BONE FORMATION

Bone is formed either by direct ossification of embryonic connective tissue (intramembranous ossification) or by replacement of hyaline cartilage (intracartilaginous or endochondral ossification). Intramembranous ossification takes place in the so-called membrane bones of the skull, while endochondral ossification is characteristic of the bones of the trunk and extremities.

1. Intramembranous bone formation. Examine the section of fetal head on slide B35 (H&E) and compare it to Figure 8 below. Irregularly shaped trabeculae of spongy bone are present within the mesenchyme lateral and dorsal to the developing nasal chambers. Locate the osseous tissue (staining pale red) and note that it is called spongy bone or cancellous bone and is characterized by many spaces between the trabeculae. Osteoblasts line the trabecular surfaces. Some of these cells have become entrapped within lacunae in the newly deposited bone matrix and have thus become osteocytes. In some sections, one may observe resorption of bone from the ends of the trabeculae or occupying shallow excavations in their surface (Howship's lacunae).

Figure 20: Intramembranous Ossification. Taken from: Gartner and Hiatt, Color Textbook of Histology, p. 122, Figure 7-12.

There are several events, which take place in intramembranous bone formation. The events are show in Figure 20 above and listed as follows:

a. Increased vascularity of tissue.

b. Active proliferation of mesenchymal cells. The mesenchymal cells give rise to osteogenic cells, which develop into osteoblasts.

c. Osteoblasts begin to lay down osteoid. Osteoid is the organic part of bone without the inorganic constituent.

d. Osteoblasts either retreat or become entrapped as osteocytes in the osteoid.

e. The osteoid calcifies to form spicules of spongy bone. The spicules unite to form trabeculae. The inorganic salts carried in by the blood vessels supposedly bring about calcification. The salts are deposited in an orderly fashion as fine crystals (hydroxyapatite crystals) intimately associated with the collagenous fibers. These crystals are only visible with the electron microscope.

f. Bone remodeling occurs. Periosteum and compact bone are formed.

2. Intracartilaginous (endochondral) bone formation. This type of ossification involves the replacement of a cartilaginous model by bone and is best observed in long bones, such as the humerus or femur. Events of endochondral ossification can be seen in Figure 21 below and include the following:

a. Primary ossification center. The first change indicative of beginning ossification takes place about the center of the future bone shaft. Here the cartilage cells hypertrophy and the cartilage matrix becomes calcified. Subsequently, part of the calcified matrix disintegrates, opening cavities that communicate with the connective tissue and vessels at the surface.

b.Bone collar. The bone collar forms concurrently with the primary ossification center. Cells of the perichondrium begin to form bone. The bone collar holds together the shaft, which has been weakened by the disintegration of the cartilage. The connective tissue about the bone collar, previously a perichondrium, is now called periosteum.

c. Periosteal buds. These are connective tissue buds or "sprouts" containing mesenchymal cells (which give rise to osteogenic cells) and blood vessels, which grow from the periosteum to reach the primary ossification center. Osteoblasts attach to spicules of calcified cartilage in the primary ossification center and begin to produce osteoid. Thus, bone is formed and the process continues toward both epiphyses while this is occurring, the cartilage outside the primary ossification center increases in size by interstitial and appositional growth.

d. Secondary ossification centers. About the time of birth, a secondary ossification center appears in each end (epiphysis) of long bones. Periosteal buds carry mesenchyme and blood vessels in and the process is similar to that occurring in a primary ossification center. The cartilage between the primary and secondary ossification canters is called the epiphyseal plate, and it continues to form new cartilage, which is replaced by bone, a process that results in an increase in length of the bone. Growth continues until the individual is about 21 years old or until the cartilage in the plate is replaced by bone. The point of union of the primary and secondary ossification centers is called the epiphyseal line.

Figure 21: Prenatal long bone development. Taken from: Stevens and Lowe, Human Histology, p. 246, Figure 13.24.

Examine the section on slide A34, fetal finger, longitudinal section, human (Trichrome), which shows portions of three developing bones and their primary ossification centers. Find the collar and, if present, the osteoid and osteoblasts on its outer surface. Osteoblasts in this location are engaged in periosteal bone formation, a type of intramembranous ossification, which is responsible for the growth in thickness of long bones. Remember there are bones in the body, which are formed exclusively by intramembranous ossification, but there is NO bone in the body, which is the product of endochondral ossification alone. Nuclei, erythrocytes and bone matrix are red or orange; collagen and cartilage matrix are blue. Identify the fibrous and cellular layer of the periosteum and note there is no sharp boundary between the fibrous periosteum and the surrounding connective tissue. Many trabeculae are present in the marrow cavity. Calcified cartilage occupies the center of some of the trabeculae. With the aid of Figure 22 below, identify and study the five zones of cartilage associated with endochondral ossification.

