Histology_Ross

Histology_Ross. At the molecular level, cartilage repair is a tentative balance between deposition of type I collagen in the form of scar tissue and repair by expression of the cartilagespecific collagens. However, in adults, new blood vessels commonly develop at the site of the healing wound that stimulate the growth of bone rather than actual cartilage repair. The limited ability of cartilage to repair itself can cause significant problems in cardiothoracic surgery, such as coronary artery bypass graft surgery, when costal cartilage must be cut to enter the chest cavity. A variety of treatments may improve the healing of articular cartilage, including perichondral grafts, cell transplantation, insertion of artificial matrices, and application of growth factors. When hyaline cartilage calcifies, it is replaced by bone.


Histology_Ross

Histology_Ross. Hyaline cartilage is prone to calcification, a process in which calcium phosphate crystals become embedded in the tumors characterized by secretion of cartilage matrix. Ap-proximately 3.6% of primary bone tumors that are diagnosed in the United States each year are chondrosarcomas. These tumors are the second most common matrix-producing tu-mors of bone after osteosarcomas (malignant bone-forming tumors). They occur more commonly in men than women and affect individuals that are usually age 45 and older. Chondrosarcomas originate predominantly in the axial skele-ton (and most commonly involve vertebrae, pelvic bones, ribs, scapulae, and the sternum), and in metaphyses of prox-imal ends of long bones (most often the femur and humerus). The most common symptom reported by patients is a deep pain, often present for months and typically dull in character. Since cartilaginous tissue is compressed inside the bone, in most cases initial growth of a tumor cannot be palpated. Radiographs, CT,


Histology_Ross

Histology_Ross. pain, often present for months and typically dull in character. Since cartilaginous tissue is compressed inside the bone, in most cases initial growth of a tumor cannot be palpated. Radiographs, CT, and MRI scans are essential for the initial diagnosis and later for the evaluation of the extent of deep intramedullary tumors. Chondrosarcomas are classified by grades that strongly correlate with a patient’s prognosis. Micro-scopically, grade 1 represents the least aggressive and grade 3 represents the most aggressive tumor. Most chondrosarcomas (90%) are pathologically classified as conventional (grades 1 and 2); they rarely metastasize and are composed of hyaline cartilage that infiltrates bone marrow cavity and surrounds existing bony trabe-culae (Fig. F7.2.1). Multiple chondroblasts that are often binucleated with pleomorphic and hyperchromatic nu-clear patterns are frequently seen in a single lacuna. Cartilaginous matrix may also undergo mineralization and subsequent endochondral


Histology_Ross

Histology_Ross. are often binucleated with pleomorphic and hyperchromatic nu-clear patterns are frequently seen in a single lacuna. Cartilaginous matrix may also undergo mineralization and subsequent endochondral ossification. Metastatic spread to lungs and lymph nodes is more frequently asso-ciated with grade 3 lesions. Recently, immunohistochemical localization of collagen types has been used to determine stage of tissue differenti-ation, which in fact correlates with a patient’s prognosis. The presence of collagen types II and X and the proteogly-can aggrecan in biopsies indicates mature tumors associ-ated with good prognosis. On the other hand, the presence of collagen type I indicates changes in the extracellular ma-trix toward dedifferentiated (fibrous) types of tumor with poorer prognosis. In addition, transcription factor SOX9, which is essential for differentiation of mesenchymal cells into chondroblasts during normal fetal development, is expressed in chondrosarcomas. Treatment of


Histology_Ross

Histology_Ross. In addition, transcription factor SOX9, which is essential for differentiation of mesenchymal cells into chondroblasts during normal fetal development, is expressed in chondrosarcomas. Treatment of chondrosarcoma is primary surgical: The tumor is widely excised. Chemotherapy and radiation play limited roles in treatment. Patients with adequately re-sected low-grade tumors have an excellent survival rate. FIGURE F7.2.1 • Photomicrograph of a chondrosarcoma (grade 1) from the epiphysis of the long bone, stained with H&E. This photomicrograph shows a tissue mass of chondrosarcoma infiltrating intertrabecular spaces of the bone marrow. Note the presence of malignant chondrocytes in various stages of maturity. Small area of active bone marrow is visible in the upper left corner of the image. 240. (Courtesy of Dr. Fabiola Medeiros.) marrowmarrowmarrowbonebonebonechondrosarcomachondrosarcomachondrosarcoma  FOLDER 7.2 Clinical Correlation: Malignant Tumors of the Cartilage; Chondrosarcomas


Histology_Ross

Histology_Ross. Chondrosarcomas are generally slow-growing malignant cartilage matrix. The matrix of hyaline cartilage undergoes calcification as a regular occurrence in three well-defined situations:  The portion of articular cartilage that is in contact with bone tissue in growing and adult bones, but not the surface portion, is calcified.  Calcification always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during an individual’s growth period.  Hyaline cartilage in the adult calcifies with time as part of the aging process.


Histology_Ross

Histology_Ross.  Hyaline cartilage in the adult calcifies with time as part of the aging process. In most of these situations, given sufficient time, cartilage that calcifies is replaced by bone. For example, in older individuals, portions of the cartilage rings in the trachea are often replaced by bone tissue (Fig. 7.13). Chondrocytes normally derive all of their nutrients and dispose of wastes by diffusion of materials through the matrix. When the matrix becomes heavily calcified, diffusion is impeded and the chondrocytes swell and die. The ultimate consequence of this event is removal of the calcified matrix and its replacement by bone. A number of investigators believe the process of cartilage removal involves a specific cell type designated as a chondroclast. This cell is described as resembling an osteoclast in both


Histology_Ross

Histology_Ross. A number of investigators believe the process of cartilage removal involves a specific cell type designated as a chondroclast. This cell is described as resembling an osteoclast in both FIGURE 7.13 • Photomicrograph of a tracheal ring from an elderly individual, stained with H&E. The darker, somewhat basophilic areas on the left side of the micrograph represent normal cartilage matrix (C). The lighter and more eosinophilic areas represent bone tissue (B) that has replaced the original cartilage matrix. A large marrow cavity has formed within the cartilage structure and is visible in the center of the micrograph. 75.


Histology_Ross

Histology_Ross. morphology and lytic function. It is thought that these cells enter cartilage along newly sprouting blood vessels and may, in fact, be derived from perivascular or bone marrow stem cells. However, the precise origin of these cells is unknown. Early studies of chondroclast structure and function were carried out on the developing mandible, in which the resorption of Meckel’s cartilage is not followed by bone replacement (endochondral ossification). It is likely that chondroclasts are cells that occur wherever cartilage is being removed. Their role is discussed in regard to endochondral bone formation.


Histology_Ross

Histology_Ross. Cartilage is an avascular form of connective tissue composed of cells called chondrocytes and a highly specialized extracellular matrix. Three kinds of cartilage are described on the basis of characteristics of the matrix: hyaline cartilage (described here), elastic cartilage (described in Plate 9), and fibrocartilage (described in Plate 10). Hyaline cartilage has a homogeneous-appearing amorphous matrix. It contains type II collagen. Type II collagen appears with the transmission electron microscope (TEM) as thin fibrils, ~20 nm in diameter, in which the character-istic 68-nm banding may not be obvious. The fibrils are arranged in a three-dimensional felt-like pattern. The matrix also contains large amounts of glycosaminoglycans, most of which form proteoglycans and proteoglycan aggregates. Hyaline cartilage is found in the adult as the structural framework for the larynx, trachea, and bronchi; it is found on the articular ends of the ribs and on the surfaces of synovial joints. In


Histology_Ross

Histology_Ross. Hyaline cartilage is found in the adult as the structural framework for the larynx, trachea, and bronchi; it is found on the articular ends of the ribs and on the surfaces of synovial joints. In addition, hyaline cartilage constitutes much of the fetal skeleton and plays an important role in the growth of most bones. Hyaline cartilage displays both appositional growth, the addition of new cartilage at its surface, and interstitial growth, the division and differentiation of chondrocytes within its substance.


Histology_Ross

Histology_Ross. Hyaline cartilage, trachea, human, H&E ×450. This micrograph reveals hyaline cartilage from the trachea as seen in a routinely prepared specimen. The cartilage appears as an avascular expanse of matrix material and a population of cells called chondrocytes (Ch). The chondrocytes produce the matrix; the space each chondrocyte occupies is called a lacuna (L). Surrounding the cartilage and in immediate apposition to it is a cover of connective tissue, the perichondrium (P). The perichondrium serves as a source of new chondrocytes during appositional growth of the cartilage. Often, the perichondrium reveals two distinctive layers: an outer, more fibrous layer and an inner, more cellular layer. The inner, more cellular layer is chondrogenic and provides for external growth. Cartilage matrix contains collagenous fibrils masked by ground substance in which they are embedded; thus, the fibrils are not evident. The Hyaline cartilage, trachea, human, H&E ×850.


Histology_Ross

Histology_Ross. This higher magnification micrograph reveals the area within the rectangle in lower left figure. The chondrocytes (Ch) in the upper part of the micrograph represent an isogenous group and are producing matrix material for interstitial growth. A prominent capsule is not yet evident. The lightly stained ba sophilic area reveals immature chondrocytes (arrows) within the perichondrium (P). Closest to the cartilage matrix, within the perichondrium (P), matrix also contains, among other components, sulfated glycosaminoglycans that exhibit basophilia with hematoxylin or other basic dyes. Also, the matrix material immediately surrounding a lacuna tends to stain more intensely with basic dyes. This region is referred to as a capsule (Cap). Not uncommonly, the matrix may appear to stain more intensely in localized areas (asterisks) that look much like the capsule matrix. This results from inclusion of a capsule within the thickness of the section, but not the lacuna it surrounds.


Histology_Ross

Histology_Ross. Frequently, two or more chondrocytes are located extremely close to one another, separated by only a thin partition of matrix. These are isogenous cell clusters that arise from a single predecessor cell. The proliferation of new chondrocytes by this means with the consequent addition of matrix results in interstitial growth of the cartilage. Hyaline cartilage, trachea, human, H&E ×160.


Histology_Ross

Histology_Ross. Hyaline cartilage, trachea, human, H&E ×160. The hyaline cartilage in this micrograph is from a specimen obtained shortly after death and kept cool during fixation. The procedure reduces the loss of its negatively charged sulfate groups; thus, the matrix is stained more heavily with hematoxylin. Also, note the very distinct and deeply stained capsules (arrows) surrounding the chondrocytes. The capsule represents the site where the sulfated glycosaminoglycans are most concentrated. In contrast to the basophilia of the cartilage matrix, the perichondrium (P) is stained with eosin. The lightly stained region between the perichondrium and the deeply stained matrix is matrix that has not yet matured. It has fewer sulfate groups.


