As the main constituent of the adult skeleton, bone tissue (Figure 8–1) provides solid support for the body, protects vital organs such as those in the cranial and thoracic cavities, and encloses internal (medullary) cavities containing bone marrow where blood cells are formed. Bone (or osseous) tissue also serves as a reservoir of calcium, phosphate, and other ions that can be released or stored in a controlled fashion to maintain constant concentrations in body fluids. Show
FIGURE 8–1 Components of bone.
A schematic overview of the basic features of bone, including the three key cell types: osteocytes, osteoblasts, and osteoclasts; their usual locations; and the typical lamellar organization of bone. Osteoblasts secrete the matrix that then hardens by calcification, trapping the differentiating cells now called osteocytes in individual lacunae. Osteocytes maintain the calcified matrix and receive nutrients from microvasculature in the central canals of the osteons via very small channels called canaliculi that interconnect the lacunae. Osteoclasts are monocyte-derived cells in bone required for bone remodeling. The periosteum consists of dense connective tissue, with a primarily fibrous layer covering a more cellular layer. Bone is vascularized by small vessels that penetrate the matrix from the periosteum. Endosteum covers all trabeculae around the marrow cavities. (Reproduced, with permission, from McKinley M, O'Loughlin VD. Human Anatomy. 2nd ed. New York, NY: McGraw-Hill; 2008; McKinley M, O'Loughlin VD. Human Anatomy. 3rd ed. New York, NY: McGraw-Hill; 2012; McKinley MP, O'Loughlin VD, Bidle TS. Anatomy & Physiology: An Integrative Approach. New York, NY: McGraw-Hill; 2013; McKinley MP, O'Loughlin VD, Bidle TS. Anatomy & Physiology: An Integrative Approach. 2nd ed. New York, NY: McGraw-Hill; 2016). In addition, bones form a system of levers that multiply the forces generated during skeletal muscle contraction and transform them into bodily movements. This mineralized tissue therefore confers mechanical and metabolic functions to the skeleton. Bone is a specialized connective tissue composed of calcified extracellular material, the bone matrix, and following three major cell types (Figure 8–2):
FIGURE 8–2 Bone tissue.
Newly formed bone tissue decalcified for sectioning and stained with trichrome in which the collagen-rich ECM appears bright blue. The tissue is a combination of mesenchymal regions (M) containing capillaries, fibroblasts, and osteoprogenitor stem cells and regions of normally calcified matrix with varying amounts of collagen and the three major cell types found in all bone tissue. Bone-forming osteoblasts (Ob) differentiate from osteoprogenitor cells in the periosteum and endosteum, and cover the surfaces of existing bone matrix. Osteoblasts secrete osteoid rich in collagen type I, but also containing proteoglycans and other molecules. As osteoid undergoes calcification and hardens, it entraps some osteoblasts that then differentiate further as osteocytes (Oc) occupying lacunae surrounded by bony matrix. The much less numerous large, multinuclear osteoclasts (Ocl), produced by the fusion of blood monocytes, reside on bony surfaces and erode the matrix during bone remodeling. (400X; Mallory trichrome) Because metabolites are unable to diffuse through the calcified matrix of bone, the exchanges between osteocytes and blood capillaries depend on communication through the very thin, cylindrical spaces of the canaliculi. All bones are lined on their internal and external surfaces by layers of connective tissue containing osteogenic cells—endosteum on the internal surface surrounding the marrow cavity and periosteum on the external surface. Because of its hardness, bone cannot be sectioned routinely. Bone matrix is usually softened by immersion in a decalcifying solution before paraffin embedding, or embedded in plastic after fixation and sectioned with a specialized microtome. OsteoblastsOriginating from mesenchymal stem cells, osteoblasts produce the organic components of bone matrix, including type I collagen fibers, proteoglycans, and matricellular glycoproteins such as osteonectin. Deposition of the inorganic components of bone also depends on osteoblast activity. Active osteoblasts are located exclusively at the surfaces of bone matrix, to which they are bound by integrins, typically forming a single layer of cuboidal cells joined by adherent and gap junctions (Figure 8–3). When their synthetic activity is completed, some osteoblasts differentiate as osteocytes entrapped in matrix-bound lacunae, some flatten and cover the matrix surface as bone lining cells, and the majority undergo apoptosis. FIGURE 8–3 Osteoblasts, osteocytes, and osteoclasts.
During the processes of matrix synthesis and calcification, osteoblasts are polarized cells with ultrastructural features denoting active protein synthesis and secretion. Matrix components are secreted at the cell surface in contact with existing bone matrix, producing a layer of unique collagen-rich material called osteoid between the osteoblast layer and the preexisting bone surface (Figure 8–3). This process of bone appositional growth is completed by subsequent deposition of calcium salts into the newly formed matrix. The process of matrix mineralization is not completely understood, but basic aspects of the process are shown in Figure 8–4. Prominent among the noncollagen proteins secreted by osteoblasts is the vitamin K-dependent polypeptide osteocalcin, which together with various glycoproteins binds Ca2+ ions and concentrates this mineral locally. Osteoblasts also release membrane-enclosed matrix vesicles rich in alkaline phosphatase and other enzymes whose activity raises the local concentration of PO43− ions. In the microenvironment with high concentrations of both these ions, matrix vesicles serve as foci for the formation of hydroxyapatite [Ca10(PO4)6(OH)2] crystals, the first visible step in calcification. These crystals grow rapidly by accretion of more mineral and eventually produce a confluent mass of calcified material embedding the collagen fibers and proteoglycans (Figure 8–4). FIGURE 8–4 Mineralization in bone matrix.
