stimulating them to proliferate and to express cell membrane RANK receptors. simultaneously, osteoblasts express receptors for RAnK, RANKL, allowing osteoclast precursors to bind to osteoblasts.
• the RAnK-RAnKl interaction induces the trimerization of RAnK on the surface of the osteoclast precursor cell, activating its adaptor molecules to trigger nuclear transcription.
• the nuclear factors that are produced convert the mononuclear osteoclast precursor into an inactive multinuclear osteoclast, which detaches from the osteoblast.
• osteoblasts also manufacture osteoprotegerin (OPG), a ligand that possesses a strong affinity for RAnKl, blocking its availability for RAnK and preventing the binding of osteoclast precursor to an osteoblast, preventing osteoclast formation.
• in the presence of PTH, osteoblasts
manufacture more RAnKl than oPg, and in this manner they facilitate osteoclastogenesis (development of osteoclasts).
• inactive osteoclasts express avb3 integrins allowing these cells to adhere to the bone surface.
• After osteoblasts remove osteoid from the bone surface, they leave, and their previous location becomes populated by inactive osteoclasts that, by adhering to the bone surface, become active osteoclasts. shallow depressions located on the bone surface, called Howship’s lacunae, house these active
osteoclasts.
osteoclasts have four recognizable regions when resorbing bone (Fig. 7.4):
• the basal zone contains most of the organelles of the osteoclast except for the mitochondria, which preferentially concentrate at the ruffled border.
• the ruffled border is located at the osteoclast- bone interface where resorption occurs. the osteoclast exhibits motile finger-like cytoplasmic extensions whose plasma membrane is thickened to protect the cell as it is resorbing bone forming a subosteoclastic compartment.
• the clear zone, the organelle-free region at the periphery of the ruffled border, expresses avb3
integrins whose extracellular aspect binds with osteopontin on the bone surface to form a sealing zone, isolating the microenvironment of the subosteoclastic compartment. intracellularly, the integrin molecules contact actin filaments that form an actin ring.
• the vesicular zone, the region of the osteoclast located between the basal zone and the ruffled border, is rich in exocytotic and endocytotic vesicles. the former transport cathepsin K, which degrades collagens and other proteins of the bone matrix, into the subosteoclastic compartment, whereas the latter transport degraded bone products into the osteoclast.
MEcHANISM of boNE RESoRPTIoN
the acidic environment leaches the inorganic compo- nents from the bone matrix, and the dissolved miner- als enter the osteoclast cytoplasm, where they are exocytosed for delivery into the local capillaries in the vicinity of the basal zone. osteoclasts secrete cathep- sin K into the subosteoclastic compartment to degrade the organic components of the bone matrix. the re- sultant partially degraded materials are endocytosed by the osteoclasts, where they undergo fur ther degra- dation before their release at the basal re gion (see Fig 7.4).
cELLS of boNE (cont.)
Chapter Cart Ilage and Bone
7
81
Figure 7.4 osteoclastic function. ReR, rough endoplasmic reticulum. (From Gartner LP, Hiatt JL, Strum JM: Cell Biology and Histology [Board Review Series]. Philadelphia, Lippincott Williams & Wilkins, 1998, p. 100.)
_
Actin filaments
Ruffled border Bone
Endocytic vesicle
CO2 + H2O H2CO3 H+ HCO3
Lysosomes RER Golgi Nucleolus
Nucleus OSTEOCLAST
Microenvironment of low pH and lysosomal enzymes
Section of circumferential clear zone
Capillary Mitochondria
+
cLINIcAL coNSIDERATIoNS
osteopetrosis results from a genetic defect in which osteoclasts are formed that are unable to resorb bone because they cannot form a ruffled border. Patients with osteopetrosis present with very dense bones and possibly anemia because of a reduced volume of marrow cavity. These individuals are also susceptible to blindness, deafness, and cranial nerve anomalies as a consequence of narrowing of the foramina through which cranial nerves exit the skull.
