Histology_Ross

Histology_Ross. With the establishment of the periosteal bony collar, the chondrocytes in the midregion of the cartilage model become hypertrophic. As the chondrocytes enlarge, their surrounding cartilage matrix is resorbed, forming thin irregular cartilage plates between the hypertrophic cells. The hypertrophic cells begin to synthesize alkaline phosphatase; concomitantly, the surrounding cartilage matrix undergoes calcification (see illustration 3 of Fig. 8.17). The calcification of the cartilage matrix should not be confused with mineralization that occurs in bone tissue.


Histology_Ross

Histology_Ross. FIGURE 8.17 • Schematic diagram of developing long bone. Illustrations 1 to 10 depict longitudinal sections through the long bone. The process begins with the formation of a cartilage model (1); next, a periosteal (perichondrial) collar of bone forms around the diaphysis (shaft) of the cartilage model (2); then, the cartilaginous matrix in the shaft begins to calcify (3). Blood vessels and connective tissue cells then erode and invade the calcified cartilage (4), creating a primitive marrow cavity in which remnant spicules of calcified cartilage remain at the two ends of the cavity. As a primary center of ossification develops, the endochondral bone is formed on spicules of calcified cartilage. The bone at the ends of the developing marrow cavity constitutes the metaphysis. Periosteal bone continues to form (5); the periosteal bone is formed as the result of intramembranous ossification. It can be recognized histologically because it is not accompanied by local cartilage erosion, nor


Histology_Ross

Histology_Ross. continues to form (5); the periosteal bone is formed as the result of intramembranous ossification. It can be recognized histologically because it is not accompanied by local cartilage erosion, nor is the bone deposited on spicules of calcified cartilage. Blood vessels and perivascular cells invade the proximal epiphyseal cartilage (6), and a secondary center of ossification is established in the proximal epiphysis (7). A similar epiphyseal (secondary) ossification center forms at the distal end of the bone (8), and an epiphyseal cartilage is thus formed between each epiphysis and the diaphysis. With continued growth of the long bone, the distal epiphyseal cartilage disappears (9), and finally, with cessation of growth, the proximal epiphyseal cartilage disappears (10). The metaphysis then becomes continuous with the epiphysis. Epiphyseal lines remain where the epiphyseal plate last existed.


Histology_Ross

Histology_Ross. The calcified cartilage matrix inhibits diffusion of nutrients, causing the death of the chondrocytes in the cartilage model. With the death of the chondrocytes, much of the matrix breaks down, and neighboring lacunae become confluent, producing an increasingly large cavity. While these events are occurring, one or several blood vessels grow through the thin diaphyseal bony collar to vascularize the cavity (see illustration 4 of Fig. 8.17). Mesenchymal stem cells migrate into the cavity along the growing blood vessels.


Histology_Ross

Histology_Ross. Mesenchymal stem cells residing in the developing periosteum migrate along the penetrating blood vessels and differentiate into osteoprogenitor cells in the bone marrow cavity. Hemopoietic stem cells (HSCs) also gain access to the cavity via the new vasculature, leaving the circulation to give rise to the marrow including all the blood cell lineages. As the calcified cartilage breaks down and is partially removed, some remains as irregular spicules. When the osteoprogenitor cells come in apposition to the remaining calcified cartilage spicules, they become osteoblasts and begin to lay down bone matrix (osteoid) on the spicule framework. Thus, the bone formed in this manner may be described as endochondral bone. This first site where bone begins to form in the diaphysis of a long bone is called the primary ossification center (see illustration 5 of Fig. 8.17). The combination of bone, which is initially only a thin layer, and the underlying calcified cartilage is described as a mixed


Histology_Ross

Histology_Ross. called the primary ossification center (see illustration 5 of Fig. 8.17). The combination of bone, which is initially only a thin layer, and the underlying calcified cartilage is described as a mixed spicule.


Histology_Ross

Histology_Ross. Histologically, mixed spicules can be recognized by their staining characteristics. Calcified cartilage tends to be basophilic, whereas bone is distinctly eosinophilic. With the Mallory stain, bone stains a deep blue, and calcified cartilage stains light blue (Fig. 8.18). Also, calcified cartilage no longer contains cells, whereas the newly produced bone may reveal osteocytes in the bone matrix. Such spicules persist for a short time before the calcified cartilage component is removed. The remaining bone component of the spicule may continue to grow by appositional growth, thus becoming larger and stronger, or it may undergo resorption as new spicules are formed. Growth of Endochondral Bone Endochondral bone growth begins in the second trimester of fetal life and continues into early adulthood.


Histology_Ross

Histology_Ross. Growth of Endochondral Bone Endochondral bone growth begins in the second trimester of fetal life and continues into early adulthood. The events described previously represent the early stage of endochondral bone formation that occurs in the fetus, beginning at about the twelfth week of gestation. The continuing growth process that lasts into early adulthood is described in the following section. Growth in length of long bones depends on the presence of epiphyseal cartilage. As the diaphyseal marrow cavity enlarges (see illustration 6 of Fig. 8.17), a distinct zonation can be recognized in the cartilage at both ends of the cavity. This remaining cartilage, referred to as epiphyseal cartilage, exhibits distinct


Histology_Ross

Histology_Ross. FIGURE 8.18 • Photomicrograph of a mixed bone spicule formed during endochondral bone formation. In this MalloryAzan–stained section, bone has been deposited on calcified cartilage spicules. In the center of the photomicrograph, the spicules have already grown to create an anastomosing trabecula. The initial trabecula still contains remnants of calcified cartilage, as shown by the light-blue staining of the calcified matrix compared with the dark-blue staining of the bone. In the upper part of the spicule, note a lone osteoclast (arrow) aligned near the surface of the spicule, where remodeling is about to be initiated. 275.


Histology_Ross

Histology_Ross. zones as illustrated in Figure 8.19 and Plate 14, page 250. During endochondral bone formation, the avascular cartilage is gradually replaced by vascularized bone tissue. This replacement is initiated by vascular endothelial growth factor (VEGF) and is accompanied by expression of genes responsible for production of type X collagen and matrix metalloproteases (enzymes responsible for degradation of cartilage matrix). The zones in the epiphyseal cartilage, beginning with the zone most distal to the diaphyseal center of ossification and proceeding toward that center, are the following.  The zone of reserve cartilage exhibits no cellular prolif eration or active matrix production.  The zone of proliferation is adjacent to the zone of re serve cartilage in the direction of the diaphysis. In this zone of reserve cartilage zone of proliferation zone of hypertrophy zone of calcified cartilage zone of resorption bone osteoclast blood vessel osteoblasts


Histology_Ross

Histology_Ross. FIGURE 8.19 • Longitudinal section through the distal end of a metatarsal bone of a 2-month-old infant. The epiphyseal (secondary) ossification center is well formed. Bone formation is taking place at both the epiphyseal and the diaphyseal surface of the epiphyseal plate. The zonation is apparent on the diaphyseal side because the growth rate there is so much greater than the epiphyseal ossification center. Because both centers are active, the zone of reserve cartilage is relatively narrow. H&E 280. (Reprinted with permission from Kelly DE, Wood RL, Enders AC. Bailey’s Textbook of Microscopic Anatomy. Baltimore: Williams & Wilkins, 1978.) zone, the cartilage cells undergo division and organize into distinct columns. These cells are larger than those in the reserve zone and actively produce collagen (mainly types II and XI) and other cartilage matrix proteins.


Histology_Ross

Histology_Ross.  The zone of hypertrophy contains greatly enlarged (hypertrophic) cartilage cells. The cytoplasm of these cells is clear, a reflection of the glycogen that they normally accumulate (which is lost during tissue preparation). Chondrocytes in this zone remain metabolically active; they continue to secrete type I collagen while increasing their secretion of type X collagen. Hypertrophic chondrocytes also secrete VEGF, which initiates vascular invasion. The cartilage matrix is compressed to form linear bands between the columns of hypertrophied cartilage cells.  In the zone of calcified cartilage, the hypertrophied cells begin to degenerate and the cartilage matrix becomes calcified. The calcified cartilage then serves as an initial scaffold for deposition of new bone. Chondrocytes positioned in the more proximal part of this zone undergo apoptosis.


Histology_Ross

Histology_Ross.  The zone of resorption is the zone nearest the diaphysis. The calcified cartilage here is in direct contact with the connective tissue of the marrow cavity. In this zone, small blood vessels and accompanying connective tissue invade the region previously occupied by the dying chondrocytes. They form a series of spearheads, leaving the calcified cartilage as longitudinal spicules. In a cross section, the calcified cartilage appears as a honeycomb because of the absence of the cartilage cells. The invading blood vessels are the source of osteoprogenitor cells, which will differentiate into bone-producing cells. Bone deposition occurs on the cartilage spicules in the same manner as described for the formation of the initial ossification center.


Histology_Ross

Histology_Ross. Bone deposition occurs on the cartilage spicules in the same manner as described for the formation of the initial ossification center. As bone is laid down on the calcified spicules, the cartilage is resorbed, ultimately leaving a primary spongy bone. This spongy bone undergoes reorganization through osteoclastic activity and addition of new bone tissue, thus accommodating the continued growth and physical stresses placed on the bone.


Histology_Ross

Histology_Ross. Shortly after birth, a secondary ossification center develops in the proximal epiphysis. The cartilage cells undergo hypertrophy and degenerate. As in the diaphysis, calcification of the matrix occurs, and blood vessels and osteogenic cells from the perichondrium invade the region, creating a new marrow cavity (see illustration 7 of Fig. 8.17). Later, a similar epiphyseal ossification center forms at the distal end of the bone (see illustration 8 of Fig. 8.17). This center is also regarded as a secondary ossification center, although it develops later. With the development of the secondary ossification centers, the only cartilage that remains from the original model is the articular cartilage at the ends of the bone and a transverse disc of cartilage, known as the epiphyseal growth plate, which separates the epiphyseal and diaphyseal cavities (Plate 13, page 248). Cartilage of the epiphyseal growth plate is responsible for maintaining the growth process.


Histology_Ross

Histology_Ross. Cartilage of the epiphyseal growth plate is responsible for maintaining the growth process. For a bone to retain proper proportions and its unique shape, both external and internal remodeling must occur as the bone grows in length. The proliferative zone of the epiphyseal plate gives rise to the cartilage on which bone is later laid down. In reviewing the growth process, it is important to realize the following:  The thickness of the epiphyseal plate remains relatively constant during growth.  The amount of new cartilage produced (zone of prolifera tion) equals the amount resorbed (zone of resorption).  The resorbed cartilage is, of course, replaced by spongy bone.