From their ends adjacent to the bone matrix, osteoblasts secrete type I collagen, several glycoproteins, and proteoglycans. Some of these factors, notably osteocalcin and certain glycoproteins, bind Ca2+ with high affinity, raising the local concentration of these ions. Osteoblasts also release very small membrane-enclosed matrix vesicles containing alkaline phosphatase and other enzymes. These enzymes remove PO4- ions from various matrix macromolecules, creating a high concentration of these ions locally. The high Ca2+ and PO4- ion concentrations cause calcified nanocrystals to form in and around the matrix vesicles. The crystals grow and mineralize further with formation of small growing masses of calcium hydroxyapatite [Ca10(PO4)6(OH)2], which surround the collagen fibers and all other macromolecules. Eventually the masses of hydroxyapatite merge as a confluent solid bony matrix as calcification of the matrix is completed. MEDICAL APPLICATION Cancer originating directly from bone cells (a primary bone tumor) is fairly uncommon (0.5% of all cancer deaths), although a cancer called osteosarcoma can arise in osteoprogenitor cells. The skeleton is often the site of secondary, metastatic tumors, however, arising when cancer cells move into bones via small blood or lymphatic vessels from malignancies in other organs, most commonly the breast, lung, prostate gland, kidney, or thyroid gland. OsteocytesAs mentioned some osteoblasts become surrounded by the material they secrete and then differentiate as osteocytes enclosed singly within the lacunae spaced throughout the mineralized matrix. During the transition from osteoblasts to osteocytes, the cells extend many long dendritic processes, which also become surrounded by calcifying matrix. The processes thus come to occupy the many canaliculi, 250-300 nm in diameter, radiating from each lacuna (Figures 8–5 and 8–1b). FIGURE 8–5 Osteocytes in lacunae.
(Figure 8–5c, used with permission from Dr Matt Allen, Indiana University School of Medicine, Indianapolis.) Diffusion of metabolites between osteocytes and blood vessels occurs through the small amount of interstitial fluid in the canaliculi between the bone matrix and the osteocytes and their processes. Osteocytes also communicate with one another and ultimately with nearby osteoblasts and bone lining cells via gap junctions at the ends of their processes. These connections between osteocyte processes and nearly all other bone cells in the extensive lacunar-canalicular network allow osteocytes to serve as mechanosensors detecting the mechanical load on the bone as well as stress- or fatigue-induced microdamage and to trigger remedial activity in osteoblasts and osteoclasts. Normally the most abundant cells in bone, osteocytes exhibit significantly less RER, smaller Golgi complexes, and more condensed nuclear chromatin than osteoblasts (Figure 8–5a). Osteocytes maintain the calcified matrix and their death is followed by rapid matrix resorption. While sharing most matrix-related activities with osteoblasts, osteocytes also express many different proteins, including factors with paracrine and endocrine effects that help regulate bone remodeling. MEDICAL APPLICATION The extensive network of osteocyte dendritic processes and other bone cells has been called a “mechanostat,” monitoring mechanical loads within bones and signaling cells to adjust ion levels and maintain the adjacent bone matrix accordingly. Resistance exercise can produce increased bone density and thickness in affected regions, while lack of exercise (or the weightlessness experienced by astronauts) leads to decreased bone density, due in part to the lack of mechanical stimulation of the bone cells. OsteoclastsOsteoclasts are very large, motile cells with multiple nuclei (Figure 8–6) that are essential for matrix resorption during bone growth and remodeling. The large size and multinucleated condition of osteoclasts are due to their origin from the fusion of bone marrow-derived monocytes. Osteoclast development requires two polypeptides produced by osteoblasts: macrophage-colony-stimulating factor (M-CSF; discussed with hemopoiesis, Chapter 13) and the receptor activator of nuclear factor-κB ligand (RANKL). In areas of bone undergoing resorption, osteoclasts on the bone surface lie within enzymatically etched depressions or cavities in the matrix known as resorption lacunae (or Howship lacunae). FIGURE 8–6 Osteoclasts and their activity.
Osteoclasts are large multinucleated cells that are derived by the fusion in bone of several blood-derived monocytes. (a) Photo of bone showing two osteoclasts (Ocl) digesting and resorbing bone matrix (B) in relatively large resorption cavities (or Howship lacunae) on the matrix surface. An osteocyte (Oc) in its smaller lacuna is also shown. (X400; H&E) (b) Diagram showing an osteoclast’s circumferential sealing zone where integrins tightly bind the cell to the bone matrix. The sealing zone surrounds a ruffled border of microvilli and other cytoplasmic projections close to this matrix. The sealed space between the cell and the matrix is acidified to ~pH 4.5 by proton pumps in the ruffled part of the cell membrane and receives secreted matrix metalloproteases and other hydrolytic enzymes. Acidification of the sealed space promotes dissolution of hydroxyapatite from bone and stimulates activity of the protein hydrolases, producing localized matrix resorption. The breakdown products of collagen fibers and other polypeptides are endocytosed by the osteoclast and further degraded in lysosomes, while Ca2+ and other ions are released directly and taken up by the blood. (c) SEM showing an active osteoclast cultured on a flat substrate of bone. A trench is formed on the bone surface by the slowly migrating osteoclast. (X5000) (Figure 8–6c, used with permission from Alan Boyde, Centre for Oral Growth and Development, University of London.) In an active osteoclast, the membrane domain that contacts the bone forms a circular sealing zone that binds the cell tightly to the bone matrix and surrounds an area with many surface projections, called the ruffled border. This circumferential sealing zone allows the formation of a specialized microenvironment between the osteoclast and the matrix in which bone resorption occurs (Figure 8–6b). Into this subcellular pocket the osteoclast pumps protons to acidify and promote dissolution of the adjacent hydroxyapatite, and releases matrix metalloproteinases and other hydrolytic enzymes from lysosome-related secretory vesicles for the localized digestion of matrix proteins. Osteoclast activity is controlled by local signaling factors from other bone cells. Osteoblasts activated by parathyroid hormone produce M-CSF, RANKL, and other factors that regulate the formation and activity of osteoclasts. MEDICAL APPLICATION In the genetic disease osteopetrosis, which is characterized by dense, heavy bones (“marble bones”), the osteoclasts lack ruffled borders and bone resorption is defective. This disorder results in overgrowth and thickening of bones, often with obliteration of the marrow cavities, depressing blood cell formation and causing anemia and the loss of white blood cells. The defective osteoclasts in most patients with osteopetrosis have mutations in genes for the cells’ proton-ATPase pumps or chloride channels. About 50% of the dry weight of bone matrix is inorganic materials. Calcium hydroxyapatite is most abundant, but bicarbonate, citrate, magnesium, potassium, and sodium ions are also found. Significant quantities of noncrystalline calcium phosphate are also present. The surface of hydroxyapatite crystals are hydrated, facilitating the exchange of ions between the mineral and body fluids. The organic matter