Bone

Long bones

These bones are long and slender. They are longer than they are wider. The long bones include the bones of the arm—humerus, radius, ulna; leg—femur, tibia, fibula; fingers and toes—each phalanx (individual bone of finger or toe, all together the phalanges); palms of hands and soles of feet—metacarpals and metatarsals.

The long bones have a tubular diaphysis or the shaft, which is made of compact bone at the periphery, surrounding a central medullary cavity, which contains yellow marrow. The two bulky ends of the long bone are called the epiphysis, which are made of compact bone at the periphery and spongy bone at the center. The articular surface of the epiphysis is covered with hyaline cartilage, which provides a cushioning effect to the opposing bone ends during joint movements and absorbs stress. Epiphyseal line is present between the diaphysis and each epiphysis of an adult long bone. This line is a remnant of the epiphyseal plate.

Short bones

These bones are usually cube shaped of nearly equal length and width. They consist of spongy bone which is covered by a thin layer of compact bone. They contain bone marrow, but no marrow cavity. These bones include bones of wrist—carpals and ankle—tarsals.

Flat bones

These bones are thin, flat, curved, with no marrow cavity. Spongy bone is present between upper and lower layer of compact bone. Flat bones include bones of sternum, ribs, scapula, clavicle, and bones that form roof of the skull, parietal, frontal, temporal and occipital.

Irregular bones

These bones have complex shapes, notched or with ridges and are not included in any of the above categories. These bones are primarily made of spongy bone, which is covered with a thin layer of compact bone, with bone marrow, but no marrow cavity. The irregular bones include bones of vertebrae, facial bones (ethmoid, sphenoid) pelvic bones (ischium and pubis), calcareous (heel bone) and mandible.

Sesamoid bones

These bones develop in tendons, where there is considerable pressure, tension or friction. Patella (knee cap) is a good example.

Endochondral bones

These bones are formed by replacement of hyaline cartilage with bony tissue. This type of ossification occurs in bones of trunk and extremities.

Intramembranous bones

These bones are formed by replacement of sheet like connective tissue membrane with bony tissue. This ossification occurs in the cranial and facial flat bones of the skull, mandible and clavicles.

Spongy bone

Spongy bone and compact bone have the same cells and intercellular matrix, but differ in the arrangement of components. Spongy bone looks like a poorly organized tissue in contrast to compact bone.

The bony substance consists of large slender spicules called trabeculae. The trabeculae are up to 50 μm thick. The trabeculae are oriented along lines of stress to withstand the forces applied to bone (Fig. 9.2). The marrow spaces are large. The trabeculae surround the marrow spaces from where they derive their nutrition through diffusion.

Morphology

Osteoblasts are basophilic, plump cuboidal or slightly flattened cells. These cells are found on the forming surfaces of growing or remodeling bone. These cells produce the organic matrix of bone (Osteoid) which primarily consists of type I collagen and the balance being noncollagenous proteins (Fig. 9.6).

Osteoblasts exhibit abundant and well developed protein synthetic organelles. The intense cytoplasmic basophilia is due to an abundance of rough endoplasmic reticulum. The procollagen and other organic constituents of bone matrix synthesized by this organelle, enter its lumen and are carried by transfer vesicles to the Golgi complex and assembled within Golgi complex in secretory granules. A pale juxtanuclear area indicates the site of the Golgi complex. These granules release their contents along the surface of cell opposed to forming bone which, assemble extracellularly as fibrils to form osteoid. The presence of the noncollagenous proteins within the secretory collagen granule or in a distinct population of granules is debatable. But, the noncollagenous proteins are also released along the surface of osteoblasts apposed to osteoid and diffuse from osteoblast surface, towards the mineralization front, where they participate in regulating mineral deposition. Nucleus is situated eccentrically in the part of the cell that is farthest away from the adjacent bone surface.

Organic matrix is deposited around the cell bodies and their cytoplasmic processes resulting in the formation of canaliculi. The cytoplasmic processes are not seen in H and E sections, but in other preparations, it is known that, they are in contact with one another and also with processes of osteocytes in lacunae beneath them. The cells contact one another by adherens and gap junctions. These are functionally connected to microfilaments and enzymes associated with intracellular secondary messenger systems. This arrangement provides for intercellular adhesion and cell to cell communication and ensures that osteoblast layer completely covers the osteoid surface and functions in a coordinated manner.

Osteoblasts also contain prominent bundles of actin, myosin and cytoskeletal proteins which are associated with maintenance of cell shape, attachment and motility.

Formation

Osteoblasts are derived from undifferentiated pluripotent mesenchymal stem cells. Osteoprogenitor cells are divided into two types, determined (DOPCs) and inducible osteogenic precursor cells (IOPCs). These DOPCs are present in the bone marrow, endosteum and periosteum and differentiate into osteoblasts under the influence of systemic and bone derived growth factors. The IOPCs represent mesenchymal cells present in other organs and tissues that may differentiate into bone forming cells when stimulated.

The osteoprogenitor cells express transcription factors cbfa1/Runx-2 and osterix which are essential for osteoblast differentiation. cbfa1 is a member of runt related family of transcription factors. It triggers the expression of BSP, osteopontin, osteocalcin and type I collagen. Osterix, a zinc fingercontaining transcription factor, is similar to Runx-2.

Functions

The main function of osteoblast is the formation of new bone via synthesis of various proteins and polysaccharides. Other functions include the regulation of bone remodeling and mineral metabolism. Osteoblasts also play a significant role in the mineralization of osteoid. Osteoblasts secrete type I collagen which is widely distributed and not unique to osteoblasts whereas, osteocalcin and cbfa-1 (osteoblast specific transcription factor) are specific to cells of osteoblast lineage. These provide useful markers of osteoblast phenotype. Osteoblasts also secrete small amounts of type V collagen, osteonectin, osteopontin, RANKL, osteoprotegerin, proteoglycans, latent proteases and growth factors including bone morphogenetic proteins. Osteoblasts exhibit high levels of alkaline phosphatase on outer surface of plasma membrane which is used as a cytochemical marker to distinguish preosteoblasts from fibroblasts. Total alkaline phosphatase activity has been recognized as a reliable indicator of osteoblast function. Osteoblasts express receptors for various hormones including PTH, vitamin D₃, estrogen and glucocorticoids, which are involved in the regulation of osteoblast differentiation. The osteoblasts recognize the resorptive signal and transmit it to the osteoclast.

