Chapter

Bone Development and Growth

Rosy Setiawati and Paulus Rahardjo

Abstract

The process of bone formation is called osteogenesis or ossification. After

progenitor cells form osteoblastic lines, they proceed with three stages of develop-
ment of cell differentiation, called proliferation, maturation of matrix, and min-
eralization. Based on its embryological origin, there are two types of ossification,
called intramembranous ossification that occurs in mesenchymal cells that dif-
ferentiate into osteoblast in the ossification center directly without prior cartilage
formation and endochondral ossification in which bone tissue mineralization is
formed through cartilage formation first. In intramembranous ossification, bone
development occurs directly. In this process, mesenchymal cells proliferate into
areas that have high vascularization in embryonic connective tissue in the formation
of cell condensation or primary ossification centers. This cell will synthesize bone
matrix in the periphery and the mesenchymal cells continue to differentiate into
osteoblasts. After that, the bone will be reshaped and replaced by mature lamellar
bone. Endochondral ossification will form the center of primary ossification, and
the cartilage extends by proliferation of chondrocytes and deposition of cartilage
matrix. After this formation, chondrocytes in the central region of the cartilage
start to proceed with maturation into hypertrophic chondrocytes. After the pri-
mary ossification center is formed, the marrow cavity begins to expand toward the
epiphysis. Then the subsequent stages of endochondral ossification will take place
in several zones of the bone.

Keywords: osteogenesis, ossification, bone formation, intramembranous

ossification, endochondral ossification

1. Introduction

Bone is living tissue that is the hardest among other connective tissues in the
body, consists of 50% water. The solid part remainder consisting of various miner-
als, especially 76% of calcium salt and 33% of cellular material. Bone has vascular
tissue and cellular activity products, especially during growth which is very depen-
dent on the blood supply as basic source and hormones that greatly regulate this
growth process. Bone-forming cells, osteoblasts, osteoclast play an important role
in determining bone growth, thickness of the cortical layer and structural arrange-
ment of the lamellae.
Bone continues to change its internal structure to reach the functional needs and
these changes occur through the activity of osteoclasts and osteoblasts. The bone
seen from its development can be divided into two processes: first is the intramem-
branous ossification in which bones form directly in the form of primitive mesen-
chymal connective tissue, such as the mandible, maxilla and skull bones. Second is
the endochondral ossification in which bone tissue replaces a preexisting hyaline

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cartilage, for example during skull base formation. The same formative cells form
two types of bone formation and the final structure is not much different.
Bone growth depends on genetic and environmental factors, including hor-
monal effects, diet and mechanical factors. The growth rate is not always the
same in all parts, for example, faster in the proximal end than the distal humerus
because the internal pattern of the spongiosum depends on the direction of bone
pressure. The direction of bone formation in the epiphysis plane is determined by
the direction and distribution of the pressure line. Increased thickness or width
of the bone is caused by deposition of new bone in the form of circumferential
lamellae under the periosteum. If bone growth continues, the lamella will be
embedded behind the new bone surface and be replaced by the haversian canal
system.

2. Bone cells and matrix

Bone is a tissue in which the extracellular matrix has been hardened to accom-
modate a supporting function. The fundamental components of bone, like all con-
nective tissues, are cells and matrix. Although bone cells compose a small amount
of the bone volume, they are crucial to the function of bones. Four types of cells are
found within bone tissue: osteoblasts, osteocytes, osteogenic cells, and osteoclasts.
They each unique functions and are derived from two different cell lines (Figure 1
and Table 1) [1–7].

• Osteoblast synthesizes the bone matrix and are responsible for its mineraliza-
tion. They are derived from osteoprogenitor cells, a mesenchymal stem cell
line.

• Osteocytes are inactive osteoblasts that have become trapped within the bone
they have formed.

• Osteoclasts break down bone matrix through phagocytosis. Predictably, they

ruffled border, and the space between the osteoblast and the bone is known as
Howship’s lacuna.

The balance between osteoblast and osteoclast activity governs bone turnover
and ensures that bone is neither overproduced nor overdegraded. These cells build
up and break down bone matrix, which is composed of:

• Osteoid, which is the unmineralized matrix composed of type I collagen and

gylcosaminoglycans (GAGs).

• Calcium hydroxyapatite, a calcium salt crystal that give bone its strength and
rigidity.

Bone is divided into two types that are different structurally and function-
ally. Most bones of the body consist of both types of bone tissue (Figure 2)
[1, 2, 8, 9]:

• Compact bone, or cortical bone, mainly serves a mechanical function. This

is the area of bone to which ligaments and tendons attach. It is thick and
dense.