Chapter Cart Ilage and Bone
7
82 GRoSS obSERvATIoN of boNE
Based on their external morphology, bones are cat- egorized as:
• Long bones—composed of a slender shaft, diaphysis, and two heads, epiphyses
• Short bones—length and width are similar
• Flat bones—composed of two flat plates of compact bone sandwiching a layer of spongy bone
• Irregular bones—no definitive morphology
• Sesamoid bones—formed within the substance of tendons
Based on density, bone may be dense, as in com- pact bone, or spongelike, as in cancellous (spongy) bone. spongy bone is always surrounded by com- pact bone. the marrow cavity of long bones, lined by a thin layer of cancellous bone, houses red mar- row in young individuals, but it accumulates fat deposits as one ages and becomes known as yellow marrow in adults. Red marrow produces blood cells, whereas yellow marrow does not produce blood cells, but does retain its hematopoietic potential.
cancellous bone has resting osteoblast-lined marrow spaces that contain red marrow, and the bone tissue that forms the perimeter of the marrow spaces has smaller and larger irregular lamellae of bone—
spicules and trabeculae.
the articulating surfaces of the epiphyses are com- posed of a thin layer of compact bone that overlies spongy bone and is covered by hyaline cartilage. in individuals still growing, an epiphyseal plate of hyaline cartilage is interposed between the epiphysis and the diaphysis. the metaphysis is a flared zone of the shaft, located between the diaphysis and the epiphyseal plate.
the external surface of the diaphysis and the non- articulating surfaces of the epiphyses are covered by a two-layered periosteum that is inserted into the bone via collagen fibers, Sharpey’s fibers (Fig. 7.5).
• the outer fibrous layer of the periosteum is composed of dense irregular fibrous connective tissue whose neurovascular elements serve the outer region of compact bone.
• the inner cellular layer possesses osteoprogenitor cells and osteoblasts.
Bones of the calvaria (skull cap) are composed of the outer and inner tables of compact bone with a layer of spongy bone known as the diploë interposed
between them. the periosteum covering the outer table of the bones of the cranium is known as the pericranium, but the periosteum covering the inner table of the bones of the calvaria is the dura mater, the outermost layer of the meninges covering and protecting the brain. the dura also serves as the peri- osteum of the inner table.
boNE TyPES bASED oN MIcRoScoPIc obSERvATIoNS
two types of bone may be observed from micro- scopic studies—primary bone and secondary bone.
• Primary bone (immature or woven bone) is the first bone to be formed and it is the bone formed initially during bone repair. Primary bone is more cellular, it is less calcified, and its collagen fiber arrangement is haphazard. it is replaced by secondary bone except in the alveoli of teeth and tendon insertions.
• Secondary bone (mature or lamellar bone) is highly organized into concentric bony lamellae (3 to 7 µm thick), and because it is more calcified and has a precise arrangement of collagen fiber bundles, it is stronger than primary bone. Osteocytes housed in lacunae are
distributed at regular intervals between, or infrequently within, lamellae (see Fig. 7.5). these cells communicate with one another via their osteocytic processes that form gap junctions with each other in narrow channels known as canaliculi.
Lamellar Systems of compact bone
compact bone consists of very thin bony layers called lamellae, arranged in four lamellar systems—
outer and inner circumferential lamellae, interstitial lamellae, and osteons (haversian canal systems)—
that are readily observable in long bones (see Fig.
7.5).
• the outermost calcified layer of the diaphysis, located just deep to the periosteum, is the outer circumferential lamellar system, into which sharpey’s fibers insert.
• lamellae of bone that encircle the marrow cavity are known as the inner circumferential lamellar system. spongy bone lining this lamellar system extends trabeculae and spicules into the marrow cavity.
Chapter Cart Ilage and Bone
7
83
Figure 7.5 Diagram of bone illustrating compact cortical bone, osteons, lamellae, Volkmann’s canals, haversian canals, lacunae, canaliculi, and spongy bone. (From Gartner LP, Hiatt JL: Color Textbook of Histology, 3rd ed. Philadelphia, Saunders, 2007, p 144.)
