OSTEOGENESIS

Bone formation (osteogenesis or ossification)

Bone develops by replacement of a preexisting connective tissue. The two processes of bone formation or osteogenesis observed in the embryo are: (1) intramembranous bone formation, in which bone tissue is laid down directly in primitive connective tissue or mesenchyme (Figures 5-1 and 5-2), and (2) endochondral bone formation, in which bone tissue replaces a preexisting hyaline cartilage, the template or anlage of the future bone (Figures 5-3 to 5-5).

Figure 5-1 Intramembranous ossification

Figure 5-2 Intramembranous ossification

Figure 5-3 Endochondral ossification: Primary ossification center

Figure 5-4 Endochondral ossification: Secondary ossification centers

Figure 5-5 Endochondral ossification: Four major zones

The mechanism of bone matrix deposition during intramembranous and endochondral ossification is essentially the same: A primary trabecular network or primary spongiosa is first laid down and then transformed into mature bone. But there is a difference: In endochondral ossification, cartilage is replaced by bone matrix.

Intramembranous bone formation

Membrane bones such as the flat bones of the skull develop by intramembranous ossification. Intramembranous ossification occurs in the following sequence (see Figure 5-1):

Osteocytes remain connected to each other by cytoplasmic processes enclosed within canaliculi, and new osteoblasts are generated from osteoprogenitor cells adjacent to the blood vessels.

The final developmental events include:

At birth, bone development is not complete, and the bones of the skull are separated by spaces (fontanelles) housing osteogenic tissue. The bones of a young child contain both woven and lamellar bony matrix.

Endochondral ossification

Endochondral ossification is the process by which skeletal cartilage templates are replaced by bone. As you may recall, intramembranous ossification is the process by which a skeletal mesenchymal template is replaced by bone without passing through the cartilage stage. Bones of the extremities, vertebral column, and pelvis derive from a hyaline cartilage template.

As in intramembranous ossification, a primary ossification center is formed during endochondral ossification (Figure 5-3). Unlike intramembranous ossification, this center of ossification derives from proliferated chondrocytes that have deposited an extracellular matrix containing type II collagen.

Shortly thereafter, chondrocytes in the central region of the cartilage undergo maturation to hypertrophy and synthesize a matrix containing type X collagen, a marker for hypertrophic chondrocytes. Angiogenic factors secreted by hypertrophic chondrocytes (vascular endothelial cell growth factor [VEGF]) induce the formation of blood vessels from the perichondrium. Osteoprogenitor and hematopoietic cells arrive with the newly formed blood vessels.

These events result in the formation of the primary ossification center. Hypertrophic chondrocytes undergo apoptosis as calcification of the matrix in the middle of the shaft of the cartilage template takes place.

At the same time, the inner perichondrial cells exhibit their osteogenic potential, and a thin periosteal collar of bone is formed around the midpoint of the shaft, the diaphysis. Consequently, the primary ossification center ends up located inside a cylinder of bone. The periosteal collar formed under the periosteum by intramembranous ossification consists of woven bone. As we will discuss later on, the periosteal collar is converted into compact bone.

The following sequence of events defines the next steps of endochondral ossification (Figure 5-4):

The growth in length of the long bones depends on the interstitial growth of the hyaline cartilage while the center of the cartilage is being replaced by bone at the equidistant zones of ossification.

Secondary centers of ossification and the epiphyseal growth plate

Up to this point, we have analyzed the development of primary centers of ossification in the diaphysis of long bones that occurs by the third month of fetal life.

After birth, secondary centers of ossification develop in the epiphyses (see Figure 5-4). As in the diaphysis, the space occupied by hypertrophic chondrocytes is invaded by blood vessels and osteoprogenitor cells from the perichondrium. Most of the hyaline cartilage of the epiphyses is replaced by the spongy bone, except for the articular cartilage and a thin disk, the epiphyseal growth plate, located between the epiphyses and the diaphysis. The epiphyseal growth plate is responsible for subsequent growth in length of the bone.

