Endochondral Ossification Process 6 Steps Of Critical Thinking

Some of the most obvious structures derived from the paraxial mesoderm are bones. We can only begin to outline the mechanisms of bone formation here; students wishing further details are invited to consult histology textbooks that devote entire chapters to this topic.

There are three distinct lineages that generate the skeleton. The somites generate the axial skeleton, the lateral plate mesoderm generates the limb skeleton, and the cranial neural crest gives rise to the branchial arch and craniofacial bones and cartilage.* There are two major modes of bone formation, or osteogenesis, and both involve the transformation of a preexisting mesenchymal tissue into bone tissue. The direct conversion of mesenchymal tissue into bone is called intramembranous ossification. This process occurs primarily in the bones of the skull. In other cases, the mesenchymal cells differentiate into cartilage, and this cartilage is later replaced by bone. The process by which a cartilage intermediate is formed and replaced by bone cells is called endochondral ossification.

Intramembranous ossification

Intramembranous ossification is the characteristic way in which the flat bones of the skull and the turtle shell are formed. During intramembranous ossification in the skull, neural crest-derived mesenchymal cells proliferate and condense into compact nodules. (Thus, intramembranous ossification is not occurring from sclerotome-derived cells.) Some of these cells develop into capillaries; others change their shape to become osteoblasts, committed bone precursor cells (Figure 14.11A). The osteoblasts secrete a collagen-proteoglycan matrix that is able to bind calcium salts. Through this binding, the prebone (osteoid) matrix becomes calcified. In most cases, osteoblasts are separated from the region of calcification by a layer of the osteoid matrix they secrete. Occasionally, though, osteoblasts become trapped in the calcified matrix and become osteocytes—bone cells. As calcification proceeds, bony spicules radiate out from the region where ossification began (Figure 14.11B). Furthermore, the entire region of calcified spicules becomes surrounded by compact mesenchymal cells that form the periosteum (a membrane that surrounds the bone). The cells on the inner surface of the periosteum also become osteoblasts and deposit osteoid matrix parallel to that of the existing spicules. In this manner, many layers of bone are formed.

Figure 14.11

Schematic diagram of intramembranous ossification. (A) Mesenchymal cells condense to produce osteoblasts, which deposit osteoid matrix. These osteoblasts become arrayed along the calcified region of the matrix. Osteoblasts that are trapped within the (more...)

The mechanism of intramembranous ossification involves bone morphogenetic proteins and the activation of a transcription factor called CBFA1. Bone morphogenetic proteins (probably BMP2, BMP4, and BMP7) from the head epidermis are thought to instruct the neural crest-derived mesenchymal cells to become bone cells directly (Hall 1988). The BMPs activate the Cbfa1 gene in the mesenchymal cells. Just as the myogenic bHLH family of transcription factors is competent to transform primitive mesenchyme cells (or just about any other cell) into muscle-forming myoblasts, the CBFA1 transcription factor appears to be able to transform mesenchyme cells into osteoblasts. Ducy and her colleagues (1997) found that the mRNA for mouse CBFA1 is severely restricted to the mesenchymal condensations that form bone, and is limited to the osteoblast lineage. The protein appears to activate the genes for osteocalcin, osteopontin, and other bone-specific extracellular matrix proteins.

Confirmation and extension of this conclusion was obtained from gene targeting experiments wherein the mouse Cbfa1 gene was knocked out (Komori et al. 1997; Otto et al. 1997). Mice homozygous for this deletion died shortly after birth without taking a breath, and their skeletons completely lacked bone. The mutants had only the cartilaginous skeletal model (Figure 14.12). In these mice, both endochondral and intramembranous ossification had been eliminated. The osteoblasts were in an arrested state of development, expressing neither osteocalcin nor osteopontin.

Figure 14.12

Gene targeting of Cbfa1 in mice causes lack of bone formation. Newborn mice (wild-type and homozygotes for Cbfa1) were stained with alcian blue (for cartilage) and alizarin red (for bone). Cartilage development in both mice was normal. (A) Wild-type littermate. (more...)