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Figure 1.
Development of bone precursor cells. Bone precursor cells are divided into developmental stages, which are 1.
mesenchymal stem cell, 2. pre-osteoblast, 3. osteoblast, and 4. mature osteocytes, and 5. osteoclast.

Table 1.
Bone cells, their function, and locations [1–7].

• Trabecular bone, also known as cancellous bone or spongy bone, mainly serves
a metabolic function. This type of bone is located between layers of compact
bone and is thin porous. Location within the trabeculae is the bone marrow.

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Figure 2.
Structure of a long bone.

3. Bone structure

3.1 Macroscopic bone structure

Long bones are composed of both cortical and cancellous bone tissue. They
consist of several areas (Figure 3) [3, 4]:

• The epiphysis is located at the end of the long bone and is the parts of the bone
that participate in joint surfaces.

• The diaphysis is the shaft of the bone and has walls of cortical bone and an
underlying network of trabecular bone.

• The epiphyseal growth plate lies at the interface between the shaft and the
epiphysis and is the region in which cartilage proliferates to cause the elonga-
tion of the bone.

• The metaphysis is the area in which the shaft of the bone joins the epiphyseal
growth plate.

Different areas of the bone are covered by different tissue [4]:

• The epiphysis is lined by a layer of articular cartilage, a specialized form of

hyaline cartilage, which serves as protection against friction in the joints.

• The outside of the diaphysis is lined by periosteum, a fibrous external layer

onto which muscles, ligaments, and tendons attach.

• The inside of the diaphysis, at the border between the cortical and cancellous
bone and lining the trabeculae, is lined by endosteum.

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Figure 3.
Bone macrostructure. (a) Growing long bone showing epiphyses, epiphyseal plates, metaphysis and diaphysis.
(b) Mature long bone showing epiphyseal lines.

3.2 Microscopic bone structure

Compact bone is organized as parallel columns, known as Haversian systems,

which run lengthwise down the axis of long bones. These columns are composed
of lamellae, concentric rings of bone, surrounding a central channel, or Haversian
canal, that contains the nerves, blood vessels, and lymphatic system of the bone.
The parallel Haversian canals are connected to one another by the perpendicular
Volkmann’s canals.
The lamellae of the Haversian systems are created by osteoblasts. As these cells
secrete matrix, they become trapped in spaces called lacunae and become known as
osteocytes. Osteocytes communicate with the Haversian canal through cytoplasmic
extensions that run through canaliculi, small interconnecting canals (Figure 4)
[1, 2, 8, 9]:
The layers of a long bone, beginning at the external surface, are therefore:

• Periosteal surface of compact bone

• Outer circumferential lamellae

• Compact bone (Haversian systems)

• Inner circumferential lamellae

• Endosteal surface of compact bone

• Trabecular bone

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Figure 4.
Bone microstructure. Compact and spongy bone structures.

4. Bone formation

Bone development begins with the replacement of collagenous mesenchymal

tissue by bone. This results in the formation of woven bone, a primitive form of
bone with randomly organized collagen fibers that is further remodeled into mature
lamellar bone, which possesses regular parallel rings of collagen. Lamellar bone is
then constantly remodeled by osteoclasts and osteoblasts. Based on the develop-
ment of bone formation can be divided into two parts, called endochondral and
intramembranous bone formation/ossification [1–3, 8].

4.1 Intramembranous bone formation

During intramembranous bone formation, the connective tissue membrane of

undifferentiated mesenchymal cells changes into bone and matrix bone cells [10]. In the
craniofacial cartilage bones, intramembranous ossification originates from nerve crest
cells. The earliest evidence of intramembranous bone formation of the skull occurs in
the mandible during the sixth prenatal week. In the eighth week, reinforcement center
appears in the calvarial and facial areas in areas where there is a mild stress strength [11].
Intramembranous bone formation is found in the growth of the skull and is
also found in the sphenoid and mandible even though it consists of endochondral
elements, where the endochondral and intramembranous growth process occurs in
the same bone. The basis for either bone formation or bone resorption is the same,
regardless of the type of membrane involved.
Sometimes according to where the formation of bone tissue is classified as “peri-
osteal” or “endosteal”. Periosteal bone always originates from intramembranous,
but endosteal bone can originate from intramembranous as well as endochondral
ossification, depending on the location and the way it is formed [3, 12].

