Structure and Classification of Bone

1
STRUCTURE OF BONE
BY
Dr. Bhuvan Nagpal
B.D.S. (Hons.), M.D.S. (Oral Pathology)
(Gold Medalist)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
Dept. of Oral Pathology & Microbiology,
JSS Dental College & Hospital,
JSS University,
Mysuru, Karnataka, India
Dr. Archana S.
B.D.S., M.D.S. (Oral Pathology)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
Dept. of Oral Pathology & Microbiology,
JSS Dental College & Hospital,
JSS University,
Mysuru, Karnataka, India
Dr. Anupam Nagpal
B.D.S. (Hons.)
(Gold Medalist)
House Surgeon
Teerthanker Mahaveer Dental College & Research Centre,
Teerthanker Mahaveer University,
Moradabad, Uttar Pradesh, India

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S.No CONTENTS Page No.
1. INTRODUCTION 3-6
2. CLASSIFICATION OF BONE 7-14
3. MECHANICAL PROPERTIES OF BONE 15
4. COMPOSITION OF BONE 16-22
5. ANATOMY OF BONE 23-34
6. MICROSCOPIC STRUCTURE OF BONE 34-53
7. HISTOGENESIS OF BONE 53-64
8. BONE DYNAMICS 64-97
9. FUNCTIONS OF BONE 98-99
10. CALCIUM & PHOSPHATE METABOLISM 99-110
11. BONE MINERALISATION 111-117
12. BLOOD SUPPLY & NUTRITION OF BONE 118-121
13. APPLIED ANATOMY 122-124
14. VENOUS & LYMPHATIC DRIANAGE OF BONE 125
15. NERVE SUPPLY OF BONE 125
16. DEVELOPMENT OF FACIAL BONES 126-132
17. MAXILLA 133-145
18. MANDIBLE 145-156
19. ALVEOLAR BONE 157-166
20. REFERENCES 167-169

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INTRODUCTION
The human skeleton is bilaterally symmetrical with the typical vertebrate
pattern of an axis, divided into segments for flexibility, and of two pairs of limbs,
pectoral and pelvic, also divided into jointed parts for locomotion, grasping etc. The
skull is the expanded and modified cranial end of the axis.Osseocartiliginous sesamoid
bones develop in some tendons and ligaments. All these elements are collectively
termed the’skeleton’.The skeletal system is composed of 206 bones that vary in size
and shape. The bones are interconnected by a variety of joints that allow for a wide
range of movement while maintaining stability1,2
The human skeleton is internal to the muscles with which it has evolved called
as ‘endoskeleton’.which has got a protective role except in the vault of skull and
spinal cord.The maxilla, mandible, clavicle and dentine of teeth are dermal
derivatives, All are vestiges of more extensive assemblies of dermal bones from which
they have been modified to form a human ‘exoskeleton’.1,2
Teeth is hardest and most stable of tissues.In mammals, where skeletal growth
is typically limited to an early period of life, there are generally two dentitions, the
first deciduous and other permanent, the condition is known as diphyodonty.The
evolution of mammals was associated with the posterosuperior downgrowth of
dentary bone, rearrangement of jaw muscles to move the mandible transversely and
change in the shape of the teeth. Teeth are selectively fossilized and preserved and
thus provide best evolutionary record. They are the excellent models of studying the

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relations between ontogeny and progeny.The durability of teeth to fire and bacterial
decomposition makes them invalulable in identification of otherwise unrecognizable
bodies, a point of great forensic importance.1,2,3
Bone is a specialized mineralized connective tissue made up of an organic
matrix of collagen fibrils embedded in an amorphous substance with mineral crystals
precipitated within the matrix. It is highly vascular, living, constantly changing
mineralized connective tissue with complicated hierarchical architecture. It is
remarkable for its hardness, resilience, regenerative property as well as its
characteristic growth mechanisms. Morphologically bone tissue appears to be under
the control of bone cells. Its surfaces are enveloped by active and resting osteoblasts
and osteoclasts, and it is permeated by an interconnected canalicular system in which
osteocytes are found. These cells control the composition of the extracellular fluids of
mineralized bone matrix within very narrow limits, and at the same time they can
remove and replace the mineralized tissue to meet the anatomical needs of a mature
skeleton.1,2,3
Throughout life, bones change in size, shape, and position. Two processes guide
these changes—modeling and remodeling. When a bone is formed at one site and
broken down in a different site, its shape and position is changed. This is called
modeling. This process allows individual bones to grow in size and to shift in
space.Remodeling repairs the damage to the skeleton that can result from repeated
stress by replacing small cracks or deformities in areas of cell damage. Remodeling