Canaliculi Concentric
lamellae Osteon
Lacuna
Haversian canal Volkmann’s canal (with blood vessel) Haversian
canal
Sharpey’s fibers Periosteum Blood vessels
Outer circumferential lamellae
Compact bone
Inner circumferential lamellae
Cancellous bone (spongy bone) Marrow cavity
Chapter Cart Ilage and Bone
7
84 • Haversian canal systems (osteons), about 20 to 100 µm in diameter, constitute the predominant lamellar system in compact bone. osteons are composed of wafer-thin lamellae of calcified bone that form concentric cylinders whose central, haversian canal contains a neurovascular supply and are lined by osteoprogenitor cells and osteoblasts (Fig. 7.6). As the vascular supply branches and bifurcates, osteons mirror this organization. osteons are delimited by a boundary, known as a cementing line, composed of calcified ground substance containing only few collagen fibers.
• the helical arrangement of collagen fibers is strictly organized, so that, when viewed in cross section, the fibers parallel each other within a particular lamella, but are
perpendicular to collagen fibers of adjacent lamellae. this pattern is created by varying the pitch of the helix, lessening the chances of bone fracture.
• haversian canals are connected to the canals of their neighboring osteon via oblique channels, Volkmann’s canals, which allow blood vessels access to other haversian canals (see Fig. 7.6).
• osteons are formed as follows: the outermost lamella, the one bordering the cementing line, is formed first; succeeding lamellae line the last one that was formed; and the innermost lamella, bordering the haversian canal, is the last one to be formed. Because osteocytes depend on the inefficient canaliculi for their sustenance, the thickness of each osteon is limited to approximately 20 lamellae.
• Bone is being continually remodeled as osteons are resorbed by osteoclasts and are replaced by osteoblasts. this process leaves remnants of the old osteons, which appear as arc-shaped fragments of lamellae, known as interstitial lamellae, trapped among unresorbed osteons.
HISToGENESIS of boNE
Bone develops in the embryo either by intramem- branous bone formation or by endochondral bone formation. Although these two methods are grossly different, histologically, the final products are indistin-
guishable from each other. Regardless of the mode of development, primary bone is the first to form;
this is resorbed and replaced by secondary bone, which is mature bone that continues to be resorbed and remodeled as it responds to environmental forces placed on it throughout life (Fig. 7.7).
• Intramembranous bone formation is the method by which most of the flat bones develop.
• Formation begins in a highly vascular environment of mesenchymal tissue in which mesenchymal cells maintain contact with each other.
• these mesenchymal cells express the osteogenic master regulators, transcription factors Cbfa1/Runx2 and the zinc finger transcription factor osterix, and differentiate into osteoblasts, which secrete bone matrix.
• in the absence of osterix, the mesenchymal cells differentiate into preosteoblasts, but cannot make the transition into fully competent, matrix-secreting osteoblasts.
• osteogenesis begins as the initial matrix forms trabecular complexes whose surfaces are occupied by osteoblasts. this area now represents a primary ossification center forming primary bone.
• When osteoid is secreted, calcification begins trapping osteoblasts in lacunae. these cells, surrounded by their matrix, are now known as osteocytes. the matrix calcifies, and canaliculi are formed around processes of osteocytes.
• Trabeculae enlarge and increase in number forming networks around the vascular elements, which become transformed into bone marrow.
• Additional ossification centers are necessary in the larger flat bones, such as those of the skull. As bone formation continues, these ossification centers fuse forming a single bone.
An exception is in the fontanelles of the newborn skull, where the ossification centers of the frontal and parietal bones do not fuse until after birth when the membranous soft spots are replaced by bone.
• Regions of the mesenchymal connective tissue that do not participate in bone formation become transformed into the periosteum and endosteum.
Lamellar Systems of compact bone (cont.)
Chapter Cart Ilage and Bone
7
85
Figure 7.6 Diagram of bone illustrating compact cortical bone, osteons, lamellae, Volkmann’s canals, haversian canals, lacunae, canaliculi, and spongy bone. (From Gartner LP, Hiatt JL: Color Textbook of Histology, 3rd ed.