4.1.1 The stage of intramembranous bone formation

The statement below is the stage of intramembrane bone formation (Figure 5)

[3, 4, 11, 12]:

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1. An ossification center appears in the fibrous connective tissue membrane.

Mesenchymal cells in the embryonic skeleton gather together and begin to
differentiate into specialized cells. Some of these cells differentiate into capil-
laries, while others will become osteogenic cells and osteoblasts, then forming
an ossification center.

2. Bone matrix (osteoid) is secreted within the fibrous membrane. Osteoblasts

produce osteoid tissue, by means of differentiating osteoblasts from the
ectomesenchyme condensation center and producing bone fibrous matrix
(osteoid). Then osteoid is mineralized within a few days and trapped osteo-
blast become osteocytes.

3. Woven bone and periosteum form. The encapsulation of cells and blood ves-
sels occur. When osteoid deposition by osteoblasts continues, the encased cells
develop into osteocytes. Accumulating osteoid is laid down between embry-
onic blood vessels, which form a random network (instead of lamellae) of
trabecular. Vascularized mesenchyme condenses on external face of the woven
bone and becomes the periosteum.

4. Production of osteoid tissue by membrane cells: osteocytes lose their ability to

contribute directly to an increase in bone size, but osteoblasts on the perios-
teum surface produce more osteoid tissue that thickens the tissue layer on the
existing bone surface (for example, appositional bone growth). Formation of a
woven bone collar that is later replaced by mature lamellar bone. Spongy bone
(diploe), consisting of distinct trabeculae, persists internally and its vascular
tissue becomes red marrow.

5. Osteoid calcification: The occurrence of bone matrix mineralization makes

bones relatively impermeable to nutrients and metabolic waste. Trapped blood
vessels function to supply nutrients to osteocytes as well as bone tissue and
eliminate waste products.

6. The formation of an essential membrane of bone which includes a membrane

outside the bone called the bone endosteum. Bone endosteum is very impor-
tant for bone survival. Disruption of the membrane or its vascular tissue can
cause bone cell death and bone loss. Bones are very sensitive to pressure. The
calcified bones are hard and relatively inflexible.

The matrix or intercellular substance of the bone becomes calcified and becomes
a bone in the end. Bone tissue that is found in the periosteum, endosteum, suture,
and periodontal membrane (ligaments) is an example of intramembranous bone
formation [3, 13].
Intramembranous bone formation occurs in two types of bone: bundle bone
and lamellar bone. The bone bundle develops directly in connective tissue that has
not been calcified. Osteoblasts, which are differentiated from the mesenchyme,
secrete an intercellular substance containing collagen fibrils. This osteoid matrix
calcifies by precipitating apatite crystals. Primary ossification centers only show
minimal bone calcification density. The apatite crystal deposits are mostly irregular
and structured like nets that are contained in the medullary and cortical regions.
Mineralization occurs very quickly (several tens of thousands of millimeters per
day) and can occur simultaneously in large areas. These apatite deposits increase
with time. Bone tissue is only considered mature when the crystalized area is
arranged in the same direction as collagen fibrils.

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Figure 5.
The stage of intramembranous ossification. The following stages are (a) Mesenchymal cells group into
clusters, and ossification centers form. (b) Secreted osteoid traps osteoblasts, which then become osteocytes.
(c) Trabecular matrix and periosteum form. (d) Compact bone develops superficial to the trabecular bone,
and crowded blood vessels condense into red marrow.

Bone tissue is divided into two, called the outer cortical and medullary
regions, these two areas are destroyed by the resorption process; which goes
along with further bone formation. The surrounding connective tissue will
differentiate into the periosteum. The lining in the periosteum is rich in cells,
has osteogenic function and contributes to the formation of thick bones as in the
endosteum.
In adults, the bundle bone is usually only formed during rapid bone remodeling.
This is reinforced by the presence of lamellar bone. Unlike bundle bone formation,
lamellar bone development occurs only in mineralized matrix (e.g., cartilage that
has calcified or bundle bone spicules). The nets in the bone bundle are filled to
strengthen the lamellar bone, until compact bone is formed. Osteoblasts appear in
the mineralized matrix, which then form a circle with intercellular matter sur-
rounding the central vessels in several layers (Haversian system). Lamella bone

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is formed from 0.7 to 1.5 microns per day. The network is formed from complex
fiber arrangements, responsible for its mechanical properties. The arrangement
of apatites in the concentric layer of fibrils finally meets functional requirements.
Lamellar bone depends on ongoing deposition and resorption which can be influ-
enced by environmental factors, one of this which is orthodontic treatment.