embedded in the calcified matrix is 90% type I collagen, but also includes mostly small proteoglycans and multiadhesive glycoproteins such as osteonectin. Calcium-binding proteins, notably osteocalcin, and the phosphatases released from cells in matrix vesicles promote calcification of the matrix. Other tissues rich in type I collagen lack osteocalcin and matrix vesicles and therefore do not normally become calcified. The association of minerals with collagen fibers during calcification provides the hardness and resistance required for bone function. If a bone is decalcified by a histologist, its shape is preserved but it becomes soft and pliable like other connective tissues. Because of its high collagen content, decalcified bone matrix is usually acidophilic. External and internal surfaces of all bones are covered by connective tissue of the periosteum and endosteum, respectively (Figures 8–1a and 8–1c). The periosteum is organized much like the perichondrium of cartilage, with an outer fibrous layer of dense connective tissue, containing mostly bundled type I collagen, but also fibroblasts and blood vessels. Bundles of periosteal collagen, called perforating (or Sharpey) fibers, penetrate the bone matrix and bind the periosteum to the bone. Periosteal blood vessels branch and penetrate the bone, carrying metabolites to and from bone cells. The periosteum’s inner layer is more cellular and includes osteoblasts, bone lining cells, and mesenchymal stem cells referred to as osteoprogenitor cells. With the potential to proliferate extensively and produce many new osteoblasts, osteoprogenitor cells play a prominent role in bone growth and repair. Internally the very thin endosteum covers small trabeculae of bony matrix that project into the marrow cavities (Figure 8–1). The endosteum also contains osteoprogenitor cells, osteoblasts, and bone lining cells, but within a sparse, delicate matrix of collagen fibers. MEDICAL APPLICATION Osteoporosis, frequently found in immobilized patients and in postmenopausal women, is an imbalance in skeletal turnover so that bone resorption exceeds bone formation. This leads to calcium loss from bones and reduced bone mineral density (BMD). Individuals at risk for osteoporosis are routinely tested for BMD by dual-energy x-ray absorptiometry (DEXA scans). Gross observation of a bone in cross section (Figure 8–7) shows a dense area near the surface corresponding to compact (cortical) bone, which represents 80% of the total bone mass, and deeper areas with numerous interconnecting cavities, called cancellous (trabecular) bone, constituting about 20% of total bone mass. Histological features and important locations of the major types of bone are summarized in Table 8–1. TABLE 8–1Summary of bone types and their organization. View Table||Download (.pdf) TABLE 8–1 Summary of bone types and their organization.
FIGURE 8–7 Compact and cancellous bone.
Macroscopic photo of a thick section of bone showing the cortical compact bone and the lattice of trabeculae in cancellous bone at the bone’s interior. The small trabeculae that make up highly porous cancellous bone serve as supportive struts, collectively providing considerable strength, without greatly increasing the bone’s weight. The compact bone is normally covered externally with periosteum and all trabecular surfaces of the cancellous bone are covered with endosteum. (X10) In long bones, the bulbous ends—called epiphyses (Gr. epiphysis, an excrescence)—are composed of cancellous bone covered by a thin layer of compact cortical bone. The cylindrical part—the diaphysis (Gr. diaphysis, a growing between)—is almost totally dense compact bone, with a thin region of cancellous bone on the inner surface around the central marrow cavity (Figure 8–1). Short bones such as those of the wrist and ankle usually have cores of cancellous bone surrounded completely by compact bone. The flat bones that form the calvaria (skullcap) have two layers of compact bone called plates, separated by a thicker layer of cancellous bone called the diploë. At the microscopic level both compact and cancellous bones typically show two types of organization: mature lamellar bone, with matrix existing as discrete sheets, and woven bone, newly formed with randomly arranged components. Lamellar BoneMost bone in adults, compact or cancellous, is organized as lamellar bone, characterized by multiple layers or lamellae of calcified matrix, each 3-7 μm thick. The lamellae are organized as parallel sheets or concentrically around a central canal. In each lamella, type I collagen fibers are aligned, with the pitch of the fibers’ orientation shifted orthogonally (by about 90 degrees) in successive lamellae (Figure 8–1a). This highly ordered organization of collagen within lamellar bone causes birefringence with polarizing light microscopy; the alternating bright and dark layers are due to the changing orientation of collagen fibers in the lamellae (Figure 8–8). Like the orientation of wood fibers in plywood the highly ordered, alternating organization of collagen fibers in lamellae adds greatly to the strength of lamellar bone. FIGURE 8–8 Lamellar bone.
Two photographs of the same area of an unstained section of compact bone, showing osteons with concentric lamellae around central canals. Lamellae are seen only faintly by bright-field microscopy (a), but they appear as alternating bright and dark bands under the polarizing light microscope (b). Bright bands are due to birefringence from the highly ordered collagen fibers in a lamella. Alternating bright and dark bands indicate that fibers in successive lamellae have different orientations, an organization that makes lamellar bone very strong. (Both X100) (Used with permission from Dr Matt Allen, Indiana University School of Medicine, Indianapolis.) An osteon (or Haversian system) refers to the complex of concentric lamellae, typically 100-250 μm in diameter, surrounding a central canal that contains small blood vessels, nerves, and endosteum (Figures 8–1 and 8–9). Between successive lamellae are lacunae, each with one osteocyte, all interconnected by the canaliculi containing the cells’ dendritic processes (Figure 8–9). Processes of adjacent cells are in contact via gap junctions, and all cells of an osteon receive nutrients and oxygen from vessels in the central canal (Figure 8–1). The outer boundary of each osteon is a layer called the cement line that includes many more noncollagen proteins in addition to mineral and collagen. FIGURE 8–9 An osteon.
Osteons (Haversian systems) constitute most of the compact bone. Shown here is an osteon with four to five concentric lamellae (L) surrounding the central canal (CC). Osteocytes (O) in lacunae are in communication with each other and with the central canal and periphery of the osteon via through hundreds of dendritic processes located within fine canaliculi (C). Also shown are the partial, interstitial lamellae (I) of an osteon that was eroded when the intact osteon was formed. (Ground bone; X500) (Reproduced, with permission, from Berman I. Color Atlas of Basic Histology. 3rd ed. New York, NY: McGraw-Hill; 2003). Each osteon is a long, sometimes bifurcated, cylinder generally parallel to the long axis of the diaphysis. Each has 5-20 concentric lamellae around the central canal that communicates with the marrow cavity and the periosteum. Canals also communicate with one another through transverse perforating canals (or Volkmann canals) that have few, if any, concentric lamellae (Figures 8–1 and 8–10). All central osteonic canals and perforating canals form when matrix is laid down around areas with preexisting blood vessels. FIGURE 8–10 Lamellar bone: Perforating canals and interstitial lamellae.