Histology_Ross

Histology_Ross. Actual lengthening of the bone occurs when new cartilage matrix is produced at the epiphyseal plate. Production of new cartilage matrix pushes the epiphysis away from the diaphysis, elongating the bone. The events that follow this incremental growth—namely, hypertrophy, calcification, resorption, and ossification—simply involve the mechanism by which the newly formed cartilage is replaced by bone tissue during development. Bone increases in width or diameter when appositional growth of new bone occurs between the cortical lamellae and the periosteum. The marrow cavity then enlarges by resorption of bone on the endosteal surface of the cortex of the bone. As bones elongate, remodeling is required. It consists of preferential epiphysis enlarges by growth of epiphyseal


Histology_Ross

Histology_Ross. FIGURE 8.20 • Diagram of external remodeling of a long bone. This diagram shows two periods during the growth of the bone. The younger bone profile (before remodeling) is shown on the right; the older (after remodeling), on the left. Superimposed on the left side of the figure is the shape of the bone (left half only) as it appeared at an earlier time. The bone is now longer, but it has retained its general shape. To grow in length and retain the general shape of the particular bone, bone resorption occurs on some surfaces, and bone deposition occurs on other surfaces, as indicated in the diagram. (Based on Ham AW. J Bone Joint Surg Am 1952;34:701.) resorption of bone in some areas and deposition of bone in other areas, as described previously and as outlined in Figure 8.20. When an individual achieves maximal growth, proliferation of new cartilage within the epiphyseal plate terminates.


Histology_Ross

Histology_Ross. When an individual achieves maximal growth, proliferation of new cartilage within the epiphyseal plate terminates. When the proliferation of new cartilage ceases, the cartilage that has already been produced in the epiphyseal plate continues to undergo the changes that lead to the deposition of new bone until, finally, there is no remaining cartilage. At this point, the epiphyseal and diaphyseal marrow cavities become confluent. The elimination of the epiphyseal plate is referred to as epiphyseal closure. In illustration 9 of Figure 8.17, the lower epiphyseal cartilage is no longer present; in illustration 10, both epiphyseal cartilages are gone. Growth is now complete, and the only remaining cartilage is found on the articular surfaces of the bone. Vestigial evidence of the site of the epiphyseal plate is reflected by an epiphyseal line consisting of bone tissue (see Fig. 8.2). Development of the Osteonal (Haversian) System Osteons typically develop in preexisting compact bone.


Histology_Ross

Histology_Ross. Development of the Osteonal (Haversian) System Osteons typically develop in preexisting compact bone. Compact bone can take several different forms. Compact bone may be formed from fetal spongy bone by continued deposition of bone on the spongy bone spicules; it may be deposited directly as adult compact bone (e.g., the circumferential lamellae of an adult bone); or it might be older compact bone consisting of osteons and interstitial lamellae. The process in which new osteons are formed is referred to as internal remodeling. During the development of new osteons, osteoclasts bore a tunnel, the resorption cavity, through compact bone.


Histology_Ross

Histology_Ross. During the development of new osteons, osteoclasts bore a tunnel, the resorption cavity, through compact bone. Formation of a new osteons in compact bone initially involves the creation of a tunnel-like space, the resorption cavity, by osteoclast activity. This resorption cavity will have the dimensions of the new osteon. When osteoclasts have produced an appropriately sized cylindrical tunnel by resorption of compact bone, blood vessels and their surrounding connective tissue occupy the tunnel. As the tunnel is occupied, new bone deposition on its wall begins almost immediately. These two aspects of cellular activity—namely, osteoclast resorption and osteoblast synthesis—constitute a bone-remodeling unit. A bone-remodeling unit consists of two distinct parts: an advancing cutting cone (also called a resorption canal) and a closing cone (Fig. 8.21). The tip of the cutting cone consists of advancing osteoclasts closely followed by an advancing capillary loop and pericytes. It


Histology_Ross

Histology_Ross. FIGURE 8.21 • Diagram of a bone-remodeling unit. A bone-remodeling unit consists of an advancing cutting cone and a closing cone. The cutting cone formed by osteoclasts is responsible for boring the tunnel or resorption cavity through the compact bone. Its action is initiated within the Haversian canal at the left of the diagram (in the area corresponding to section a). The cutting cone moves along the Haversian canal, in the direction indicated by the arrow, to the area corresponding to section d. Section d shows the cross section through the cutting cone. The resorption cavity is the site where the future osteon is formed by the action of the closing cone, which consists of osteoblasts. These cells begin to deposit the osteoid on the walls of the canal in successive lamellae. Gradual formation of the new bone fills the resorption cavity. Note the deposition of the osteoid deep to the osteoblasts seen in sections b and c. As successive lamellae of bone are deposited, the canal


Histology_Ross

Histology_Ross. formation of the new bone fills the resorption cavity. Note the deposition of the osteoid deep to the osteoblasts seen in sections b and c. As successive lamellae of bone are deposited, the canal ultimately attains the relatively narrow diameter of the mature Haversian canal, like that shown in section a. The growth-reversal line that appears at the outer limits of a newly formed osteon represents a border between the resorption activity of the cutting cone and the bony matrix not remodeled by this activity.


Histology_Ross

Histology_Ross. also contains numerous dividing cells that give rise to osteoblasts, additional pericytes, and endothelial cells. (Recall that osteoclasts are derived from mononuclear hemopoietic progenitor cells.) The osteoclasts drill a canal about 200 min diameter. This canal establishes the diameter of the future osteonal (Haversian) system. The cutting cone constitutes only a small fraction of the length of the bone-remodeling unit; thus, it is seen much less frequently than the closing cone. After the diameter of the future Haversian system is established, osteoblasts begin to fill the canal by depositing the organic matrix of bone (osteoid) on its walls in successive lamellae. With time, the bone matrix in each of the lamellae becomes mineralized. As the successive lamellae of bone are deposited, from the periphery inward, the canal ultimately attains the relatively narrow diameter of the adult osteonal canal. Compact adult bone contains Haversian systems of varying age and size.


Histology_Ross

Histology_Ross. Compact adult bone contains Haversian systems of varying age and size. Microradiographic examination of a ground section of bone reveals that younger Haversian systems are less completely mineralized than older systems (Fig. 8.22). They undergo a progressive secondary mineralization that continues (up to a point) even after the osteon has been fully formed. Figure 8.22 also illustrates the dynamic internal remodeling of compact bone. In the adult, deposition balances resorption. In the aged, resorption often exceeds deposition. If this imbalance becomes excessive, then osteoporosis develops (see Folder 8.2). Biologic mineralization is a cell-regulated extracellular event.


Histology_Ross

Histology_Ross. Biologic mineralization is a cell-regulated extracellular event. Mineralization occurs in the extracellular matrix of bone, cartilage and in the dentin, cementum, and enamel of teeth. The matrices of all of these structures except enamel contain collagen fibrils and ground substance. Mineralization is initiated in the same time within the collagen fibrils and in the ground substance surrounding them. In enamel, mineralization occurs within the extracellular matrix secreted by the enamel organ. Despite the extracellular location of biologic mineralization and the fact that physicochemical factors are basic to the process, biologic mineralization is a cell-regulated event. Mineralization involves the secretion of matrix vesicles into the bony matrix.


Histology_Ross

Histology_Ross. Mineralization involves the secretion of matrix vesicles into the bony matrix. In places where the mineralization of bone, cartilage, dentin, and cementum is initiated, the local concentration of Ca2 and PO4 ions in the matrix must exceed the normal threshold level. Several events are responsible for this mineralization:  The binding of extracellular Ca2 by osteocalcin and other sialoproteins creates a high local concentration of this ion.  The high Ca2 concentration stimulates the osteoblasts to secrete alkaline phosphatase (ALP), which increases the local concentration of PO4 ions. The high PO4 concentra where mineralization will be initiated.


Histology_Ross

Histology_Ross. FIGURE 8.22 • Microradiograph of the cross section of a bone. This 200-m-thick cross section of bone from a healthy 19-year-old male shows various degrees of mineralization in different osteons. Mature compact bone is actively replacing immature bone, which is seen on the periosteal (upper) surface. The degree of mineralization is reflected by the shade of light and dark in the microradiograph. Thus, very light areas represent the highly mineralized tissue that deflects the X-rays and prevents them from striking the photographic film. Conversely, dark areas contain less mineral and, thus, are less effective in deflecting the X-rays. Note that the interstitial lamellae (the older bone) are very light, whereas some of the osteons are very dark (these are the most newly formed). The Haversian canals appear black, as they represent only soft tissue. 157. (Courtesy of Dr. Jenifer Jowsey.)  At this stage of high extracellular Ca2 and PO4 concentration, the osteoblasts release small (50to


Histology_Ross

Histology_Ross. canals appear black, as they represent only soft tissue. 157. (Courtesy of Dr. Jenifer Jowsey.)  At this stage of high extracellular Ca2 and PO4 concentration, the osteoblasts release small (50to 200-nm) matrix vesicles into the bony matrix by exocytosis. The matrix vesicles contain ALP and pyrophosphatase that cleave PO 4 ions from other molecules of the matrix.


Histology_Ross

Histology_Ross.  The matrix vesicles that accumulate Ca2 and cleave PO4 ions cause the local isoelectric point to increase, which results in crystallization of CaPO4 in the surrounding matrix vesicles.  The CaPO4 crystals initiate matrix mineralization by the formation and deposition of hydroxyapatite crystals [Ca10(PO4)6(OH)2] in the matrix surrounding the osteoblasts. The osteoblast-derived matrix vesicles are the essential factors in controlling the initial site of mineral deposition in osteoid. Once the initial crystals of hydroxyapatite have precipitated, they grow rapidly by accretion until they join neighboring crystals produced around other matrix vesicles. In this way, a wave of mineralization sweeps through the osteoid. Other cells that produce osteoid are the ameloblasts and odontoblasts of developing teeth. Bone serves as a reservoir for body calcium.


Histology_Ross

Histology_Ross. Bone serves as a reservoir for body calcium. The maintenance of normal blood calcium levels is critical to health and life. Calcium may be delivered from the bone matrix to the blood if the circulating blood levels of calcium fall below a critical point (physiologic calcium concentration in the human ranges from 8.9 to 10.1 mg/dL). Conversely, excess blood calcium may be removed from the blood and stored in bone. These processes are regulated by parathyroid hormone (PTH), secreted by the parathyroid gland, and calcitonin, secreted by the parafollicular cells of the thyroid gland (see Folder 8.4).  PTH acts on the bone to raise low blood calcium levels to normal.  Calcitonin acts to lower elevated blood calcium levels to normal.