4.1.2 Factors that influence intramembranous bone formation

Intramembranous bone formation from desmocranium (suture and periosteum)

is mediated by mesenchymal skeletogenetic structures and is achieved through
bone deposition and resorption [8]. This development is almost entirely controlled
through local epigenetic factors and local environmental factors (i.e. by muscle
strength, external local pressure, brain, eyes, tongue, nerves, and indirectly by
endochondral ossification). Genetic factors only have a nonspecific morphogenetic
effect on intramembranous bone formation and only determine external limits and
increase the number of growth periods. Anomaly disorder (especially genetically
produced) can affect endochondral bone formation, so local epigenetic factors and
local environmental factors, including steps of orthodontic therapy, can directly
affect intramembranous bone formation [3, 11].

4.2 Endochondral bone formation

During endochondral ossification, the tissue that will become bone is firstly
formed from cartilage, separated from the joint and epiphysis, surrounded by
perichondrium which then forms the periosteum [11]. Based on the location of
mineralization, it can be divided into: Perichondral Ossification and Endochondral
Ossification. Both types of ossification play an essential role in the formation
of long bones where only endochondral ossification takes place in short bones.
Perichondral ossification begins in the perichondrium. Mesenchymal cells from
the tissue differentiate into osteoblasts, which surround bony diaphyseal before
endochondral ossification, indirectly affect its direction [3, 8, 12]. Cartilage is
transformed into bone is craniofacial bone that forms at the eigth prenatal week.
Only bone on the cranial base and part of the skull bone derived from endochondral
bone formation. Regarding to differentiate endochondral bone formation from
chondrogenesis and intramembranous bone formation, five sequences of bone
formation steps were determined [3].

4.2.1 The stages of endochondral bone formation

The statements below are the stages of endochondral bone formation

(Figure 6) [4, 12]:

1. Mesenchymal cells group to form a shape template of the future bone.

2. Mesenchymal cells differentiate into chondrocytes (cartilage cells).

3. Hypertrophy of chondrocytes and calcified matrix with calcified central car-

tilage primordium matrix formed. Chondrocytes show hypertrophic changes
and calcification from the cartilage matrix continues.

4. Entry of blood vessels and connective tissue cells. The nutrient artery supplies
the perichondrium, breaks through the nutrient foramen at the mid-region
and stimulates the osteoprogenitor cells in the perichondrium to produce

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osteoblasts, which changes the perichondrium to the periosteum and starts the
formation of ossification centers.

5. The periosteum continues its development and the division of cells (chon-
drocytes) continues as well, thereby increasing matrix production (this helps
produce more length of bone).

6. The perichondrial membrane surrounds the surface and develops new

chondroblasts.

7. Chondroblasts produce growth in width (appositional growth).

8. Cells at the center of the cartilage lyse (break apart) triggers calcification.

During endochondral bone formation, mesenchymal tissue firstly differ-

entiates into cartilage tissue. Endochondral bone formation is morphogenetic
adaptation (normal organ development) which produces continuous bone
in certain areas that are prominently stressed. Therefore, this endochondral
bone formation can be found in the bones associated with joint movements
and some parts of the skull base. In hypertrophic cartilage cells, the matrix
calcifies and the cells undergo degeneration. In cranial synchondrosis, there is
proliferation in the formation of bones on both sides of the bone plate, this is
distinguished by the formation of long bone epiphyses which only occurs on
one side only [2, 14].
As the cartilage grows, capillaries penetrate it. This penetration initiates the
transformation of the perichondrium into the bone-producing periosteum. Here,
the osteoblasts form a periosteal collar of compact bone around the cartilage of the
diaphysis. By the second or third month of fetal life, bone cell development and
ossification ramps up and creates the primary ossification center, a region deep in
the periosteal collar where ossification begins [4, 10].
While these deep changes occur, chondrocytes and cartilage continue to grow
at the ends of the bone (the future epiphyses), which increase the bone length and
at the same time bone also replaces cartilage in the diaphysis. By the time the fetal
skeleton is fully formed, cartilage only remains at the joint surface as articular
cartilage and between the diaphysis and epiphysis as the epiphyseal plate, the latter
of which is responsible for the longitudinal growth of bones. After birth, this same
sequence of events (matrix mineralization, death of chondrocytes, invasion of
blood vessels from the periosteum, and seeding with osteogenic cells that become
osteoblasts) occur in the epiphyseal regions, and each of these centers of activity is
referred to as a secondary ossification center [4, 8, 10].
There are four important things about cartilage in endochondral bone
formation:

1. Cartilage has a rigid and firm structure, but not usually calcified nature, giving
three basic functions of growth (a) its flexibility can support an appropriate
network structure (nose), (b) pressure tolerance in a particular place where
compression occurs, (c) the location of growth in conjunction with enlarging
bone (synchondrosis of the skull base and condyle cartilage).