Scattered among the intact osteons are numerous irregularly shaped groups of parallel lamellae called interstitial lamellae. These structures are lamellae remaining from osteons partially destroyed by osteoclasts during growth and remodeling of bone (Figure 8–10). Compact bone (eg, in the diaphysis of long bones) also includes parallel lamellae organized as multiple external circumferential lamellae immediately beneath the periosteum and fewer inner circumferential lamellae around the marrow cavity (Figure 8–1a). The lamellae of these outer and innermost areas of compact bone enclose and strengthen the middle region containing vascularized osteons. Bone remodeling occurs continuously throughout life. In compact bone, remodeling resorbs parts of old osteons and produces new ones. As shown in Figure 8–11 osteoclasts remove old bone and form small, tunnel-like cavities. Such tunnels are quickly invaded by osteoprogenitor cells from the endosteum or periosteum and sprouting loops of capillaries. Osteoblasts develop, line the wall of the tunnels, and begin to secrete osteoid in a cyclic manner, forming a new osteon with concentric lamellae of bone and trapped osteocytes (Figure 8–11). In healthy adults 5%-10% of the bone turns over annually. FIGURE 8–11 Development of an osteon.
During remodeling of compact bone, osteoclasts act as a cutting cone that tunnels into existing bone matrix. Behind the osteoclasts, a population of osteoblast progenitors enters the newly formed tunnel and lines its walls. The osteoblasts secrete osteoid in a cyclic manner, producing layers of new matrix (lamellae), and trapping some cells (future osteocytes) in lacunae. The tunnel becomes constricted with multiple concentric layers of new matrix, and its lumen finally exists as only a narrow central canal with small blood vessels. The dashed lines in (a) indicate the levels of the structures shown in cross section (b). An x-ray image (c) shows the different degrees of mineralization in osteons and in interstitial lamellae (I). MEDICAL APPLICATION The antibiotic tetracycline is a fluorescent molecule that binds newly deposited osteoid matrix during mineralization with high affinity and specifically labels new bone under the UV microscope (Figure 8–12). This discovery led to methods for measuring the rate of bone growth, an important parameter in the diagnosis of certain bone disorders. In one technique tetracycline is administered twice to patients, with an intervening interval of 11-14 days. A bone biopsy is then performed, sectioned without decalcification, and examined. Bone formed while tetracycline was present appears as fluorescent lamellae and the distance between the labeled layers is proportional to the rate of bone appositional growth. This procedure is of diagnostic importance in such diseases as osteomalacia, in which mineralization is impaired, and osteitis fibrosa cystica, in which increased osteoclast activity results in removal of bone matrix and fibrous degeneration. FIGURE 8–12
Newly formed bone can be labeled with tetracycline, which forms fluorescent complexes with calcium at ossification sites and provides an in vivo tracer by which newly formed bone can be localized. A group of osteons in bone after tetracycline incorporation in vivo seen with bright-field (a) and fluorescent microscopy (b) reveals active ossification in one osteon (center) and in the external circumferential lamellae (upper right). (Used with permission from Dr Matt Allen, Indiana University School of Medicine, Indianapolis.) Woven BoneWoven bone is nonlamellar and characterized by random disposition of type I collagen fibers and is the first bone tissue to appear in embryonic development and in fracture repair. Woven bone is usually temporary and is replaced in adults by lamellar bone, except in a very few places in the body, for example, near the sutures of the calvaria and in the insertions of some tendons. In addition to the irregular, interwoven array of collagen fibers, woven bone typically has a lower mineral content (it is more easily penetrated by x-rays) and a higher proportion of osteocytes than mature lamellar bone. These features reflect the facts that immature woven bone forms more quickly but has less strength than lamellar bone. Bone development or osteogenesis occurs by one of two processes:
The names refer to the mechanisms by which the bone forms initially; in both processes woven bone is produced first and is soon replaced by stronger lamellar bone. During growth of all bones, areas of woven bone, areas of bone resorption, and areas of lamellar bone all exist contiguous to one another. MEDICAL APPLICATION Osteogenesis imperfecta, or “brittle bone disease,” refers to a group of related congenital disorders in which the osteoblasts produce deficient amounts of type I collagen or defective type I collagen due to genetic mutations. Such defects lead to a spectrum of disorders, all characterized by significant fragility of the bones. The fragility reflects the deficit in normal collagen, which normally reinforces and adds a degree of resiliency to the mineralized bone matrix. Intramembranous OssificationIntramembranous ossification, by which most flat bones begin to form, takes place within condensed sheets (“membranes”) of embryonic mesenchymal tissue. Most bones of the skull and jaws, as well as the scapula and clavicle, are formed embryonically by intramembranous ossification. Within the condensed mesenchyme bone formation begins in ossification centers, areas in which osteoprogenitor cells arise, proliferate, and form incomplete layers of osteoblasts around a network of developing capillaries. Osteoid secreted by the osteoblasts calcifies as described earlier, forming small irregular areas of woven bone with osteocytes in lacunae and canaliculi (Figure 8–13). Continued matrix secretion and calcification enlarges these areas and leads to the fusion of neighboring ossification centers. The anatomical bone forms gradually as woven bone matrix is replaced by compact bone that encloses a region of cancellous bone with marrow and larger blood vessels. Mesenchymal regions that do not undergo ossification give rise to the endosteum and the periosteum of the new bone. FIGURE 8–13 Intramembranous ossification.
A section of fetal pig mandible developing by intramembranous ossification. (a) Areas of typical mesenchyme (M) and condensed mesenchyme (CM) are adjacent to layers of new osteoblasts (O). Some osteoblasts have secreted matrices of bone (B), the surfaces of which remain covered by osteoblasts. Between these thin regions of new woven bone are areas with small blood vessels (V). (X40; H&E) (Reproduced, with permission, from Berman I. Color Atlas of Basic Histology. 3rd ed. New York, NY: McGraw-Hill; 2003). (b) At higher magnification another section shows these same structures, but also includes the developing periosteum (P) adjacent to the masses of woven bone that will soon merge to form a continuous plate of bone. The larger mesenchyme-filled region at the top is part of the developing marrow cavity. Osteocytes in lacunae can be seen within the bony matrix. (X100; H&E) In cranial flat bones, lamellar bone formation predominates over bone resorption at both the internal and external surfaces. Internal and external plates of compact bone arise, while the central portion (diploë) maintains its cancellous nature. The fontanelles or “soft spots” on the heads of newborn infants are areas of the skull in which the membranous tissue is not yet ossified. Endochondral OssificationEndochondral (Gr. endon, within + chondros, cartilage) ossification takes place within hyaline cartilage shaped as a small version, or model, of the bone to be formed. This type of ossification forms most bones of the body and is especially well studied in developing long bones, where it consists of the sequence of events shown in Figure 8–14. FIGURE 8–14 Osteogenesis of long bones by endochondral ossification.