Histology_Ross

Histology_Ross. are several chondrocytes that exhibit just barely detectable cytoplasm and elongate nuclei (FCh). These cells are formative chondrocytes that are just beginning to, or will shortly, produce matrix material. In contrast, the nuclei near the bottom edge of the micrograph are fibroblast nuclei (Fib); they belong to the outer layer of the perichondrium. Note how attenuated their nuclei are compared with the formative chondroblast nuclei of the inner perichondrial layer. KEY Cap, capsule Ch, chondrocytes FCh, formative chondrocytes Fib, fibroblasts L, lacuna P, perichondrium arrows, immature chondrocytes asterisk, capsule of a lacuna, but with lacuna and contained chondrocyte not included within the thickness of the section PLATE 8 Cartilage and the Developing Skeleton


Histology_Ross

Histology_Ross. PLATE 8 Cartilage and the Developing Skeleton Hyaline cartilage is present as a precursor to bones in the fetus. This cartilage is replaced by bone tissue except where one bone contacts another, as in a movable joint. In these locations, cartilage persists and covers the end of each bone as articular cartilage, providing a smooth, well-lubricated surface against which the end of one bone moves on the other in the joint. In addition, cartilage, being capable of interstitial growth, persists in weight-supporting bones and other long bones as a growth plate as long as growth in length occurs. The role of hyaline cartilage in bone growth is considered briefly below and in more detail in Plates 13 and 14. Developing skeleton, fetal foot, rat, H&E ×85.


Histology_Ross

Histology_Ross. This section shows the cartilages that will ultimately become the bones of the foot. In several places, developing ligaments (L) can be seen where they join the cartilages. The nuclei of the fibroblasts within the ligaments are just barely perceptible. They are aligned in rows and are separated from other rows of fibroblasts by collagenous material. The hue and intensity of color of the cartilage matrix, except at the periphery, are due to the combined uptake of 212 the H&E. The collagen of the matrix stains with eosin; however, the presence of sulfated glycosaminoglycans results in staining by hematoxylin. The matrix of cartilage that is about to be replaced by bone, such as that shown here, becomes impregnated with calcium salts, and the calcium is also receptive to staining with hematoxylin. The many enlarged lacunae (seen as light spaces within the matrix where the chondrocytes have fallen out of the lacunae) are due to hypertrophy of the chondrocytes, an event associated with


Histology_Ross

Histology_Ross. The many enlarged lacunae (seen as light spaces within the matrix where the chondrocytes have fallen out of the lacunae) are due to hypertrophy of the chondrocytes, an event associated with calcification of the matrix. Thus, where these large lacunae are present, i.e., in the center region of the cartilage, the matrix is heavily stained.


Histology_Ross

Histology_Ross. This figure also shows that the cartilage is surrounded by perichondrium, except where it faces a joint cavity (JC). Here, the bare cartilage forms a surface. Note that the joint cavity is a space between the cartilages whose boundaries are completed by connective tissue (CT). The connective tissue at the surface of the cavity is special. It will constitute the synovial membrane in the adult and contribute to the formation of a lubricating fluid (synovial fluid) that is present in the joint cavity. Therefore, all the surfaces that will enclose the adult joint cavity are derived originally from the mesenchyme. Synovial fluid is a viscous substance containing, among other things, glycosaminoglycans; it can be considered an exudate of interstitial fluid. The synovial fluid could be considered an extension of the extracellular matrix, as the joint cavity is not lined by an epithelium.


Histology_Ross

Histology_Ross. CARTI LAG E AN D TH E DEVE LOPI NG S KE LETON KEY B, bone C, cartilage CT, connective tissue JC, joint cavity L, ligament MC, marrow cavity arrowhead, calcified cartilage Developing skeleton, fetal fnger, human, thionine-picric acid ×30.


Histology_Ross

Histology_Ross. This figure shows a developing long bone of the finger and its articulation with the distal and proximal bones. Before the stage shown here, each bone consisted entirely of a hyaline cartilaginous structure similar to the cartilages seen in figure above but shaped like the long bones into which they would develop. Here, only the ends, or epiphyses, of the bone remain as cartilage, the epiphyseal cartilage (C). The shaft, or diaphysis, has become a cylinder of bone tissue (B) surrounding the marrow cavity (MC). The dark region at the ends of the marrow cavity is calcified cartilage (arrowhead) that is being replaced by bone. The bone at the ends of the marrow cavity constitutes the metaphysis. With this staining method, the calcified cartilage appears dark brown. The newly formed metaphyseal bone, which is admixed with this degenerating calcified cartilage and is difficult to define at this low magnification, has the same yellow-brown color as the diaphyseal bone. By the continued


Histology_Ross

Histology_Ross. bone, which is admixed with this degenerating calcified cartilage and is difficult to define at this low magnification, has the same yellow-brown color as the diaphyseal bone. By the continued proliferation of cartilage, the bone grows in length. Later, the cartilage becomes calcified; bone is then produced and occupies the site of the resorbed cartilage. With the cessation of cartilage proliferation and its replacement by bone, growth of the bone stops, and only the cartilage at the articular surface remains. The details of this process are explained under endochondral bone formation (Plates 13 and 14).


Histology_Ross

Histology_Ross. Elastic cartilage has a matrix containing elastic fibers and elastic lamellae in addition to type II collagen. It is found in the auricle of the exter-nal ear, in the auditory tube, in the epiglottis, and in part of the larynx. The elastic material imparts properties of elasticity, as distinguished from resiliency, which are not shared by hyaline cartilage. Elastic cartilage is surrounded by perichondrium, and it, too, increases in size by both ap-positional and interstitial growth. Unlike hyaline cartilage, however, elastic cartilage does not normally calcify. Elastic cartilage, epiglottis, human, H&E and orcein stains ×80. This section of the epiglottis contains elastic cartilage (EC) as the centrally located structure. The essential components of the cartilage, namely, the matrix that stains deep blue and the light, unstained lacunae surrounded by matrix, are evident in this low-magnification micrograph. The perimeter of the cartilage is covered


Histology_Ross

Histology_Ross. PLATE 9 • E LASTIC CARTI LAG E KEY AT, adipose tissue E, elastic fiber EC, elastic cartilage MG, mucous gland PC, perichondrium SE, stratified squamous epithelium Elastic cartilage, epiglottis, human, H&E and orcein stains ×250; inset ×400. This shows an area of the elastic cartilage at higher magnification. The elastic fibers appear as the blue, elongate profiles within the matrix. They are most evident at the edges of the cartilage, but they are obscured in some deeper parts of the matrix, where they blend with the elastic material that forms a honeycomb about the lacunae. Elastic fibers (E) are also apparent in the adipose tissue (AT), between the adipocytes.


Histology_Ross

Histology_Ross. Some of the lacunae in the cartilage are arranged in pairs separated by a thin plate of matrix. The plate of matrix appears as a bar between the adjacent lacunae. This is a reflection of interstitial growth by the cartilage, in that the adjacent cartilage cells are derived from the same parent cell. They have moved away from each other and secreted a plate of cartilage matrix by perichondrium (PC); its fibrous character is just barely visible in this figure. Also note the adipose tissue (AT) within the boundaries of the elastic cartilage. Both above and below the elastic cartilage is connective tissue, and each surface of the epiglottis is formed by stratified squamous epithelium (SE). Mucous glands (MG) are in the connective tissue in the bottom of this figure.


Histology_Ross

Histology_Ross. between them to form two lacunae. Most chondrocytes shown in this figure occupy only part of the lacuna. This is, in part, due to shrinkage, but it is also due to the fact that older chondrocytes contain lipid in large droplets that is lost during the processing of the tissue. The shrinkage of chondrocytes within the lacunae or their loss due to dropping out of the section during preparation causes the lacunae to stand out as light, unstained areas against the darkly stained matrix. The inset shows the elastic cartilage at still higher magnification. Here, the elastic fibers (E) are again evident as elongate profiles, chiefly at the edges of the cartilage. Most chondrocytes in this part of the specimen show little shrinkage. Many of the cells display a typically rounded nucleus, and the cytoplasm is evident. Note, again, that some lacunae contain two chondrocytes, indicating interstitial growth.


Histology_Ross

Histology_Ross. Fibrocartilage is a combination of dense connective tissue and cartilage. It has a matrix with large bundles of type I collagen in addition to type II collagen. The amount of cartilage varies, but in most locations the cartilage cells and their matrix occupy a lesser portion of the tissue mass. Fibrocartilage is found at the intervertebral discs, the symphysis pubis, the knee joint, the mandibular joint, the sternoclavicular joint, and the shoulder joint. It may also be present along the grooves or insertions for tendons and ligaments. Its presence is associated with sites where resilience is required in dense connective tissue to help absorb sudden physical impact, i.e., where resistance to both compressive and shearing forces is required in the tissue. Histologically, fibrocartilage appears as small fields of cartilage blending almost imperceptibly with regions of dense fibrous connective tissue. It is usually identified by the presence of aggregates of rounded chondrocytes


Histology_Ross

Histology_Ross. appears as small fields of cartilage blending almost imperceptibly with regions of dense fibrous connective tissue. It is usually identified by the presence of aggregates of rounded chondrocytes (isogenous groups) among bundles of collagen fibers and by the basophilic staining of the capsular matrix material and territorial matrix secreted by these cells. No perichondrium is present.


Histology_Ross

Histology_Ross. Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×160. This is a low-magnification view of fibrocartilage. The Mallory method stains collagen light blue. The tissue has a fibrous appearance, and at this low magnification the nuclei of the fibroblasts (F) appear as small, elongate, or spindle-shaped bodies. There are relatively few fibroblasts present, as is characteristic of dense connective tissue. The chondrocytes (C) are more numerous and exhibit close spatial groupings, i.e., isogenous groups. Some of the chondrocytes appear as elongate clusters of cells, whereas others appear in single-file rows. The matrix material immediately surrounding the chondrocytes has a homogeneous appearance and is, thereby, distinguishable from the fibrous connective tissue. PLATE 10 • FIBROCARTILAGE KEY C, chondrocytes F, fibroblast arrow, lacuna Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×700.


Histology_Ross

Histology_Ross. PLATE 10 • FIBROCARTILAGE KEY C, chondrocytes F, fibroblast arrow, lacuna Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×700. This figure shows the area circumscribed by the rectangle in figure above at higher magnification. The chondrocytes are contained within lacunae (arrows), and their cytoplasm stains deeply. The surrounding cartilage matrix material is scant and blends into the dense connective tissue. Cartilage matrix material can be detected best by observing the larger group of chondrocytes at the left of this figure and then observing this same area in figure above. Note the light homogeneous area around the cell nest in the lower-power view. This is the region of cartilage matrix. At the greater magnification of this figure, it is possible to see that some of the collagen fibers are incorporated in the matrix, where they appear as wispy bundles.