2. Cartilage grows in two adjacent places (by the activity of the chondrogenic
membrane) and grows in the tissues (chondrocyte cell division and the addi-
tion of its intercellular matrix).

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Figure 6.
The stage of endochondral ossification. The following stages are: (a) Mesenchymal cells differentiate into
chondrocytes. (b) The cartilage model of the future bony skeleton and the perichondrium form. (c) Capillaries
penetrate cartilage. Perichondrium transforms into periosteum. Periosteal collar develops. Primary ossification
center develops. (d) Cartilage and chondrocytes continue to grow at ends of the bone. (e) Secondary ossification
centers develop. ( f) Cartilage remains at epiphyseal (growth) plate and at joint surface as articular cartilage.

3. Bone tissue is not the same as cartilage in terms of its tension adaptation and
cannot grow directly in areas of high compression because its growth depends
on the vascularization of bone formation covering the membrane.

4. Cartilage growth arises where linear growth is required toward the pressure
direction, which allows the bone to lengthen to the area of strength and has not
yet grown elsewhere by membrane ossification in conjunction with all peri-
osteal and endosteal surfaces.

4.2.2 Factors that influence endochondral ossification

Membrane disorders or vascular supply problem of these essential membranes

can directly result in bone cell death and ultimately bone damage. Calcified bones
are generally hard and relatively inflexible and sensitive to pressure [12].
Cranial synchondrosis (e.g., spheno ethmoidal and spheno occipital growth)
and endochondral ossification are further determined by chondrogenesis.
Chondrogenesis is mainly influenced by genetic factors, similar to facial mesenchy-
mal growth during initial embryogenesis to the differentiation phase of cartilage
and cranial bone tissue.
This process is only slightly affected by local epigenetic and environmental
factors. This can explain the fact that the cranial base is more resistant to deforma-
tion than desmocranium. Local epigenetic and environmental factors cannot trigger
or inhibit the amount of cartilage formation. Both of these have little effect on the
shape and direction of endochondral ossification. This has been analyzed especially
during mandibular condyle growth.

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Local epigenetics and environmental factors only affect the shape and direction
of cartilage formation during endochondral ossification Considering the fact that
condyle cartilage is a secondary cartilage, it is assumed that local factors provide a
greater influence on the growth of mandibular condyle.

4.2.3 Chondrogenesis

Chondrogenesis is the process by which cartilage is formed from condensed

mesenchyme tissue, which differentiates into chondrocytes and begins secreting the
molecules that form the extracellular matrix [5, 14].
The statement below is five steps of chondrogenesis [8, 14]:

1. Chondroblasts produce a matrix: the extracellular matrix produced by cartilage

cells, which is firm but flexible and capable of providing a rigid support.

2. Cells become embed in a matrix: when the chondroblast changes to be com-

pletely embed in its own matrix material, cartilage cells turn into chondro-
cytes. The new chondroblasts are distinguished from the membrane surface
(perichondrium), this will result in the addition of cartilage size (cartilage can
increase in size through apposition growth).

3. Chondrocytes enlarge, divide and produce a matrix. Cell growth continues and
produces a matrix, which causes an increase in the size of cartilage mass from
within. Growth that causes size increase from the inside is called interstitial
growth.

4. The matrix remains uncalcified: cartilage matrix is rich of chondroitin sulfate

which is associated with non-collagen proteins. Nutrition and metabolic waste
are discharged directly through the soft matrix to and from the cell. Therefore,
blood vessels aren’t needed in cartilage.

5. The membrane covers the surface but is not essential: cartilage has a closed
membrane vascularization called perichondrium, but cartilage can exist with-
out any of these. This property makes cartilage able to grow and adapt where it
needs pressure (in the joints), so that cartilage can receive pressure.