Philadelphia, Saunders, 2007, p 144.) Canaliculi
Concentric lamellae Osteon
Lacuna
Haversian canal Volkmann’s canal (with blood vessel) Haversian
canal
Sharpey’s fibers Periosteum Blood vessels
Outer circumferential lamellae
Compact bone
Inner circumferential lamellae
Cancellous bone (spongy bone) Marrow cavity
Figure 7.7 intramembranous bone formation. (From Gartner LP, Hiatt JL: Color Textbook of Histology, 3rd ed. Philadelphia, Saunders, 2007, p 146.)
Skin Connective tissue
Spongy bone
Collagen
fiber Primary bone
tissue (trabeculae) Osteoid
Mesenchyme Osteoblasts Osteocytes
Connective tissue
Chapter Cart Ilage and Bone
7
86 Endochondral bone formation
With the exception of the flat bones, most of the bones of the body are developed via endochondral bone formation, a method employing several phases.
these processes are presented graphically in Figure 7.8 and summarized in table 7.3.
• A hyaline cartilage model becomes a scaffold for development of bone.
• As the bone being formed becomes stable enough to support the body, the cartilage model is resorbed and replaced by the forming bone.
• the first area of the cartilage model to be replaced is the diaphysis, the primary center of ossification, to be followed by bone formation in the epiphyses, the secondary centers of ossification.
the process of endochondral bone formation is represented by a dynamic series of interrelated events that begin in fetal life and continue into adulthood and beyond as bone may need repair. even in adult- hood, these processes are in action as bone is in a dynamic state and must be remodeled constantly to accommodate environmental forces.
Table 7.3 EVENTS IN ENDOCHONDRAL BONE FORMATION
Event Description
hyaline cartilage model formed Miniature hyaline cartilage model formed in region of embryo where bone is to develop. some chondrocytes mature, hypertrophy, and die. cartilage matrix becomes calcified Primary Center of Ossification
Perichondrium at midriff of diaphysis becomes
vascularized Vascularization of perichondrium changes it to periosteum.
chondrogenic cells become osteoprogenitor cells osteoblasts secrete matrix, forming subperiosteal bone
collar subperiosteal bone collar is formed of primary bone
(intramembranous bone formation) chondrocytes within diaphysis core hypertrophy, die,
and degenerate Presence of periosteum and bone prevents diffusion of
nutrients to chondrocytes. their degeneration leaves lacunae, opening large spaces in septa of cartilage
osteoclasts etch holes in subperiosteal bone collar,
permitting entrance of osteogenic bud holes permit osteoprogenitor cells and capillaries to invade cartilage model, now calcified, and begin elaborating bone matrix
Formation of calcified cartilage/calcified bone complex Bone matrix laid down on septa of calcified cartilage forms this complex. histologically, calcified cartilage stains blue, calcified bone stains red
osteoclasts resorbing calcified cartilage/calcified bone
complex Destruction of calcified cartilage/calcified bone complex
enlarges marrow cavity subperiosteal bone collar thickens, begins growing
toward epiphyses this event, over time, completely replaces diaphyseal cartilage with bone
Secondary Center of Ossification
ossification begins at epiphysis Begins in same way as primary center except there is no bone collar. osteoblasts lay down bone matrix on calcified cartilage scaffold
growth of bone at epiphyseal plate cartilaginous articular surface of bone remains. epiphyseal plate persists—growth added at epiphyseal end of plate.
Bone added at diaphyseal end of plate
epiphysis and diaphysis become continuous At end of bone growth, cartilage of epiphyseal plate ceases proliferation. Bone development continues to unite diaphysis and epiphysis
Chapter Cart Ilage and Bone
7
87
Figure 7.8 endochondral bone formation. Blue represents the cartilage model on which bone is formed. Bone then replaces cartilage. A, hyaline cartilage model. B, cartilage at the midriff (diaphysis) is invaded by vascular elements. C, subperiosteal bone collar is formed. D, Bone collar prevents nutrients from reaching cartilage cells, so they die leaving confluent lacunae.