Histology_Ross

Histology_Ross. OVERVIEW OF BONE / 218 BONES AND BONE TISSUE / 219 GENERAL STRUCTURE OF BONES / 220 Outer Surface of Bones / 220 Bone Cavities / 221 Mature Bone / 221 Immature Bone / 223 CELLS OF BONE TISSUE / 223 Osteoprogenitor Cells / 225 Osteoblasts / 225 Osteocytes / 227 Bone-Lining Cells / 227 Osteoclasts / 227 BONE FORMATION / 232 Intramembranous Ossification / 234 Endochondral Ossification / 235 Growth of Endochondral Bone / 237 Development of the Osteonal (Haversian) System / 240 BIOLOGIC MINERALIZATION AND MATRIX VESICLES / 241 PHYSIOLOGIC ASPECTS OF BONE / 242 Folder 8.1 Clinical Correlation: Joint Diseases / 221 Folder 8.2 Clinical Correlation: Osteoporosis / 233 Folder 8.3 Clinical Correlation: Nutritional Factors in Bone Formation / 234 Folder 8.4 Functional Considerations: Hormonal Regulation of Bone Growth / 242 Bone is a connective tissue characterized by a mineralized extracellular matrix.


Histology_Ross

Histology_Ross. Bone is a connective tissue characterized by a mineralized extracellular matrix. Bone is a specialized form of connective tissue that, like other connective tissues, consists of cells and extracellular matrix. The feature that distinguishes bone from other connective tissues is the mineralization of its matrix, which produces an extremely hard tissue capable of providing support and protection. The mineral is calcium phosphate in the form of hydroxyapatite crystals [Ca10(PO4)6(OH)2]. By virtue of its mineral content, bone also serves as a storage site for calcium and phosphate. Both calcium and phosphate can be mobilized from the bone matrix and taken up by the blood as needed to maintain appropriate levels throughout the body. Thus, in addition to support and protection, bone plays an important secondary role in the homeostatic regulation of blood calcium levels. Bone matrix contains mainly type I collagen along with other matrix (noncollagenous) proteins.


Histology_Ross

Histology_Ross. Bone matrix contains mainly type I collagen along with other matrix (noncollagenous) proteins. The major structural component of bone matrix is type I collagen and, to a lesser extent, type V collagen. Trace amounts of other types such as type III, XI, and XIII collagens have also been found in the matrix. All collagen molecules constitute about 90% of the total weight of the bone matrix proteins.


Histology_Ross

Histology_Ross. The matrix also contains other matrix (noncollagenous) proteins that constitute the ground substance of bone. As a minor component of bone, constituting only 10% of the total weight of bone matrix proteins, they are essential to bone development, growth, remodeling, and repair. Both the collagen and the ground substance become mineralized to form bone tissue. The four main groups of noncollagenous proteins found in the bone matrix are the following:  Proteoglycan macromolecules contain a core protein with various numbers of covalently attached side chains of glycosaminoglycans (hyaluronan, chondroitin sulfate, and keratan sulfate). They contribute to the compressive strength of bone. They are also responsible for binding growth factors and may inhibit mineralization. Proteoglycans are described in detail in Chapter 6 (Table 6.3, page 176).


Histology_Ross

Histology_Ross.  Multiadhesive glycoproteins are responsible for attachment of bone cells and collagen fibers to the mineralized ground substance. Some of the more important glycoproteins are osteonectin (which serves as a glue between the collagen and hydroxyapatite crystals) and sialoproteins such as osteopontin (which mediates attachment of cells to bone matrix) and sialoprotein I and II (which mediate cell attachment and initiate calcium phosphate formation during the mineralization process).  Bone-specific, vitamin K–dependent proteins, which include osteocalcin (which captures calcium from the circulation and attracts and stimulates osteoclasts in bone remodeling), protein S (which assists in the removal of cells undergoing apoptosis), and matrix Gla-protein (MGP) (which participates in the development of vascular calcifications).


Histology_Ross

Histology_Ross.  Growth factors and cytokines, which are small regulatory proteins including insulinlike growth factors (IGFs), tumor necrosis factor (TNF-), transforming growth factor (TGF-), platelet-derived growth factors (PDGFs), bone morphogenic proteins (BMPs), and interleukins (IL-1, IL-6). The most unique members of this group are BMPs, because they induce the differentiation of mesenchymal cells into osteoblasts, the bone-producing cells. Recombinant human BMP-7, also known as osteogenic protein-1 (OP-1), is now used clinically to induce bone growth after bone surgery involving large bone defects, spinal fusions, or implantation of graft materials. Bone matrix contains lacunae connected by a network of canaliculi. Within the bone matrix are spaces called lacunae (sing., lacuna), each of which contains a bone cell, or osteocyte.


Histology_Ross

Histology_Ross. Bone matrix contains lacunae connected by a network of canaliculi. Within the bone matrix are spaces called lacunae (sing., lacuna), each of which contains a bone cell, or osteocyte. The osteocyte extends numerous processes into small tunnels called canaliculi. Canaliculi course through the mineralized matrix, connecting adjacent lacunae and allowing contact be tween the cell processes of neighboring osteocytes (Plate 11, page 244). In this manner, a continuous network of canaliculi and lacunae-containing cells and their processes is formed throughout the entire mass of mineralized tissue. Electron gap junctions. Bone tissue depends on the osteocytes to maintain viability. In addition to osteocytes, four other cell types are associ ated with bone.  Osteoprogenitor cells are cells derived from mesenchymal stem cells; they give rise to osteoblasts.


Histology_Ross

Histology_Ross.  Osteoprogenitor cells are cells derived from mesenchymal stem cells; they give rise to osteoblasts.  Osteoblasts are cells that secrete the extracellular matrix of bone; once the cell is surrounded with its secreted matrix, it is referred to as an osteocyte.  Bone-lining cells are cells that remain on the bone surface when there is no active growth. They are derived from those osteoblasts that remain after bone deposition ceases.  Osteoclasts are bone-resorbing cells present on bone surfaces where bone is being removed or remodeled (reorganized) or where bone has been damaged. Osteoprogenitor cells and osteoblasts are developmental precursors of the osteocyte. Osteoclasts are phagocytotic cells derived from fusion of hemopoietic progenitor cells in bone marrow that give rise to the neutrophilic granulocyte and monocyte lineages. Each of these cells is described in more detail below.


Histology_Ross

Histology_Ross. Bones are the organs of the skeletal system; bone tissue is the structural component of bones. Typically, a bone consists of bone tissue and other connective tissues, including hemopoietic tissue, fat tissue, blood vessels, and nerves. If the bone forms a freely movable or synovial joint, hyaline cartilage is present. The ability of the bone to perform its skeletal function is attributable to the bone tissue and, where present, the hyaline or articular cartilage. Bone tissue is classified as either compact (dense) or spongy (cancellous). If a bone is cut, two distinct structural arrangements of bone tissue can be recognized (Fig. 8.1 and Plate 12, page 246). A compact, dense layer forms the outside of the bone (compact bone); a spongelike meshwork consisting of trabe culae (thin, anastomosing spicules of bone tissue) forms the interior of the bone (spongy bone). The spaces within the meshwork are continuous and, in a living bone, are occupied by marrow and blood vessels.


Histology_Ross

Histology_Ross. Bones are classified according to shape; the location of spongy and compact bone varies with bone shape. Spongy and compact bone tissues are located in specific parts of bones. It is useful, then, to outline briefly the kinds of bones and survey where the two kinds of bone tissue are located. On the basis of shape, bones can be classified into four groups:  Long bones are longer in one dimension than other bones and consist of a shaft and two ends (e.g., the tibia and the metacarpals). A schematic diagram of a long bone sectioned longitudinally through the shaft is shown in Figure 8.2.  Short bones are nearly equal in length and diameter (e.g., the carpal bones of the hand).  Flat bones are thin and platelike (e.g., the bones of the calvaria [skull cap] and the sternum). They consist of two layers of relatively thick compact bone with an intervening layer of spongy bone.


Histology_Ross

Histology_Ross.  Irregular bones have a shape that does not fit into any one of the three groups just described; the shape may be complex (e.g., a vertebra), or the bone may contain air spaces or sinuses (e.g., the ethmoid bone). FIGURE 8.1 • Epiphysis of an adult long bone. This photo shows a longitudinally sectioned epiphysis of a long bone. The outer portion of the bone has a solid structure (arrows) and represents compact (dense) bone. The interior of the bone exhibits a spongy configuration and represents spongy (cancellous) bone. It consists of numerous interconnecting bony trabeculae separated by a labyrinth of interconnecting marrow spaces.


Histology_Ross

Histology_Ross. Long bones have a shaft, called the diaphysis, and two expanded ends, each called an epiphysis (see Fig. 8.2). The articular surface of the epiphysis is covered with hyaline cartilage. The flared portion of the bone between the diaphysis and the epiphysis is called the metaphysis. It extends from the diaphysis to the epiphyseal line. A large cavity filled with bone marrow, called the marrow or medullary cavity, forms the inner portion of the bone. In the shaft, almost the entire thickness of the bone tissue is compact; at most, only a small amount of spongy bone faces the marrow cavity. At the ends of the bone, the reverse is true. Here the spongy bone is extensive, and the compact bone consists of little more than a thin outer shell (see Fig. 8.1). Short bones possess a shell of compact bone and have spongy bone and a marrow space on the inside. Short bones usually form movable joints with their neighbors; like long


Histology_Ross

Histology_Ross. Short bones possess a shell of compact bone and have spongy bone and a marrow space on the inside. Short bones usually form movable joints with their neighbors; like long FIGURE 8.2 • Structure of a typical long bone. The diaphysis (shaft) of a long bone contains a large marrow cavity surrounded by a thick-walled tube of compact bone. A small amount of spongy bone may line the inner surface of the compact bone. The proximal and distal ends, or epiphyses, of the long bone consist chiefly of spongy bone with a thin outer shell of compact bone. The expanded or flared part of the diaphysis nearest the epiphysis is referred to as the metaphysis. Except for the articular surfaces that are covered by hyaline (articular) cartilage, indicated in blue, the outer surface of the bone is covered by a fibrous layer of connective tissue called the periosteum, indicated in pink.


Histology_Ross

Histology_Ross. bones, their articular surfaces are covered with hyaline cartilage. Elsewhere, periosteum, a fibrous connective tissue capsule covers the outer surface of the bone. Outer Surface of Bones Bones are covered by periosteum, a sheath of dense fibrous connective tissue containing osteoprogenitor cells. Bones are covered by a periosteum except in areas where they articulate with another bone. In the latter case, the articulating surface is covered by cartilage. The periosteum that covers an actively growing bone consists of an outer fibrous layer that resembles other dense connective tissues and an inner, more cellular layer that contains the osteoprogenitor cells. If active bone formation is not in progress on the bone surface, the fibrous layer is the main component of the periosteum, and the inner layer is not well defined. The relatively few cells that are present, the periosteal cells, are, however, capable of undergoing division and becoming osteoblasts under appropriate stimulus.


