor.

Osteoblasts synthesize osteoprotegerin, a protein with high binding affinity for RANKL. Osteoprotegerin is a soluble “decoy” protein that binds to RANKL and prevents RANK-RANKL interaction. Consequently, osteoprotegerin modulates the osteoclastogenic process.

Parathyroid hormone stimulates the expression of osteoclastogenic RANKL. By this mechanism, the pool of RANKL increases relative to osteoprotegerin. An excess of parathyroid hormone enhances osteoclastogenesis (see Chapter 19, Endocrine System).

Denosumab-induced inhibition of RANKL in hyperparathyroidism prevents bone loss caused by excessive production of parathyroid hormone.

We mentioned that a lack of M-CSF in the op/op mutant mouse results in osteopetrosis. For comparison, osteosclerosis is an increase in bone mass due to an increase in osteoblastic activity.

Clinical significance: Osteoporosis and osteomalacia

The realization that RANKL plays a major contribution in osteoclast development and in bone resorptive activity stimulated the development of pharmaceutical agents to arrest skeletal disorders. Osteoporosis (Greek osteon, bone; poros, pore; osis, condition) is defined as the loss of bone mass leading to bone fragility and susceptibility to fractures.

The major factor in osteoporosis is the deficiency of the sex steroid estrogen that occurs in postmenopausal women. In this condition, the amount of reabsorbed old bone—due to an increase in the number of osteoclasts—exceeds the amount of formed new bone. This accelerated turnover state can be reversed by estrogen therapy and calcium and vitamin D supplementation. Osteoporosis and osteoporotic fractures are also observed in men.

Osteoporosis is asymptomatic until it produces skeletal deformity and bone fractures (typically in the spine, hip, and wrist). The vertebral bones are predominantly trabecular bone surrounded by a thin rim of compact bone. Therefore, they may be crushed or may wedge anteriorly, resulting in pain and in a reduction in height. Elderly persons with osteoporosis are unlikely to have a hip fracture unless they fall.

The diagnosis of osteoporosis is made radiologically or, preferentially, by measuring bone density by dual-energy x-ray absorptiometry (DEXA). DEXA measures photon absorption from an x-ray source to estimate the amount of bone mineral content.

A monoclonal antibody to RANKL, called denosumab (Amgen), functions like osteoprotegerin. The antibody has been administered subcutaneously every 3 months for 1 year in postmenopausal women with severe osteoporosis determined by low bone mineral density detected by DEXA. Denosumab mimics the function of osteoprotegerin and decreases bone resorption, as determined by measuring in urine and serum of bone-collagen degradation products and increased bone mineral density at 1 year. A concern with denosumab anti-RANKL treatment is the expression of RANKL-osteoprotegerin in cells of the immune system (dendritic cells and B and T cells).

Osteomalacia (Greek osteon, bone; malakia, softness) is a disease characterized by a progressive softening and bending of the bones. Softening occurs because of a defect in the mineralization of the osteoid due to lack of vitamin D or renal tubular dysfunction (see Chapter 14, Urinary System). In the young, a defect in mineralization of cartilage in the growth plate (see Chapter 5, Osteogenesis), causes a defect called rickets (juvenile osteomalacia). Osteomalacia can result from a deficiency of vitamin D (for example, intestinal malabsorption) or heritable disorders of vitamin D activation (for example, renal 1α-hydroxylase deficiency in which calciferol is not converted to the active form of vitamin D, calcitriol; see vitamin D in Chapter 19, Endocrine System).