Histology_Ross

Histology_Ross.  PTH acts on the bone to raise low blood calcium levels to normal.  Calcitonin acts to lower elevated blood calcium levels to normal. PTH acts by stimulating both osteocytes and osteoclasts to resorb bone, allowing the release of calcium into the blood. As described previously (see page 227), resorption of bone by osteocytes constitutes osteocytic osteolysis. PTH also reduces excretion of calcium by the kidney and stimulates absorption of calcium by the small intestine. PTH further acts to maintain homeostasis by stimulating the kidney to excrete the excess phosphate produced by bone resorption. Calcitonin inhibits bone resorption, specifically inhibiting the effects of PTH on osteoclasts.


Histology_Ross

Histology_Ross. The classical concept of PTH action related to the regulation of serum calcium levels and bone resorption is more complex. For some time now, it has been known that PTH can also stimulate bone formation. In other words, it has an anabolic action (increases bone formation) in contrast to its catabolic action to cause bone resorption. In fact, clinical trials in which PTH hormone was administered to post-menopausal women with osteoporosis have shown significant increases in bone formation and bone mineral density. Increases in the amount of cancellous (trabecular) bone due to PTH treatment were shown in the ilium, vertebral bodies, and the shafts of radial and femoral bones (see Folder 8.2). The possible mechanisms behind this counterintuitive anabolic action of PTH are largely unknown. It is speculated that activation of different PTH-regulated genes might be responsible for each of the hormone’s contrasting effects. Bone can repair itself after injury.


Histology_Ross

Histology_Ross. Bone can repair itself after injury. The initial response to a fracture is similar to the response to any injury that produces tissue destruction and hemorrhage. Neutrophils are the first cells to arrive on the scene, followed by macrophages that begin to clean up the site of injury. Fibroblasts and capillaries then proliferate and grow into the site of injury. New loose connective tissue, granulation tissue, is formed, and as this tissue becomes denser, cartilage forms in parts of it. Both fibroblasts and periosteal cells participate in this phase of the healing process. The dense connective tissue and newly formed cartilage grow, covering the bone at the fracture site and producing a soft callus (Fig. 8.23). A callus will form whether or not the fractured parts of the bone are in immediate apposition to each other. The callus helps stabilize and bind together the fractured bone.  FOLDER 8.4 Functional Considerations: Hormonal Regulation of Bone Growth


Histology_Ross

Histology_Ross. Hormones other than PTH and calcitonin have major ef-fects on bone growth. One such hormone is pituitary growth hormone (GH, somatotropin). This hormone stimulates growth in general and, especially, growth of epiphyseal cartilage and bone. It acts directly on osteopro-genitor cells, stimulating them to divide and differentiate. Chondrocytes in epiphyseal growth plates are regulated by insulinlike growth factor I (IGF-I), which is primarily pro-duced by the liver in response to GH. In addition to IGF-I, insulin and thyroid hormones also stimulate chondrocyte activity. Oversecretion in childhood, caused by a defect in the mechanism regulating GH secretion or a GH-secreting tumor in the pituitary gland, leads to gigantism, an abnormal increase in the length of bones. Absence or hy-posecretion of GH in childhood leads to failure of growth of the long bones, resulting in pituitary dwarfism. Absence or severe hyposecretion of thyroid hormone dur-ing development and infancy leads to failure


Histology_Ross

Histology_Ross. of GH in childhood leads to failure of growth of the long bones, resulting in pituitary dwarfism. Absence or severe hyposecretion of thyroid hormone dur-ing development and infancy leads to failure of bone growth and dwarfism, a condition known as congenital hypothyroidism. When oversecretion of GH occurs in an adult, bones do not grow in length as a result of epiphy-seal closure. Instead, abnormal thickening and selective overgrowth of hands, feet, mandible, nose, and intramem-branous bones of the skull occurs. This condition, known as acromegaly, is caused by increased activity of osteoblasts on bone surfaces.


Histology_Ross

Histology_Ross. FIGURE 8.23 • Photomicrograph of fractured long bone undergoing repair. a. This low-magnification photomicrograph of a 3-week-old bone fracture, stained with H&E, shows parts of the bone separated from each other by the fibrocartilaginous callus. At this stage, the cartilage undergoes endochondral ossification. In addition, the osteoblasts of the periosteum are involved in secretion of new bony matrix on the outer surface of the callus. On the right of the microphotograph, the fibrocartilaginous callus is covered by periosteum, which also serves as the attachment site for the skeletal muscle. 35. b. Higher magnification of the callus from the area indicated by the upper rectangle in panel a shows osteoblasts lining bone trabeculae. Most of the original fibrous and cartilaginous matrix at this site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. 300. c. Higher magnification of the callus from the area indicated


Histology_Ross

Histology_Ross. site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. 300. c. Higher magnification of the callus from the area indicated by the lower rectangle in panel a. A fragment of old bone pulled away from the fracture site by the periosteum is now adjacent to the cartilage. It will be removed by osteoclast activity. The cartilage will calcify and be replaced by new bone spicules as seen in panel b. 300.


Histology_Ross

Histology_Ross. While the callus is forming, osteoprogenitor cells of the periosteum divide and differentiate into osteoblasts. The newly formed osteoblasts begin to deposit new bone on the outer surface of the bone at some distance from the fracture. This new formation of bone progresses toward the fracture site until new bone forms a bony sheath over the fibrocartilaginous callus. Osteogenic buds from the new bone invade the callus and begin to deposit new bone within the callus, gradually replacing the original fibrous and cartilaginous callus with a bony callus. The cartilage in the original callus calcifies and is replaced by bone as in endochondral ossification.


Histology_Ross

Histology_Ross. Endosteal proliferation and differentiation also occur in the marrow cavity, and medullary bone grows from both ends of the fracture toward the center. When this bone unites, the bony union of the fractured bone produced by the osteoblasts derived from both the periosteum and endosteum consists of spongy bone. As in normal bone formation, the spongy bone is gradually replaced by compact bone. While compact bone is being formed, the bony callus is removed by the action of osteoclasts, and gradual remodeling restores the bone to its original shape.


Histology_Ross

Histology_Ross. In healthy individuals, this process usually takes from 6 to 12 weeks, depending on the severity of the break and the particular bone that is broken. Setting the bone (i.e., reapproximating the normal structure) and holding the parts in place by internal fixation (by pins, screws, or plates) or by external fixation (by casts or by pins and screws) speeds the healing process and usually results in superior structural and functional restoration. PLATE 11 Bone, Ground Section


Histology_Ross

Histology_Ross. Bone is a specialized connective tissue characterized by a mineralized extracellular matrix. Calcium phosphate, in the form of hydroxyapatite crystals (Ca10(PO4)6OH2), is deposited along the collagen fibrils and in the proteoglycan ground substance. Bone serves as a storage site for calcium and phosphate, which can be released to the blood to maintain homeostatic levels. Osteocytes reside in lacunae in the bone ma-trix and extend fine cellular processes into canaliculi that connect the lacunae, thus forming a continuous network of cells within the mineral-ized tissue. Bones are organs of the skeletal system; bone tissue is the structural component of bones. Ground sections of bone are prepared from bone that has not been fixed but merely allowed to dry. Thin slices of the dried bone are then cut with a saw and further ground to a thinness that allows viewing in a light microscope. Slices may be treated with India ink to fill spaces that were formerly occupied by organic matter, e.g.,


Histology_Ross

Histology_Ross. cut with a saw and further ground to a thinness that allows viewing in a light microscope. Slices may be treated with India ink to fill spaces that were formerly occupied by organic matter, e.g., cells, blood vessels, and unmineralized matrix. A simpler method is to mount the ground specimen on a slide with a viscous medium that traps air in some of the spaces, as in the specimen in this plate. Here, some of the osteonal canals and a perforating canal are filled with the mounting medium, making them translucent instead of black. Specimens prepared in this manner are of value chiefly to display the architecture of the compact bone.


Histology_Ross

Histology_Ross. Ground bone, long bone, human, ×80. This figure reveals a cross-sectioned area of a long bone at low magnification and includes the outer or peripheral aspect of the bone, identified by the presence of circumferential lamellae (CL). (The exterior or periosteal surface of the bone is not included in the micrograph.) To their right are the osteons (O) or Haversian systems that appear as circular profiles. Between the osteons are interstitial lamellae (IL), the remnants of previously existing osteons.


Histology_Ross

Histology_Ross. Osteons are essentially cylindrical structures. In the shaft of a long bone, the long axes of the osteons are oriented parallel to the long axis of the bone. Thus, a cross section through the shaft of a long bone would reveal the osteons in cross section, as in this figure. At the center of each osteon is an osteonal (Haversian) canal (HC) that contains blood vessels, connective tissue, and cells lining the surface of the bone material. Because the organic material is not retained in ground sections, the Haversian canals and other spaces will appear black, as-they do here, if filled with India ink or air. Concentric layers of mineralized substance, the concentric lamellae, PLATE 11 • BONE, GROUND SECTION Ground bone-osteon, long bone, human, ×300.


Histology_Ross

Histology_Ross. PLATE 11 • BONE, GROUND SECTION Ground bone-osteon, long bone, human, ×300. This figure shows a higher-magnification micrograph of the labeled osteon from the upper figure. It includes some of the interstitial lamellae (IL) that are now seen at the bottom of the micrograph (the micrograph has been reoriented). Note the lacunae (L) and the fine thread-like profiles emanating from surround the Haversian canal and appear much the same as growth rings of a tree. The canal is also surrounded by concentric arrangements of lacunae. These appear as the small, dark, elongate structures.


Histology_Ross

Histology_Ross. During the period of bone growth and during adult life, there is constant internal remodeling of bone. This involves the destruction of osteons and formation of new ones. The breakdown of an osteon is usually not complete; however, part of the osteon may remain intact. Moreover, portions of adjacent osteons may also be partially destroyed. The space created by the breakdown process is reoccupied by a new osteon. The remnants of the previously existing osteons become the interstitial lamellae. Blood vessels reach the Haversian canals from the marrow through other tunnels called perforating (Volkmann’s) canals (VC). In some instances, as here, Volkmann’s canals travel from one Haversian canal to another. Volkmann’s canals can be distinguished from Haversian canals in that they pass through lamellae, whereas Haversian canals are surrounded by concentric rings of lamellae.


Histology_Ross

Histology_Ross. the lacunae. These thread-like profiles represent the canaliculi, spaces within the bone matrix that contained cytoplasmic processes of the osteocyte. The canaliculi of each lacuna communicate with canaliculi of neighboring lacunae to form a three-dimensional channel system throughout the bone. Ground bone, long bone, human, ×400.