Histology_Ross

Histology_Ross. In general, the collagen fibers of the periosteum are arranged parallel to the surface of the bone in the form of a capsule. The character of the periosteum is different where ligaments and tendons attach to the bone. Collagen fibers from these structures extend directly, but at an angle, into the bone tissue, where they are continuous with the collagen fibers of the extracellular matrix of the bone tissue. These fibers are called Sharpey’s fbers. Bones that articulate with neighboring bones possess movable (synovial) joints.


Histology_Ross

Histology_Ross. Bones that articulate with neighboring bones possess movable (synovial) joints. Where a bone articulates with a neighboring bone, as in synovial joints, the contact areas of the two bones are referred to as articular surfaces. The articular surfaces are covered by hyaline cartilage, also called articular cartilage because of its location and function; articular cartilage is exposed to the joint cavity. This cartilage is not covered with perichondrium. The details of articular cartilage are discussed in Chapter 7 (page 203 and in Folder 8.1 (Clinical Correlation: Joint Diseases). Bone cavities are lined by endosteum, a layer of connective tissue cells that contains osteoprogenitor cells.


Histology_Ross

Histology_Ross. Bone cavities are lined by endosteum, a layer of connective tissue cells that contains osteoprogenitor cells. The lining tissue of both the compact bone facing the marrow cavity and the trabeculae of spongy bone within the cavity is referred to as endosteum. The endosteum is often only one cell layer thick and consists of osteoprogenitor cells that can differentiate into bone matrix–secreting cells, the osteoblasts, and bone-lining cells. Osteoprogenitor cells and bone-lining cells are difficult to distinguish at the microscopic level. They are both flattened in shape with elongated nuclei and indistinguishable cytoplasmic features. Because of their location within the bone cavities they are frequently called endosteal cells. The marrow cavity and the spaces in spongy bone contain bone marrow.


Histology_Ross

Histology_Ross. The marrow cavity and the spaces in spongy bone contain bone marrow. Red bone marrow consists of blood cells in different stages of development and a network of reticular cells and fibers that serve as a supporting framework for the developing blood cells and vessels. As an individual grows, the amount of red marrow does not increase proportionately with bone growth. In later stages of growth and in adults, when the rate of blood cell formation has diminished, the tissue in the marrow cavity consists mostly of fat cells; it is then called yellow marrow. In response to appropriate stimuli, such as extreme blood loss, yellow marrow can revert to red marrow. In the adult, red marrow is normally restricted to the spaces of spongy bone in a few locations such as the sternum and the iliac crest. Diagnostic bone marrow samples and marrow for transplantation are obtained from these sites. Mature bone is composed of structural units called osteons (Haversian systems).


Histology_Ross

Histology_Ross. Mature bone is composed of structural units called osteons (Haversian systems). Mature bone is largely composed of cylindrical units called osteons or Haversian systems (Fig. 8.3). The osteons consist of concentric lamellae (sing., lamella) of bone matrix surrounding a central canal, the osteonal (Haversian) canal, which contains the vascular and nerve supply of the osteon. Canaliculi containing the processes of osteocytes are generally arranged in a radial pattern with respect to the canal (Plate 11, page 244). The system of canaliculi that opens to the osteonal canal also serves for the passage of substances between the osteocytes and blood vessels. Between the osteons are remnants of previous concentric lamellae  FOLDER 8.1 Clinical Correlation: Joint Diseases


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Histology_Ross. Inflammation of the joints or arthritis can be caused by many factors and can produce varying degrees of pain and disability, from the pathologic response of articular carti-lage to injury. Simple trauma to a joint by a single incident or by re-peated insult can so damage the articular cartilage that it calcifies and begins to be replaced by bone. This process can lead to ankylosis (i.e., bony fusion in the joint and subsequent loss of motion). The foot and knee joints of run-ners and football players and hand and finger joints of stringed instrument players are especially vulnerable to this condition. Immune responses or infectious processes that localize in joints, as in rheumatoid arthritis or tuberculosis, can also damage the articular cartilages, producing both severe joint pain and gradual ankylosis. Surgery that replaces the damaged joint with a prosthetic joint can often relieve the pain and restore joint motion in seriously debilitated individ-uals. Another common cause of


Histology_Ross

Histology_Ross. gradual ankylosis. Surgery that replaces the damaged joint with a prosthetic joint can often relieve the pain and restore joint motion in seriously debilitated individ-uals. Another common cause of damage to articular carti-lages is the deposition of crystals of uric acid in the joints, particularly those of the toes and fingers. This condition is known as gouty arthritis or, more simply, gout. Gout has become more common because of the widespread use of thiazide diuretics in the treatment of hypertension. In genet-ically predisposed individuals, gout is the most common side effect of these drugs. Gout causes severe, unbear-able pain because of the sharp crystals in the joint. The irritation also causes the formation of calcareous deposits that deform the joint and limit its motion.


Histology_Ross

lamellae of bone

Histology_Ross. lamellae of bone


Histology_Ross

Histology_Ross. FIGURE 8.3 • Diagram of a section of compact bone removed from the shaft of a long bone. The concentric lamellae and the Haversian canal that they surround constitute an osteon (Haversian system). One of the Haversian systems in this diagram is drawn as an elongated cylindrical structure rising above the plane of the bone section. It consists of several concentric lamellae that have been partially removed to show the perpendicular orientation of collagen fibers in adjacent layers. Interstitial lamellae result from bone remodeling and formation of new Haversian systems. The inner and outer surfaces of the compact bone in this diagram show additional lamellae—the outer and inner circumferential lamellae—arranged in broad layers. The inner circumferential lamella is covered by a thin layer of endosteum that faces the marrow cavity, similar to the outer surface of the bone, which is covered by periosteum. Branches of nutritional arteries accompanied by small veins are shown within the


Histology_Ross

Histology_Ross. of endosteum that faces the marrow cavity, similar to the outer surface of the bone, which is covered by periosteum. Branches of nutritional arteries accompanied by small veins are shown within the Haversian and Volkmann’s canals. These arteries also supply the periosteum, endosteum, and bone marrow.


Histology_Ross

Histology_Ross. called interstitial lamellae (see Fig. 8.3). Because of this organization, mature bone is also called lamellar bone. The long axis of an osteon is usually parallel to the long axis of the bone. The collagen fibers in the concentric lamellae in an osteon are laid down parallel to one another in any given lamella but in different directions in adjacent lamellae. This arrangement gives the cut surface of lamellar bone the appearance of plywood and imparts great strength to the osteon.


Histology_Ross

Histology_Ross. Lamellar bone is also found at sites other than the osteon. Circumferential lamellae follow the entire inner and outer circumferences of the shaft of a long bone, appearing much like the growth rings of a tree (see Fig. 8.3). Perforating canals (Volkmann’s canals) are channels in lamellar bone through which blood vessels and nerves travel from the periosteal and endosteal surfaces to reach the osteonal canal; they also connect osteonal canals to one another (Plate 11, page 244). They usually run at approximately right angles to the long axis of the osteons and of the bone (see Fig. 8.3). Volkmann’s canals are not surrounded by concentric lamellae, a key feature in their histologic identification. Mature spongy bone is structurally similar to mature compact bone.


Histology_Ross

Histology_Ross. Mature spongy bone is structurally similar to mature compact bone. Mature spongy bone is similar in structure to mature compact bone except that the tissue is arranged as trabeculae or spicules; numerous interconnecting marrow spaces of various sizes are present between the bone tissue. The matrix of the bone is lamellated. Arteries that enter the marrow cavity through the nutrient foramina supply blood to the shaft of long bones. Nutrient foramina are openings in the bone through which blood vessels pass to reach the marrow. The greatest numbers of nutrient foramina are found in the diaphysis and epiphysis (Fig. 8.4). Metaphyseal arteries supplement the blood supply to the bone. Veins that exit through the nutrient foramina


Histology_Ross

Histology_Ross. FIGURE 8.4 • Diagram showing the blood supply of an adult long bone. The nutrient artery and the epiphyseal arteries enter the bone through nutrient foramina. These openings in the bone arise developmentally as the pathways of the principal vessels of periosteal buds. Metaphyseal arteries arise from periosteal vessels that become incorporated into the metaphysis as the bone grows in diameter. or through the bone tissue of the shaft and continue out through the periosteum drain the blood from bone. The nutrient arteries that supply the diaphysis and epiphysis arise developmentally as the principal vessels of the periosteal buds. The metaphyseal arteries, in contrast, arise developmentally from periosteal vessels that become incorporated into the metaphysis during the growth process (i.e., through the widening of the bone). The blood supply to bone tissue is essentially centrifugal.


Histology_Ross

Histology_Ross. The blood supply to bone tissue is essentially centrifugal. The blood that nourishes bone tissue moves from the marrow cavity into and through the bone tissue and out via periosteal veins; thus, its flow is in a centrifugal direction. With respect to nourishment of the bone tissue itself, Volkmann’s canals provide the major route of entry for vessels to pass through the compact bone. The smaller blood vessels enter the Haversian canals, which contain a single arteriole and a venule or a single capillary. A lesser blood supply to the outermost portions of the compact bone is provided by the branches of periosteal arteries (see Fig. 8.4). Bone tissue lacks lymphatic vessels; lymphatic drainage occurs only from the periosteum.


Histology_Ross

Histology_Ross. Bone tissue initially formed in the skeleton of a developing fetus is called immature bone. It differs from mature bone in several respects (Fig. 8.5):  Immature bone does not display an organized lamellated appearance. On the basis of its collagen fiber arrangement, such bone is designated nonlamellar. Nonlamellar bone is also referred to as bundle bone or woven bone because of the interlacing arrangement of the collagen fibers.


Histology_Ross

Histology_Ross. osteoclast resorption canal osteocyte osteocyte interstitial lamellae MATURE BONE IMMATURE BONE osteon concentric lamella a b FIGURE 8.5 • Diagram of immature and mature bone. Immature bone does not display an organized lamellar appearance because of the interlacing arrangement of the collagen fibers. The cells tend to be randomly arranged, whereas the cells in mature bone are organized in a circular fashion that reflects the lamellar structure of the Haversian system. Resorption canals in mature bone have their long axes in the same direction as the Haversian canals. than does mature bone.  The cells in immature bone tend to be randomly arranged, whereas cells in mature bone are usually arranged with their long axes in the same direction as the lamellae.  The matrix of immature bone has more ground substance than does the matrix of mature bone. The matrix in im mature bone stains more intensely with hematoxylin, whereas the matrix of mature bone stains more intensely with eosin.