RANKL is a membrane bound TNF related factor, that is expressed by osteoblasts/stromal cells. The presence of RANKL is vital in osteoclast differentiation.

Cbfa1 expressed by osteoprogenitor cells, regulates the expression of OPG (osteoprotegerin) which is a potent inhibitor of osteoclast formation and function.

Regulation of osteoblast activity

The overall integrity of bone is controlled by hormones, proteins secreted by hematopoietic bone marrow cells and bone cells.

Role of parathormone (PTH)

In response to hypocalcemia, the hormone activates a mechanism for the release of calcium from bone, the principal body store house. PTH does so, by an indirect effect mediated by PTH receptors on bone stromal cells including osteoblasts, as osteoclasts are devoid of PTH receptors. PTH regulates serum calcium levels by stimulation of bone resorption and can also have anabolic effects in vivo that appear to be mediated through TGF-β and IGF-I. These opposing effects of PTH are consistent with the apparent coupling of bone formation and remodeling.

Cells of mesenchymal lineage that differentiate into osteoblasts were shown to produce PTHrP very early during differentiation, and PTHrP levels decreased with further cell maturation. PTHR1 is expressed at a later stage in differentiation, by committed preosteoblasts. PTHrP (parathyroid hormone related protein) resembles PTH in its amino terminal sequence and the two have similar structural requirements for binding and activation of their common receptor, type 1 PTH receptor (PTHR1). Persistently increased levels of local PTHrP favor increased osteoclast formation through stimulation of RANKL production. Hence, PTHrP needs to be regulated in terms of concentration, location and time, so that it is presented briefly to target cells. PTH is secreted as a hormone in response to a hypocalcemic signal in order to regulate calcium homeostasis by promoting bone resorption, whereas PTHrP functions as a bone cytokine to control bone mass.

While vitamin D₃ stimulates bone resorption, it is also essential for normal bone growth and mineralization. It also promotes calcium absorption from the intestine. It stimulates synthesis of osteocalcin and osteopontin by osteoblasts and suppresses collagen production. The action of parathormone and vitamin D₃ is that, they enhance bone resorption at high concentrations (pharmacological) and support bone formation at low (physiologic) concentrations.

Growth hormone is required for attaining normal bone mass which is mediated by the local production of IGF-I. It binds to membrane bound growth hormone receptors on activated osteoblasts.

Insulin targets osteoblasts directly and stimulates bone matrix formation and mineralization and indirectly affects bone formation through stimulation of IGF-I (insulin like growth factor) produced in the liver.

Bone morphogenetic proteins

These are the only factors that can initiate osteoblastogenesis from uncommitted progenitor cells. These proteins are expressed during embryonic development as well as in adulthood. BMPs 2, 4 and 6 direct the pluripotent cells to commit to an osteoblastic pathway. BMPs can also increase the differentiation of committed cells to the osteoblast lineage. BMPs can also upregulate cbfa1 under certain conditions during osteoblast differentiation. cbfa1, in turn, activates osteoblast-specific genes such as osteopontin, bone sialoprotein, type I collagen and osteocalcin.

TGF-β During the early stages of bone formation, the action of transforming growth factor-β is to recruit and stimulate osteoprogenitor cells to proliferate, providing a pool of early osteoblasts. In contrast, during later stages of osteoblast differentiation, TGF-β blocks differentiation and mineralization. These effects appear to be highly dependant on bone cell source, dose applied and local environment, which may be a result of inhibition of DNA synthesis at high TGF-β concentration. Additionally, it inhibits the expression of Runx-2 and osteocalcin genes, whose expression is controlled by cbfa1/ Runx-2 in osteoblast like cell lines.

IGF I and II (insulin like growth factors) increase proliferation and play a major role in stimulating mature osteoblast function. IGF-1 upregulates osterix but not cbfa1. IGF-1 along with BMP-2 acts synergistically on osterix expression.

FGF (fibroblast growth factors) are a family of structurally related polypeptides that play a critical role in angiogenesis and mesenchymal cell mitogenesis. In normal adult tissues, the most abundant proteins are FGF-1 and FGF-2. FGF-2 is expressed by osteoblasts and is more potent than FGF-1. These factors exert their effect on bone formation, primarily through increased proliferation of osteoprogenitor cells and promotion of osteogenic differentiation.

Glucocorticoids promote differentiation of osteoblasts and stimulate bone matrix formation in vitro. But, prolonged treatment with glucocorticoids in vivo, results in bone loss, which can be attributed to increased PTH production in response to the inhibitory effects of glucocorticoids on calcium absorption and depletion of osteogenic precursor cells.

PDGF (platelet derived growth factor) The isoforms of this factor have a strong chemotactic effect on osteoblasts and other connective tissue cells and may act to recruit mesenchymal cells, during bone development and remodeling. It has similar effects to fibroblast growth factor in promoting osteogenesis. It acts as a potent mitogen for all cells of mesenchymal origin. PDGF may also have direct and indirect effects on bone resorption by the upregulation of collagenase transcription and an increase in IL-6 expression in osteoblasts.

Vascular endothelial growth factor (VEGF) acts directly on osteoblasts to promote osteoblast migration, proliferation and differentiation in an autocrine manner. It is also a mediator of osteoinductive factors like TGF-β, IGF-1, FGF-2, which upregulate VEGF expression in osteoblasts. VEGF also influences osteoblasts indirectly via its effects on endothelial cells. It stimulates the production of bone forming factors for osteoblasts by endothelial cells.