Histology_Ross

Histology_Ross. Ground bone, long bone, human, ×400. In a still higher magnification, the circumferential lamellae are found around the shaft of the long bone at the outer as well as the inner surface of the bone. The osteoblasts that contribute to the formation of circumferential lamellae at these sites come from the periosteum and endosteum, respectively, whereas the osteons are constructed from osteoblasts in the canal of the developing Haversian system. This figure reveals not only the canaliculi but also the lamellae of the bone. The latter are just barely defined by the faint lines (arrows) that extend across the micrograph. Collagenous fibers in neighboring lamellae are oriented in different directions. This change in orientation accounts for the faint line or interface between adjacent lamellae. KEY CL, circumferential lamellae HC, Haversian canal IL, interstitial lamellae L, lacuna VC, Volkmann’s canal arrow, lamellar boundary


Histology_Ross

Histology_Ross. KEY CL, circumferential lamellae HC, Haversian canal IL, interstitial lamellae L, lacuna VC, Volkmann’s canal arrow, lamellar boundary PLATE 11 • BONE, GROUND SECTION VCVCILILHCHCCLCLOO CL HC IL ILILIL VC LL LL LL LL HCHCHC HCHCHC


Histology_Ross

Histology_Ross. Bone represents one of the specialized connective tissues. It is characterized by a mineralized extracellular matrix. It is the mineralization of the matrix that sets bone tissue apart from the other connective tissues and results in an extremely hard tissue that is capable of providing support and protection to the body. The mineral is calcium phosphate in the form of hydroxyapatite crystals. In addition to its supporting role, bone also provides a storage site for calcium and phosphate. Both can be mobilized from the bone matrix and taken up by the blood as needed to maintain normal levels. Bone matrix contains type I collagen and, in small amounts, a number of other types of collagen, i.e., types V, III, XI, and XIII. Other matrix proteins that constitute the ground substance of bone such as proteoglycan macromolecules, multiadhesive glycoproteins, growth factors, and cytokines are also present. Bone is typically studied in histological prepara-tions by removing the calcium content


Histology_Ross

Histology_Ross. as proteoglycan macromolecules, multiadhesive glycoproteins, growth factors, and cytokines are also present. Bone is typically studied in histological prepara-tions by removing the calcium content of the bone (decalcified bone), thus allowing it to be sectioned like other soft tissues. ORIENTATION MICROGRAPH: The orientation micrograph shows the upper end of a decal-cified humerus from an infant. The interior of the head of the bone, the epiphysis (E), consists of spongy cancellous bone made up of an anastomosing network of trabeculae (T) in the form of spicules of bone tissue. The outer portion consists of a dense layer of bone tissue known as compact bone (CB). Its thickness varies in different parts of the bone. The shaft of this bone, the diaphysis (D), is also made up of compact bone (CB) and in its interior, spongy bone (SB). Also within the shaft of the bone is bone marrow (BM), which at this stage of life is in the form of hemopoetic tissue. Lastly, cartilage is also a


Histology_Ross

Histology_Ross. bone (CB) and in its interior, spongy bone (SB). Also within the shaft of the bone is bone marrow (BM), which at this stage of life is in the form of hemopoetic tissue. Lastly, cartilage is also a component of the bone where it is present as an articular surface (AS) and as a growth plate (GP). The latter is described in a later plate. ASECBDCBCBSBBMCBTGPAS E CB D CB CB SB BM CB T GP


Histology_Ross

Histology_Ross. Compact bone, long bone, human, H&E, ×178. The area in the rectangle in the orientation micrograph containing compact bone in the epiphysis is shown here at higher magnification. The lighter staining area is cartilage (C). It serves as the articular surface of the epiphysis. Note the presence of isogenous groups of chondrocytes (Ch), a charac teristic feature of a growing cartilage. Below this is bone tissue (BT). It can Compact bone, long bone, human, H&E, ×135.


Histology_Ross

Histology_Ross. Compact bone, long bone, human, H&E, ×135. Bone from the diaphysis within the far right rectangle of the oritentation miocrograph is shown here at higher magnification. The outer surface of the bone is covered by dense connective tissue known as periosteum (P). The remaining tissue in be distinguished from the cartilage by the arrangement of its cells, the osteocytes (Oc). The osteocytes lie within the bone matrix, but are typically recognized only by their nuclei. Because bone matrix is laid down in layers (lamellae), bone characteristically shows linear or circular patterns that appear as striations. The irregular spaces seen within the bone tissue are vascular channels (VC) that contain, in addition to vessels, bone-forming tissue.


Histology_Ross

Histology_Ross. the micrograph is compact bone. The osteocytes (Oc) are again recognized by their nuclei within the bone matrix. Another feature worth noting in this growing bone is the presence of bone resorbing cells known as osteoclasts (Ocl). They are large multinucleated cells found at sites in bone where remodeling is taking place (see Plate 14). Spongy bone, long bone, human, H&E, ×135.


Histology_Ross

Histology_Ross. Spongy bone, long bone, human, H&E, ×135. The area in the rectangle in the orientation micrograph containing spongy bone in the epiphysis is shown here at higher magnification. Although the bone tissue at this site forms a three dimensional structure consisting of branching trabeculae, its structural organization and components are the same as that seen in compact bone. Note the osteocyte nuclei (N). As bone matures, the bone tissue becomes reorganized and forms osteons (O), which consist of a central vascular channel and surrounding layers (lamellae) of bone matrix. The two circular spaces are sites in which bone tissue has been removed and will be replaced by new tissue in the form of osteons. The spaces surrounding the spongy bone contain bone marrow consisting mainly of adipocytes. Other cells that have the capacity to form bone or hemopoetic tissue are also present.


Histology_Ross

Histology_Ross. KEY AS, articular surface BM, bone marrow BT, bone tissue C, cartilage CB, compact bone Ch, chondrocytes E, epiphysis GP, growth plate N, nuclei O, osteons Oc, osteocytes Ocl, osteoclasts P, periosteum SB, spongy bone T, trabeculae VC, vascular channels


Histology_Ross

Histology_Ross. Endochondral bone formation involves the continuing growth of a cartilage precursor, which serves as a fetal skeleton, and the simultane-ous removal of the cartilage and its replacement with bone tissue. In addition, as a bone grows, some of the bone tissue is removed while newer bone tissue is being laid down, a process called remodeling. Remodeling that alters the shape of the bone is called external remodel-ing; that which does not alter the shape of the bone, as in the formation of Haversian systems, is called internal remodeling. Two specialized cell types are identified with the process of bone growth and remodeling. The osteoblast is engaged in the formation of bone. Although the removal of bone is not as well described as its formation, it has been established that multinucleated cells, called osteo-clasts, are engaged in the removal of bone. Osteocytes, also, can alter and resorb bone in their immediate vicinity. The process is called os-teocytic osteolysis. It is important


Histology_Ross

Histology_Ross. cells, called osteo-clasts, are engaged in the removal of bone. Osteocytes, also, can alter and resorb bone in their immediate vicinity. The process is called os-teocytic osteolysis. It is important in calcium homeostasis, i.e., the maintenance of normal blood calcium concentrations.


Histology_Ross

Histology_Ross. Developing bone, short bone, monkey, H&E ×240. The early steps of endochondral bone formation are shown in this figure. The structure seen here is the cartilage model of the bone about to be formed. The steps of bone formation are 1. The cartilage (C) cells in the center of the cartilage model become hypertrophic (HC). 2. The matrix of the cartilage becomes calcified (CM). (The calcified matrix stains intensely with hematoxylin and appears as the darker condensed matrix material between the enlarged cartilage cells.) Developing bone, fetal fnger, human, H&E ×60.


Histology_Ross

Histology_Ross. Developing bone, fetal fnger, human, H&E ×60. The bone in this figure shows later events and a continuation of the earlier ones just described. A vascular bud (not shown) and accompanying perivascular cells from the periosteum have invaded the shaft of the cartilage model, resulting in the formation of a cavity (Cav). Examination at higher magnification would reveal that the cavity contains fat cells, hematopoietic tissue (the dark-blue–staining component), and other connective tissue elements. While the new steps of bone formation occur, the earlier steps continue: 1. Cartilage (C) cells proliferate at the epiphyses. They are responsible for production of new matrix material. It is this process that creates lengthening of the bone. 2. Periosteal bone (PB) continues to form. Developing bone, long bone, human, H&E ×60; inset ×200.


Histology_Ross

Histology_Ross. 2. Periosteal bone (PB) continues to form. Developing bone, long bone, human, H&E ×60; inset ×200. This shows an early stage after the invasion of the epiphysis. A secondary ossification center (Os) has formed, and along with this event, the head of the long bone will develop a marrow cavity similar in its content to that of the diaphysis. The 3. A collar of bone forms around the circumference of the center of the cartilage model. This bone is called periosteal bone (PB) because the osteoblasts that have produced the bone material develop from the periosteum. (Note that the periosteal bone is, in fact, intramembranous bone [see Plate 15], because it develops within the connective tissue membrane that immediately surrounds the developing bone and not on a spicule of calcified cartilage.)


Histology_Ross

Histology_Ross. PLATE 13 • E N DOCHON DRAL BON E FOR MATION I KEY C, cartilage Cav, marrow cavity CC, calcified cartilage CM, calcified matrix EB, endochondral bone EP, epiphyseal plate HC, hypertrophic cartilage cell JC, joint cavity Os, secondary ossification center PB, periosteal bone 3. Cartilage cells facing the cavity become hypertrophic. 4. Cartilage matrix becomes calcified. 5. Erosion of cartilage occurs, creating spicules of cartilage. 6. Bone forms on the spicules of the calcified cartilage at the erosion front; this bone is endochondral bone (EB).


Histology_Ross

Histology_Ross. 5. Erosion of cartilage occurs, creating spicules of cartilage. 6. Bone forms on the spicules of the calcified cartilage at the erosion front; this bone is endochondral bone (EB). As these processes continue in the shaft of the bone, one end of the cartilage model (the epiphysis) is invaded by blood vessels and connective tissue from the periosteum (periosteal bud), and it undergoes the same changes that occurred earlier in the shaft (except that no periosteal bone forms). This same process then occurs at the other end of the bone. Consequently, at each end of the developing long bone, a cartilaginous plate (epiphyseal plate) is created that lies between two sites of bone formation.


Histology_Ross

Histology_Ross. cartilage separating the two cavities is the epiphyseal plate (EP). At the early stage shown in this figure, the plate is not well defined. Despite the enlargement of the epiphyseal cavity, the remaining cartilage between the two cavities persists as a disc or plate until growth ceases. The inset shows some calcified cartilage as well as the deposition of endochondral bone (EB) within the secondary ossification center.


Histology_Ross

Histology_Ross. Endochondral bone formation is the principal process by which the long bones, e.g., the bones of the axial skeleton and the appendages and digits, increase in length to achieve their adult dimensions. So long as epiphyseal cartilage exists between the diaphyseal and epiphy-seal ossification centers, the bone will continue to grow. Cessation of bone growth is the result of the cessation of interstitial growth of the epiphyseal cartilages. X-ray examination of the bones of late adolescents can determine whether there is still an epiphyseal cartilage plate and, therefore, determine the potential for further growth in bone length and body height. Endochondral bone formation, epiphysis of long bone, human, H&E ×80; inset ×380.