Histology_Ross

Histology_Ross. Although not evident in typical histologic sections (Fig. 8.6), immature bone is not heavily mineralized when it is initially formed, whereas mature bone undergoes prolonged secondary mineralization. The secondary mineralization of mature bone is evident in microradiographs of ground sections that show younger Haversian systems to be less mineralized than older Haversian systems (see Fig. 8.22). Immature bone forms more rapidly than mature bone. Although mature bone is clearly the major bone type in the adult and immature bone is the major bone type in the developing fetus, areas of immature bone are present in adults, especially where bone is being remodeled. Areas of immature bone are common in the alveolar sockets of the adult oral cavity and where tendons insert into bones. It is this immature bone in the alveolar sockets that makes it possible to make orthodontic corrections even in adults.


Histology_Ross

Histology_Ross. As noted previously, five designated cell types are associated with bone tissue: osteoprogenitor cells, osteoblasts, osteocytes, bone-lining cells, and osteoclasts. With the exception of the osteoclast, each of these cells may be regarded as a bone). In contrast, the osteoclast originates from a different differentiated form of the same basic cell type (Fig. 8.7). Each cell line and is responsible for bone resorption, an activity as-undergoes transformation from a less mature form to a more sociated with bone remodeling. mature form in relation to functional activity (growth of


Histology_Ross

Histology_Ross. FIGURE 8.6 • Photomicrographs of decalcified immature and mature bone. a. Decalcified immature bone, stained with H&E, showing the relationship of cells to the extracellular matrix. The immature bone has more cells, and the matrix is not layered in osteonal arrays. 130. b. This cross section of decalcified mature compact bone stained with H&E shows several osteons (O) with concentric lamellae. The Haversian canals contain blood vessels and connective tissue. Osteocytes undergo considerable shrinkage during routine slide preparation, revealing empty lacunae with a small nucleus attached to their walls. Mature bone has fewer osteocytes per unit area than immature bone. Note the presence of interstitial lamellae between neighboring osteons. 160. active inactive granulocyte/monocyte osteoclasts osteoclast progenitor (GMP, CFU-GM) endosteal cells


Histology_Ross

Histology_Ross. active inactive granulocyte/monocyte osteoclasts osteoclast progenitor (GMP, CFU-GM) endosteal cells FIGURE 8.7 • Schematic drawing of cells associated with bone. All cells except osteoclasts originate from the mesenchymal stem cells, which differentiate into osteoprogenitor cells, osteoblasts, and finally osteocytes and bone-lining cells. Bone-lining cells on external bone surfaces are part of the periosteum, hence the term periosteal cells. Bone-lining cells on internal bone surfaces are frequently called endosteal cells. Note that osteoprogenitor cells and bone-lining cells have a similar microscopic appearance and are often difficult to distinguish from each other. Osteoclasts originate from hemopoietic progenitor cells, which differentiate into boneresorbing cells. The specific details of osteoclast differentiation are illustrated in Figure 8.13. The osteoprogenitor cell is derived from mesenchymal stem cells.


Histology_Ross

Histology_Ross. The osteoprogenitor cell is derived from mesenchymal stem cells. Osteogenesis, the process of new bone formation, is essential to normal bone function. It requires a population of renewable osteoprogenitor cells (osteoblast precursor cells) that are responsive to molecular stimuli that transform them into bone-forming cells. Osteoprogenitor cells are derived from mesenchymal stem cells in the bone marrow that have the potential to differentiate into many different cell types including fibroblasts, osteoblasts, adipocytes, chondrocytes, and muscle cells. The key factor that triggers differentiation of osteoprogenitor cells is a transcription factor called core binding factor alpha-1 (CBFA1). This protein prompts the expression of genes that are characteristic of the phenotype of the osteoblast. As noted on page 219, bone morphogenic proteins also play a role in the differentiation of osteoblasts.


Histology_Ross

Histology_Ross. The osteoprogenitor cell is a resting cell that can differentiate into an osteoblast and secrete bone matrix.


Histology_Ross

Histology_Ross. Osteoprogenitor cells are found on the external and internal surfaces of bones and may also reside in the microvasculature supplying bone. Morphologically, they comprise the periosteal cells that form the innermost layer of the periosteum and the endosteal cells that line the marrow cavities, the osteonal (Haversian) canals, and the perforating (Volkmann’s) canals. In growing bones, osteoprogenitor cells appear as flattened or squamous cells with lightly staining, elongate, or ovoid nuclei and inconspicuous acidophilic or slightly basophilic cytoplasm. Electron micrographs reveal profiles of rough-surfaced endoplasmic reticulum (rER) and free ribosomes as well as a small Golgi apparatus and other organelles. The morphology of the osteoprogenitor cell is consistent with the finding that its stimulation leads to differentiation into a more active secretory cell, the osteoblast. The osteoblast is the differentiated bone-forming cell that secretes bone matrix.


Histology_Ross

Histology_Ross. The osteoblast is the differentiated bone-forming cell that secretes bone matrix. Like its close relatives, the fibroblast and the chondroblast, the osteoblast is a versatile secretory cell that retains the ability to divide. It secretes both type I collagen (which constitutes 90% of the protein in bone) and bone matrix proteins (BMPs), which constitute the initial unmineralized bone, or osteoid. The bone matrix proteins produced by the osteoblast include calcium-binding proteins such as osteocalcin and osteonectin; multiadhesive glycoproteins such as bone sialoproteins I and II, osteopontin, and thrombospondin, various proteoglycans and their aggregates, and alkaline phosphatase (ALP). Circulating levels of ALP and osteocalcin are used clinically as markers of osteoblast activity.


Histology_Ross

Histology_Ross. The osteoblast is also responsible for the calcification of bone matrix. The calcification process appears to be initiated by the osteoblast through the secretion into the matrix of small, 50to 250-nm, membrane-limited matrix vesicles. The vesicles are rich in ALP and are actively secreted only during the period in which the cell produces the bone matrix. The role of these vesicles is discussed later in this chapter (page 241). Osteoblasts are recognized in the light microscope by their cuboidal or polygonal shape and their aggregation into a single layer of cells lying in apposition to the forming bone (Fig. 8.8). The newly deposited matrix is not immediately calcified. It stains lightly or not at all compared with the mature mineralized matrix, which stains heavily with eosin. Because of this staining property of the newly formed matrix, osteoblasts appear to be separated from the bone by a light band. This band represents the osteoid, the nonmineralized matrix.


Histology_Ross

Histology_Ross. FIGURE 8.8 • Photomicrograph of a growing bone spicule stained with Mallory-Azan. Osteocytes are embedded within the bone matrix of the spicule, which is stained dark blue. These cells are metabolically active, laying down the unmineralized bone matrix (osteoid). A number of osteoblasts are aligned on the right side of the spicule. Between these cells and the calcified bone spicule is a thin, light-blue–stained layer of osteoid. This is the uncalcified matrix material produced by the osteoblasts. One of the cells (arrow) has virtually surrounded itself by its osteoid product; thus it can now be called an osteocyte. On the left side of the spicule, the nongrowing part, are inactive osteoblasts. The cells exhibit flattened nuclei and attenuated cytoplasm. 550.


Histology_Ross

Histology_Ross. The cytoplasm of the osteoblast is markedly basophilic, and the Golgi apparatus, because of its size, is sometimes observed as a clear area adjacent to the nucleus. Small, periodic acid–Schiff (PAS)-positive granules are observed in the cytoplasm, and a strong ALP reaction associated with the cell membrane can be detected by appropriate histochemical staining. In contrast to the secreting osteoblasts found in active matrix deposition, inactive osteoblasts are flat or attenuated cells that cover the bone surface. These cells resemble osteoprogenitor cells. Osteoblasts respond to mechanical stimuli to mediate the changes in bone growth and bone remodeling. As osteoid deposition occurs, the osteoblast is eventually surrounded by osteoid matrix and then becomes an osteocyte. Osteoblast processes communicate with other osteoblasts and with osteocytes by gap junctions.


Histology_Ross

Histology_Ross. Osteoblast processes communicate with other osteoblasts and with osteocytes by gap junctions. At the electron microscope level, osteoblasts exhibit thin cytoplasmic processes that penetrate the adjacent osteoid produced by the cell and are joined to similar processes of adjacent osteocytes by gap junctions. This early establishment of junctions between an osteoblast and adjacent osteocytes (as well as between adjacent osteoblasts) allows neighboring cells within the bone tissue to communicate. The osteoblast cytoplasm is characterized by abundant rER and free ribosomes (Fig. 8.9). These features are consistent with its basophilia observed in the light microscope as well as with its role in the production of collagen and proteoglycans


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Histology_Ross. FIGURE 8.9 • Electron micrograph showing active bone formation. This electron micrograph is similar to the growing surface of the bone spicule in the preceding light micrograph (Fig. 8.8). The marrow cavity (M) with its developing blood cells is seen in the lower right corner. Osteoprogenitor cells (Opc) are evident between the marrow and the osteoblasts (Ob). They exhibit elongated or ovoid nuclei. The osteoblasts are aligned along the growing portion of the bone, which is covered by a layer of osteoid (Os). In this same region, one of the cells (upper right corner) embedded within the osteoid exhibits a small process (arrow). This cell, because of its location within the osteoid, can now be called an osteocyte (Oc). The remainder of the micrograph (upper left) is composed of calcified bone matrix (CB). Within the matrix are canaliculi (C) containing osteocyte processes. The boundary between two adjacent lamellae (L) of previously formed bone is evident as an irregular dark line.


Histology_Ross

Histology_Ross. bone matrix (CB). Within the matrix are canaliculi (C) containing osteocyte processes. The boundary between two adjacent lamellae (L) of previously formed bone is evident as an irregular dark line. 9,000.


Histology_Ross

Histology_Ross. for the extracellular matrix. The Golgi apparatus and surrounding regions of the cytoplasm contain numerous vesicles with a flocculent content that is presumed to consist of matrix precursors. These vesicles are the PAS-staining granules seen in light microscopy. The matrix vesicles, also produced by the osteoblast, appear to arise by a different pathway, originating as spherelike outgrowths that pinch off from the plasma membrane to become free in the matrix. Other cell organelles include numerous rod-shaped mitochondria and occasional dense bodies and lysosomes. The osteocyte is the mature bone cell enclosed by bone matrix that it previously secreted as an osteoblast.