The Wnt/β-catenin signaling pathway has been shown to have important roles in the maintenance of self-renewal of stem cells in epidermal and hematopoietic cells, and regulation of bone formation. This pathway is proposed to directly promote osteogenesis through actions on Runx-2 gene.

Bone lining cells

Once osteoblasts have completed their function, they are either entrapped in the bone matrix and become osteocytes or remain on the surface as lining cells. Osteoblasts flatten, when bone is not forming and extend along the bone surface and hence the name. These cells contain very few organelles but, retain gap junctions with osteocytes, while retaining its vitality.

But 50–70% of osteoblasts present at the remodeling site cannot be accounted for after enumeration of lining cells and osteocytes. It has been proposed that missing osteoblasts die by apoptosis. Growth factors and cytokines produced in bone microenvironment influence this process. Tumor necrosis factor (TNF) promotes apoptosis. TGF-β and IL-6 have antiapoptotic effects. Glucocorticoids and estrogen withdrawal promote apoptosis in osteoblasts and osteocytes.

Morphology

Osteoclasts lie in resorption bays called Howship's lacunae. Osteoclast is a large cell approximately 40–100 μm in diameter with 15 to 20 closely packed nuclei. Osteoclasts with many nuclei resorb more bone than osteoclasts with few nuclei (Fig. 9.6). The different nuclei are proposed to be of different ages and there is evidence of apoptosis. These cells are variable in shape due to their motility. The cytoplasm of the osteoclast shows acid phosphatase containing vesicles and vacuoles. The presence of acid phosphatase distinguishes the osteoclast from other multinucleated giant cells. Mitochondria are extensive and distributed throughout the cytoplasm, except below the ruffled border. Rough endoplasmic reticulum is relatively sparse for the size of the cell. Golgi complex is extensive and arranged in stacks. The cytoplasm also contains microtubules, which transport vesicles between Golgi stacks and ruffled membrane. Cathepsin containing vesicles and vacuoles are present close to the ruffled border indicating resorptive activity of these cells.

Formation of osteoclast

Multinucleated giant cells are derived from hemopoietic cells of monocyte macrophage lineage. The earliest identifiable hematopoietic precursor that can form osteoclast is the granulocytemacrophage colony forming unit (CFU-GM). The early precursor cells proliferate and differentiate to form post mitotic committed precursor cells. These committed precursors then differentiate and fuse to form immature multinucleated giant cells. These are activated to form bone resorbing osteoclasts. The differentiation into osteoclasts is through a mechanism involving cell–cell interaction with osteoblast stromal cells.

The formation of osteoclast requires the presence of RANK ligand (receptor activator of nuclear factor κB) and M-CSF (macrophage colony stimulating factor). These two membrane bound proteins are produced by neighboring stromal cells and osteoblasts, thus requiring direct contact between these cells and osteoclast precursors.

M-CSF acts through its receptor on osteoclast precursors c-Fms (colony stimulating factor 1 receptor) and thereby provides signals required for proliferation. M-CSF also enhances osteoclast activity by preventing osteoclast apoptosis.

RANKL belongs to the TNF (tumor necrosis factor) family of ligands. RANKL is also called osteoclast differentiation factor (ODF), TNF related induced cytokine (TRANCE), or osteoprotegerin ligand (OPGL).

RANK is homotrimeric TNF receptor family member. As RANKL binds to RANK on surface of M-CSF triggered osteoclast precursors, a number of signaling pathways are activated committing the cell to osteoclast lineage. These are initially mediated by TRAF 6 TNF (receptor-associated factor), leading to activation of NF-κβ, the (AP)-1 transcription factor complex and ultimately NFATc1 nuclear factor of activated T cells.

RANKL has been implicated in the fusion of osteoclast precursors into multinucleated giant cells, their differentiation into mature osteoclasts, their attachment to bone surface and their activation to resorb bone.

Osteoprotegerin (OPG) is a member of the TNF receptor family and is expressed by osteoblasts. It recognizes RANKL, and blocks the interaction between RANK and RANKL, leading to an inhibition of osteoclast differentiation and activation. Cbfa1 contributes to the expression of OPG. The balance between RANKL-RANK signaling and the levels of biologically active OPG, regulates development and activation of osteoclasts and bone metabolism.

Regulation of osteoclast activity

Estrogen suppresses the production of bone resorbing cytokines including IL-1 and IL-6. Estrogen deficiency results in marked bone resorption by increasing osteoclast activity. It is suggested that estrogen prevents excessive bone loss before and after menopause by decreasing the life span of osteoclast, by promoting apoptosis. Estrogen increases production of TGF-β by osteoblasts which stimulate apoptosis of osteoclasts.

Vitamin D₃ and parathyroid hormone (PTH) Vitamin D3 promotes the differentiation of osteoclasts from monocyte macrophage stem cell precursors in vitro. In vivo, enhanced osteoclastic bone resorption has been observed when vitamin D is applied at high dose. This effect is caused by the stimulation of RANKL production by osteoclasts. PTH is secreted in response to changes in blood calcium and affects bone formation and resorption. PTH binds to osteoblasts and induces the production of M-CSF and RANKL that stimulate the maturation and action of osteoclasts.

Calcitonin is a particularly potent inhibitor of osteoclast activity, but its effects are transient likely, due to downregulation of calcitonin receptor on osteoclast in the sustained presence of hormone. Calcitonin inhibits proliferation and differentiation of osteoclast precursors. It reduces the dimension of ruffled border and their dissociation into monocytic cells. Calcitonin also promotes osteoclast apoptosis.

Several factors expressed by osteoblasts/lymphocytes have an impact on osteoclastogenesis to enhance (IL-1, IL-6, IL-8 and IL-11) or limit (IL-4, IL-10, IL-12, IL-13, IL-18) osteoclast formation.