E, calcified bone/calcified cartilage complex at the epiphyseal ends of the growing bone. F, enlargement of the epiphyseal plate at the end of the bone where bone replaces cartilage. (From Gartner LP, Hiatt JL: Color Textbook of Histology, 3rd ed.
Philadelphia, Saunders, 2007, p 147.) A
E
F
B C D
Chapter Cart Ilage and Bone
7
88 Bone Growth in Length
the proliferation of chondrocytes located in the epiphyseal plate is responsible for bone elongation.
the epiphyseal side of the plate is cartilaginous, whereas at the diaphyseal side of the plate, bone is replacing cartilage. the epiphyseal plate presents five distinct zones beginning at the epiphyseal side of the epiphyseal plate as follows (Fig. 7.9):
• Zone of reserve cartilage: Mitotically active chondrocytes are haphazardly arranged.
• Zone of proliferation: chondrocytes secrete the protein Indian hedgehog, which hinders hypertrophy of chondrocytes and induces the release of PTH-related protein (PTH-RP), which promotes cell division among the chondrocytes of the zone of proliferation. the proliferating chondrocytes form parallel rows aligned in the direction of bone growth.
• Zone of maturation and hypertrophy: Maturing chondrocytes amass glycogen and express the transcription factors Cbfa1/Runx2, which permits them to hypertrophy. these chondrocytes also release type X collagen and vascular endothelial growth factor, which promotes vascular
incursion.
• Zone of calcification: hypertrophied cells attract macrophages to destroy the calcified walls between their adjacent, enlarged lacunae;
chondrocytes undergo apoptosis and die.
• Zone of ossification: osteoprogenitor cells enter the zone of ossification and form osteoblasts, which deposit bone matrix that becomes calcified on the surface of the calcified cartilage. the calcified cartilage/calcified bone complex becomes resorbed and is replaced by bone.
Bone continues to grow in length as long as there is a balance between the zone of proliferation and the rate of resorption in the zone of ossification. By the time the individual reaches 20 or so years of age, the mitotic rate in the proliferation zone is sur- passed by the resorption rate in the zone of ossifica- tion depleting the zone of reserve cartilage. When the last calcified cartilage/calcified bone complex is resorbed, the epiphyseal plate no longer separates the epiphysis from the diaphysis, the marrow cavities of the two regions become continuous, and the bone is unable to continue growing in length.
Bone Growth in Width
Bone growth in length occurs by interstitial growth of the cartilage of the epiphyseal plate, whereas bone growth in width is accomplished by appositional growth occurring deep to the periosteum. osteo- blasts derived from osteoprogenitor cells of the periosteum secrete bone matrix on the bone surface, a process known as subperiosteal intramembran- ous bone formation, which continues during bone development and growth.
throughout life, the processes of bone resorption and bone deposition must be in balance. Bone for- mation on the external surface of the diaphysis must be balanced with osteoclastic activity resorbing the internal aspect to enlarge the marrow cavity.
cALcIfIcATIoN of boNE
Although the process of calcification is not fully understood, it is known that proteoglycans, osteo- nectin, and bone sialoprotein stimulate calcification.
the calcification theory currently accepted involves release of membrane-bound matrix vesicles (100 to 200 nm in diameter) by osteoblasts.
• Matrix vesicles contain high concentrations of ca++ and Po43− ions, adenosine triphosphate (AtP), alkaline phosphatase, cyclic adenosine monophosphate, AtPase, pyrophosphatase, calcium-binding proteins, and phosphoserine.
• Matrix vesicle membranes have calcium pumps that transport ca++ ions into the vesicle; increased concentrations of ca++ ions cause the formation of calcium hydroxyapatite crystals that grow in size and eventually puncture the vesicle membrane causing it to disperse its contents.
• Freed calcium hydroxyapatite crystals serve as nidi of crystallization within the matrix.
• Enzymes released from matrix vesicles liberate phosphate ions that combine with calcium ions and calcify the matrix around the nidi of crystallization.
• Water is resorbed from the matrix, and hydroxyapatite crystals are deposited within the gap regions of the collagen molecules.
• the various nidi of mineralization enlarge and fuse with each other, and the entire matrix becomes calcified.