Histology_Ross

Histology_Ross. This is a photomicrograph of an epiphysis at higher magnification than that seen in Plate 13. Different zones of the cartilage of the epiphyseal plate reflect the progressive changes that occur in active growth of endochondral bone. These zones are not sharply delineated, and the boundaries between them are somewhat arbitrary. They lead toward the marrow cavity (M), so that the first zone is fur 250 thest from the cavity. There are five zones:  Zone of reserve cartilage (RC). The cartilage cells of this zone have not yet begun to participate in the growth of the bone; thus, they are reserve cells. These cells are small, usually only one to a lacuna, and not grouped. At some time, some of these cells will proliferate and undergo the changes outlined for the next zone.  Zone of proliferating cartilage (PC). The cells of this zone are increasing in number; they are slightly larger than the reserve cells and close to their neighbors; they begin to form rows.


Histology_Ross

Histology_Ross.  Zone of hypertrophic cartilage (HC). The cells of this zone are aligned in rows and are significantly larger than the cells in the preceding zone.  Zone of calcified matrix (CM). In this zone the cartilage matrix is impregnated with calcium salts.  Zone of resorption (R). This zone is represented by eroded cartilage that is in direct contact with the connective tissue of the marrow cavity. Spicules (actually a honeycomb at the level of the advancing blood vessels) of cartilage are formed because the pericapillary cells invade and resorb in spearheads rather than along a straight front. Specifically, the pericapillary cells break into the rows of hypertrophied chondrocytes, temporarily leaving the calcified cartilage (C) between the rows of cells. In this manner, spicules of calcified cartilage are formed. Endochondral bone (EB) is then deposited on the surfaces of these calcified cartilage spicules by osteoblasts (Ob), thus forming mixed spicules as seen in the inset.


Histology_Ross

Histology_Ross. Endochondral bone formation, epiphysis of long bone, human, H&E ×150; inset ×380. This is a higher magnification of the lower middle area of upper figure. It shows calcified cartilage spicules on which bone has been deposited. In the lower portion of the figure, the spicules have already grown to create anastomosing bone trabeculae. These initial trabeculae still contain remnants of calcified cartilage, as shown by the bluish color of the cartilage matrix (compared with the red staining of the bone). Osteoblasts (Ob) are aligned on the surface of the spicules, where bone formation is active. The upper inset shows the surface of several spicules from the left circle in lower figure, at higher magnification. Note the osteoblasts (Ob), some of which are just beginning to produce bone in apposition to the calcified cartilage (C). The lower right of the inset shows bone (EB) with an osteocyte (Oc) already embedded in the bone matrix.


Histology_Ross

Histology_Ross. The lower inset, an enlargement of the right circle in lower figure, reveals several osteoclasts (Ocl). They are in apposition to the spicule, which is mostly cartilage. A small amount of bone is evident, based on the red-staining material in this inset. Note the light area (arrow) representing the ruffled border of the osteoclast. Examination of lower figure reveals a number of other osteoclasts (Ocl). • KEY CM, zone of calcified matrix C, calcified cartilage EB, endochondral bone HC, zone of hypertrophic cartilage M, marrow Ob, osteoblast Oc, osteocyte Ocl, osteoclast PC, zone of proliferating cartilage RC, zone of reserve cartilage arrow, ruffled border of osteoclast R, zone of resorption


Histology_Ross

Histology_Ross. Intramembranous bone formation is limited to those bones that are not required to perform an early supporting function, e.g., the flat bones of the skull. This process requires the proliferation and differentiation of cells of the mesenchyme to become osteoblasts, the bone-forming cells. They produce ground substance and collagen. This initial matrix, called osteoid, calcifies to form bone. As the osteoblasts continue to secrete their product, some are entrapped within their matrix and are then known as osteocytes. They are responsible for maintenance of the newly formed bone tissue. The remaining osteoblasts continue the bone deposition process at the bone surface. They are capable of reproducing to maintain an adequate population for continued growth. This newly formed bone appears first as spicules that enlarge and interconnect as growth proceeds, creating a three-dimensional trabecu-lar structure similar in shape to the future mature bone. The interstices contain blood vessels and


Histology_Ross

Histology_Ross. as spicules that enlarge and interconnect as growth proceeds, creating a three-dimensional trabecu-lar structure similar in shape to the future mature bone. The interstices contain blood vessels and connective tissue (mesenchyme). As the bone continues to grow, remodeling occurs. This involves resorption of localized areas of bone tissue by osteoclasts in order to maintain ap-propriate shape in relation to size and to permit vascular nourishment during the growth process.


Histology_Ross

Histology_Ross. Intramembranous bone formation, fetal head, human, Mallory trichrome ×45. A cross section of the developing mandible, as seen at this relatively early stage of development, consists of bone spicules (BS) of various sizes and shapes. The bone spicules interconnect and, in three dimensions, have the general shape of the mandible. Other structures present that will assist in orientation include Intramembranous bone formation, fetal head, human, Mallory trichrome ×350.


Histology_Ross

Histology_Ross. This higher-magnification micrograph of a portion of the field in lower left figure shows to advantage the distinction between newly deposited osteoid, which stains blue, and mineralized bone, which stains red. Osteoblasts are seen in two different levels of activity. Those that are relatively inactive and are in apposition to well-formed osteoid (Ob1) exhibit elongate nuclear profiles and appear to be flattened on the surface of the osteoid. Those osteoblasts developing teeth (DT), the tip of Meckel’s cartilage (MC), also referred to as the mandibular process, seen on the left side, and the oral cavity (OC). The bottom surface of the specimen shows the epidermis (Ep) of the underside of the chin. A large portion of the developing tongue is seen in the upper half of the figure. The tongue consists largely of developing striated visceral muscle fibers arranged in a three-dimensional orthogonal array that is characteristic of this organ.


Histology_Ross

Histology_Ross. Intramembranous bone formation, fetal head, human, Mallory trichrome ×175. This higher-magnification view of the boxed area in upper figure shows the interconnections of the bone spicules (BS) of the developing mandible. Within and around the spaces enclosed by the developing spicules is mesenchymal tissue. These mesenchymal cells will give rise to new osteoblasts as well as to the cells that will form the vascular components of the bone. The more dense connective tissue (CT) will differentiate into the periosteum on one side of the developing mandible. Other structures shown in the field include numerous blood vessels (BV) and the enamel organ of a developing tooth (DT).


Histology_Ross

Histology_Ross. (Ob2) that are actively secreting new osteoid appear as tall, columnar-like cells adjacent to osteoid. One of the spicules shows a cell completely surrounded by bone matrix; this is an osteoblast that has become trapped in its own secretions and is now an osteocyte (OC). At this magnification, the very loose connective tissue characteristics of the mesenchyme and the sparseness of the mesenchymal cells (MC) are well demonstrated. The highly cellular connective tissue (CT) on the right margin of the figure is the developing perichondrium. Some of its cells will also develop into osteoblasts to allow growth of the bone at its surface. • KEY BS, bone spicules BV, blood vessels CT, connective tissue DT, developing tooth EO, enamel organ Ep, epithelium MC, Meckel’s cartilage (Fig. 1.) MC, mesenchymal cells (Fig. 3.) Ob1 , inactive osteoblast Ob2 , active osteoblast OC, oral cavity (upper figure) OC, osteocyte (lower right figure)


Histology_Ross

Histology_Ross. OVERVIEW OF ADIPOSE TISSUE / 254 WHITE ADIPOSE TISSUE / 254 Function of White Adipose Tissue / 254 Differentiation of Adipocytes / 255 Structure of Adipocytes and Adipose Tissue / 256 Regulation of Adipose Tissue / 257 BROWN ADIPOSE TISSUE / 259 Folder 9.1 Clinical Correlation: Obesity / 261 Folder 9.2 Clinical Correlation: Adipose Tissue Tumors / 262 Folder 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference / 264 Adipose tissue is a specialized connective tissue that plays an important role in energy homeostasis. Individual fat cells, or adipocytes, and groups of adipocytes are found throughout loose connective tissue. Tissues in which adipocytes are the primary cell type are designated adipose tissue. Adipocytes play a key role in energy homeostasis.


Histology_Ross

Histology_Ross. For its survival, the body needs to ensure a continuous delivery of energy despite highly variable supplies of nutrients from the external environment. To meet the body’s energy demands when nutrient supplies are low, adipose tissue efficiently stores excess energy. The body has a limited capacity to store carbohydrate and protein, therefore energy reserves are stored within lipid droplets of adipocytes in the form of triglycerides. Triglycerides represent a dynamic form of energy storage that is added to when food intake is greater than energy expenditure and is tapped when energy expenditure is greater than food intake. The energy stored in adipocytes can be rapidly released for use at other sites in the body.


Histology_Ross

Histology_Ross. Triglycerides are the most concentrated form of metabolic energy storage available to humans. Because triglycerides lack water, they have about twice the energy density of carbohydrates and proteins. The energy density of triglycerides is approximately 37.7 kJ/g (9 cal/g), whereas the density of carbohydrates and proteins is 16.8 kJ/g (4 cal/g). In the event of food deprivation, triglycerides are an essential source of water and energy. Some animals can rely solely on metabolic water obtained from fatty-acid oxidation for maintenance of their water balance. For instance, the hump of a camel consists largely of adipose tissue and is a source of water and energy for this desert animal.


Histology_Ross

Histology_Ross. Adipocytes perform other functions in addition to their role as fat-storage containers. They also regulate energy metabolism by secreting paracrine and endocrine substances. The newly discovered secretory functions of adipocytes have shifted views on adipose tissue, which is now considered a major endocrine organ. Considerable evidence already exists to link increased endocrine activity of adipocytes to the metabolic and cardiovascular complications associated with obesity. There are two types of adipose tissue: white (unilocular) and brown (multilocular). The two types of adipose tissue, white adipose tissue and brown adipose tissue, are so named because of their color in the living state.  White adipose tissue is the predominant type in adult humans.  Brown adipose tissue is present in humans during fetal life but diminishes during the first decade after birth. Function of White Adipose Tissue


Histology_Ross

Histology_Ross. Function of White Adipose Tissue Functions of white adipose tissue include energy storage, insulation, cushioning of vital organs, and secretion of hormones.


