Endochondral ossification

Endochondral ossification is one of two types of bone formation (ossification) and is the process responsible for much of the bone growth in vertebrate skeletons, especially in long bones. As the name might suggest (endo - within, chondro - root for cartilage), endochondral ossification occurs by replacement of hyaline cartilage.

Development of the cartilage model
During endochondral ossification in the developing fetus, mesenchymal cells aggregate to form a compact grouping of cells in a process called prechondrogenic condensation. Prechondrogenic condensation begins the process of endochondral ossification and is required for subsequent skeletal development. Following prechondrogenic condensation, the mesenchymal cells differentiate into chondrocytes and begin to produce a cartilaginous matrix. At the end of its life cycle, the chondrocyte calcifies its matrix before undergoing programmed cell death or apoptosis. At this point, blood vessels penetrate this calcified matrix, bringing in osteoblasts. The osteoblasts use the calcified cartilage matrix as a template to build bone, thus completing endochondral ossification.

Growth of the cartilage model
The cartilage model would 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 would also grow in thickness which is due to the addition of more extracellular matrix on the periphery 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). The following steps then occur:


 * Formation of periosteum:Once vascularized, the perichondrium becomes the periosteum. The periosteum contains a layer of undifferentiated cells which later become osteoblasts.
 * Formation of bone collar: The osteoblast secretes osteoid against the shaft of the cartilage model. 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. Nutrients can no longer diffuse if the matrix becomes sufficiently calcified and the chondrocytes subsequently die. This creates cavities within the bone.
 * Invasion of periosteal bud: A periosteal bud, which consists of blood vessels, lymph vessels and nerves, invades the cavity left by the chondrocytes. The vascularization utlimately carries hemopoietic cells, osteoblasts and osteoclasts inside the cavity. The hemopoietic cells will later form the bone marrow.
 * Formation of trabeculae: Osteoblasts use the calcified matrix as a scaffold and begin to secrete osteoid, which forms the bone trabecula. Osteoclasts break down spongy bone to form the medullary (bone marrow) cavity.

Secondary center of ossification
Cartilage is retained in the epiphyseal plate, located between the diaphysis (the shaft) and the epiphysis (end) of the bone. These areas of cartilage are known as secondary centers of ossification. Cartilage cells undergo the same transformation as above. As growth progresses, the proliferation of cartilage cells in the epiphyseal plate slows and eventually stops. The continuous replacement of cartilage by bone results in the obliteration of the epiphyseal plate, termed the closure of the epiphysis. Only articular cartilage remains. Mineralisation of articular cartilage and its replacement by bone continues in the adult, though at a much reduced rate than in growing animals.

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, four 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.