Mice that were heterozygous for Cbfa1 showed skeletal defects similar to those of a human syndrome called cleidocranial dysplasia (CCD). In this syndrome, the skull sutures fail to close (adults retain the fontanel associated with young infants), growth is stunted, and the clavicle (collarbone) is often absent or deformed. When DNA from patients with CCD was analyzed, each patient had either deletions or point mutations in the CBFA1 gene. Control individuals did not have such mutations. Therefore, it appears that cleidocranial dysplasia is caused by heterozygosity of the CBFA1 gene (Mundlos et al. 1997).


14.5 Human dermal ossification syndromes. The human dermis appears to remain responsive to BMPs throughout life. The misexpression of BMP genes in children and adults can retrigger the embryonic bone growth pathway and lead to progressive and debilitating ossification of the skin. http://www.devbio.com/chap14/link1405.shtml

Endochondral ossification

Endochondral ossification involves the formation of cartilage tissue from aggregated mesenchymal cells, and the subsequent replacement of cartilage tissue by bone (Horton 1990). The process of endochondral ossification can be divided into five stages (Figure 14.13). First, the mesenchymal cells are commited to become cartilage cells. This committment is caused by paracrine factors that induce the nearby mesodermal cells to express two transcription factors, Pax1 and Scleraxis. These transcription factors are thought to activate cartilage-specific genes (Cserjesi et al. 1995; Sosic et al. 1997). Thus, Scleraxis is expressed in the mesenchyme from the sclerotome, in the facial mesenchyme that forms cartilaginous precursors to bone, and in the limb mesenchyme (Figure 14.14).

Figure 14.13

Schematic diagram of endochondral ossification. (A, B) Mesenchymal cells condense and differentiate into chondrocytes to form the cartilaginous model of the bone. (C) Chondrocytes in the center of the shaft undergo hypertrophy and apoptosis while they (more...)

Figure 14.14

Localization of the scleraxis message (light areas) at the sites of chondrocyte formation. (A) Expression of scleraxis in the somites of a 12.5-day mouse embryo. This section was cut tangentially, and the neural tube runs along the anterior-posterior (more...)

During the second phase of endochondral ossification, the committed mesenchyme cells condense into compact nodules and differentiate into chondrocytes, the cartilage cells. N-cadherin appears to be important in the initiation of these condensations, and N-CAM seems to be critical for maintaining them (Oberlender and Tuan 1994; Hall and Miyake 1995). In humans, the SOX9 gene, which encodes a DNA-binding protein, is expressed in the precartilaginous condensations. Mutations of the SOX9 gene cause camptomelic dysplasia, a rare disorder of skeletal developmentthat results in deformities of most of the bones of the body. Most affected babies die from respiratory failure due to poorly formed tracheal and rib cartilage (Wright et al. 1995).

During the third phase of endochondral ossification, the chondrocytes proliferate rapidly to form the model for the bone. As they divide, the chondrocytes secrete a cartilage-specific extracellular matrix. In the fourth phase, the chondrocytes stop dividing and increase their volume dramatically, becoming hypertrophic chondrocytes. These large chondrocytes alter the matrix they produce (by adding collagen X and more fibronectin) to enable it to become mineralized by calcium carbonate. The fifth phase involves the invasion of the cartilage model by blood vessels. The hypertrophic chondrocytes die by apoptosis. This space will become bone marrow. As the cartilage cells die, a group of cells that have surrounded the cartilage model differentiate into osteoblasts. The ostoblasts begin forming bone matrix on the partially degraded cartilage (Bruder and Caplan 1989; Hatori et al. 1995). Eventually, all the cartilage is replaced by bone. Thus, the cartilage tissue serves as a model for the bone that follows. The skeletal components of the vertebral column, the pelvis, and the limbs are first formed of cartilage and later become bone.