Endochondral ossification begins with characteristic changes in cartilage bone

cells (hypertrophic cartilage) and the environment of the intercellular matrix
(calcium laying), the formation which is called as primary spongiosa. Blood vessels
and mesenchymal tissues then penetrate into this area from the perichondrium.
The binding tissue cells then differentiate into osteoblasts and cells. Chondroblasts
erode cartilage in a cave-like pattern (cavity). The remnants of mineralized cartilage
the central part of laying the lamellar bone layer.
The osteoid layer is deposited on the calcified spicules remaining from the
cartilage and then mineralized to form spongiosa bone, with fine reticular struc-
tures that resemble nets that possess cartilage fragments between the spicular
bones. Spongy bones can turn into compact bones by filling empty cavities. Both
endochondral and perichondral bone growth both take place toward epiphyses and
joints. In the bone lengthening process during endochondral ossification depends
on the growth of epiphyseal cartilage. When the epiphyseal line has been closed,
the bone will not increase in length. Unlike bone, cartilage bone growth is based

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on apposition and interstitial growth. In areas where cartilage bone is covered by

bone, various variations of zone characteristics, based on the developmental stages
of each individual, can differentiate which then continuously merge with each
other during the conversion process. Environmental influences (co: mechanism of
orthopedic functional tools) have a strong effect on condylar cartilage because the
bone is located more superficially [5].

5. Bone growth

Cartilage bone height development occurs during the third month of intra
uterine life. Cartilage plate extends from the nasal bone capsule posteriorly to the
foramen magnum at the base of the skull. It should be noted that cartilages which
close to avascular tissue have internal cells obtained from the diffusion process
from the outermost layer. This means that the cartilage must be flatter. In the early
stages of development, the size of a very small embryo can form a chondroskel-
eton easily in which the further growth preparation occurs without internal blood
supply [1].
During the fourth month in the uterus, the development of vascular elements
to various points of the chondrocranium (and other parts of the early cartilage
skeleton) becomes an ossification center, where the cartilage changes into an ossi-
fication center, and bone forms around the cartilage. Cartilage continues to grow
rapidly but it is replaced by bone, resulting in the rapid increase of bone amount.
Finally, the old chondrocranium amount will decrease in the area of cartilage and
large portions of bone, assumed to be typical in ethmoid, sphenoid, and basioccipi-
tal bones. The cartilage growth in relation to skeletal bone is similar as the growth of
the limbs [1, 3].
Longitudinal bone growth is accompanied by remodeling which includes apposi-
tional growth to thicken the bone. This process consists of bone formation and
reabsorption. Bone growth stops around the age of 21 for males and the age of 18 for
females when the epiphyses and diaphysis have fused (epiphyseal plate closure).
Normal bone growth is dependent on proper dietary intake of protein, minerals
and vitamins. A deficiency of vitamin D prevents calcium absorption from the GI
tract resulting in rickets (children) or osteomalacia (adults). Osteoid is produced
but calcium salts are not deposited, so bones soften and weaken.

5.1 Oppositional bone growth

At the length of the long bones, the reinforcement plane appears in the
middle and at the end of the bone, finally produces the central axis that is called
the diaphysis and the bony cap at the end of the bone is called the epiphysis.
Between epiphyses and diaphysis is a calcified area that is not calcified called
the epiphyseal plate. Epiphyseal plate of the long bone cartilage is a major
center for growth, and in fact, this cartilage is responsible for almost all the
long growths of the bones. This is a layer of hyaline cartilage where ossification
occurs in immature bones. On the epiphyseal side of the epiphyseal plate, the
cartilage is formed. On the diaphyseal side, cartilage is ossified, and the diaphy-
sis then grows in length. The epiphyseal plate is composed of five zones of cells
and activity [3, 4].
Near the outer end of each epiphyseal plate is the active zone dividing the
cartilage cells. Some of them, pushed toward diaphysis with proliferative activity,

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develop hypertrophy, secrete an extracellular matrix, and finally the matrix begins
to fill with minerals and then is quickly replaced by bone. As long as cartilage
cells multiply growth will continue. Finally, toward the end of the normal growth
period, the rate of maturation exceeds the proliferation level, the latter of the
cartilage is replaced by bone, and the epiphyseal plate disappears. At that time,
bone growth is complete, except for surface changes in thickness, which can be
produced by the periosteum [4]. Bones continue to grow in length until early
adulthood. The lengthening is stopped in the end of adolescence which chon-
drocytes stop mitosis and plate thins out and replaced by bone, then diaphysis
and epiphyses fuse to be one bone (Figure 7). The rate of growth is controlled by
hormones. When the chondrocytes in the epiphyseal plate cease their proliferation
and bone replaces the cartilage, longitudinal growth stops. All that remains of
the epiphyseal plate is the epiphyseal line. Epiphyseal plate closure will occur in
18-year old females or 21-year old males.

