OSTEOGENESIS

5 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):

1. The embryonic mesenchyme changes into a highly vascularized connective tissue. Fibroblast-like mesenchymal cells, embedded in a gelatinous extracellular matrix containing collagen fibers, aggregate.

2. Mesenchymal cells acquire the typical columnar form of osteoblasts and begin to secrete bone matrix (see Box 5-A). Numerous ossification centers develop and eventually fuse, forming a network of anastomosing trabeculae resembling a sponge, the so-called spongy bone or primary spongiosa.

3. Because collagen fibers in the newly formed trabeculae are randomly oriented, the early intramembranous bone is described as woven bone—in contrast with lamellar bone formed later during bone remodeling.

4. Calcium phosphate is deposited in the bone matrix, which is laid down by apposition. Interstitial bone growth does not occur.

5. Bone matrix mineralization leads to two new developments (see Figure 5-2): the entrapment of osteoblasts as osteocytes, as trabeculae thicken, and the partial closing of the perivascular channels, which assume the new role of hematopoiesis by conversion of mesenchymal cells into blood-forming cells.

Box 5-A From osteoblasts to osteocytes

• Mesenchymal cells differentiate to osteoblasts when the transcription factors Cbfa1/Runx2 and osterix are expressed.

• The differentiation of osteoblasts to osteocytes also requires the expression of Cbfa1/Runx2 and osterix.

• The differentiation of mesenchymal cells to chondrocytes occurs when the gene encoding Sox9 is expressed. During endochondral ossification (as we will see later), chondrocytes undergo considerable enlargement and become hypertrophic. The transition from chondrocyte to hypertrophic chondrocyte is stimulated by Cbfa1/Runx2 but inhibited by Sox9.

• Putting things together, Cbfa1/Runx2 has a role in both chondrocytic and osteoblastic differentiation. Osterix specifies the differentiation of osteoblasts to osteocytes. A lack of osterix gene expression affects osteoblastic differentiation but not chondrocyte maturation. An example is cleidocranial dysplasia with defects in both intramembranous and endochondral ossification.

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:

1. The conversion of woven bone to lamellar bone. In lamellar bone, the newly synthesized collagen fibers are aligned into regular bundles. Lamellae arranged in concentric rings around a central blood vessel occupying the haversian canal form osteons or haversian systems. Membrane bones remain as spongy bone in the center, the diploë, enclosed by an outer and an inner layer of compact bone.

2. The condensation of the external and internal connective tissue layers to form the periosteum and endosteum, respectively, containing fusiform cells with osteoprogenitor cell potential.

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):

1. Blood vessels invade the space formerly occupied by the hypertrophic chondrocytes, and they branch and project toward either end of the center of ossification. Blind capillary ends extend into the cavities formed within the calcified cartilage.

2. Osteoprogenitor cells and hematopoietic stem cells reach the core of the calcified cartilage through the perivascular connective tissue surrounding the invading blood vessels. Then, osteoprogenitor cells differentiate into osteoblasts that aggregate on the surfaces of the calcified cartilage and begin to deposit bone matrix (osteoid).

3. At this developmental step, a primary center of ossification—defined by both the periosteal collar (intramembranous ossification type) and the center of ossification in the interior of the cartilage template—is organized at the diaphysis. Secondary centers of ossification develop later in the epiphyses.

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.