Clinical significance: The epiphyseal growth plate and dwarfism

Indian hedgehog (Ihh), a member of the hedgehog family of proteins secreted by chondrocytes, regulates chondrocyte proliferation of the growth plate in a paracrine fashion and delays chondrocyte hypertrophy (see Figure 5-9). Ihh also regulates bone formation in the perichondrial collar. A lack of expression of Ihh protein in mutant mice results in dwarfism and lack of endochondral ossification. Essentially, Ihh maintains the pool of proliferating chondrocytes in the growth plate by delaying their hypertrophy. In addition, Ihh stimulates the expression of parathyroid hormone-related peptide (PTH-RP) in perichondrial chondrocytes adjacent to the articular surface. A feedback loop between Ihh and PTH-RP regulates the balance between proliferating and hypertrophic chondrocytes.

Figure 5-6 Endochondral ossification: Zones of proliferation, hypertrophy, and vascular invasion

Figure 5-7 Endochondral ossification: Zones of proliferation and hypertrophy

Figure 5-8 Endochondral ossification: Zones of hypertrophy and vascular invasion

Figure 5-9 Growth plates and bone growth in length

At the end of the growing period, the epiphyseal growth plate is gradually eliminated by a continuum established between the diaphysis and the epiphyses. No further growth in length of the bone is possible once the epiphyseal growth plate disappears at puberty.

Zones of endochondral ossification

As we have seen, the deposition of bone in the center of the diaphysis is preceded by an erosion process in the hyaline cartilage template (see Figure 5-4). This center of erosion, defined as the primary ossification center, extends in both directions of the template, in parallel with the formation of a bony collar.

The bony collar provides strength to the midsection of the diaphysis or shaft as the cartilage is weakened by the gradual removal of the cartilage before its replacement by bone.

The continuing process of cartilage erosion and bone deposition can be visualized histologically (Figure 5-5). Four major zones can be distinguished, starting at the end of the cartilage and approaching the zone of erosion:

3. The hypertrophic zone is defined by both chondrocyte apoptosis and calcification of the territorial matrix surrounding the columns of previously proliferated chondrocytes (see Figures 5-6 and 5-7). Despite their “unhealthy”, hypertrophic chondrocytes play an important role in bone growth. Hypertrophic chondrocytes have the following functional characteristics: They (1) direct the mineralization of the surrounding cartilage matrix, (2) attract blood vessels through the secretion of vascular endothelial growth factor (VEGF), (3) recruit macrophages (called chondroclasts) to degrade the cartilage matrix, (4) instruct adjacent chondrocytes of the perichondrium to change into osteoblasts forming the bone collar, (5) produce type X collagen, a marker of hypertrophic chondrocytes, and, when their task is accomplished, (6) they undergo apoptosis.

Figure 5-10 Bone growth in length involves an osteoclastic “chase” and chondrocytic “run” sequence

Chondrocytes in this zone are significantly enlarged (hypertrophic). As a result, the septa separating adjacent columns appear thinner due to a compression effect mediated by the hypertrophic chondrocytes. A provisional calcification begins in the longitudinal septa. The deepest layer, proximal to the vascular invasion zone, displays the blind end of capillary sprouts (Figure 5-8) derived from the developing bone marrow cavity occupied by hematopoietic cells (see Chapter 6, Blood and Hematopoiesis).

Osteoprogenitor cells give rise to osteoblasts that begin lining the surfaces of the exposed cores of calcified cartilage (stained blue—basophilic—in the light microscopy photograph in Figure 5-8) and initiate the deposition of osteoid (stained pink—acidophilic—in Figure 5-8). The osteoid contains abundant type I collagen fibers embedded in the extracellular matrix.

The cartilage struts are gradually replaced by bone. The deposit of osteoid denotes the beginning of osteogenesis and results in the formation of bone spicules and, later, in trabeculae. As a consequence, cancellous bone appears in the midsection of the template.

As the ossification process advances toward the adjacent proliferative zones (a “chase” effect), the bone marrow cavity increases in size owing to loss of cartilage and erosion of newly formed bone spicules by osteoclasts (Figure 5-10).

The periosteal collar grows in length and thickness (by appositional growth) at the midsection of the shaft and compensates for the loss of endochondral bone, while also strengthening the gradually eroding cartilage template.

The reserve zone persists by continuous cell division and is responsible for a continued growth in length by the epiphyseal growth plate, which remains between the diaphysis and epiphysis of the bone. The epiphyseal growth plate becomes reduced to an epiphyseal line from puberty to maturity, and the long bone no longer grows in length.