Histology_Ross

Histology_Ross. White (unilocular) adipose tissue forms a layer called the panniculus adiposus [Lat. panniculus, a little garment; adipatus, fatty] or hypodermis in the connective tissue under the skin. Since the thermal conductivity of adipose tissue is only about half that of skeletal muscle, the subcutaneous layer of connective tissue provides a significant thermal insulation against cold by reducing the rate of heat loss. Concentrations of adipose tissue are found in the connective tissue under the skin of the abdomen, buttocks, axilla, and thigh. Sex differences in the thickness of this fatty layer in the skin of different parts of the body account, in part, for the differences in body contour between females and males. In both sexes, the mammary fat pad is a preferential site for accumulation of adipose tissue; the nonlactating female breast is composed primarily of this tissue. In the lactating female, mammary fat pad plays an important role in supporting breast function. It provides lipids


Histology_Ross

Histology_Ross. adipose tissue; the nonlactating female breast is composed primarily of this tissue. In the lactating female, mammary fat pad plays an important role in supporting breast function. It provides lipids and energy for milk production, but it is also a site for the synthesis of different growth factors that modulate responses to different steroid and proteins and hormones acting on mammary gland function.


Histology_Ross

Histology_Ross. Internally, adipose tissue is preferentially located in the greater omentum, mesentery, and retroperitoneal space and is usually abundant around the kidneys. It is also found in bone marrow and between other tissues, where it fills in spaces. In the palms of the hands and the soles of the feet, beneath the visceral pericardium (around the outside of the heart), and in the orbits around the eyeballs, adipose tissue functions as a cushion. It retains this structural function even during reduced caloric intake; when adipose tissue elsewhere becomes depleted of lipid, this structural adipose tissue remains undiminished. White adipose tissue produces a variety of hormones, growth factors, and cytokines.


Histology_Ross

Histology_Ross. White adipose tissue produces a variety of hormones, growth factors, and cytokines. Adipocytes actively synthesize and secrete hormones, growth factors, and cytokines. Leptin [Gr. leptos, thin], a 16-kilodalton peptide hormone involved in the regulation of energy homeostasis, is exclusively secreted by adipocytes. Leptin inhibits food intake and loss of body weight and stimulates metabolic rate. Thus, leptin fulfills the criteria for a circulating satiety factor that controls food intake when the body’s store of energy is sufficient. Leptin also participates in an endocrine signaling pathway that communicates the energy state of adipose tissue to brain centers that regulate food uptake. It acts on the central nervous system by binding to specific receptors, mainly in the hypothalamus. In addition, leptin communicates the fuel state of adipocytes from fat-storage sites to other metabolically active tissues (i.e., from adipose tissue to muscle at a different site).


Histology_Ross

Histology_Ross. In addition to leptin, adipose tissue secretes other hormones, including angiotensinogen (AGE), adiponectin, and resistin, and produces steroid hormones (testosterone, estrogens, and glucocorticoids). AGE is synthesized in other tissues, including the liver; increased production of this peptide contributes to hypertension (elevation of blood pressure), which is a frequent complication of obesity. Sex hormones and glucocorticoids are not synthesized de novo; instead they are converted from inactive forms by specific enzymes expressed in adipocytes. These enzymes can therefore inﬂuence the sex steroid profiles of obese individuals. Obesity-increased secretion of growth factors (tumor necrosis factor a [TNF-a], transforming growth factor b [TGF-b], and insulinlike growth factor I [IGF-I]) and cytokines (interleukin 6 and prostaglandins) may be linked to metabolic abnormalities and development of diabetes. Table 9.1 presents a summary of the molecules produced by adipocytes and their


Histology_Ross

Histology_Ross. and cytokines (interleukin 6 and prostaglandins) may be linked to metabolic abnormalities and development of diabetes. Table 9.1 presents a summary of the molecules produced by adipocytes and their functions.


Histology_Ross

Histology_Ross. Differentiation of Adipocytes White adipocytes differentiate from mesenchymal stem cells under the control of PPAR/RXR transcription factors.


Histology_Ross

Histology_Ross. Early histologists debated whether adipose tissue was a specific tissue, distinct from connective tissue, or ordinary connective tissue in which fibroblasts store fat globules. The current consensus is that adipocytes are a specific cell type derived from undifferentiated mesenchymal stem cells associated with the adventitia of small venules (Fig. 9.1). Current evidence suggests that a transcription factor called peroxisome proliferator–activated receptor gamma (PPAR) in complex with the retinoid X receptor (RXR) play a critical role in adipocyte differentiation and initiation of lipid metabolism. It induces the maturation of early lipoblasts (adipoblasts) or preadipocytes into fat cells of white adipose tissue. Most of the PPAR target genes in adipose tissue influence lipogenic pathways and initiate the storage of triglycerides. Therefore, PPAR/RXR is regarded as the “master switch” regulator in the white adipocytes’ differentiation.


Histology_Ross

Histology_Ross. White adipose tissue begins to form midway through fetal development. Lipoblasts initially develop from stromal-vascular cell along the small blood vessels in the fetus and are free of lipids. These cells are committed to becoming adipocytes at this early stage by expressing PPAR/RXR transcription factors. Collections of such cells are sometimes called primitive fat organs. They are characterized by proliferating early lipoblasts and proliferating capillaries. Lipid accumulation in lipoblasts produces the typical morphology of adipocytes. Early lipoblasts look like fibroblasts but develop small lipid inclusions and a thin external lamina.


Histology_Ross

Histology_Ross. Early lipoblasts look like fibroblasts but develop small lipid inclusions and a thin external lamina. Transmission electron microscopy (TEM) studies reveal that early lipoblasts have an elongated configuration, multiple cytoplasmic processes, and abundant endoplasmic reticulum and Golgi apparatus. As lipoblastic differentiation begins, vesicles increase in number, with a corresponding decrease in rough-surfaced endoplasmic reticulum (rER). Small lipid inclusions appear at one pole of the cytoplasm. Pinocytotic vesicles and an external lamina also appear. The presence of an external lamina is a feature that further distinguishes adipocytes from proper connective tissue cells. Midstage lipoblasts become ovoid as lipid accumulation changes the cell dimensions.


Histology_Ross

Histology_Ross. Midstage lipoblasts become ovoid as lipid accumulation changes the cell dimensions. With continued development, the early lipoblasts assume an oval configuration. The most characteristic feature at this stage is an extensive concentration of vesicles and small lipid droplets around the nucleus and extending toward both poles of the cell. Glycogen particles appear at the periphery of the lipid droplets, and pinocytotic vesicles and basal lamina become more apparent. These cells are designated midstage lipoblasts.


Histology_Ross

Histology_Ross. TABLE Summary of Molecules Synthesized and Secreted by Adipose Tissue and Their Functions 9.1 Molecule Major Function or Effect Acylation-stimulating Influences the rate of triglyceride synthesis in adipose tissue protein (ASP) Adiponectin, adipocyte Stimulates fatty-acid oxidation complement–related Decreases plasma triglycerides and glucose concentrations and increases insulin protein (ACRP30) sensitivity in cells Plays a role in the pathogenesis of familial combined hyperlipidemia Correlated with insulin resistance and hyperinsulinemia Adipophilin Serves as a specific marker for lipid accumulation in cells Adipsin Serine proteinase that regulates adipose tissue metabolism by facilitating fatty-acid storage and stimulating triglyceride synthesis Angiotensinogen (AGE) ACG is the precursor of vasoactive angiotensin II (AngII) that regulates blood pressure and Angiotensin II and electrolyte levels in the serum and is also involved in the metabolism and (AngII) differentiation of


Histology_Ross

Histology_Ross. of vasoactive angiotensin II (AngII) that regulates blood pressure and Angiotensin II and electrolyte levels in the serum and is also involved in the metabolism and (AngII) differentiation of adipose tissue During development, AngII inhibits differentiation of lipoblasts; in mature adipocytes it regulates lipid storage Insulinlike growth factor I Stimulates proliferation of a wide variety of cells and mediates many of the effects (IGF-I) of growth hormone Interleukin 6 (IL-6) Interacts with cells of immune system and regulates glucose and lipid metabolism Decreases activity of adipose tissue in cancer and other wasting disorders Leptin Regulates appetite and body energy expenditure Signals to the brain about body fat stores Increases formation of new vessels (angiogenesis) Involved in blood pressure control by regulating vascular tone Potent inhibitor of bone formation Plasminogen activator Inhibits the fibrinolytic system inhibitor-1 (PAI-1) Elevated levels are associated with


Histology_Ross

Histology_Ross. in blood pressure control by regulating vascular tone Potent inhibitor of bone formation Plasminogen activator Inhibits the fibrinolytic system inhibitor-1 (PAI-1) Elevated levels are associated with increased formation of blood clots Prostaglandins I2 and Helps regulate inflammation, blood clotting, ovulation, menstruation, F2, (PGI2 and PGF2) and acid secretion Resistin Increases insulin resistance Linked to obesity and to type 2 diabetes Transforming growth Regulates a wide variety of biologic responses, including proliferation, differentiation, factor (TGF-) apoptosis, and development Tumor necrosis factor Interferes with insulin receptor signaling and is a possible cause of development (TNF-) of insulin resistance in obesity (Modified from Frbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab 2001;280:E827–E847.)


Histology_Ross

Histology_Ross. The mature adipocyte is characterized by a single, large lipid inclusion surrounded by a thin rim of cytoplasm. In the late stage of differentiation, the cells increase in size and become more spherical. Small lipid droplets coalesce to form a single large lipid droplet that occupies the central portion of the cytoplasm. Smooth-surfaced endoplasmic reticulum (sER) is abundant, whereas rER is less prominent. These cells are designated late lipoblasts. Eventually, the lipid mass compresses the nucleus to an eccentric position, producing a signet-ring appearance in hematoxylin-and-eosin (H&E) preparations. Because these cells have a single lipid droplet, they are designated unilocular [Lat. unus, single; loculus, a little place] adipocytes or mature lipocytes. Structure of Adipocytes and Adipose Tissue Unilocular adipocytes are large cells, sometimes 100 m or more in diameter.


Histology_Ross

Histology_Ross. Structure of Adipocytes and Adipose Tissue Unilocular adipocytes are large cells, sometimes 100 m or more in diameter. When isolated, white adipocytes are spherical, but they may appear polyhedral or oval when crowded together in adipose are surrounded by reticular fibers (type III collagen), which are mesechymal stem cell secreted by the adipocytes. Special stains also reveal the pres ence of unmyelinated nerve fibers and numerous mast cells. A summary of white adipose tissue features is listed in Table 9.2.


Histology_Ross

Histology_Ross. FIGURE 9.1 • Development of adipose tissue cells. Like all connective tissue cells, adipocytes are derived from undifferentiated mesenchymal stem cells. By expressing PPAR-/ RXR transcription factors, they become committed to becoming early lipoblasts (preadipocytes) committed to white adipocyte lineage development. By expressing PRDM16/PGC-1 transcription factors, these cells will differentiate into early lipoblasts committed to brown adipocyte lineage development. Lipoblasts develop an external (basal) lamina and begin to accumulate numerous lipid droplets in their cytoplasm. In white adipose tissue, these droplets fuse to form a single large lipid droplet that ultimately fills the mature cell, compressing the nucleus, cytoplasm, and cytoplasmic organelles into a thin rim around the droplet. In brown adipose tissue, the individual lipid droplets remain separate.


