Endochondral ossification

Endochondral ossification is one of the two essential processes during fetal development of the mammalian skeletal system by which bone tissue is created. Unlike intramembranous ossification, which is the other process by which bone tissue is created, cartilage is present during endochondral ossification. It is also an essential process during the rudimentary formation of long bones, the growth of the length of long bones, and the natural healing of bone fractures.

Growth of the cartilage model
The cartilage model will grow in length by continuous cell division of chondrocytes, which is accompanied by further secretion of extracellular matrix. This is called interstitial growth. The process of appositional growth occurs when the cartilage model also grows in thickness due to the addition of more extracellular matrix on the peripheral cartilage surface, which is accompanied by new chondroblasts that develop from the perichondrium.

Primary center of ossification
The first site of ossification occurs in the primary center of ossification, which is in the middle of diaphysis (shaft). Then:


 * Formation of periosteum: The perichondrium becomes the periosteum. The periosteum contains a layer of undifferentiated cells (osteoprogenitor cells) which later become osteoblasts.
 * Formation of bone collar: The osteoblasts secrete osteoid against the shaft of the cartilage model (Appositional Growth). This serves as support for the new bone.
 * Calcification of matrix: Chondrocytes in the primary center of ossification begin to grow (hypertrophy). They stop secreting collagen and other proteoglycans and begin secreting alkaline phosphatase, an enzyme essential for mineral deposition. Then calcification of the matrix occurs and apoptosis of the hypertrophic chondrocytes occurs. This creates cavities within the bone. The exact mechanism of chondrocyte hypertrophy and apoptosis is currently unknown.
 * Invasion of periosteal bud: The hypertrophic chondrocytes (before apoptosis) secrete Vascular Endothelial Cell Growth Factor that induces the sprouting of blood vessels from the perichondrium. Blood vessels forming the periosteal bud invade the cavity left by the chondrocytes and branch in opposite directions along the length of the shaft. The blood vessels carry hemopoietic cells, osteoprogenitor cells and other cells inside the cavity. The hemopoietic cells will later form the bone marrow.
 * Formation of trabeculae: Osteoblasts, differentiated from the osteoprogenitor cells that entered the cavity via the periosteal bud, use the calcified matrix as a scaffold and begin to secrete osteoid, which forms the bone trabecula. Osteoclasts, formed from macrophages, break down spongy bone to form the medullary (bone marrow) cavity.

Secondary center of ossification
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 centers 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 26 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.

Appositional bone growth
The growth in diameter of bones around the diaphysis occurs by deposition of bone beneath the periosteum. Osteoclasts in the interior cavity continue to degrade bone until its ultimate thickness is achieved, at which point the rate of formation on the outside and degradation from the inside is constant.

Histology
During endochondral ossification, five distinct zones can be seen at the light-microscope level.
 * 1) Zone of resting cartilage. This zone contains normal, resting hyaline cartilage.
 * 2) Zone of proliferation. In this zone, chondrocytes undergo rapid mitosis, forming distinctive looking stacks.
 * 3) Zone of maturation / hypertrophy. It is during this zone that the chondrocytes undergo hypertrophy (become enlarged). Chondrocytes contain large amounts of glycogen and begin to secrete alkaline phosphatase.
 * 4) Zone of calcification. In this zone, chondrocytes are either dying or dead, leaving cavities that will later become invaded by bone-forming cells. Chondrocytes here die when they can no longer receive nutrients or eliminate wastes via diffusion.  This is because the calcified matrix is much less hydrated than hyaline cartilage.
 * 5) Zone of ossification. Osteoprogenitor cells invade the area and differentiate into osteoblasts, which elaborate matrix that becomes calcified on the surface of calcified cartilage. This is followed by resorption of the calcified cartilage/calcified bone complex.

Fracture healing
During fracture healing, cartilage is often formed and is called callus. This cartilage ultimately develops into new bone tissue through the process of endochondral ossification.