This process, by which most bones form initially, begins with embryonic models of the skeletal elements made of hyaline cartilage (1). Late in the first trimester, a bone collar develops beneath the perichondrium around the middle of the cartilage model, causing chondrocyte hypertrophy in the underlying cartilage (2). This is followed by invasion of that cartilage by capillaries and osteoprogenitor cells from what is now the periosteum to produce a primary ossification center in the diaphysis (3). Here osteoid is deposited by the new osteoblasts, undergoes calcification into woven bone, and is then remodeled as compact bone. (4) Around the time of birth secondary ossification centers begin to develop by a similar process in the bone’s epiphyses. During childhood the primary and secondary ossification centers gradually come to be separated only by the epiphyseal plate (5) that provides for continued bone elongation. The two ossification centers do not merge until the epiphyseal plate disappears (6) when full stature is achieved. (Reproduced, with permission, from McKinley M, O'Loughlin VD. Human Anatomy. 2nd ed. New York, NY: McGraw-Hill; 2008). In this process ossification first occurs within a bone collar produced by osteoblasts that differentiate within the perichondrium (transitioning to periosteum) around the cartilage model diaphysis. The collar impedes diffusion of oxygen and nutrients into the underlying cartilage, causing local chondrocytes to swell up (hypertrophy), compress the surrounding matrix, and initiate its calcification by releasing osteocalcin and alkaline phosphatase. The hypertrophic chondrocytes eventually die, creating empty spaces within the calcified matrix. One or more blood vessels from the perichondrium (now the periosteum) penetrate the bone collar, bringing osteoprogenitor cells to the porous central region. Along with the vasculature newly formed osteoblasts move into all available spaces and produce woven bone. The remnants of calcified cartilage at this stage are basophilic and the new bone is more acidophilic (Figure 8–15). FIGURE 8–15 Cells and matrices of a primary ossification center.
A small region of a primary ossification center showing key features of endochondral ossification. Compressed remnants of calcified cartilage matrix (C) are basophilic and devoid of chondrocytes. This material becomes enclosed by more lightly stained osteoid and woven bone (B) that contains osteocytes in lacunae. The new bone is produced by active osteoblasts (O) arranged as a layer on the remnants of old cartilage. (X200; Pararosaniline–toluidine blue) This process in the diaphysis forms the primary ossification center (Figure 8–14), beginning in many embryonic bones as early as the first trimester. Secondary ossification centers appear later at the epiphyses of the cartilage model and develop in a similar manner. During their expansion and remodeling both the primary and secondary ossification centers produce cavities that are gradually filled with bone marrow and trabeculae of cancellous bone. With the primary and secondary ossification centers, two regions of cartilage remain:
The epiphyseal cartilage is responsible for the growth in length of the bone and disappears upon completion of bone development at adulthood. Elimination of these epiphyseal plates (“epiphyseal closure”) occurs at various times with different bones and by about age 20 is complete in all bones, making further growth in bone length no longer possible. In forensics or through x-ray examination of the growing skeleton, it is possible to determine the “bone age” of a young person, by noting which epiphyses have completed closure. An epiphyseal growth plate shows distinct regions of cellular activity and is often discussed in terms of overlapping but histologically distinct zones (Figures 8–16 and 8–17), starting with the cartilage farthest from the ossification center in the diaphysis:
FIGURE 8–16 Epiphyseal growth plate: Locations and zones of activity.
The large and growing primary ossification center in long bone diaphyses and the secondary ossification centers in epiphyses are separated in each developing bone by a plate of cartilage called the epiphyseal plate.
(Reproduced, with permission, from McKinley M, O'Loughlin VD. Human Anatomy. 2nd ed. New York, NY: McGraw-Hill; 2008). FIGURE 8–17 Details of the epiphyseal growth plate.
In summary, longitudinal growth of a bone occurs by cell proliferation in the epiphyseal plate cartilage. At the same time, chondrocytes in the diaphysis side of the plate undergo hypertrophy, their matrix becomes calcified, and the cells die. Osteoblasts lay down a layer of new bone on the calcified cartilage matrix. Because the rates of these two opposing events (proliferation and destruction) are approximately equal, the epiphyseal plate does not change thickness, but is instead displaced away from the center of the diaphysis as the length of the bone increases. Growth in the circumference of long bones does not involve endochondral ossification but occurs through the activity of osteoblasts developing from osteoprogenitor cells in the periosteum by a process of appositional growth which begins with formation of the bone collar on the cartilaginous diaphysis. As shown in Figure 8–18, the increasing bone circumference is accompanied by enlargement of the central marrow cavity by the activity of osteoclasts in the endosteum. FIGURE 8–18 Appositional bone growth.