5.1.1 Epiphyseal plate growth

The cartilage found in the epiphyseal gap has a defined hierarchical structure,
directly beneath the secondary ossification center of the epiphysis. By close exami-
nation of the epiphyseal plate, it appears to be divided into five zones (starting from
the epiphysis side) (Figure 8) [4]:

1. The resting zone: it contains hyaline cartilage with few chondrocytes, which
means no morphological changes in the cells.

2. The proliferative zone: chondrocytes with a higher number of cells divide

rapidly and form columns of stacked cells parallel to the long axis of the bone.

3. The hypertrophic cartilage zone: it contains large chondrocytes with cells

increasing in volume and modifying the matrix, effectively elongating bone

Figure 7.
Oppositional bone growth and remodeling. The epiphyseal plate is responsible for longitudinal bone growth.

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Figure 8.
Epiphyseal plate growth. Five zones of epiphyseal growth plate includes: 1. resting zone, 2. proliferation zone,
3. hypertrophic cartilage zone, 4. calcified cartilage zone, and 5. ossification zone.

whose cytoplasm has accumulated glycogen. The resorbed matrix is reduced to

thin septa between the chondrocytes.

4. The calcified cartilage zone: chondrocytes undergo apoptosis, the thin septa of
cartilage matrix become calcified.

5. The ossification zone: endochondral bone tissue appears. Blood capillaries

and osteoprogenitor cells (from the periosteum) invade the cavities left by the
chondrocytes. The osteoprogenitor cells form osteoblasts, which deposit bone
matrix over the three-dimensional calcified cartilage matrix.

5.2 Appositional bone growth

When bones are increasing in length, they are also increasing in diameter;
diameter growth can continue even after longitudinal growth stops. This is called
appositional growth. The bone is absorbed on the endosteal surface and added to
the periosteal surface. Osteoblasts and osteoclasts play an essential role in appo-
sitional bone growth where osteoblasts secrete a bone matrix to the external bone
surface from diaphysis, while osteoclasts on the diaphysis endosteal surface remove
bone from the internal surface of diaphysis. The more bone around the medul-
lary cavity is destroyed, the more yellow marrow moves into empty space and fills
space. Osteoclasts resorb the old bone lining the medullary cavity, while osteoblasts
through intramembrane ossification produce new bone tissue beneath the perios-
teum. Periosteum on the bone surface also plays an important role in increasing
thickness and in reshaping the external contour. The erosion of old bone along the
medullary cavity and new bone deposition under the periosteum not only increases
the diameter of the diaphysis but also increases the diameter of the medullary cav-
ity. This process is called modeling (Figure 9) [3, 4, 15].

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Osteogenesis and Bone Regeneration

Figure 9.
Appositional bone growth. Bone deposit by osteoblast as bone resorption by osteoclast.

6. The role of mesenchymal stem cell migration and differentiation in

bone formation

Recent research reported that bone microstructure is also the principle of bone
function, which regulates its mechanical function. Bone tissue function influenced
by many factors, such as hormones, growth factors, and mechanical loading. The
microstructure of bone tissue is distribution and alignment of biological apatite
(BAp) crystallites. This is determined by the direction of bone cell behavior, for
example cell migration and cell regulation. Ozasa et al. found that artificial control
the direction of mesenchymal stem cell (MSCs) migration and osteoblast alignment
can reconstruct bone microstructure, which guide an appropriate bone formation
during bone remodeling and regeneration [16].
Bone development begins with the replacement of collagenous mesenchymal
tissue by bone. Generally, bone is formed by endochondral or intramembranous
ossification. Intramembranous ossification is essential in the bone such as skull,
facial bones, and pelvis which MSCs directly differentiate to osteoblasts. While,
endochondral ossification plays an important role in most bones in the human
skeleton, including long, short, and irregular bones, which MSCs firstly experi-
ence to condensate and then differentiate into chondrocytes to form the cartilage
growth plate and the growth plate is then gradually replaced by new bone tissue
[3, 8, 12].
MSC migration and differentiation are two important physiological processes
in bone formation. MSCs migration raise as an essential step of bone formation
because MSCs initially need to migrate to the bone surface and then contribute in
bone formation process, although MSCs differentiation into osteogenic cells is also
crucial. MSC migration during bone formation has attracted more attention. Some
studies show that MSC migration to the bone surface is crucial for bone formation
[17]. Bone marrow and periosteum are the main sources of MSCs that participate in
bone formation [18].
In the intramembranous ossification, MSCs undergo proliferation and dif-
ferentiation along the osteoblastic lineage to form bone directly without first
forming cartilage. MSC and preosteoblast migration is involved in this process and
are mediated by plentiful factors in vivo and in vitro. MSCs initially differentiate
into preosteoblasts which proliferate near the bone surface and secrete ALP. Then
they become mature osteoblasts and then form osteocytes which embedded in
an extracellular matrix (ECM). Other factors also regulate the intramembranous
ossification of MSCs such as Runx2, special AT-rich sequence binding protein 2
(SATB 2), and Osterix as well as pathways, like the wnt/β-catenin pathway and
bone morphogenetic protein (BMP) pathway [17, 19].