After endochondral ossification, the general organization of a long bone is remodeled by combined reabsorption mediated by osteoclasts in certain areas and the deposition of new bone by osteoblasts in others. As a result, spongy bone is replaced by compact bone by a process in which osteoblasts produce overlapping layers of bone or lamellae on the surface of longitudinal cavities occupied by blood vessels. Consequently, a concentric arrangement of bone lamellae encircles a blood vessel entrapped within a canal to form a primitive haversian system.

Some variation exists in the literature concerning the classification of the zones of endochondral ossification. The reserve, proliferative, hypertrophic, and vascular invasion zones summarized earlier provide a simple way to guide you through the complexity of bone formation and the understanding of the mechanisms of bone repair.

Finally, it is important to stress that local regulatory molecules (bone morphogenetic proteins, hedgehog proteins, the RANK-RANKL signaling pathway, and fibroblast growth factors) and blood circulating proteins (insulin-like growth factor-1 [IGF-1], thyroid hormone, estrogens, androgens, vitamin D, retinoids, and glucocorticoids) control both bone development and remodeling throughout life. We will emphasize the specific function of these biological agents as we discuss them. Their impact on skeletal biology and the therapeutic opportunities confronting an increasing number of genetic and degenerative diseases must be appreciated.

Growth in width of the diaphysis

As the bone grows in length, new layers of bone are added to the outer portions of the diaphysis by appositional growth. As a result, the thickness of the diaphysis increases. Simultaneous erosion of the inner wall of the diaphysis results in enlargement of the marrow cavity.

New bone in the form of haversian systems is added beneath the periosteum by its osteogenic layer. The surface of the diaphysis has longitudinal ridges with grooves between them. The periosteum contains blood vessels.

The following sequence is observed (Figure 5-11):

Figure 5-11 Periosteal bone growth

Bone remodeling

Bone remodeling consists in the replacement of newly formed and old bone by a resorption-production sequence with the participation of osteoclasts and osteoblasts. Bone remodeling is a continuous process throughout life and occurs at random locations. The purpose of remodeling is to establish the optimum of bone strength by repairing microscopic damage (called microcracking) and to maintain calcium homeostasis.

Microcracking is limited to a region of the osteon (for example, damage to canaliculi, which disrupts osteocyte cell-cell communication leading to cell death). It can be repaired by the osteoclast-osteoblast remodeling process. When the architecture of the osteon is defective, microcracking becomes widespread and a complete bone fracture may occur.

Under normal conditions, the same amount of resorbed bone is replaced by the same volume of new bone. If the volume of resorbed bone is not completely replaced by new bone, the tissue becomes weakened and a risk of spontaneous fractures arises.

There are two forms of bone remodeling: (1) cortical bone remodeling, and (2) trabecular bone remodeling. Cortical bone remodeling is the resorption of an old haversian system followed by the organization of a new haversian system (Figure 5-12). Trabecular bone remodeling occurs on the bone surface (see Figure 5-12), in contrast to cortical bone remodeling, which occurs inside an osteon. The trabecular endosteal surface is remodeled by a mechanism similar to cortical bone remodeling.

Figure 5-12 Bone remodeling

Clinical significance: Hereditary and degenerative bone disorders

Ossification includes growth, modeling, and remodeling of the bone, processes mediated by osteoblasts and osteoclasts under the control of local regulatory factors and blood-borne signaling molecules, including parathyroid hormone and vitamin D₃. A number of conditions can alter the skeleton by affecting cell-mediated bone remodeling or disturbing the mineralization of the extracellular matrix.

Rickets and osteomalacia are a group of bone diseases characterized by a defect in the mineralization of the bone matrix (osteoid), most often caused by alack of vitamin D₃. Rickets is observed in children and produces skeletal deformities. Osteomalacia is observed in adults and is caused by poor mineralization of the bone matrix.

We have already stressed the importance of the RANK-RANKL signaling pathway as a pharmacologic target in the treatment of osteoporosis by controlling osteoclastogenesis.

Osteopetrosis (“stonelike bone”) includes a group of hereditary diseases characterized by abnormal osteoclast function. The bone is abnormally brittle and breaks like a soft stone. The marrow canal is not developed, and most of the bone is woven because of absent remodeling.