Histology_Ross

Histology_Ross. The osteocyte is the mature bone cell enclosed by bone matrix that it previously secreted as an osteoblast. When completely surrounded by osteoid or bone matrix, the osteoblast is referred to as an osteocyte (see Fig. 8.8). Osteocytes are the cells responsible for maintaining the bone matrix. One of the roles of osteocytes is mechanotransduction, the process by which the osteocyte responds to mechanical forces applied to the bone. Different mechanical stimuli (e.g., weightlessness or increased mechanical loading) alter not only gene expression but also the cell’s apoptotic mechanism. Osteocytes can synthesize new matrix, as well as participate in matrix degradation. Such activities help to maintain calcium homeostasis. The death of osteocytes through trauma (e.g., a fracture), cell senescence, or apoptosis results in resorption of the bone matrix by osteoclast activity, followed by repair or remodeling of the bone tissue by osteoblast activity.


Histology_Ross

Histology_Ross. Each osteocyte occupies a space, or lacuna, that conforms to the shape of the cell. Osteocytes extend cytoplasmic processes through the canaliculi in the matrix to contact processes of neighboring osteocytes and bone-lining cells by means of gap junctions. Osteocytes can also communicate indirectly with distant osteoblasts, pericytes of blood vessels, and other bone cells through the expression of various signaling molecules such as nitric oxide and glutamate transporters. In hematoxylin and eosin (H&E)–stained sections, the canaliculi and the processes they contain are not discernible. In ground sections, the canaliculi are readily evident (Plate 11, page 244). Osteocytes are typically smaller than their precursors because of their reduced perinuclear cytoplasm. Often, in routinely prepared microscopic specimens, the cell is highly distorted by shrinkage and other artifacts that result from decalcifying the matrix before sectioning the bone. In such instances, the nucleus may be the


Histology_Ross

Histology_Ross. microscopic specimens, the cell is highly distorted by shrinkage and other artifacts that result from decalcifying the matrix before sectioning the bone. In such instances, the nucleus may be the only prominent feature. In wellpreserved specimens, osteocytes exhibit less cytoplasmic basophilia than osteoblasts, but little additional cytoplasmic detail can be seen (Plate 12, page 246).


Histology_Ross

Histology_Ross. Electron microscopy has revealed osteocytes in various functional states. Indeed, there is histologic and microradiologic evidence (i.e., enlarged lacunae and reduced radiodensity) that the osteocyte can modify the surrounding bone matrix. Three functional states, each with a characteristic morphology, have been described:  Quiescent osteocytes exhibit a paucity of rER and a markedly diminished Golgi apparatus (Fig. 8.10a). An osmiophilic lamina representing mature calcified matrix is seen in close apposition to the cell membrane. Formative osteocytes show evidence of matrix deposi tion and exhibit certain characteristics similar to those of osteoblasts. Thus, the rER and Golgi apparatus are more abundant, and there is evidence of osteoid in the pericel lular space within the lacuna (Fig. 8.10b).  Resorptive osteocytes, like formative osteocytes, con tain numerous profiles of endoplasmic reticulum and a well-developed Golgi apparatus. Moreover, lysosomes are conspicuous (Fig.


Histology_Ross

Histology_Ross. (Fig. 8.10b).  Resorptive osteocytes, like formative osteocytes, con tain numerous profiles of endoplasmic reticulum and a well-developed Golgi apparatus. Moreover, lysosomes are conspicuous (Fig. 8.10c).


Histology_Ross

Histology_Ross. The “resorptive” function of the osteocyte is not precisely defined and is supported mainly by the observation that the pericellular space is devoid of collagen fibrils and contains a flocculent material suggestive of a breakdown product. These observed changes could be explained by enzymatic degradation of collagen by osteocyte-secreted matrix metalloproteinases (MMPs). It has been shown under experimental conditions that a reduced load on bone initiates expression of MMP mRNA in the osteocyte. Degradation of bone by MMPs is called osteocytic osteolysis. The current concept of osteocytic osteolysis is that the lytic role of osteocytes is not related to bone matrix remodeling but functions to maintain blood calcium level. Bone-lining cells are derived from osteoblasts and cover bone that is not remodeling.


Histology_Ross

Histology_Ross. In sites where remodeling is not occurring, the bone surface is covered by a layer of flat cells with attenuated cytoplasm and a paucity of organelles beyond the perinuclear region (see Fig. 8.11a). These cells are designated simply as bone-lining cells. Bone-lining cells on external bone surfaces are called periosteal cells, and those lining internal bone surfaces are often called endosteal cells (see Fig. 8.7) Gap junctions are present where the bone-lining cell processes contact one another (Fig. 8.11b). Bone-lining cells represent a population of cells that are derived from osteoblasts. They are thought to function in the maintenance and nutritional support of the osteocytes embedded in the underlying bone matrix and regulate the movement of calcium and phosphate into and out of the bone. These suggested roles are based on the observation that the cell processes of bone-lining cells extend into the canalicular channels of the adjacent bone (see Fig. 8.11b) and communicate by means


Histology_Ross

Histology_Ross. These suggested roles are based on the observation that the cell processes of bone-lining cells extend into the canalicular channels of the adjacent bone (see Fig. 8.11b) and communicate by means of gap junctions with osteocytic processes. In these respects, bone-lining cells are somewhat comparable to osteocytes.


Histology_Ross

Histology_Ross. The osteoclast is responsible for bone resorption. Osteoclasts are large, multinucleated cells found at sites where bone is being removed. They rest directly on the bone tissue where resorption is taking place (Fig. 8.12). As a result of osteoclast activity, a shallow bay called a resorption bay FIGURE 8.10 • Electron micrographs of three different functional stages of an osteocyte. a. Relatively quiescent osteocyte that contains only a few profiles of rER and a few mitochondria (M). The cell virtually fills the lacuna that it occupies; the arrows indicate where cytoplasmic processes extend into canaliculi. Hydroxyapatite crystals have been lost from the matrix, which is ordinarily mineralized (MM), but some hydroxyapatite crystals fill the pericellular space. The hydroxyapatite crystals obscure the other substances within the pericellular space. The dark band marking the boundary of the lacuna is the osmiophilic lamina (OL). 25,000.


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Histology_Ross. b. A formative osteocyte containing larger amounts of rER and a large Golgi apparatus (G). Of equal importance is the presence of a small amount of osteoid in the pericellular space within the lacuna. The osteoid shows profiles of collagen fibrils (arrows) not yet mineralized. The lacuna of a formative osteocyte is not bounded by an osmiophilic lamina. 25,000. c. A resorptive osteocyte containing a substantial amount of rER, a large Golgi apparatus, mitochondria (M), and lysosomes (L). The pericellular space is devoid of collagen fibrils and may contain some flocculent material. The lacuna containing a resorptive osteocyte is bounded by a less conspicuous osmiophilic lamina (OL). 25,000.


Histology_Ross

Histology_Ross. (Howship’s lacuna) can be observed in the bone directly under the osteoclast. The cell is conspicuous not only because of its large size but also because of its marked acidophilia. It also exhibits a strong histochemical reaction for acid phosphatase because of the numerous lysosomes that it contains. One of these enzymes, the 35-kilodalton iron-containing tartrate-resistant acid phosphatase (TRAP), is used clinically as a marker of osteoclast activity and differentiation. Osteoclasts are derived from the fusion of mononuclear hemopoietic progenitor cells under the influence of multiple cytokines.


Histology_Ross

Histology_Ross. Contrary to what was once thought, osteoclasts are not related to osteoblasts. They are derived from the fusion of mononuclear hemopoietic cells, namely, granulocyte/ macrophage progenitor cells (GMP, CFU-GM) that give rise to granulocyte and monocyte cell lineages (see Fig. 10.16). Osteoclast formation occurs in close association with stromal cells in bone marrow. These cells secrete essential cytokines for differentiation of both osteoclasts and macrophages from GMP progenitor cells, including monocyte colony-stimulating factor (M-CSF), TNF, and several interleukins. Initially, cells committed to become osteoclasts (osteoclast precursors) express two important transcription factors, c-fos and NFB; later, a receptor molecule called receptor activator of nuclear factor B (RANK) is expressed on their surface. The RANK receptor interacts with RANK ligand molecule (RANKL) produced and expressed on the stromal cell surface (Fig. 8.13). The RANK–RANKL signaling mechanism is essential for


Histology_Ross

Histology_Ross. on their surface. The RANK receptor interacts with RANK ligand molecule (RANKL) produced and expressed on the stromal cell surface (Fig. 8.13). The RANK–RANKL signaling mechanism is essential for osteoclast differentiation and maturation. Alternatively, during inﬂammation, activated T lymphocytes can produce both membrane-bound and soluble RANKL molecules. Therefore, inﬂammatory processes can stimulate osteoclast-mediated bone resorption. This pathway can be blocked by osteoprotegerin (OPG), which serves as a “decoy” receptor for RANKL. Lack of available ligand affects the RANK–RANKL signaling pathway and acts as a potent inhibitor of osteoclast formation. OPG is produced mainly by osteoblasts and is regulated by many bone metabolic regulators, such as IL-1, TNF, TGF, vitamin D, and prostaglandin E2. Recent studies indicate


Histology_Ross

Histology_Ross. FIGURE 8.11 • Electron micrograph of bone-lining cells. a. The cytoplasm of a bone-lining cell located on the surface of a spicule of mature bone is very attenuated and contains small amounts of rER and free ribosomes. A gap junction is seen between the two adjacent bone-lining cells. In addition, cytoplasmic processes are clearly seen where they pass through the matrix of unmineralized bone (osteoid). A fat cell of the marrow is also present. 8,900. (Reprinted with permission from Miller SC, Bowman BM, Smith JM, Jee WS. Characterization of endosteal bone-lining cells from fatty marrow bone sites in adult beagles. Anat Rec 1980;198:163–173.) Inset. High-magnification photomicrograph of a similar bone spicule stained with H&E, included for orientation purposes. The bone-lining cells (endosteal cells) on the surface of the spicule are indicated by the arrows. 350. b. Electron micrograph of the cytoplasm of two bone-lining cells observed at higher magnification. The gap junction is


Histology_Ross

Histology_Ross. cells) on the surface of the spicule are indicated by the arrows. 350. b. Electron micrograph of the cytoplasm of two bone-lining cells observed at higher magnification. The gap junction is clearly seen where the two cells are in apposition. The edge of a fat cell is seen at the top of the electron micrograph; its lipid, thin rim of cytoplasm, plasma membrane, and external lamina are also evident. 27,000.


Histology_Ross

Histology_Ross. that substances that promote osteoclast differentiation and bone resorption act through the OPG/RANKL system in the bone marrow. Both OPG and RANKL are detected in a free form in the blood, and their concentrations can be measured for diagnostic purposes and to monitor therapy of many bone diseases. Newly formed osteoclasts undergo an activation process to become bone-resorbing cells.