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DOI: http://dx.doi.org/10.5772/intechopen.82452

In the endochondral ossification, MSCs are first condensed to initiate cartilage

model formation. The process is mediated by BMPs through phosphorylating
and activating receptor SMADs to transduce signals. During condensation, the
central part of MSCs differentiates into chondrocytes and secretes cartilage
matrix. While, other cells in the periphery, form the perichondrium that continues
expressing type I collagen and other important factors, such as proteoglycans and
ALP. Chondrocytes undergo rapid proliferation. Chondrocytes in the center become
maturation, accompanied with an invasion of hypertrophic cartilage by the vas-
culature, followed by differentiation of osteoblasts within the perichondrium and
marrow cavity. The inner perichondrium cells differentiate into osteoblasts, which
secrete bone matrix to form the bone collar after vascularization in the hypertrophic
cartilage. Many factors that regulate endochondral ossification are growth factors
(GFs), transforming growth factor-β (TGF-β), Sry-related high-mobility group
box 9 (Sox9) and Cell-to-cell interaction [17, 19].

7. Conclusions

• Osteogenesis/ossification is the process in which new layers of bone tissue are

placed by osteoblasts.

• During bone formation, woven bone (haphazard arrangement of collagen

fibers) is remodeled into lamellar bones (parallel bundles of collagen in a layer
known as lamellae)

• Periosteum is a connective tissue layer on the outer surface of the bone; the
endosteum is a thin layer (generally only one layer of cell) that coats all the
internal surfaces of the bone

• Major cell of bone include: osteoblasts (from osteoprogenitor cells, forming

osteoid that allow matrix mineralization to occur), osteocytes (from osteo-
blasts; closed to lacunae and retaining the matrix) and osteoclasts (from hemo-
poietic lineages; locally erodes matrix during bone formation and remodeling.

• The process of bone formation occurs through two basic mechanisms:

○○ Intramembranous bone formation occurs when bone forms inside the mes-
enchymal membrane. Bone tissue is directly laid on primitive connective
tissue referred to mesenchyma without intermediate cartilage involvement.
It forms bone of the skull and jaw; especially only occurs during develop-
ment as well as the fracture repair.
○○ Endochondral bone formation occurs when hyaline cartilage is used as a
precursor to bone formation, then bone replaces hyaline cartilage, forms
and grows all other bones, occurs during development and throughout life.

• During interstitial epiphyseal growth (elongation of the bone), the growth

plate with zonal organization of endochondral ossification, allows bone to
lengthen without epiphyseal growth plates enlarging zones include:
○○ Zone of resting.
○○ Zone of proliferation.
○○ Zone of hypertrophy.

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Osteogenesis and Bone Regeneration

○○ Zone of calcification.
○○ Zone of ossification and resorption.

• During appositional growth, osteoclasts resorb old bone that lines the medul-
lary cavity, while osteoblasts, via intramembranous ossification, produce new
bone tissue beneath the periosteum.

• Mesenchymal stem cell migration and differentiation are two important physi-
ological processes in bone formation.

Acknowledgements

The author is grateful to Zahrona Kusuma Dewi for assistance with preparation
of the manuscript.

Conflict of interest

The authors declare that there is no conflict of interests regarding the publica-
tion of this paper.

Acronyms and abbreviations

ALP alkaline phosphatase

Bap biological apatite
BMP bone morphogenetic protein
ECM extracellular matrix
GFs growth factors
MSCs mesenchymal stem cells
Runx2 runt-related transcription factor 2
SATB 2 special AT-rich sequence binding protein 2
Sox9 sry-related high-mobility group box 9
TGF-β transforming growth factor-β

Author details

Rosy Setiawati* and Paulus Rahardjo

Department of Radiology, Faculty of Medicine Airlangga University, Dr. Soetomo
General Hospital, Surabaya, East Java, Indonesia

*Address all correspondence to: [email protected]

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Bone Development and Growth
DOI: http://dx.doi.org/10.5772/intechopen.82452

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