Bones increase in diameter as new bone tissue is added beneath the periosteum in a process of appositional growth. Also called radial bone growth, such growth in long bones begins with formation of the bone collar early in endochondral ossification. During radial bone growth formation of new bone at the periosteal surface occurs concurrently with bone removal at the endosteal surface around the large medullary, enlarging this marrow-filled region and not greatly increasing the bone’s weight. (Reproduced, with permission, from McKinley M, O'Loughlin VD. Human Anatomy. 2nd ed. New York, NY: McGraw-Hill; 2008). MEDICAL APPLICATION Calcium deficiency in children can lead to rickets, a disease in which the bone matrix does not calcify normally and the epiphyseal plate can become distorted by the normal strains of body weight and muscular activity. Ossification processes are consequently impeded, which causes bones to grow more slowly and often become deformed. The deficiency can be due either to insufficient calcium in the diet or a failure to produce the steroid prohormone vitamin D, which is important for the absorption of Ca2+ by cells of the small intestine. In adults calcium deficiency can give rise to osteomalacia (osteon + Gr. malakia, softness), characterized by deficient calcification of recently formed bone and partial decalcification of already calcified matrix. Bone growth involves both the continuous resorption of bone tissue formed earlier and the simultaneous laying down of new bone at a rate exceeding that of bone removal. The sum of osteoblast and osteoclast activities in a growing bone constitutes osteogenesis or the process of bone modeling, which maintains each bone’s general shape while increasing its mass. The rate of bone turnover is very active in young children, where it can be 200 times faster than that of adults. In adults the skeleton is also renewed continuously in a process of bone remodeling that involves the coordinated, localized cellular activities for bone resorption and bone formation shown in the diagram of Figure 8–11. The constant remodeling of bone ensures that, despite its hardness, this tissue remains plastic and capable of adapting its internal structure in the face of changing stresses. A well-known example of bone plasticity is the ability of the positions of teeth in the jawbone to be modified by the lateral pressures produced by orthodontic appliances. Bone forms on the side where traction is applied and is resorbed on the opposite side where pressure is exerted. In this way, teeth are moved within the jaw while the bone is being remodeled. Because it contains osteoprogenitor stem cells in the periosteum, endosteum, and marrow and is very well vascularized, bone normally has an excellent capacity for repair. Bone repair after a fracture or other damage uses cells, signaling molecules, and processes already active in bone remodeling. Surgically created gaps in bone can be filled with new bone, especially when periosteum is left in place. The major phases that occur typically during bone fracture repair include initial formation of fibrocartilage and its replacement with a temporary callus of woven bone, as shown in Figure 8–19. FIGURE 8–19 Main features of bone fracture repair.
Repair of a fractured bone occurs through several stages but utilizes the cells and mechanisms already in place for bone growth and remodeling. (1) Blood vessels torn within the fracture release blood that clots to produce a large fracture hematoma. (2) This is gradually removed by macrophages and replaced by a soft fibrocartilage-like mass called procallus tissue. If torn by the break the periosteum reestablishes its continuity over this tissue. (3) The procallus is invaded by regenerating blood vessels and proliferating osteoblasts. In the next few weeks the fibrocartilage is gradually replaced by woven bone that forms a hard callus throughout the original area of fracture. (4) The woven bone is then remodeled as compact and cancellous bone in continuity with the adjacent uninjured areas and fully functional vasculature is reestablished. (Reproduced, with permission, from McKinley M, O'Loughlin VD. Human Anatomy. 2nd ed. New York, NY: McGraw-Hill; 2008). MEDICAL APPLICATION Bone fractures are repaired by a developmental process involving fibrocartilage formation and osteogenic activity of the major bone cells (Figure 8–19). Bone fractures disrupt blood vessels, causing bone cells near the break to die. The damaged blood vessels produce a localized hemorrhage or hematoma. Clotted blood is removed along with tissue debris by macrophages and the matrix of damaged, cell-free bone is resorbed by osteoclasts. The periosteum and the endosteum at the fracture site respond with intense proliferation and produce a soft callus of fibrocartilage-like tissue that surrounds the fracture and covers the extremities of the fractured bone. The fibrocartilaginous callus is gradually replaced in a process that resembles a combination of endochondral and intramembranous ossification. This produces a hard callus of woven bone around the fractured ends of bone. Stresses imposed on the bone during repair and during the patient’s gradual return to activity serve to remodel the bone callus. The immature, woven bone of the callus is gradually resorbed and replaced by lamellar bone, remodeling and restoring the original bone structure. Calcium ions are required for the activity of many enzymes and many proteins mediating cell adhesion, cytoskeletal movements, exocytosis, membrane permeability, and other cellular functions. The skeleton serves as the calcium reservoir, containing 99% of the body’s total calcium in hydroxyapatite crystals. The concentration of calcium in the blood (9-10 mg/dL) and tissues is generally quite stable because of a continuous interchange between blood calcium and bone calcium. The principal mechanism for raising blood calcium levels is the mobilization of ions from hydroxyapatite to interstitial fluid, primarily in cancellous bone. Ca2+ mobilization is regulated mainly by paracrine interactions among bone cells, many of which are not well understood, but two polypeptide hormones also target bone cells to influence calcium homeostasis:
MEDICAL APPLICATION In addition to PTH and calcitonin, several other hormones act on bone. The anterior lobe of the pituitary synthesizes growth hormone (GH or somatotropin), which stimulates the liver to produce insulin-like growth factor-1 (IGF-1 or somatomedin). IGF has an overall growth-promoting effect, especially on the epiphyseal cartilage. Consequently, lack of growth hormone during the growing years causes pituitary dwarfism; an excess of growth hormone causes excessive growth of the long bones, resulting in gigantism. Adult bones cannot increase in length even with excess IGF because they lack epiphyseal cartilage, but they do increase in width by periosteal growth. In adults, an increase in GH causes acromegaly, a disease in which the bones—mainly the long ones—become very thick. MEDICAL APPLICATION In rheumatoid arthritis, chronic inflammation of the synovial membrane causes thickening of this connective tissue and stimulates the macrophages to release collagenases and other hydrolytic enzymes. Such enzymes eventually cause destruction of the articular cartilage, allowing direct contact of the bones projecting into the joint. Joints are regions where adjacent bones are capped and held together firmly by other connective tissues. The type of joint determines the degree of movement between the bones. Joints classified as synarthroses (Gr. syn, together + arthrosis, articulation) allow very limited or no movement and are subdivided into fibrous and cartilaginous joints, depending on the type of tissue joining the bones. Major subtypes of synarthroses include the following:
Intervertebral discs (Figure 8–20) are large symphyses between the articular surfaces of successive bony vertebral bodies. Held in place by ligaments these discoid components of the intervertebral joints cushion the bones and facilitate limited movements of the vertebral column. Each disc has an outer portion, the annulus fibrosus, consisting of concentric fibrocartilage laminae in which collagen bundles are arranged orthogonally in adjacent layers. The multiple lamellae of fibrocartilage produce a disc with unusual toughness able to withstand pressures and torsion generated within the vertebral column. FIGURE 8–20 Intervertebral disc.