Histology_Ross

Histology_Ross. Newly formed osteoclasts undergo an activation process to become bone-resorbing cells. The newly formed osteoclast must be activated to become a bone-resorbing cell. During this process, it becomes highly polarized. When actively resorbing bone, osteoclasts exhibit three specialized regions:  The ruffled border is the part of the cell in direct contact with bone. It contains numerous deep plasma membrane infoldings forming microvillous-type structures responsible for increasing surface area for the exocytosis of hydrolytic enzymes and secretion of protons by ATP–dependent proton pumps, as well as endocytosis of degradation products and bone debris. The ruffled border stains less intensely than the remainder of the cell and often appears as a light band adjacent to the bone at


Histology_Ross

Histology_Ross. FIGURE 8.12 • Photomicrograph of an osteoclast on a bone spicule. This Mallory-stained specimen shows a spicule made of calcified cartilage (stained light blue) and a covering of bone tissue (stained dark blue). An osteoclast on the left side of the spicule has resorbed bone tissue and lies in a depression (Howship’s lacuna) in the spicule. The light band between the osteoclast and the bone spicule corresponds to the ruffled border of the osteoclast. The arrows on the nongrowing surface indicate cytoplasm of inactive bone-lining cells (osteoprogenitor cells). In contrast, bone is being deposited on the opposite side of the spicule, as evidenced by the presence of osteoblasts on this surface and newly formed osteocytes just below the surface of the spicule. 550. common myeloid progenitor (CMP, CFU-GEMM) granulocyte/ monocyte progenitors (GMP, CFU-GM)


Histology_Ross

Histology_Ross. FIGURE 8.13 • The origin of osteoclasts. Osteoclasts are derived from fusion of granulocyte/monocyte progenitor cells (GMP, CFUGM), which originate from multipotential common myeloid progenitor cells (CMP, CFU-GEMM). GMP cells also give rise to the granulocyte and monocyte cell lineages such as neutrophil progenitor (NoP, CFU-G) and monocyte progenitor (MoP, CFU-M) cells. Osteoclast formation occurs in close association with stromal cells in bone marrow, which secrete monocyte colony-stimulating factor (M-CSF ), tumor necrosis factor (TNF ), and several interleukins (ILs). Osteoclast precursors express c-fos and NFB, and receptor molecules called RANK (receptor activator of nuclear factor B). The signal generated by the interaction of the RANK receptor with the RANK ligand (RANKL) molecule is essential for osteoclast differentiation and maturation. During inflammation, T lymphocytes produce both soluble and membrane-bound RANKL molecules, which increase bone resorption. These


Histology_Ross

Histology_Ross. molecule is essential for osteoclast differentiation and maturation. During inflammation, T lymphocytes produce both soluble and membrane-bound RANKL molecules, which increase bone resorption. These pathways can be blocked by osteoprotegerin (OPG). Note that activated T lymphocytes can stimulate formation of osteoclasts by producing both membrane-bound and soluble RANKL molecules.


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Histology_Ross. FIGURE 8.14 • Electron micrograph of an osteoclast. This micrograph shows a segment of bone surface (B) and a portion of an osteoclast that is in apposition to the partially digested bone. The resorption front (RF) of the osteoclast possesses numerous infoldings of the plasma membrane. When viewed in the light microscope, these infoldings are evident as the ruffled border. When the plane of section is parallel to the infoldings (asterisks), a broad, nonspecialized expanse of cytoplasm is seen. The cytoplasm of the osteoclast contains numerous mitochondria (M), lysosomes, and Golgi apparatus, all of which are functionally linked with the resorption and degradation of the bone matrix. In the upper part of the figure, some collagen fibrils are evident; the arrows indicate where 68-nm cross-banding is visible. 10,000.


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Histology_Ross. the resorption site (see Fig. 8.12). At the electron microscopic level, hydroxyapatite crystals from the bone substance are observed between the processes of the ruffled border (Fig. 8.14). Internal to the ruffled border and in close proximity are numerous mitochondria and lysosomes. The nuclei are typically located in the part of the cell more removed from the bone surface. In this same region are profiles of rER, multiple stacks of Golgi apparatus, and many vesicles.


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Histology_Ross.  The clear zone (sealing zone) is a ringlike perimeter of cytoplasm adjacent to the ruffled border that demarcates the bone area being resorbed. Essentially, the clear zone is a compartment at the site of the ruffled border where resorption and degradation of the matrix occurs. It contains abundant actin filaments but essentially lacks other organelles. The actin filaments are arranged in a ringlike structure surrounded on both sides by actin-binding proteins such as vinculin and talin (Fig. 8.15). The plasma membrane at the site of the clear zone contains cell and extracellular matrix adhesion molecules that are responsible for providing a tight seal between the plasma membrane and mineralized matrix of the bone. Several classes of integrin extracellular receptors (i.e., v 3 vitronectin receptor, 2 1 collagen receptor, or v 1 receptor) help maintain the seal.


Histology_Ross

Histology_Ross.  The basolateral region functions in the exocytosis of digested material (see Fig. 8.15). Transport vesicles containing degraded bone material endocytosed at the ruffled border fuse here with the cell membrane to release their contents. TRAP has been found within these vesicles, suggesting its role in the fragmentation of endocytosed material. Osteoclasts resorb bone tissue by releasing protons and lysosomal hydrolases into the constricted microenvironment of the extracellular space. Some, if not most, of the vesicles in the osteoclast are lysosomes. Their contents are released into the extracellular space in the clefts between the cytoplasmic processes of the ruffled border, a clear example of lysosomal enzymes functioning outside the cell. Once liberated, these hydrolytic enzymes, which include cathepsin K (a cysteine protease) and matrix metalloproteinases, degrade collagen and other proteins of the bone matrix.


Histology_Ross

Histology_Ross. Before digestion can occur, however, the bone matrix must be decalcified through acidification of the bony surface, which initiates dissolution of the mineral matrix. The cytoplasm of the osteoclast contains carbonic anhydrase II, which produces carbonic acid (H2CO3) from carbon dioxide and water. Subsequently, the carbonic acid dissociates to bicarbonate (HCO3) and a proton (H ). With the help of ATP-dependent proton pumps, protons are transported through the ruffled border, generating a low pH (4 to 5) in the microenvironment of the resorption bay. This local acidic environment created in the extracellular space between the bone and the osteoclast is protected by the clear zone. Chloride channels coupled with proton pumps facilitate the electroneutrality of the ruffled border membrane (see Fig. 8.15). Excess bicarbonate is removed by passive exchange with chloride ions via chloride–carbonate protein exchangers located at the basolateral membrane.


Histology_Ross

Histology_Ross. The acidic environment initiates the degradation of the mineral component of bone (composed primarily of hydroxyapatites) to calcium ions, soluble inorganic phosphates, and water. When resorption of designated bone tissue is completed, osteoclasts undergo apoptosis. Recent studies indicate that many drugs used to inhibit bone exocytosis of digested material lysosomes actin filaments vinculin, talin v 3 integrin receptors carbonic anhydrase chloride channel clear zone ruffled border ATP dependent proton pump Cl HCO3 HCO3 Cl H H CO2 H2O H2CO3 K cathepsin matrixmetalloproteinases


Histology_Ross

Histology_Ross. FIGURE 8.15 • Schematic drawing of an osteoclast. This drawing shows the structure of the osteoclasts and its three regions: the ruffled border, clear zone, and basolateral region. Note that the clear zone contains abundant actin filaments arranged in a ringlike structure surrounded on both sides by actin-binding proteins such as vinculin and talin. The plasma membrane at the site of the clear zone contains cell-to–extracellular matrix adhesion molecules (integrin receptors) that provide a tight seal between the plasma membrane and mineralized matrix of the bone. The pathways for proton and chloride transport are described in the text. resorption in osteoporosis (i.e., bisphosphonates and estrogens) promote osteoclast apoptosis (Folder 8.2). The phagocytotic function of osteoclasts is regulated by many factors.


Histology_Ross

Histology_Ross. resorption in osteoporosis (i.e., bisphosphonates and estrogens) promote osteoclast apoptosis (Folder 8.2). The phagocytotic function of osteoclasts is regulated by many factors. Numerous coated pits and coated vesicles are also present at the ruffled border, suggesting endocytotic activity. Osteoclasts are observed at sites where bone remodeling is in progress. (The process of remodeling is described in more detail shortly.) Thus, in sites where osteons are being altered or where a bone is undergoing change during the growth process, osteoclasts are relatively numerous.


Histology_Ross

Histology_Ross. An increase in parathyroid hormone (PTH) level promotes bone resorption and has a demonstrable effect on osteoclast activity, in addition to its effects on osteocytes, described previously. In contrast, calcitonin, secreted by parafollicular cells of the thyroid gland, has a counterbalancing effect, reducing osteoclast activity. PTH has also anabolic effect on bone either through a direct stimulatory effect on osteoblasts or indirectly through a mechanism that requires it to increase activity of osteoclasts. For example, PTH might stimulate bone formation directly by increasing the local production of IGF I or other bone-stimulating growth factors.


Histology_Ross

Histology_Ross. Other molecules that play an important role in regulating osteoclast activity include cathepsin K, carbonic anhydrase II, and proteins encoding the proton pump (TCIRG1). Deficiency of these proteins causes osteopetrosis, a congenital disease characterized by increased bone density and defective osteoclast function. In individuals with osteopetrosis, osteoclasts do not function properly, which causes bones to appear dense on X-ray; however, they are actually very fragile and break easily. The development of a bone is traditionally classified as endochondral or intramembranous.


Histology_Ross

Histology_Ross. The development of a bone is traditionally classified as endochondral or intramembranous. The distinction between endochondral and intramembranous formation rests on whether a cartilage model serves as the precursor of the bone (endochondral ossification) or whether the bone is formed by a simpler method, without the intervention of a cartilage precursor (intramembranous ossification). The bones of the extremities and those parts of the axial skeleton that bear weight (e.g., vertebrae) develop by endochondral ossification. The flat bones of the skull and face, the mandible, and the clavicle develop by intramembranous ossification.