IL-6 is produced by stromal or osteoblast lineage cells in response to PTH and vitamin D3 and on stimulation by IL-1, TNFα, TGFβ, PDGF and IGF-2. IL-6 alone or in concert with other agents stimulates osteoclastogenesis. But it is a much less potent stimulator of osteoclast generation than IL-1 and TNF-α.

Osteoclast precursors interact with bone matrix to trigger their own differentiation by producing IL-1. IL-1 can also induce osteoclast formation by increasing expression of RANKL on surface of osteoblasts and marrow stromal cells.

TNFα stimulates differentiation of osteoclast progenitors into osteoclasts in the presence of M-CSF independent of RANKL-RANK interaction.

OCIL (osteoclast inhibitory lectin) has been recognized as an inhibitor of osteoclast formation. OCIL and its related proteins OCILrP1 and OCILrP2 are type II membrane bound C-lectins expressed by osteoblasts.

TGF-β and interferon-γ inhibit proliferation and differentiation of committed precursors into mature osteoclasts. TGF-β also promotes apoptosis of osteoclasts. But, TGF-β is also believed to enhance osteoclast differentiation in hematopoietic cells stimulated with RANKL and M-CSF.

IFN-γ interferes with the osteoclast differentiation induced by RANKL and this mechanism is critical for the suppression of pathological bone resorption associated with inflammation.

Bisphosphonates suppress bone resorption via injury to osteoclasts when they solubilize bisphosphonate contaminated bone. These do not suppress osteoclastogenesis. They are capable of inducing osteoclast apoptosis. Bisphosphonates suppress bone resorption without consistent reduction in osteoclast numbers which suggests that these compounds might act through a mechanism that is distinct from OPG. These are used as antibone resorbing agents in various diseases associated with stimulated bone resorption.

PGE2 (Prostaglandins of E series) can act as powerful mediators of bone resorption and can also influence bone formation. The prostaglandins, induce osteoclast formation through increased expression of RANKL on the surface of immature osteoblasts and stromal cells. PGE2 can also stimulate bone formation when administered systemically.

Formation of woven bone

The first small mass of newly formed bone matrix is an irregular shaped spicule. The bony spicules gradually lengthen into longer anastomosing structures called trabeculae. The trabeculae extend in a radial pattern leading to an anastomosing network of trabeculae characteristic of spongy bone. The spicules and trabeculae are easily recognized in Hematoxylin and Eosin sections, because the matrix stains a bright pink color, and they are also covered with large rounded osteoblasts that have intensely basophilic cytoplasm. These trabeculae enclose local blood vessels. This early membrane bone is termed woven bone.

At this stage, few mesenchymal cells remain undifferentiated. But, before these cells disappear, they leave a layer of flat cells called osteogenic cells on trabeculae which do not have osteoblasts. In richly vascular areas, these osteogenic cells give rise to osteoblasts that form bone matrix. In areas, with no capillary blood supply, they form chondroblasts which lay down cartilage.

Appositional growth mechanism and formation of compact bone plates

The new layers of bone matrix are deposited on preexisting bone surfaces. The osteogenic cells on the surface of spicules and trabeculae are always in a superficial position repeating the process again and again. This is appositional growth, which results in build up of bone tissue one layer at a time. Every generation of osteoblasts produce their own canaliculi. Hence, all the new osteocytes remain linked through canaliculi to bone surface above and to osteocytes below. As the trabeculae increase in width due to appositional growth, neighboring capillaries are incorporated to provide nutrition to osteocytes in deeper layers. New bone is deposited on some surfaces and resorbed at other sites leading to remodeling of trabeculae. This remodeling maintains shape and size of bone throughout life.

Continued appositional growth and remodeling of trabeculae converts cancellous bone to compact bone. Cancellous bone is in the central part of bones as the trabeculae do not increase in size. The vascular tissue in cancellous bone differentiates into red marrow.

Formation of osteon

As layers of bone tissue build up by apposition, the trabeculae thicken and the soft tissue spaces get narrowed. This process converts cancellous bone to compact bone. As cancellous bone gets converted to compact bone, a number of narrow canals are formed lined by osteogenic cells. These canals enclose vessels that were present in soft tissue spaces of cancellous network. The consecutive lamellae of bone become added to the bony walls of spaces in cancellous bone, which is called osteon or haversian system. These osteons are called primitive osteons as they are short, compared to those in long bones.

While these changes occur, external to the woven bone, there is condensation of vascular mesenchyme called the periosteum.

The mechanism of intramembranous ossification involves bone morphogenetic proteins (BMPs) and activation of transcription factor called cbfa1. BMPs activate cbfa1 gene in mesenchymal cells. The cbfa1 transcription factor transforms mesenchymal cells into osteoblasts. It is believed that the proteins activate genes for osteocalcin, osteopontin and other bone specific extracellular matrix proteins.

Formation of a cartilaginous model

At the site where a limb will later emerge, the embryo shows outgrowth of mesoderm covered by ectoderm. The mesenchymal cells in this site condense, differentiate into chondroblasts and form the cartilage matrix, resulting in the development of a hyaline cartilage model. This process begins late in the second month of development. The model is surrounded by a perichondrium, made up of an inner chondrogenic layer and outer fibrous layer. No osteoblasts are produced by the cells in the chondrogenic layer, because differentiation is taking place in an avascular environment. Fibroblasts in fibrous layer produce collagen and a dense fibrous covering is formed.

The growth of the cartilage model is by interstitial and appositional growth. Increase in the length is by interstitial growth, due to repeated division of chondrocytes, along with production of additional matrix by the daughter cells. Widening of the model is due to further addition of matrix to its periphery by new chondroblasts, derived from the chondrogenic layer of the perichondrium. This is called appositional growth. In case of long bones as the differentiation of cartilage cells moves towards the metaphysis, the cells organize into longitudinal columns which are subdivided into three zones.