Section of a rat tail showing an intervertebral disc and the two adjacent vertebrae with bone marrow (BM) cavities. The disc consists of concentric layers of fibrocartilage, comprising the annulus fibrosus (AF), which surrounds the nucleus pulposus (NP). The nucleus pulposus contains scattered residual cells of the embryonic notochord embedded in abundant gel-like matrix. The intervertebral discs function primarily as shock absorbers within the spinal column and allow greater mobility within the spinal column. (X40; PSH) Situated in the center of the annulus fibrosus, a gel-like body called the nucleus pulposus allows each disc to function as a shock absorber (Figure 8–20). The nucleus pulposus consists of a viscous fluid matrix rich in hyaluronan and type II collagen fibers, but also contains scattered, vacuolated cells derived from the embryonic notochord, the only cells of that structure to persist postnatally. The nucleus pulposus is large in children, but these structures gradually become smaller with age and are partially replaced by fibrocartilage. MEDICAL APPLICATION Within an intervertebral disc, collagen loss or other degenerative changes in the annulus fibrosus are often accompanied by displacement of the nucleus pulposus, a condition variously called a slipped or herniated disc. This occurs most frequently on the posterior region of the intervertebral disc where there are fewer collagen bundles. The affected disc frequently dislocates or shifts slightly from its normal position. If it moves toward nerve plexuses, it can compress the nerves and result in severe pain and other neurologic disturbances. The pain accompanying a slipped disc may be perceived in areas innervated by the compressed nerve fibers—usually the lower lumbar region. Joints classified as diarthroses permit free bone movement. Diarthroses (Figure 8–21) such as the elbow and knee generally unite long bones and allow great mobility. In a diarthrosis ligaments and a capsule of dense connective tissue maintain proper alignment of the bones. The capsule encloses a sealed joint cavity containing a clear, viscous liquid called synovial fluid. The joint cavity is lined, not by epithelium, but by a specialized connective tissue called the synovial membrane that extends folds and villi into the joint cavity and produces the lubricant synovial fluid. FIGURE 8–21 Diarthroses or synovial joints.
Diarthroses are joints that allow free movement of the attached bones, such as knuckles, knees, and elbows. (a) Diagram showing major components of a diarthrosis, including the articular capsule continuous with a ligament inserting into the periosteum of both bones; the joint cavity containing synovial fluid lubricant; and the ends of epiphyses covered by articular cartilage. The synovial membrane lines the capsule and produces the synovial fluid. (b) Longitudinal section through a diarthrosis with the growing bones of a mouse knee, showing the position near the boundaries of the capsule (C) of the epiphyseal growth plate (E) where endochondral ossification occurs. Also shown are the articular cartilage (A) and the folds of synovial membrane (SM), which extend prominently into the joint cavity from connective tissue of the capsule for production of synovial fluid. (X10; PSH stain) In different diarthrotic joints the synovial membrane may have prominent regions with dense connective tissue or fat. The superficial regions of this tissue however are usually well vascularized, with many porous (fenestrated) capillaries. Besides having cells typical of connective tissue proper and a changing population of leukocytes, this area of a synovial membrane is characterized by two specialized cells with distinctly different functions and origins (Figure 8–22):
FIGURE 8–22 Synovial membrane.
The synovial membrane is a specialized connective tissue that lines capsules of synovial joints and contacts the synovial fluid lubricant, which it is primarily responsible for maintaining.
The collagen fibers of the hyaline articular cartilage are disposed as arches with their tops near the exposed surface which, unlike most hyaline cartilage, is not covered by perichondrium (Figure 8–23). This arrangement of collagen helps distribute more evenly the forces generated by pressure on joints. The resilient articular cartilage efficiently absorbs the intermittent mechanical pressures to which many joints are subjected. FIGURE 8–23 Articular cartilage.
(a) Articular surfaces of a diarthrosis are made of hyaline cartilage that lacks the usual perichondrium covering (X40; H&E). (b) The top diagram here shows a small region of articular cartilage in which type II collagen fibers run perpendicular to the tissue surface and then bend gradually in a broad arch. The lower left diagram shows a 3D view of arched collagen fibers in articular cartilage. Proteoglycan aggregates bound to hyaluronan fill the space among the collagen fibers and form a hydrated megacomplex that acts as a biomechanical spring. When pressure is applied, a small amount of water is forced out of the cartilage matrix into the synovial fluid. When pressure is released, water is attracted back into the interstices of the matrix. Such movements of water occur constantly with normal use of the joint and are essential for nutrition of the articular cartilage and for facilitating the interchange of O2, CO2, and metabolites between synovial fluid and chondrocytes. Bone SUMMARY OF KEY POINTS
Major Cells & Matrix Components of Bone
Periosteum & Endosteum
Types & Organization of Bone
Osteogenesis
Bone Growth, Remodeling, & Repair
Metabolic Role of Bone
Joints
Specialized connective/supporting tissues include cartilage and bone, which derive from embryonic mesenchyme and are related both structurally and functionally. In fact, as we will see, most of the bones in the body develop from one type of cartilage. Both cells and fibers are prominent in most kinds of these supportive tissues, but the relative rigidity of their matrices is the special quality that distinguishes them from other types of connective tissue. In spite of their similarities, however, cartilage is distinguishable from bone on the bases of matrix hardness, density, and avascularity. Unlike cartilage, bone has a more extensive set of cell types, extracellular specializations that produce calcification, and the presence of blood vessels. All of these features of bone permit greater possibilities for the tissue’s development and remodeling, compared to cartilage. Bone development (or osteogenesis) can occur in two ways, termed intramembranous and endochondral ossification. Intramembranous ossification gives rise to so-called membrane bones, a term for the developing flat bones of the skull and parts of the mandible. This process involves the deposition of bone matrix (osteoid) directly in thin, “membranous” regions of embryonic mesenchyme. Bones of the extremities, pelvis, and vertebral column (cartilage bones) are formed by endochondral ossification. This process involves the replacement of an embryonic hyaline cartilage model of these skeletal elements with bone. Regardless of the ossification process, the histological structure of the bone is the same. Finally we examine histological aspects of two dissimilar types of joints, which allow movements between bony skeletal components. LABORATORY SESSION LEARNING OBJECTIVES
TERMS TO UNDERSTAND & USE
SLIDES & MICROGRAPHSCartilageCartilage is a specialized, semirigid, durable type of connective/supporting tissue that is characterized mainly by extensive extracellular matrix (ECM) containing high concentrations of proteoglycans and glycosaminoglycans (GAGs) interacting with collagen (type II) and elastic fibers. Variations in the amounts and types of these ECM components give rise to three broad categories of cartilage. Features of these three types are summarized in Table 7–1.