Histology_Ross

Histology_Ross. tissue. Their large size is due to the accumulated lipid in the cell. The nucleus is flattened and displaced to one side of the lipid mass; the cytoplasm forms a thin rim around the lipid. In routine histologic sections, the lipid is lost through extraction by organic solvents such as xylene; consequently, adipose tissue appears as a delicate meshwork of polygonal profiles (Fig. 9.2). The thin strand of meshwork that separates adjacent adipocytes represents the cytoplasm of both cells and the extracellular matrix. The strand is usually so thin, however, that it is not possible to resolve its component parts in the light microscope. Adipose tissue is richly supplied with blood vessels, and capillaries are found at the angles of the meshwork where adjacent adipocytes meet. Silver stains show that adipocytes The lipid mass in the adipocyte is not membrane bounded.


Histology_Ross

Histology_Ross. The lipid mass in the adipocyte is not membrane bounded. The TEM reveals that the interface between the contained lipid and surrounding cytoplasm of the adipocyte is composed of a 5-nm-thick condensed layer of lipid reinforced by parallel vimentin flaments measuring 5 to 10 nm in diameter. This layer separates the hydrophobic contents of the lipid droplet from the hydrophilic cytoplasmic matrix. The perinuclear cytoplasm of the adipocyte contains a small Golgi apparatus, free ribosomes, short profiles of rER, microfilaments, and intermediate filaments. Filamentous mitochondria and multiple profiles of sER are also found in the thin rim of cytoplasm surrounding the lipid droplet (Fig. 9.3). Regulation of Adipose Tissue


Histology_Ross

Histology_Ross. Regulation of Adipose Tissue It is almost impossible to separate regulation of adipose tissue from digestive processes and functions of the central nervous system. These interconected hormonal and neural signals emanating from the adipose tissue, alimentary tract, and central nervous system form the brain–gut–adipose axis that regulates appetite, hunger, satiety, and energy homeostasis (Fig 9. 4). The amount of an individual’s adipose tissue is determined by two physiologic systems: One associated with short-term weight regulation, the other with long-term weight regulation.


Histology_Ross

Histology_Ross. The amount of an individual’s adipose tissue is determined by two physiologic systems: One associated with short-term weight regulation, the other with long-term weight regulation. The amount of adipose tissue in an individual is regulated by two physiologic systems. The first system, which is associated with short-term weight regulation, controls appetite and metabolism on a daily basis. Recently, two small peptide hormones produced in the gastrointestinal tract— ghrelin, an appetite stimulant, and peptide YY (PYY), an appetite suppressant—have been linked to this system. The second system, which is associated with long-term weight regulation, controls appetite and metabolism on a continual basis (over months and years). Two major hormones influence this system, leptin and insulin, along with other hormones, including thyroid hormone, glucocorticoids, and hormones of the pituitary gland (see Fig. 9.4).


Histology_Ross

Histology_Ross. Ghrelin and peptide YY control appetite as part of the short-term weight control system. The recently discovered potent appetite stimulant ghrelin is a small, 28-amino-acid polypeptide produced by gastric epithelial cells. In addition to its appetite stimulatory role, it acts on the anterior lobe of the pituitary gland to release growth hormone. In humans, ghrelin functions through receptors located in the hypothalamus, increasing the sense of hunger. As such it is considered a “meal initiator” factor. A genetic mutation in chromosome 15 causes Prader-Willi syndrome, in which an overproduction of ghrelin leads to morbid obesity. In individuals with this syndrome, compulsive eating and an obsession with food usually arise at an early age. The urge to eat


Histology_Ross

Histology_Ross. FIGURE 9.2 • White adipose tissue. a. Photomicrograph of white adipose tissue, showing its characteristic meshwork in an H&E–stained paraffin preparation. Each space represents a single large drop of lipid before its dissolution from the cell during tissue preparation. The surrounding eosin-stained material represents the cytoplasm of the adjoining cells and some intervening connective tissue. 320. b. High-power photomicrograph of a glutaraldehyde-preserved, plastic-embedded specimen of white adipose tissue. The cytoplasm of the individual adipose cells is recognizable in some areas, and part of the nucleus of one of the cells is included in the plane of section. A second nucleus (arrow), which appears intimately related to one of the adipose cells, may actually belong to a fibroblast; it is difficult to tell with assurance. Because of the large size of adipose cells, the nucleus is infrequently observed in a given cell. A capillary and a small venule are also evident in the


Histology_Ross

Histology_Ross. it is difficult to tell with assurance. Because of the large size of adipose cells, the nucleus is infrequently observed in a given cell. A capillary and a small venule are also evident in the photomicrograph. 950.


Histology_Ross

Histology_Ross. in these individuals is physiologic, overwhelming, and very difficult to control. If not treated, these individuals often die before age 30 of complications attributable to obesity. The small, 36-amino-acid long gastrointestinal hormone peptide YY is produced by the small intestine and plays an important role in promoting and maintaining weight loss by inducing a greater sense of fullness soon after a meal. It also acts through receptors in the hypothalamus that suppress appetite. It decreases food intake in individuals by inducing satiety or a sense of fullness and the desire to stop eating. In experimental clinical studies, the infusion of PYY into humans has been shown to reduce food intake by 33% over a period of 24 hours. Two hormones, leptin and insulin, are responsible for long-term regulation of body weight.


Histology_Ross

Histology_Ross. The discovery of the leptin (ob) gene, which encodes a fat-specific messenger RNA (mRNA) for leptin, has given some insight into the mechanism of energy homeostasis. In experimental animal models, the addition of recombinant leptin to obese, leptin-deficient ob/ob mice causes them to reduce their food intake and lose about 30% of their total body weight after 2 weeks of treatment. Unlike mutant mice, in most obese humans, levels of leptin mRNA in adipose tissue as well as serum levels of leptin are elevated. This was observed in all types of obesity, regardless of whether it is caused by genetic factors, hypothalamic lesions, or increased efficiency of food utilization. For unknown reasons, adipocytes in these obese individuals are resistant to leptin’s action, and administration of leptin does not reduce the amount of adipose tissue. Conversely, studies of individuals who have lost weight and those with anorexia nervosa show that leptin mRNA levels in their adipose tissue and serum


Histology_Ross

Histology_Ross. does not reduce the amount of adipose tissue. Conversely, studies of individuals who have lost weight and those with anorexia nervosa show that leptin mRNA levels in their adipose tissue and serum levels of leptin are significantly reduced. Recent clinical findings indicate that leptin most likely protects the body against weight loss in times of food deprivation.


Histology_Ross

Histology_Ross. Insulin, the pancreatic hormone that regulates blood glucose levels, is also involved in regulation of adipose tissue metabolism. It enhances the conversion of glucose into FIGURE 9.3 • Electron micrograph showing portions of two adjacent adipose cells. The cytoplasm of the adipose cells reveals mitochondria (M) and glycogen (the latter appears as the very dark particles). 15,000. Upper inset. Attenuated cytoplasm (Cy) of two adjoining adipose cells. Each cell is separated by a narrow space containing external (basal) lamina and an extremely attenuated process of a fibroblast. 65,000. Lower inset. The external (basal) lamina (BL) of the adipose cells appears as a discrete layer by which the cells are adequately separated from one another. F, fibroblast processes. 30,000.


Histology_Ross

Histology_Ross. the triglycerides of the lipid droplet by the adipocyte. Like leptin, insulin regulates weight by acting on brain centers in the hypothalamus. In contrast to leptin, insulin is required for the accumulation of adipose tissue. Antiobesity drug research is currently focusing on substances that can inhibit insulin and leptin signaling in the hypothalamus. Deposition and mobilization of lipid are influenced by neural and hormonal factors.


Histology_Ross

Histology_Ross. Deposition and mobilization of lipid are influenced by neural and hormonal factors. One of the major metabolic functions of adipose tissue involves the uptake of fatty acids from the blood and their conversion to triglyceride within the adipocyte. Triglyceride is then stored within the cell’s lipid droplet. When adipose tissue is stimulated by neural or hormonal mechanisms, triglycerides are broken down into glycerol and fatty acids, a process called mobilization. The fatty acids pass through the adipocyte cell membrane to enter a capillary. Here they are bound to the carrier protein albumin and transported to other cells, which use fatty acids as metabolic fuel.


Histology_Ross

Histology_Ross. Neural mobilization is particularly important during periods of fasting and exposure to severe cold. During the early stages of experimental starvation in rodents, adipose cells in a denervated fat pad continue to deposit fat. Adipose cells in the intact contralateral fat pad mobilize fat. It is now known that norepinephrine (which is liberated by the endings of nerve cells of the sympathetic nervous system) initiates a series of metabolic steps that lead to the activation of lipase. This enzyme splits triglycerides, which constitute more than 90% of the lipids stored in the adipocyte. This enzymatic activity is an early step in the mobilization of lipids.


Histology_Ross

Histology_Ross. Hormonal mobilization involves a complex system of hormones and enzymes that controls fatty-acid release from adipocytes. These include insulin, thyroid hormones, and adrenal steroids. Insulin is an important hormone that promotes lipid synthesis by stimulating lipid synthesis enzymes (fatty-acid synthase, acetyl-CoA carboxylase) and suppresses lipid degradation by inhibiting the action of hormone-sensitive lipase and thus blocking the release of fatty acids. Glucagon, another pancreatic hormone, and growth hormone from the pituitary gland both increase lipid utilization (lipolysis). In addition, elevated levels of tumor necrosis factor (TNF-) have been implicated as a causative factor in the development of insulin resistance associated with obesity and diabetes. Adipocytes of brown, multilocular adipose tissue contain numerous fat droplets. The cells of brown (multilocular) adipose tissue are smaller than those of white adipose tissue. The nucleus of a muscle, other tissues


Histology_Ross

Histology_Ross. The cells of brown (multilocular) adipose tissue are smaller than those of white adipose tissue. The nucleus of a muscle, other tissues FIGURE 9.4 • Regulation of energy homeostasis. This schematic diagram shows the relationship of adipose tissue to the central nervous system and gastrointestinal system within the brain– gut–adipose axis that is responsible for regulating energy homeostasis.