Zone of proliferation. The cells are small and flat, and constitute a source of new cells.

Zone of hypertrophy and maturation. This is the broadest zone. The chondrocytes hypertrophy, and in the early stages secrete type II collagen. As hypertrophy proceeds, proteoglycans are secreted. The increased cell size and increased cell secretion, lead to an increase in the size of the cartilaginous model. As the chondrocytes reach maximum size, they secrete type X collagen and noncollagenous proteins. Subsequently, there is partial breakdown of proteoglycans, creating a matrix environment receptive for mineral deposition.

Zone of provisional mineralization. Matrix mineralization begins in the zone of mineralization by formation of matrix vesicles. These membrane bound vesicles bud off from the cell and form independent units in the longitudinal septa of the cartilage.

Formation of bone collar

The capillaries grow into the perichondrium that surrounds midsection of the model. The cells in the inner layer of the perichondrium differentiate into osteoblasts in a vascular environment and form a thin collar of bone matrix around the mid region of the model. At this stage, periochondrium is referred to as periosteum as the differentiation of cells from the inner layer of the perichondrium is giving rise to bone. Vascularization of the middle of the cartilage occurs, and chondroclasts resorb most of the mineralized cartilage matrix. The bone collar holds together the shaft, which has been weakened by disintegration of the cartilage. Hence, more space is created for vascular ingrowth.

Formation of periosteal bud

Periosteal capillaries accompanied by osteogenic cells invade the calcified cartilage in the middle of the model and supply its interior. The osteogenic cells and the vessels comprise a structure called the periosteal bud. The periosteal capillaries grow into the cartilage model and initiate development of a primary ossification center. Osteogenic cells in the periosteal bud give rise to osteoblasts that deposit bone matrix on the residual calcified cartilage. This results in the formation of cancellous bone that has remnants of calcified cartilage. This is the mixed spicule. The network of mixed spicules is called primary spongiosa. The calcified cartilage in the trabeculae in hematoxylin and eosin sections stains pale blue to mauve, whereas bone matrix appears bright pink to red.

Formation of medullary cavity

As the primary ossification center enlarges, spreading proximally and distally, osteoclasts break down the newly formed spongy bone and open up a medullary cavity in the center of the shaft. Hematopoietic stem cells enter the medullary cavity giving rise to myeloid tissue.

The two ends of the developing bone are at this stage still composed entirely of cartilage. The midsection of the bone becomes the diaphysis and the cartilaginous ends of bone become the epiphysis. Hence, the primary center of ossification is the diaphyseal center of ossification.

Formation of secondary ossification center

At birth, most of the long bones have a bony diaphysis surrounding remnants of spongy bone, a widening medullary cavity, and two cartilaginous epiphysis. Shortly before or after birth, secondary ossification centers appear in one or both epiphysis. Initially chondrocytes in the middle of the epiphysis hypertrophy and mature, and the matrix partitions between their lacunae calcify.

Periosteal buds carry mesenchymal cells and blood vessels and process is same as that occurring in a primary ossification center, except that the spongy bone in the interior is retained and no medullary cavity forms in the epiphysis. The ossification spreads from secondary center in all directions. Eventually, the cartilage in the middle of epiphysis gradually gets replaced by cancellous bone. When secondary ossification is complete, hyaline cartilage remains at two places—on the articular surface as articular cartilage and at the junction of the diaphysis and epiphysis, where it forms the epiphyseal plates.

This plate continues to form new cartilage, which is replaced by bone, a process that increases the length of the bone. Long bones have one or two secondary ossification centers. Short bones have one ossification center. The union of primary and secondary ossification center is called epiphyseal line.

Long bones development depends on endochondral bone formation, which requires balance between hypertrophic cartilage (HC) formation and its ossification. Dysregulation of this process may result in skeletal dysplasia and heterotopic ossification. Endochondral ossification requires the precise orchestration of HC, vascularization, extracellular matrix remodeling, and the recruitment of osteoclasts and osteoblasts. Matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and osteoclasts have all been shown to regulate endochondral ossification.

Calcification is the process of deposition of insoluble calcium salts in a tissue.

Mechanism of calcification. It is not entirely clear if the first formed solid phase is amorphous or crystalline. It is widely conceded that the first formed solid phase is amorphous. This initial phase is subsequently transformed to hydroxyapatite.

Amorphous calcium phosphate appears as microscopic spheres 30–100 nm in diameter, comprising randomly packed apatite crystals each of about 0.95 nm diameter.

Under normal conditions, there is insufficient concentration of available calcium and phosphate ions in blood and tissue fluid, for calcium phosphate to crystallize or precipitate spontaneously. The critical factor is the local (Ca²⁺) x. (Pi) ion product. Pi is the total free inorganic orthophosphate. When factors operate locally, to raise this ion product, calcium phosphate separates out in the solid phase and undergoes solid phase transition to a number of crystalline arrangements. Once the microcrystals have begun to form, they continue to grow and also catalyse further crystallization of calcium phosphate even at sites where the Ca²⁺. Pi product does not exceed the plasma level.

Theories of calcification

Traditionally, calcification has been treated from the point of view of precipitation dynamics, with initially the only ions needed being calcium and phosphate. The extracellular fluid is supersaturated with respect to the basic calcium phosphate, yet the mineralization is not a widespread phenomenon. Thus for precipitation to occur, the conditions in the osteoid matrix must be in some way especially favorable to this process. Interplay of various factors may contribute to conditions that may favor calcification.

Inhibitors of calcification

Collagen and other potential nucleating agents occur in tissues that do not calcify. The probable reason is that, the collagen molecules are packed closely together in soft tissues than in bone, which impede phosphate ion access to intrafibrillary nucleation sites. Pyrophosphate, diphosphonates or adenosine triphosphate can delay or prevent the transformation of amorphous calcium phosphate to hydroxyapatite. Other potential inhibitors include citrate, magnesium, and proteins like albumin. Components of the bone matrix may act locally and inhibit mineralization e.g. proteoglycans.