The replacement of chondrocytes by bone cells is dependent on the mineralization of the extracellular matrix. This is clearly illustrated in the developing skeleton of the chick embryo, which utilizes the calcium carbonate of the eggshell as its calcium source. During development, the circulatory system of the chick embryo translocates about 120 mg of calcium from the shell to the skeleton (Tuan 1987). When chick embryos are removed from their shells at day 3 and grown in shell-less cultures (in plastic wrap) for the duration of their development, much of the cartilaginous skeleton fails to mature into bony tissue (Figure 14.15; Tuan and Lynch 1983). A number of events lead to the hypertrophy and mineralization of the chondrocytes, including an initial switch from aerobic to anaerobic respiration, which alters their cell metabolism and mitochondrial energy potential (Shapiro et al. 1992). Hypertrophic chondrocytes secrete numerous small membrane-bound vesicles into the extracellular matrix. These vesicles contain enzymes that are active in the generation of calcium and phosphate ions and initiate the mineralization process within the cartilaginous matrix (Wu et al. 1997). The hypertrophic chondrocytes, their metabolism and mitochondrial membranes altered, then die by apoptosis (Hatori et al. 1995; Rajpurohit et al. 1999).

Figure 14.15

Skeletal mineralization in 19-day chick embryos that developed (A) in shell-less culture and (B) inside the egg during normal incubation. The embryos were fixed and stained with alizarin red to show the calcified bone matrix. (From Tuan and Lynch 1983; (more...)

In the long bones of many mammals (including humans), endochondral ossification spreads outward in both directions from the center of the bone (see Figure 14.13). If all of our cartilage were turned into bone before birth, we would not grow any larger, and our bones would be only as large as the original cartilaginous model. However, as the ossification front nears the ends of the cartilage model, the chondrocytes near the ossification front proliferate prior to undergoing hypertrophy, pushing out the cartilaginous ends of the bone. These cartilaginous areas at the ends of the long bones are called epiphyseal growth plates. These plates contain three regions: a region of chondrocyte proliferation, a region of mature chondrocytes, and a region of hypertrophic chondrocytes (Figure 14.16; Chen et al. 1995). As the inner cartilage hypertrophies and the ossification front extends farther outward, the remaining cartilage in the epiphyseal growth plate proliferates. As long as the epiphyseal growth plates are able to produce chondrocytes, the bone continues to grow.

Figure 14.16

Proliferation of cells in the epiphyseal growth plate in response to growth hormone. (A) Epiphyseal growth plate in a young rat that was made growth hormone-deficient by removal of its pituitary. (B) Same region in the rat after injection of growth hormone. (more...)


14.6 Paracrine factors, their receptors, and human bone growth. Mutations in the genes encoding paracrine factors and their receptors cause numerous skeletal anomalies in humans and mice. The FGF and Hedgehog pathways are especially important. http://www.devbio.com/chap14/link1406.shtml


Control of Cartilage Maturation at the Growth Plate. Recent discoveries of human and murine mutations resulting in abnormal skeletal development have provided remarkable insights into how the differentiation, proliferation, and patterning of chondrocytes (more...)


As new bone material is added peripherally from the internal surface of the periosteum, there is a hollowing out of the internal region to form the bone marrow cavity. This destruction of bone tissue is due to osteoclasts, multinucleated cells that enter the bone through the blood vessels (Kahn and Simmons 1975; Manolagas and Jilka 1995). Osteoclasts are probably derived from the same precursors as macrophage blood cells, and they dissolve both the inorganic and the protein portions of the bone matrix (Ash et al. 1980; Blair et al. 1986). Each osteoclast extends numerous cellular processes into the matrix and pumps out hydrogen ions onto the surrounding material, thereby acidifying and solubilizing it (Figure 14.17; Baron et al. 1985, 1986). The blood vessels also import the blood-forming cells that will reside in the marrow for the duration of the organism's life. The number and activity of osteoclasts must be tightly regulated. If there are too many active osteoclasts, too much bone will be dissolved, and osteoporosis will result. Conversely, if not enough osteoblasts are produced, the bones are not hollowed out for the marrow, and osteopetrosis results (Tondravi et al. 1997).

Figure 14.17

Osteoclast activity on the bone matrix. (A) Electron micrograph of the ruffled membrane of a chick osteoclast cultured on reconstituted bone matrix. (B) Section of ruffled membrane stained for the presence of an ATPase capable of transporting hydrogen (more...)