Histology_Ross

Histology_Ross. mature multilocular adipocyte is typically in an eccentric position within the cell, but it is not flattened as is the nucleus of a unilocular adipocyte. In routine H&E–stained sections, the cytoplasm of the multilocular adipocyte consists largely of empty vacuoles because the lipid that ordinarily occupies the vacuolated spaces is lost during preparation (Fig. 9.5). Multilocular adipocytes depleted of their lipid bear a closer resemblance to epithelial cells than to connective tissue cells. The multilocular adipocyte contains numerous mitochondria, a small Golgi apparatus, and only small amounts of rER and sER. The mitochondria contain large amounts of cytochrome oxidase, which imparts the brown color to the cells. Brown adipose tissue features are listed in Table 9.2. Brown adipose tissue, abundant in newborns, is markedly reduced in adults.


Histology_Ross

Histology_Ross. Brown adipose tissue, abundant in newborns, is markedly reduced in adults. Brown adipose tissue is present in large amounts in the newborn, which helps offset the extensive heat loss that results from the newborn’s high surface-to-mass ratio and avoid lethal hypothermia (a major risk of death for premature babies). In newborns, brown adipose tissue makes up about 5% of the total body mass and is located on the back, along the upper half of the spine, and toward the shoulders. The amount of brown adipose tissue gradually decreases as the body grows, but it remains widely distributed throughout the first decade of life in the cervical, axillary, paravertebral, mediastinal, sternal, and abdominal regions of the body. It then disappears from most sites except for regions around the kidney, adrenal glands, large vessels (i.e., aorta), and regions of the neck (deep cervical and supraclavicular), regions of the back (interscapular and paravertebral), and thorax (mediastinum).


Histology_Ross

Histology_Ross. Brown adipose tissue is subdivided into lobules by partitions of connective tissue, but the connective tissue stroma between individual cells within the lobules is sparse. The tissue has a rich supply of capillaries that enhance its color. Numerous unmyelinated nerve fibers are present among the fat cells. Brown adipocytes differentiate from mesenchymal stem cells under the control of PRDM16/PGC-1 transcription factors in the presence of catecholamines.


Histology_Ross

Histology_Ross. Brown adipocytes are derived from undifferentiated mesenchymal stem cells. In contrast to white adipocytes, differentiation of brown adipocytes is under the influence of a different pair of transcription factors. When the zinc-finger protein known as PR domain containing 16 (PRDM16) is activated, mesenchymal stem cells synthesize several members of the PPAR coactivator-1 (PGC-1) family of transcription factors. Therefore, PRDM16/PGC-1 is regarded as a “master switch” regulator in the brown adipocytes’ differentiation. These factors in turn regulate expression of genes (i.e., UPC-1) that control brown fat differentiation. The UPC-1 gene encodes a specific mitochondrial protein called uncoupling protein (UCP-1) or thermogenin (a 33-kilodalton inner mitochondrial membrane protein) is essential for brown adipocytes metabolism (thermogenesis). Clinical observations confirm that under normal conditions brown adipose tissue can expand in response to increased blood levels of norepinephrine.


Histology_Ross

Histology_Ross. for brown adipocytes metabolism (thermogenesis). Clinical observations confirm that under normal conditions brown adipose tissue can expand in response to increased blood levels of norepinephrine. This becomes evident in patients with pheochromocytoma, an endocrine tumor of adrenal medulla secreting excessive amounts of epinephrine and norepinephrine. In these individuals, the  FOLDER 9.1


Histology_Ross

Clinical Correlation: Obesity

Histology_Ross. Clinical Correlation: Obesity


Histology_Ross

Histology_Ross. Obesity is epidemic in the United States. According to current estimates by the National Institutes of Health (NIH), about two-thirds of Americans are considered to be obese, and 300,000 die annually from obesity-related metabolic diseases (i.e., diabetes, hypertension, cardiovascular dis-eases, and cancer). An individual is considered obese when the percentage of body fat exceeds the average per-centage for the individual’s age and sex. The prevalence of obesity has increased in the last decade from 12% to 18%. The increases are seen in both sexes and at all so-cioeconomic levels, with the greatest increase reported in the 18to 29-year-old age group. The body mass index (BMI), expressed as weight/ height2, is closely correlated with the total amount of body fat and is commonly used to classify overweight and obe-sity among adults. A BMI of approximately 25 kg/m2 is considered normal. A BMI greater than 27 kg/m2, which correlates to an excess body weight of approximately 20%, is


Histology_Ross

Histology_Ross. to classify overweight and obe-sity among adults. A BMI of approximately 25 kg/m2 is considered normal. A BMI greater than 27 kg/m2, which correlates to an excess body weight of approximately 20%, is considered a health risk. Obesity is associated with an increased risk of mortality as well as with many diseases such as hypertension, car-diovascular diseases, diabetes, and cancer. It is a chronic condition that develops as a result of an interaction be-tween a person’s genetic makeup and his or her environ-ment. Obesity genes encode the molecular components of the short-term and long-term weight regulation systems, which include leptin, ghrelin, and other factors that regulate energy balance. In addition, several of these factors modu-late glucose metabolism by adipose tissue and contribute to the development of insulin resistance, which is associ-ated with type 2 diabetes. Intensive research directed to-ward adipocyte-derived proteins may in the future provide potential drugs for


Histology_Ross

Histology_Ross. to the development of insulin resistance, which is associ-ated with type 2 diabetes. Intensive research directed to-ward adipocyte-derived proteins may in the future provide potential drugs for reducing obesity and overcoming insulin resistance. blood vesselblood vessel connectiveconnective tissue septatissue septa


Histology_Ross

Histology_Ross. FIGURE 9.5 • Brown adipose tissue. a. Photomicrograph of brown adipose tissue from a newborn in an H&E–stained paraffin preparation. The cells contain fat droplets of varying size. 150. b. This photomicrograph, obtained at a higher magnification, shows the brown adipose cells with round and often centrally located nuclei. Most of the cells are polygonal and closely packed, with numerous lipid droplets. In some cells, large lipid droplets displace nuclei toward the cell periphery. A network of collagen fibers and capillaries surrounds the brown adipose cells. 320. UCP-1 gene is activated by norepinephrine stimulation, which also protects brown adipocytes by inhibiting apoptosis.


Histology_Ross

Histology_Ross. UCP-1 gene is activated by norepinephrine stimulation, which also protects brown adipocytes by inhibiting apoptosis. In the past, it was thought that uncoupling proteins were expressed only in brown adipose tissue. Recently, several similar uncoupling proteins have been discovered in other tissues. UCP-2 is linked to hyperinsulimenia and obesity and may be involved in regulation of body weight. UPC-3 is expressed in skeletal muscles and may account for the thermogenic effects of thyroid hormone. UPC-4 is a brain-specific molecule. Metabolism of lipid in brown adipose tissue generates heat in process known as thermogenesis. Hibernating animals have large amounts of brown adipose tissue. The tissue serves as a ready source of lipid. When oxidized, it produces heat to warm the blood flowing through the brown fat on arousal from hibernation and in the maintenance of body temperature in the cold. This type of heat production is known as nonshivering thermogenesis.


Histology_Ross

Histology_Ross. Brown adipose tissue is also present in nonhibernating animals and humans and again serves as a source of heat. As in the mobilization of lipid in white adipose tissue, lipid is mobilized, and heat is generated by brown adipocytes when they are stimulated by the sympathetic nervous system. Therefore, normally present brown adipose tissue can most likely be induced and function in the context of human adaptive thermogenesis. Future research is being directed toward finding mechanisms for increased brown fat differentiation, which may potentially be an attractive treatment in both diet-induced and genetically acquired obesity.


Histology_Ross

Histology_Ross. The study of the numerous varieties of benign and malig-nant adipose tissue tumors provides further insight into, and confirmation of, the sequence of adipose tissue differ-entiation described above. As with epithelial tumors and tu-mors of fibroblast origin, the variety of adipose tissue tumors reflects the normal pattern of adipose tissue differ-entiation; that is, discrete tumor types can be described that consist primarily of cells resembling a given stage in normal adipose tissue differentiation. The most common benign tumor of adipose tissue of adulthood is the lipoma. It is more common than all other soft tissue tumors combined. Lipomas are subclassified by the morphology of the predominant cell in the tumor. For in-stance, the conventional lipoma consists of mature white adipocytes, whereas a fbrolipoma has adipocytes sur-rounded by an excess of fibrous tissue and an angio lipoma contains adipocytes separated by an unusually large num-ber of vascular channels. The majority of


Histology_Ross

Histology_Ross. whereas a fbrolipoma has adipocytes sur-rounded by an excess of fibrous tissue and an angio lipoma contains adipocytes separated by an unusually large num-ber of vascular channels. The majority of lipomas show structural chromosome aberrations that include balanced re-arrangements, often involving chromosome 12. Lipomas are usually found in subcutaneous tissues in middle-aged and elderly individuals. They are characterized as well-defined, soft, and painless masses of mature adipocytes usually found in the subcutaneous fascia of the back, thorax, and proximal parts of the upper and lower limbs. Treatment of lipomas usually involves a simple surgical excision. Malignant tumors of adipose tissue, called liposarco-mas, are rare. They are typically detected in older individu-als and are mainly found in the deep adipose tissues of the lower limbs, abdomen, and the shoulder area. Liposarco-mas may contain both well-differentiated, mature adipocytes and early, undifferentiated cells (Fig.


Histology_Ross

Histology_Ross. found in the deep adipose tissues of the lower limbs, abdomen, and the shoulder area. Liposarco-mas may contain both well-differentiated, mature adipocytes and early, undifferentiated cells (Fig. F9.2.1). Tumors containing more cells in earlier stages of differenti-ation are more aggressive and more frequently metasta-size. Typically, liposarcomas are surgically removed, but if a tumor has already metastasized, then both chemotherapy and radiation therapy can be utilized as a preor postsurgi-cal treatment. Although the term lipoma relates primarily to white adi-pose tissue tumors, tumors of brown adipose tissue are also found. Not surprisingly, these are called hibernomas. They are rare, benign, and slow-growing soft tissue tumors of brown fat most commonly arising in the periscapular region, axillary fossa, neck, or mediastinum. Most hibernomas contain a mixture of white and brown adipose tissue; pure hibernomas are very rare.


Histology_Ross

Histology_Ross.  FOLDER 9.2 Clinical Correlation: Adipose Tissue Tumors FIGURE F9.2.1 • Well-differentiated liposarcoma. This photomicrograph was obtained from a tumor surgically removed from the retroperitoneal space of the abdomen. Well-differentiated liposarcoma is characterized by a predominance of mature adipocytes that vary in size and shape. They are interspersed between broad fibrous septa of connective tissue containing cells (majority of them are fibroblasts) with atypical hyperchromatic nuclei. A relatively few scattered spindle cells with hyperchromatic and pleomorphic nuclei are found within connective tissue. 340. (Courtesy of Dr. Fabiola Medeiros.) Thermogenic activity of brown adipose tissue is facilitated by UCP-1 that is found at the inner mitochondrial membrane.