Sequence of events of bone resorption

The first phase involves the formation of osteoclast progenitors in the hematopoietic tissues, followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in the bone itself. The second phase consists of activation of osteoclasts at the surface of mineralized bone. Osteoblasts play a major role by retracting, to expose the mineral to the osteoclast and releasing a soluble factor that activates these cells. The third phase involves the activated osteoclasts resorbing the bone.

Alterations in the osteoclast

Immediately before the resorption event, the osteoclasts undergo changes by assuming a polarity of structure and function. These changes facilitate bone resorption. The two distinct alterations are the development of a ruffled border and a sealing zone at the plasma membrane. These changes occur only in the region of the cell that is next to the bone surface. The ruffled border consists of many infoldings of the cell membrane, resulting in fingerlike projections of the cytoplasm. Thus, an extensive surface is created well suited for an intensive exchange between the cell and bone.

At the periphery of the ruffled border, the plasma membrane is smooth and apposed closely to the bone surface. The adjacent cytoplasm, devoid of cell organelles contains contractile actin microfilaments, surrounded by two vinculin rings. This region is called the clear (sealing) zone. This zone serves to attach the cell very closely to the surface of bone and creates an isolated microenvironment, in which resorption can take place without diffusion of the hydrolytic enzymes produced by the cell into adjacent tissue. When osteoclasts arrive at the resorption site, they use the sealing zone to attach themselves to the bone surface. The attachment of the osteoclast cell membrane to the bone matrix at the sealing zone is due to the presence of cell membrane proteins known as integrins, especially αVβ3. Integrins are a large family of heteromeric cell surface receptors composed of noncovalently bound α and β subunits which interact with extracellular matrix molecules, serum constituents and adhesion molecules of immunoglobulin family. αVβ3, a vitronectin receptor, is expressed by resorbing osteoclasts. These integrins bind to specific amino acid sequences present in proteins of the bone matrix, namely, RGD (Arg-Gly-Asp).

Removal of hydroxyapatite

The initial phase involves the dissolution of the mineral phase by the action of hydrochloric acid (HCl). The protons for the acid arise from the activity of cytoplasmic carbonic anhydrase II, which is synthesized in the osteoclast. The protons are then released across the ruffled border into the resorption zone by an ATP consuming proton pump. This leads to a fall in pH to 2.5–3.0 in the osteoclast resorption space. The proton pump is an absolute requirement for normal bone resorption to take place.

Degradation of organic matrix

Organic constituents of bone tissue remain after the dissolution of mineralized component. Next step involves the digestion of organic components of matrix. Proteolytic enzymes are synthesized by osteoclasts, namely, cathepsin-K and MMP-9 (matrix metalloproteinase). The enzymes are synthesized in rough endoplasmic reticulum, transported to Golgi complexes and moved to the ruffled border in transport vesicles, and the contents of the same are released into sealed compartment, creating extracellular lysosomes. As a result, a visible depression or Howship's lacunae is excavated into the bone.

Cathepsin-K is the most important enzyme in bone resorption. It is a collagenolytic papain like cysteine protease expressed in osteoclasts. In vivo studies, have shown that, activation of cathepsin-K occurs intracellularly before secretion into lacunae and onset of bone resorption. The processing of procathepsin-K to mature cathepsin-K occurs as osteoclast approaches bone. Cathepsin-K degrades major amount of type I collagen and other noncollagenous proteins, which have been demineralized by the acidic environment of the resorptive zone.

MMP-9 (collagenase B) is believed to be required for osteoclast migration. MMP-13 is proposed to be involved in bone resorption and osteoclast differentiation.

Removal of degradation products from lacunae

Once liberated from bone, the free organic and nonorganic particles of bone matrix are taken in or endocytosed from the resorption lacunae, across the ruffled border, into the osteoclast. These are then packed in membrane bound vesicles within cytoplasm of osteoclast. These vesicles and their contents pass across the cell and fuse with FSD (functional secretory domain) a specialized region of the basal membrane. Then the vesicles are released by exocytosis. The changes in the cytoplasm framework in the cell and presence of clusters of matrix fragments in the region directly outside the cell next to FSD, indicate that matrix fragments have been expelled from the cell by exocytosis into extracellular space away from bone.

Following resorption, osteoclasts undergo apoptosis, which provides a mechanism for limiting resorptive activity. Factors like TGF-β and estrogen promote apoptosis. PTH and IL-1, act as suppressors prolonging osteoclast activity.

Extracellular role of TRAP

TRAP accumulates extracellularly in bone matrix, immediately adjacent to ruffled border of resorbing osteoclasts. Osteopontin, bone sialoprotein and osteonectin act as substrates for TRAP. Osteopontin is highly expressed at the bone surface, opposite sealing zone of resorbing osteoclasts, and is essential for resorption to take place. Osteopontin enables osteoclasts to adhere to bone surface by binding with integrin αVβ3, which are abundantly present at the sealing zone. TRAP can remove phosphate groups from osteopontin, an event that consequently disrupts adhesion of osteoclasts to the bone. This suggests that the enzyme might regulate osteoclast adhesion to the bone and also enable migration of osteoclasts to adjacent sites of resorption. The ability of TRAP to degrade phosphoproteins in bone by dephosphorylation may illustrate a preliminary stage in the degradation of the bone matrix. TRAP can hydrolyse and liberate pyrophosphate from bone matrix which is an inhibitor of resorption. This hydrolysis event would enable osteoclasts to begin bone resorption activity.