We have already discussed a mutation in the colony-stimulating factor–1 gene whose expression is required for the formation of osteoclasts (see Bone in Chapter 4, Connective Tissue). A clinical variant of osteopetrosis, also known as marble bone disease, or Albers-Schönberg disease, is caused by a deficiency in carbonic anhydrase II, required by osteoclasts to accumulate H⁺ in Howship’s resorption lacunae and acidify the environment for the activation of secretory cathepsin K enzyme.

Fibrodysplasia ossificans progressiva (FOP) is a very rare autosomal dominant disorder of the connective tissue. The main clinical features are skeletal malformations (hands and feet) present at birth and the ossification of soft tissues (muscles of the neck and back) precipitated by trauma. Ectopic bone formation also occurs in ligaments, fasciae, aponeuroses, tendons, and joint capsules.

Patients with FOP have a mutation in the gene encoding activin receptor type 1A (ACVR1), a receptor for bone morphogenetic protein (BMP). BMPs are members of the transforming growth factor–β superfamily with a role in the development of bone and other tissues. The mutation consists in the substitution of histidine for arginine at position 206 of the 509-amino-acids-long ACVR1. This single amino acid substitution results in the abnormal activation of ACVR1 leading to the transformation of connective tissue and muscle tissue into a secondary skeleton.

Joints

Bones are interconnected by articulations, or joints, that permit movement. Synarthroses are the joints that permit little or no movement (cranial bones, ribs, and the sternum). Amphiarthroses enable slight movement (intervertebral disks and bodies). Diarthroses permit free movement.

In a diarthroidal joint, a capsule links the ends of the bones. The capsule is lined by a synovial membrane that encloses the articular or synovial cavity. The synovial cavity contains a fluid necessary for reducing the friction between the hyaline cartilage covering the opposing articular surfaces.

The articular cartilage is almost typical hyaline cartilage except that it lacks a perichondrium and has a unique collagen fiber organization in the form of overlapping arches. Collagen arcades sustain the mechanical stress on the joint surfaces.

The joint capsule consists of two layers: an outer layer of dense connective tissue with blood vessels and nerves, and an inner layer, called the synovial membrane. The inner surface of the synovial membrane is covered by one to two layers of synovial cells overlying the connective tissue (Figure 5-13). There are two classes of synovial cells: (1) type A macrophage-like synovial cells, and (2) type B fibroblast-like synovial cells. There is no basal lamina separating synovial cells from the connective tissue. The connective tissue contains a rich network of fenestrated capillaries.

Figure 5-13 Joints and arthritis

Synovial fluid is a combined product of the synovial cells and the ultrafiltrate of the capillaries. The fluid is rich in hyaluronic acid, glycoproteins, and leukocytes.

Clinical significance: Rheumatoid arthritis

Rheumatoid arthritis is a common chronic inflammatory and destructive disease of the joints that starts with a proliferative process of the synovial membrane, leading to the erosion of the articular cartilage and destruction of the subjacent bone.

The initial event is the activation of CD4⁺ T cells by an undetermined antigen. Activated CD4⁺ T cells stimulate the production of tumor necrosis factor–α (TNF-α), interleukin-2 (IL-2), and interleukin-6 (IL-6), and the secretion of collagenase and metalloproteinases by monocytes, macrophages, and fibroblast-like synovial cells. Activated CD4⁺ T cells stimulate B cells to differentiate into plasma cells to produce immunoglobulins and rheumatoid factor.

TNF-α, IL-1, and IL-6 are key cytokines in driving inflammation in rheumatoid arthritis (Figure 5-14). TNF-α and IL-1 can be detected in synovial fluid of patients with rheumatoid arthritis. TNF-α and IL-1 stimulate fibroblast-like synovial cells, osteoclasts, and chondrocytes to release cartilage and bone-destroying matrix metalloproteinases.

Figure 5-14 Synovial membrane in rheumatoid arthritis

The neutralization of proinflammatory cytokines by soluble receptors or monoclonal antibodies is currently used in the treatment of patients with rheumatoid arthritis. Figure 5-14 provides a summary of the major therapeutic strategies for suppressing inflammation and preventing joint damage.

Concept mapping

Osteogenesis