Osteogenesis: The Development of Bones

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 crestderived 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 osteocytesbone 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 bone matrix become osteocytes. (B) Intramembranous ossification in the
plastron (ventral shell) of the red-ear slider turtle Trachemys scripta. The plastron of a one-month-old hatchling
was stained with alcian blue (for cartilage) and alizarin red (for bone). No cartilage was seen to precede the
formation of bone. (Photograph courtesy of G. Loredo, A. Brukman, and S. F. Gilbert.)

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 crestderived 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. (B) Homozygous mutant
showing cartilage, but an absence of ossification throughout the entire body. (From Otto et al. 1997;
photographs courtesy of Cell and MIT Press.)

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.

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 change and mineralize their extracellular matrix. Their deaths allow blood
vessels to enter. (D, E) Blood vessels bring in osteoblasts, which bind to the degenerating cartilaginous matrix
and deposit bone matrix. (F-H) Bone formation and growth consist of ordered arrays of proliferating,
hypertrophic, and mineralizing chondrocytes. Secondary ossification centers also form as blood vessels enter
near the tips of the bone. (After Horton 1990.)

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 axis. (B) Section through a 11.5-day mouse embryo, in which scleraxis transcripts
are seen in the condensing cartilage of the nose and face and in the precursors of the limbs and ribs. (After
Cserjesi et al. 1995; photographs courtesy of E. Olson.)

During the second phase of endochondral ossification, the committed mesenchyme cells
condense into compact nodules and differentiate into chondrocytes, the cartilage cells. Ncadherin 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; photographs courtesy of R. Tuan.)

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. (C) Stained cartilage from specific regions of the epiphyseal growth plate.
(A, B,I. Gersh's photographs from Bloom and Fawcett 1975; C from Chen et al. 1995, photograph courtesy of P.
Goetinck.)
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 are regulated.
Fibroblast Growth Factor Receptors
The proliferation of the epiphyseal growth plate cells and facial cartilage can be halted by the presence of
fibroblast growth factors (Deng et al. 1996; Webster and Donoghue 1996). These factors appear to instruct the
cartilage precursors to differentiate rather than to divide. In humans, mutations of the receptors for fibroblast
growth factors can cause these receptors to become activated prematurely. Such mutations give rise to the major
types of human dwarfism. Achondroplasia is a dominant condition caused by mutations in the transmembrane
region of fibroblast growth factor receptor 3 (FGFR3). Roughly 95% of achondroplastic dwarfs have the same
mutation of FGFR3, a base pair substitution that converts glycine to arginine at position 380 in the
transmembrane region of the protein. In addition, mutations in the extracellular portion of the FGFR3 protein or
in the tyrosine kinase intracellular domain may result in thanatophoric dysplasia, a lethal form of dwarfism that
resembles homozygous achondroplasia (see Figure 6.22; Bellus et al. 1995; Tavormina et al. 1995). Mutations in
FGFR1 can cause Pfeiffer syndrome, characterized by limb defects and premature fusion of the cranial sutures
(craniosynostosis), resulting in abnormal skull and facial shape. Different mutations in FGFR2 can give rise to
various abnormalities of the limbs and face (Park et al. 1995; Wilkie et al. 1995).
Insulin-like Growth Factors
The epiphyseal growth plate cells are very responsive to hormones, and their proliferation is stimulated by
growth hormone and insulin-like growth factors. Nilsson and colleagues (1986) showed that growth hormone
stimulates the production of insulin-like growth factor I (IGF-I) in the epiphyseal chondrocytes, and that these

chondrocytes respond to it by proliferating. When they added growth hormone to the tibial growth plates of
young mice who could not manufacture their own growth hormone (because their pituitaries had been removed),
it stimulated the formation of IGF-I in the chondrocytes of the proliferative zone (see Figure 14.16). The
combination of growth hormone and IGF-I appears to provide an extremely strong mitotic signal. It appears that
IGF-I is essential for the normal growth spurt at puberty. The pygmies of the Ituri Forest of Zaire have normal
growth hormone and IGF-I levels until puberty. However, at puberty, their IGF-I levels fall to about one-third
that of other adolescents (Merimee et al. 1987).
Estrogen Receptors
The pubertal growth spurt and the subsequent cessation of growth are induced by sex hormones (Kaplan and
Grumbach 1990). At the end of puberty, high levels of estrogen or testosterone cause the remaining epiphyseal
plate cartilage to undergo hypertrophy. These cartilage cells grow, die, and are replaced by bone. Without any
further cartilage formation, growth of these bones ceases, a process known as growth plate closure.
In conditions of precocious puberty, there is an initial growth spurt (making the individual taller than his or her
peers), followed by the cessation of epiphyseal cell division (allowing that person's peers to catch up and surpass
his or her height). In males, it was not thought that estrogen played any role in these events. However, in 1994,
Smith and colleagues published the case history of a man whose growth was still linear despite his undergoing a
normal puberty. His epiphyseal plates had not matured, and he still had proliferating chondrocytes at 28 years of
age. His bone agethe amount of ephiphyseal cartilage he retainedwas roughly half his chronological age.
This person was found to lack any functional estrogen receptor. At present, at least three human males have been
reported who either cannot make estrogens or who lack the estrogen receptor. All three are close to 7 feet tall
and are still growing (Sharpe 1997). Therefore, estrogen plays a role in epiphyseal maturation in males as well
as in females.
Thyroid hormone and parathyroid-related hormone are also important in regulating the maturation and
hypertrophy program of the epiphyseal growth plate (Ballock and Reddi 1994). Thus, children with
hypothyroidism are prone to developing growth plate disorders.
Extracellular Matrix Proteins
The extracellular matrix of the cartilage is also critical for the proper differentiation and organization of growth
plate chondrocytes. Mutations that affect type XI collagen or the sulfation of cartilage proteoglycans can cause
severe skeletal abnormalities. Mice with deficiencies of type XI collagen die at birth with abnormalities of limb,
mandible, rib, and tracheal cartilage (Li et al. 1995). Failure to add sulfate groups to cartilage proteoglycans
causes diastrophic dysplasia, a human dwarfism characterized by severe curvature of the spine, clubfoot, and
deformed earlobes (Hstbacka et al. 1994).

Osteoclasts
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 ions from the cell. The ATPase is restricted to the membrane of the cell
process. (C) Solubilization of inorganic and collagenous matrix components (as measured by the release of
[45Ca] and [3H] proline, respectively) by 10,000 osteoclasts incubated on labeled bone fragments. (A and C from
Blair et al. 1986; B from Baron et al. 1986, photograph courtesy of the authors.)