Intracellular role of TRAP

Intracellularly TRAP has been found to be co-localized with organic products of bone degradation released from bone matrix during resorption and endocytosed into osteoclasts. It has been put forward that TRAP containing vesicles fuse with transcytotic vesicles transporting the matrix degradation products from ruffled border to FSD (functional secretory domain) of osteoclasts. In this location, TRAP is secreted out of cells together with matrix degradation products. After this stage, both entities leak into the circulation at a rate that corresponds to the amount of resorption activity being undertaken by the osteoclast.

The enzyme is synthesized to help dispose of the products of bone breakdown within transcytotic vesicles. Along with fragments of bone matrix, it is released into the extracellular environment as an active enzyme by exocytosis at the FSD. TRAP then subsequently leaks into the circulation through the interstitial fluid.

Extracellular fate of TRAP

Once secreted TRAP is exposed to physiological influences present in body fluids. It binds to α2 macroglobulin, a high molecular weight molecule in serum. It has been proposed that, α2 macroglobulin may be a carrier molecule for TRAP, that mediates clearance of enzyme from areas of bone resorption and then the circulation. Ultimately, TRAP has the fate of all circulating enzymes. Its structure becomes compromised, leading to its inactivation as a catalyst. It loses its binuclear iron center, which is then recycled, and the iron free enzyme protein is broken down by proteases in the plasma and the liver. The fragments that result from these events are eventually metabolized by the liver and/or removed in the urine.

The concentration of osteoclast derived TRAP in serum can be assessed by immunoassay, and has a quantitative and dynamic relation to amount of resorption taking place on a day by day basis.

The immunoassay would provide additional information in the hospital's clinical laboratory for diagnosis and monitoring of bone resorption conditions.

Hormones

Parathyroid hormone (PTH) is produced in the parathyroid glands in response to hypocalcemia, stimulating bone resorption. A stimulating role in bone formation has also been established through the synthesis of IGF 1 and TGF β. This dual effect of resorption and formation is explained by the fact that the continuous supply of PTH stimulates bone resorption through the synthesis of RANKL on the part of the osteoblastic cells, while at intermittent doses, it would stimulate the formation of bone, associated with an increase of the growth factors and with a decrease in the apoptosis of osteoblasts.

Calcitonin is secreted when blood calcium levels rise. It inhibits bone resorption and promotes calcium salt deposition in bone matrix, effectively reducing blood calcium levels. As blood calcium levels fall, calcitonin release wanes as well. Calcitonin also reduces the number and activity of osteoclasts.

Vitamin D metabolites

The major active metabolite of vitamin D is 1,25-dihydroxycholecalciferol. This has been shown to affect bone formation and also to cause bone resorption. Its effect on bone resorption appears to be by the differentiation of committed progenitor cells into mature cells. It favors the intestinal absorption of calcium and phosphate and therefore bone mineralization. It is necessary for normal growth of the skeleton.

Estrogen receptors are present on osteoblasts, osteocytes and osteoclasts. They favor bone formation, increasing the number and function of osteoblasts. It has also been proposed that these hormones are believed to increase the levels of OPG, which inhibits resorption.

Growth hormones act directly on the osteoblasts stimulating their activity and increasing the synthesis of collagen, osteocalcin and alkaline phosphatase. Indirect action is produced through an increase in the synthesis of IGF-I and II by osteoblasts, which stimulate the proliferation and differentiation of osteoblasts.

Glucocorticoids at high doses inhibit the synthesis of IGF-1 by osteoblasts and suppress BMP-2 and Cbfa-1 which are essential for the formation of osteoblasts. But, at physiological doses they are believed to have osteogenic capacity favoring osteoblastic differentiation.

- alkaline phosphatase (total)

- alkaline phosphatase (skeletal isoenzymes)

- osteocalcin

- procollagen I extension peptide

- urine calcium

- urinary hydroxy proline

- collagen crosslink fragments (first to be hydrolysed)

- urine N-telopeptide (N terminus of collagen fibrils)

- urine C-telopeptide (C terminus of collagen fibrils)

- urine total pyridinoline

- urine free deoxypyridinoline

Serum markers

Pathologies caused by improper control of remodeling are; osteoporosis, osteopetrosis, malignant bone tumors inflammatory joint diseases, hyperparathyroidism, Paget's disease and hyperthyroidism.

Functions of alveolar bone are

Alveolar bone proper

The alveolar bone proper consists partly of lamellated and partly of bundle bone and is about 0.1–0.4 mm thick. It surrounds the root of the tooth and gives attachment to principal fibers of the periodontal ligament.

Lamellated bone

The lamellar bone contains osteons each of which has a blood vessel in a haversian canal. Blood vessel is surrounded by concentric lamellae to form osteon. Some lamellae of the lamellated bone are arranged roughly parallel to the surface of the adjacent marrow spaces, whereas others form haversian systems.

Bundle bone

Bundle bone is that bone in which the principal fibers of the periodontal ligament are anchored. The term ‘bundle’ was chosen, because, the bundles of the principal fibers continue into the bone as Sharpey's fibers (Fig. 9.11). The bundle bone is characterized by the scarcity of the fibrils in the intercellular substance. These fibrils, more over, are all arranged at right angles to Sharpey's fibers. The bundle bone contains fewer fibrils than does lamellated bone, and therefore it appears dark in routine hematoxylin and eosin stained sections and much lighter in preparations stained with silver than does lamellated bone. These fibers are mineralized at the periphery and have a larger diameter. These fibers are less numerous than the corresponding fiber bundles in the cementum on the opposite side of the periodontal ligament. The collagen adjacent to bone is always less mature than that adjacent to cementum. In some areas, the alveolar bone proper consists mainly of bundle bone. Bundle bone is formed in areas of recent bone apposition. Lines of rest are seen in bundle bone.

Radiographically, it is also referred to as the lamina dura, because, of increased radiopacity, which is due to the presence of thick bone without trabeculations, that X-rays must penetrate and not to any increased mineral content.