1. Zone of reserve cartilage. This is typical hyaline cartilage and is a large zone in this preparation.

2. Zone of cell proliferation (ZP). The cartilage cells are small and tend to be arranged in columns, which run parallel to the long axis of the cartilage. This arrangement is indicative of their intense mitotic activity.

3. Zone of cell and lacunar maturation and hypertrophy enlargement (ZH). Chondrocytes and lacunae are larger than in the previous zone. The chondrocytes increase in size and resorb some their lacunar walls, enlarging them to such an extent that some of the lacunae become confluent.

4. Zone of calcification (ZC). This is a small zone having a slightly darker appearance than the preceding zone due to the basophilic staining of the calcified cartilage. The chondrocytes die in this zone.

5. Zone of cartilage removal and bone deposition. Osseous elements are present among the pieces of calcified cartilage.

Figure 22: Postnatal development of long bones. Taken from: Stevens and Lowe, Human Histology, p. 247, Figure 13.25.

Slide A37, endochondral ossification, fetal joint, (H & E). Calcified cartilage stains blue, while the matrix of bone is red and that of hyaline cartilage is pink (cartilage is more basophilic in most H & E slides). Good view of joint capsule with folds of synovial membrane, etc.

Slide A38, endochondral ossification, fetal joint, (Trichrome). Longitudinal section of joint between long bones of human fetus. Comparable to previous slide but more advanced in development. Note vessels in epiphysis.

Slide A36, secondary center of ossification, (H & E). This section includes the epiphysis of a long bone as well as a portion of the metaphysis and elements of the joint. Compare features

of the secondary center to those of primary sites of ossification.

Drawing of membranous bone formation

Using slide A35 (H&E), make a drawing from one area to show active osteoblasts, osteoid, and bone with osteocytes. Draw another area from the same slide to show bone associated with osteoclasts and inactive osteoblasts.

Drawing of endochondral bone formation

Draw an area toward the epiphysis of the bone in slide A34 (Trichrome), to show the destruction of cartilage and the formation of bone. Indicate and label the different zones.

SYNOVIAL JOINTS

A synovial joint is a movable joint, which contains synovial fluid in a closed cavity, the synovial cavity (a fiber-less tissue space). Synovial fluid consists largely of ground substance in highly polymerized hyaluronic acid. In addition, it contains a few cells leukocytes, macrophages, and synovial cells. The term synovial is derived from syn (together) and ovum (egg). Ovum as used here refers in particular to the white of the egg, which is a glairy fluid. The fluid acts as a lubricant to allow the free surface of the cartilage-capped bones that meet in the joint cavity to slide freely on one another. Synovial joints, then, represent a special type of joint for free movement. They are sometimes called diarthoses (di, apart, arthon, joint) because the bones are separated by a cleft (synovial cavity).

Study the synovial joint on slide A37 (H&E) and compare it with Figure 23 below to identify the various parts. The joint capsule can be seen around the joint area where it fits like a sleeve over the end of each cartilage model. The joint capsule consists of two layers (commonly called fibrous capsule of the joint) and the inner stratum is the synovial membrane. The fibrous layer is composed predominately of collagenous fibers, which extend from the periosteum of one bone of the joint to the periosteum of the other bone of the joint. Sharpey’s fibers help anchor the collagenous fibers to the underlying bone.

Figure 23: Synovial Joint. Taken from: Stevens and Lowe, Color Histology, p. 249, Figure 13.28

The synovial membrane attaches to the periphery of the articular cartilage but does not extend over the free surfaces of the articular cartilage. Prominent infoldings of the synovial membrane into the joint cavity are called synovial cells. They secrete the synovial fluid, which fills the cavity in life. The cells can undergo mitosis and completely repair the membrane in case of injury. Synovial cells, in addition to being randomly distributed in the membrane, are often concentrated and aligned along the inner surface of the membrane to produce the appearance of epithelium. Electron micrographs show, however, that the cells rest among collagenous fibers rather than on a basement membrane. The synovial membrane is richly supplied with blood and lymphatic vessels. Identify the small blood vessels in the synovial folds.