14.7 Osteoclast differentiation. Hormones regulate the production of osteoclasts, and the hormonal changes of aging may cause osteoporosis by increasing the number of osteoclasts. The conversion of a macrophage stem cell into a osteoclast is regulated by osteoprotegerin and its ligand. It is thought that signals on osteoblasts instruct the progenitor cell to become an osteoclast. http://www.devbio.com/chap14/link1407.shtml


Craniofacial cartilage development was discussed in Chapter 13 and will be revisited in Chapter 22; the development of the limbs will be detailed in Chapter 16.

CCD may have been responsible for the phenotype of Thersites, the Greek soldier described in the Iliad as having “both shoulders humped together, curving over his caved-in chest, and bobbing above them his skull warped to a point.…” (Dickman 1997).

Given the physiology of the osteoclast, we can now appreciate H. L. Mencken's (1919) prescient intuition: “Life is a struggle, not against sin, not against the Money Power, not against malicious animal magnetism, but against hydrogen ions.”

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There is a any easy way to memorize Endochodral ossification if you can remember the 5 main stages it consists of. If you remember the simplified names you will be able to fill in what these stages do. As an overview of ossification the bone starts as a solid and becomes hollow before you are born.

The 5  stages of endochondral ossification can be memorized with mnemonics. This mnemonic may be inappropriate so I have a clean version and a dirty version:

(clean)Bones Cant Pop During Exercise
(dirty)Boys Cant Pee During Erections

  1. Bone Collar Formation
  2. Cavitation
  3. Periosteal Bud Invasion
  4. Diaphysis Elongation
  5. Epiphiseal Ossification

When we talk about endochondral ossification we are talking about the development of long bones from a hyalin cartilage model.

Bone Collar Formation

The primary ossification center develops in the center of the bone, and is the source of bone development. Osteoblasts found in the bone will secrete osteoid against the walls of the diaphysis. The diaphysis ,made of hyalin cartilage, is now encased in compact bone. This bone collar gives the developing bone structural support to begin hardening.


The chondrocytes (cartilage cells) go through hypertrophication or enlargement and signal the hyalin cartilage to harden into bone. This hardening into bone is called calcification. The calcified hyalin cartilage is impermeable to the diffusion of nutrients. Since the chondrocytes cant receive any nutrients they begin to die and leave small cavities. These small cavities leave room in the hardened bone for blood vessels to travel through.

Periosteal Bud Invasion

Periosteal bud invasion is the introduction of a nutrient highway to the bone. Previously nutrients were delivered through the cartilage by simple diffusion. To support the capacity of the hardening bone, the periosteal region is invaded by a bud containing blood vessels and nerves. This allows not only nutrients, but osteoblasts and osteoclasts cells to enter into the cavities that were previously inhabited by chondrocytes. The osteoblasts secrete osteoid into the remaining hyalin cartilage and give rise to early spongy bone. Think of the periosteal bud invasion as a group of grave robbers who invaded the graves (cavities) where fallen chondrocytes used to rest.

Diaphysis Elongation

After the nutrient source is delivered to the center of the bone, the diaphysis region has the resources to elongate. The elogated region is powered by cells dividing in the primary center of ossification. This elongated region is known as the medullary cavity. The medullary cavity is where the bone marrow is contained.

Epiphyseal Ossification

Just before birth the ends of the bone or Epiphysis will develop their own centers of ossification. They cant stay made of cartilage or they would be structurally weak.  These centers are known as the secondary centers of ossification. They go through the same process as the primary center of ossification: hypertrophication, calcification, cavitation, and periosteal bud invasion.


Why not just grow completely from the center? Why do we need secondary centers? There is a region left between the primary and secondary centers of ossification known as the Epiphyseal Plate. This is more commonly known as the growth plate. This is how your bones grow after you are born.


Endochondral ossification can be summed into 5 major steps:

  1. Hypertrophication: Chondrocyte cells grow.
  2. Calcification: Hardening of hyalin cartilage matrix.
  3. Cavitation: Chrondrocytes die and leave cavities in the bone.
  4. Periosteal bud invasion: Nutrients are delivered to the bone via blood vessels, and nerves also enter.
  5. Epiphyseal Ossification: the bone ends develop (secondary) ossification centers.

There is now bone with compact bone on the outside that begun with the bone collar. The inside of the bone is spongy bone because of the cavities made by the chondrocytes. The ends still have hyalin cartilage on them which helps absorb shock.


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