The avascularity of cartilage limits its size according to the distance small molecules can diffuse through the matrix to nourish the chondrocytes. The matrix is a barrier to the entry of large proteins like immunoglobulins and also to lymphocytes, which is important for the practice of reconstructive and cosmetic surgery since cartilage can be transplanted from one individual to another without rejection by the immune system.
BoneBone is another specialized connective/supporting tissue in which the ECM is mineralized, in addition to containing much collagen and proteoglycan aggregates. A. Carefully examine gross or macroscopic specimens of bone cut transversely and longitudinally. In such specimens, note the appearance and locations of the compact bone, cancellous bone, trabeculae, and medullary cavity which normally contains blood-forming marrow (Figure 8–1). Using Table 8–1, review the meaning of the following terms that describe histological types of bone. Think about and understand the interrelationships between them and note their commonly used synonyms (Table 8–1).
Both the cells and matrix of compact bone are highly organized into the units called osteons (also commonly known as Haversian systems). Osteons form in a manner that ensures that the bone cells (osteocytes), each enclosed within a lacuna, can all receive nutrients and eliminate wastes via microscopic passages called canaliculi.
B. Examine a slide with compact bone prepared as “ground bone” (slide 32 ). Such a preparation shows unstained bone that was not decalcified for microtome sectioning, but rather was prepared as a thin wafer using a grinding stone. Cells are not preserved by this method, but the lamellae, lacunae, and canaliculi of osteons (Haversian systems) are shown very well (Figure 8–9). If available, study specimens both longitudinally and transversely (slide 32 ), noting osteons sectioned in various orientations. Identify several examples of central (Haversian) canals in osteons, and locate one or two of the much less numerous perforating (or Volkmann canals (Figure 8–10a), connecting the central canals of neighboring osteons, as diagrammed in Figure 8–1a.
C. Now examine a slide with bone that was prepared by chemical decalcification and routine sectioning and staining (slide 104 , slide 129 ). After hematoxylin and eosin (H&E) staining, the basophilic nuclei of various bone cells are readily distinguishable, with discrete locations on the eosinophilic matrix. Note osteons and their associated cells, which were absent from the ground bone preparation. Examine a stained slide (such as 34) of mostly cancellous bone, but with a thinner, cortical area of compact bone, as shown macroscopically in Figure 8–7. In the region of the compact bone, find and compare the appearance of the osteons to those you saw in the ground specimen (slide 32 ). At low or medium magnification, note the outermost periosteum of dense CT containing small blood vessels and the deep marrow cavity with many eosinophilic trabeculae. At higher magnification, identify the basophilic nuclei of osteoblasts on the surface of the bony trabeculae, using Figures 8–2 and 8–3 as guides. Just beneath osteoblasts, distinguish, if possible, the more lightly stained matrix of the new and not yet fully calcified layer of osteoid. Identify mature osteocytes (each is still in a lacuna) deeper within a trabecula (Figures 8–2 and 8–3).
Also identify the much larger, multinucleated cells called osteoclasts that may be located in depressions called Howship lacunae but in any case are almost always on the surfaces of trabeculae (Figure 8–2). Also, while examining the surfaces of trabeculae, try to identify the very thin and delicate layer of CT called the endosteum that separates the bone tissue of trabeculae from the marrow cavity but is very often difficult to identify. Its location is shown in the diagram of Figure 8–1c. Despite this high degree of organization within osteons, compact bone is always being remodeled, with demolition handled by groups of osteoclasts and reconstruction by osteoblasts, as shown diagrammatically in Figure 8–11. These remodeling processes ensure that bones are somewhat plastic and can very gradually change their overall shapes throughout life. The normal balance between bone deposition and absorption is influenced by steroid hormones and in older individuals this balance is disturbed and there is slow, progressive reduction in bone mass per unit volume. This occurs in both sexes, but it is accelerated in women after menopause and often leads to osteoporosis, in which the reduction in mass of the cortical bone and the number of trabeculae makes the bones fragile.
Mechanisms of Bone Formation during Growth
To understand the process of endochondral ossification, it is important to study carefully the gradual transitions seen here from zone to zone. Review again the diagrams (Figure 8–14). Using Figure 8–15, identify calcified remnants of the cartilage, osteoclasts, osteoblasts, and newly formed osteoid or woven bone. You will be expected to recognize and be able to identify each zone (Figure 8–16b) and the cells labeled in Figure 8–17.
Achondroplasia is an inherited disorder in which there is reduced proliferation of the chondrocytes in the epiphyseal plate of long bones. The result is a form of dwarfism in which the trunk is of normal length, but the extremities are short. JointsJoints are specialized regions of CT holding bones firmly together but allowing bone movements as needed.
Articular cartilage normally begins to break down slowly as we age, potentially leading to inflammation associated with osteoarthritis. This process can be delayed by regular exercise and full use of the joints because recurrent pressure on the cartilage and other parts of joints improves tissue maintenance. Injection of hyaluronan into the synovial cavity is a routine treatment for arthritis, and dietary supplementation with chondroitin sulfate and glucosamine has been shown to slow its progression.
ANSWERS TO STUDY QUESTIONS
What is the term used to describe the softer matrix of spongy bone in between 2 layers of dense bone?cancellous bone, also called trabecular bone or spongy bone, light, porous bone enclosing numerous large spaces that give a honeycombed or spongy appearance. The bone matrix, or framework, is organized into a three-dimensional latticework of bony processes, called trabeculae, arranged along lines of stress.
What is the name of the soft and spongy bone that makes up part of the spine?The vertebra, like all bones, has an outer shell called cortical bone that is hard and strong. The inside is made of a soft, spongy type of bone that is called cancellous bone.
What are layers of bone matrix called?The entire complex consisting of several layers of concentric lamellae around a vascular channel is known as an osteon or haversian system. Interstitial lamellae, which appear as irregularly shaped areas between adjacent osteons, consist of lamellae that are remnants of osteons destroyed during bone remodeling.
What is trabeculae in spongy bone?Introduction. Trabecular bone tissue is a hierarchical, spongy, and porous material composed of hard and soft tissue components which can be found at the epiphyses and metaphyses of long bones and in the vertebral bodies (Fig.
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The term used to describe the softer, matrix of spongy bone in-between 2 layers of dense bone is:
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