The alveolar bone proper, which forms the inner wall of the socket is perforated by many openings that carry branches of the interalveolar nerves and blood vessels into the periodontal ligament, and it is therefore called the cribriform plate (Fig. 9.12). Bone between the teeth is called interdental septum and is composed entirely of cribriform plate. The interdental and interradicular septa contain the perforating canals of Zuckerkandl and Hirschfeld (nutrient canals) which house the interdental and interradicular arteries, veins, lymph vessels and nerves (Fig. 9.13).

The supporting alveolar bone consists of two parts:

Cortical plates

Cortical plates consist of compact bone and form the outer and inner plates of the alveolar processes. The cortical plates, continuous with the compact layers of the maxillary and mandibular body, are generally much thinner in the maxilla, than in the mandible. They are thickest in the premolar and molar region of the lower jaw, especially on the buccal side. In the maxilla, the outer cortical plate is perforated by many small openings through which blood and lymph vessels pass.

In the region of the anterior teeth of both jaws, the supporting bone usually is very thin. No spongy bone is found here, and the cortical plate is fused with the alveolar bone proper (Fig. 9.14). In such areas, notably in the premolar and molar regions of the maxilla, defects of the outer alveolar wall are fairly common. Such defects, where periodontal tissues and covering mucosa fuse, do not impair the firm attachment and function of the tooth.

Bone underlying the gingiva is the cortical plate. Both cribriform plate and cortical plate are compact bone separated by spongy bone.

Histologically, the cortical plates consist of longitudinal lamellae and haversian systems (Fig. 9.4). In the lower jaw, circumferential or basic lamellae reach from the body of the mandible into the cortical plates.

Spongy bone

Spongy bone fills the area between the cortical plates and the alveolar bone proper. It contains trabeculae of lamellar bone. These are surrounded by marrow that is rich in adipocytes and pluripotent mesenchymal cells.

The trabeculae contain osteocytes in the interior and osteoblasts or osteoclasts on the surface. These trabeculae of the spongy bone buttress the functional forces to which alveolar bone proper is exposed. The cancellous component in maxilla is more than in the mandible. The study of radiographs permits the classification of the spongiosa of the alveolar process into two main types.

In type I the interdental and interradicular trabeculae are regular and horizontal in a ladder like arrangement (Fig. 9.15).

Type II shows irregularly arranged, numerous, delicate interdental and interradicular trabeculae. Both types show a variation in thickness of trabeculae and size of marrow spaces. The architecture of type I is seen most often in the mandible and fits well into the general idea of a trajectory pattern of spongy bone. Type II, although evidently functionally satisfactory, lacks a distinct trajectory pattern, which seems to be compensated for by the greater number of trabeculae in any given area. This arrangement is more common in the maxilla.

From the apical part of the socket of lower molars, trabeculae are sometimes seen radiating in a slightly distal direction.

These trabeculae are less prominent in the upper jaw, because of the proximity of the nasal cavity and the maxillary sinus. In the condylar process, in the angle of the mandible, in the maxillary tuberosity, and in other isolated foci, hematopoietic cellular marrow is found.

Synthetic bone grafting materials

Examples of synthetic materials include natural and synthetic hydroxyapatites, ceramics, calcium carbonate (natural coral), silicon-containing glasses, and synthetic polymers.

Synthetic materials carry no risk of disease transmission or immune system rejection. They help create an environment that facilitates the body's regenerative process.

A guided tissue regeneration approach has evolved which is an epithelial exclusionary technique. This technique facilitates the cells of the periodontal ligament to differentiate into osteoblasts, cementoblasts and fibroblasts resulting in regeneration of the lost periodontium. It involves use of membranes, nonresorbable and/or resorbable, which prevent the downgrowth of the epithelium thereby promoting regeneration. Example of a resorbable material is collagen and nonresorbable material is ePTFE (expanded polytetrafluoroethylene) membrane.

Biologically mediated strategies

Biologically mediated strategies include materials, such as enamel matrix proteins, that can be premixed with vehicle solution. They are intended as an adjunct to periodontal surgery for topical application onto exposed root surfaces or bone to treat intrabony defects and furcations due to moderate or severe periodontitis. Enamel matrix protein leave only a resorbable protein matrix on the root surface, which makes bone more likely to regenerate. These bone morphogenetic proteins help to initiate a cascade of events leading to the differentiation of progenitor cells into phenotypes involved in periodontal regeneration.

Bone tissue engineering has emerged as a new therapeutic alternative to promote bone healing. This approach aims to regenerate or repair bone tissue with various combinations of polymeric scaffolds, cells and inductive factors into a system that actively stimulates tissue formation.

Scaffolds are of two types:

These scaffolds are a porous composite material composed mainly of hydroxyapatite in a collagen I matrix. These materials promote osteoblasts and osteoprogenitor cell attachment and differentiation to enhance bone tissue formation.

Other materials used for bone tissue engineering include synthetic, biocompatible and biodegradable polymers such as poly (lactide-co-glycolide)

– For filling small defects, injectable polymers such as alginate or poly (ethylene glycol) are preferred.

Cells. Osteoblasts and osteoprogenitor cells have been incorporated into various scaffolds to enhance bone repair. These cells must be obtained from patient or donor. Stem cells derived from bone marrow periosteum, adipose tissue, skeletal muscle or baby teeth can be induced to differentiate into diverse cell types such as muscle, nerve, cartilage, bone and fat.

Direct or gene therapy approaches to the delivery of osteoinductive factors are also promising approaches for bone regeneration.

Controlled release of proteins from a polymeric scaffold allows localized sustained protein delivery and may be a more effective means to enhance bone formation.

Delivery of BMP-2 from absorbable collagen sponge induces bone formation and heals bony defects. BMP-2 can also be delivered from synthetic materials such as poly (ethylene glycol) hydrogels to promote repair of bone defects. Other than BMP-2, sustained release of FGF-2 and IGF-1 can also accelerate bone formation.