Histology_Ross

Histology_Ross. The existence of two distinct types of ossification does not imply that existing bone is either membrane bone or endochondral bone. These names refer only to the mechanism by which a bone is initially formed. Because of the remodeling that occurs later, the initial bone tissue laid down by endochondral formation or by intramembranous formation is soon replaced. The replacement bone is established on the preexisting bone by appositional growth and is identical in both cases. Although the long bones are classified as being formed by endochondral formation, their continued growth involves the histogenesis of both endochondral and  FOLDER 8.2 Clinical Correlation: Osteoporosis


Histology_Ross

Histology_Ross. Osteoporosis, which literally means porous bone, is the most commonly occurring bone disease characterized by progressive loss of normal bone density accompanied by the deterioration of its microarchitecture. It is caused by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone deposition, resulting in de-creased bone mass, enhanced bone fragility, and in-creased risk of fracture. In healthy individuals, osteoclast activity is primarily regulated by PTH and to a lesser degree by IL-1 and TNF. In addition, differentiation of osteoclast precursors is under the influence of M-CSF and IL-6. Fe-male hormones known as estrogens (especially estradiol) inhibit formation of these cytokines, therefore limiting the activity of osteoclasts. In postmenopausal women in whom estrogen levels are reduced, secretion of these cytokines is increased, resulting in enhanced activity of osteoclasts leading to intensified bone resorption. Osteoporosis is a disease that affects


Histology_Ross

Histology_Ross. estrogen levels are reduced, secretion of these cytokines is increased, resulting in enhanced activity of osteoclasts leading to intensified bone resorption. Osteoporosis is a disease that affects an estimated 75 million people in the United States, Europe, and Japan, including one-third of postmenopausal women and most of the elderly popula-tion. It results in more than 1.3 million fractures annually in the United States There are three general types of osteoporosis. 1. Type I primary osteoporosis occurs in post-menopausal women. Since this type appears at an earlier stage of life than type II, its long-term effect is usually more severe than osteoporosis that de-velops in the later years of life. 2. Type II primary osteoporosis occurs in elderly individuals in their seventh or eighth decade of life and is the leading cause of serious morbidity and functional loss in this age group. 3. Secondary osteoporosis develops as a result of drug therapy (i.e., corticosteroids) or disease


Histology_Ross

Histology_Ross. decade of life and is the leading cause of serious morbidity and functional loss in this age group. 3. Secondary osteoporosis develops as a result of drug therapy (i.e., corticosteroids) or disease pro-cesses that may affect bone remodeling, including malnutrition, prolonged immobilization, weightless-ness (i.e., with space travel), and metabolic bone dis-eases (i.e., hyperparathyroidism, metastatic cancers). Osteoporotic bone has normal histologic structure; however, there is less tissue mass (Fig. F8.2.1). This results in weakened bones that are more prone to fractures follow-ing even minor trauma. Femoral head and neck fractures (commonly known as hip fractures), wrist fractures, and compressed vertebrae fractures are common injuries that frequently disable and confine an elderly person to a wheelchair. Individuals suffering from fractures are at greater risk for death, not directly from the fracture, but from the complications of hospitalization because of immo-bilization and


Histology_Ross

Histology_Ross. to a wheelchair. Individuals suffering from fractures are at greater risk for death, not directly from the fracture, but from the complications of hospitalization because of immo-bilization and increased risk of pneumonia, pulmonary thrombosis, and embolism. Traditional treatment of individuals with osteoporosis in-cludes an improved diet with vitamin D and calcium sup-plementation and moderate exercise to help slow further bone loss. In addition to diet and exercise, pharmacologic therapy directed toward slowing down bone resorption is employed. Until recently, the treatment of choice in post-menopausal women with osteoporosis was hormone re-placement therapy with estrogen and progesterone. Estrogen is known to retard bone resorption, thereby di-minishing bone loss. The results of the Women’s Health Initiative have shown that hormone replacement therapy can indeed reduce the risk of fractures; however, it causes greater risk of adverse cardiovascular diseases as FIGURE F8.2.1 •


Histology_Ross

Histology_Ross. Women’s Health Initiative have shown that hormone replacement therapy can indeed reduce the risk of fractures; however, it causes greater risk of adverse cardiovascular diseases as FIGURE F8.2.1 • Scanning electron micrograph of trabecular bone. a. This image shows section from the trabecular bone obtained from a vertebral body of a healthy individual. b. This specimen was obtained from a vertebral body of elderly women showing extensive signs of osteoporosis. Compare the pattern of trabecular architecture in osteoporosis with normal vertebral bone. (Courtesy of Dr. Alan Boyd). aba b continued next page chapter 8 Bone BONE FORMATION 233 intramembranous bone, with the latter occurring through the activity of the periosteal (membrane) tissue.


Histology_Ross

Histology_Ross. In intramembranous ossification, bone is formed by differentiation of mesenchymal cells into osteoblasts.


Histology_Ross

Histology_Ross. The first evidence of intramembranous ossification is seen around the eighth week of gestation in humans. Some of the pale-staining, elongated mesenchymal cells within the mesenchyme migrate and aggregate in specific areas, the sites where bone is destined to form. This condensation of cells within the mesenchymal tissue initiates the process of intramembranous ossification (Fig. 8.16 and Plate 15, page 252). Mesenchymal cells then differentiate into osteoprogenitor cells expressing Cbfa1 transcription factor. This transcription factor is essential for osteoblast differentiation and expression of genes necessary for both intramembranous and endochondral ossification. As the process continues, the newly organized tissue at the presumptive bone site becomes more vascularized, and the aggregated mesenchymal cells become larger and rounded. The cytoplasm of osteoprogenitor cells changes from eosinophilic to basophilic, and a clear Golgi area becomes evident. These cytologic changes result


Histology_Ross

Histology_Ross. mesenchymal cells become larger and rounded. The cytoplasm of osteoprogenitor cells changes from eosinophilic to basophilic, and a clear Golgi area becomes evident. These cytologic changes result in the differentiated osteoblast, which then secretes the collagens (mainly type I collagen molecules), bone sialoproteins, osteocalcin, and other components of the bone matrix (osteoid). The osteoblasts within the bone matrix become increasingly separated from one another as the matrix is produced, but they remain attached by thin cytoplasmic processes. Because of the abundant collagen content, the bone matrix appears denser than the surrounding mesenchyme, in which the intercellular spaces reveal only delicate connective tissue fibers.


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Histology_Ross.  FOLDER 8.3 Clinical Correlation: Nutritional Factors in Bone Formation


Histology_Ross

Histology_Ross. Both nutritional and hormonal factors affect the degree of bone mineralization. Calcium deficiency during growth causes rickets, a condition in which the bone matrix does not calcify normally. Rickets may be caused by insufficient amounts of dietary calcium or insufficient vitamin D (a steroid prohormone), which is needed for absorption of calcium by the intestines. An X-ray of the child with ad-vanced rickets presents classic radiological symptoms: bowed lower limbs (outward curve of long bones of the leg and thighs) and a deformed chest and skull (often having a distinctive “square” appearance). If rickets is not treated while children are still growing, then skeletal deformities and short stature may be permanent. In adults, the same nutritional or vitamin deficiency leads to osteomalacia. Although rickets and osteomalacia are no longer major health problems in populations where nutrition is adequate, it is among the most frequent childhood diseases in many developing countries. In


Histology_Ross

Histology_Ross. Although rickets and osteomalacia are no longer major health problems in populations where nutrition is adequate, it is among the most frequent childhood diseases in many developing countries. In addition to its influence on intestinal absorption of calcium, vitamin D is also needed for normal calcification. Other vitamins known to affect bone are vitamins A and C. Vitamin A deficiency suppresses endochondral growth of bone; vitamin A excess leads to fragility and subsequent frac-tures of long bones. Vitamin C is essential for synthesis of collagen, and its deficiency leads to scurvy. The matrix pro-duced in scurvy is not calcifiable. Another form of insufficient bone mineralization often seen in postmenopausal women is the condition known as osteoporosis (see Folder 8.2).


Histology_Ross

Histology_Ross. FIGURE 8.16 • Section of mandible developing by the process of intramembranous ossification. This photomicrograph shows a section from a developing mandible stained with H&E. In this relatively early stage of development, the mandible consists of bone spicules of various sizes and shapes. The bone spicules interconnect with each other and form trabeculae, providing the general shape of the developing bone (no cartilage model is present). The numerous osteoblasts responsible for this growing region of spicules are seen at the surface of the newly deposited bone. The older, calcified portion of spicules contains osteocytes surrounded by bone matrix. In the right portion of the figure, adjacent to the bone spicules, the connective tissue is very cellular and is developing into the early periosteum. 250. Newly formed bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae.


Histology_Ross

Histology_Ross. Newly formed bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae. With time, the matrix becomes calcified, and the interconnecting cytoplasmic processes of the bone-forming cells, now termed osteocytes, are contained within canaliculi. Concomitantly, more of the surrounding mesenchymal cells in the membrane proliferate, giving rise to a population of osteoprogenitor cells. Some of the osteoprogenitor cells come into apposition with the initially formed spicules, become osteoblasts, and add more matrix. By this process, called appositional growth, the spicules enlarge and become joined in a trabecular network with the general shape of the developing bone.


Histology_Ross

Histology_Ross. Through continued mitotic activity, the osteoprogenitor cells maintain their numbers and thus provide a constant source of osteoblasts for growth of the bone spicules. The new osteoblasts, in turn, lay down bone matrix in successive layers, giving rise to woven bone. This immature bone, discussed on page 223, is characterized internally by interconnecting spaces occupied by connective tissue and blood vessels. Bone tissue formed by the process just described is referred to as membrane bone or intramembranous bone. Endochondral ossification also begins with the proliferation and aggregation of mesenchymal cells at the site of the future bone. Under the influence of different fibroblastic growth factors (FGFs) and bone morphogenic proteins (BMPs) (see page 219), the mesenchymal cells initially express type II collagen and differentiate into chondroblasts that, in turn, produce cartilage matrix. Initially, a hyaline cartilage model with the general shape of the bone is formed.


Histology_Ross

Histology_Ross. Initially, a hyaline cartilage model with the general shape of the bone is formed. Once established, the cartilage model (a miniature version of the future definitive bone) grows by interstitial and appositional growth (Plate 13, page 248). The increase in the length of the cartilage model is attributed to interstitial growth. The increase in its width is largely the result of the addition of cartilage matrix produced by new chondrocytes that differentiate from the chondrogenic layer of the perichondrium surrounding the cartilage mass. Illustrations 1 of Figure 8.17 show an early cartilage model. The first sign of ossification is the appearance of a cuff of bone around the cartilage model.


Histology_Ross

Histology_Ross. At this stage, the perichondrial cells in the midregion of the cartilage model no longer give rise to chondrocytes. Instead, bone-forming cells or osteoblasts are produced. Thus, the connective tissue surrounding this portion of the cartilage is no longer functionally a perichondrium; rather, because of its altered role, it is now called periosteum. Moreover, because the cells within this layer are differentiating into osteoblasts, an osteogenic layer can now be identified within the periosteum. Because of these changes, a layer of bone is formed around the cartilage model (Plate 13, page 248). This bone can be classified as either periosteal bone, because of its location, or intramembranous bone, because of its method of development. In the case of a long bone, a distinctive cuff of periosteal bone, the bony collar, is established around the cartilage model in the diaphyseal portion of the developing bone. The bony collar is shown in illustration 2 of Figure 8.17.