5
also prevents the accumulation of too much old bone, which can lose its resilience and
become brittle. Remodeling is also important for the function of the skeleton as the
bank for calcium and phosphorus.4
Bone health is critically important to the overall health and quality of life of an
individual. Bones serve as a storehouse for minerals that are vital to the functioning of
many other life-sustaining systems in the body.. Both genes and the environment
contribute to bone health. External factors, such as diet and physical activity, are
critically important to bone health throughout life and can be modified. The
mechanical loading of the skeleton is essential for maintenance of normal bone mass
and architecture. In addition, the skeleton needs certain nutritional elements to build
tissue.4
The growth of the skeleton, its response to mechanical forces, and its role as a
mineral storehouse are all dependent on the proper functioning of a number of
systemic or circulating hormones produced outside the skeleton that work in concert
with local regulatory factors. Eg calcium regulating hormone (parathyroid, calcitonin,
calcitriol),Sex hormones (estrogen, testosterone), growth hormone, thyroid hormone,
cortisol. This complex system of regulatory hormones responds to changes in blood
calcium and phosphorus, acting not only on bone but also on other tissues such as the
intestine and the kidney.4
Genetic abnormalities can produce weak, thin bones, or bones that are too
dense.eg osteogenesis imperfecta, osteoporosis. Nutritional deficiencies, particularly

6
of vitamin D, calcium, and phosphorus, can result in the formation of weak, poorly
mineralized bone.eg vitamin D deficiency. Many hormonal disorders can also affect
the skeleton. Overactive parathyroid glands or hyperparathyroidism can cause
excessive bone breakdown and increase the risk of fractures. In severe cases, large
holes or cystic lesions appear in the bone, which makes them particularly fragile. A
deficiency of the growth hormone/IGF-1 system can inhibit growth, leading to short
stature. Many bone disorders are local, affecting only a small region of the skeleton 4
Inflammation can lead to bone loss, probably through the production of local
resorbing factors by the inflammatory white cells. Bacterial infections, such as severe
gum inflammation or periodontal disease, can produce loss of the bones around the
teeth, and osteomyelitis can produce a loss of bone at the site of infection. This type of
bone loss is due to the direct damaging effect of bacterial products as well as
production of resorbing factors by white blood cells.
Thus bones have fascinated human beings since the dawn of time. Much of
what is known about the evolution of vertebrates is based on recovery of bones &
teeth from the soil. Remarkable progress has been gained in our understanding of the
cellular, molecular biology, and genetics of skeletal tissues in the last quarter century.
This has lead to new approaches to diagnosis, prevention, and treatment.1,2,4

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CLASSIFICATION:1,5.,6
I) Based on location, bones can be classified as follows:
x Axial skeleton - Bones of the skull, vertebral column, sternum, and ribs
x Appendicular skeleton - Bones of the pectoral, pelvis girdles, and limbs
x Acral bone - Part of the appendicular skeleton, including bones of the hands
and feet
2) Based on shape, bones can be classified as follows:
x Flat bone - Bones of the skull, sternum, pelvis, and ribs
xTubular bone - Long tubular bone, including bones of the limbs; short tubular
bone, including bones of the hands and feet, such as phalanges, metacarpals,
and metatarsals
xIrregular bone - Bones of the face and vertebral column

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xSesamoid bone - Bones developing in specific tendons, the largest example of
which is the patella
xAccessory bone or supernumerary bone - Extra bones developing in additional
ossification centers
3) Based on size, bones can be classified as follows:
x Long bone - Tubular in shape with a hollow shaft and two ends, including
bones of the limbs

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x Short bone - Cuboidal in shape, located only in the foot (tarsal bones) and wrist
(carpal bones)
4) Based on texture of cross sections, bone tissue can be classified as follows:
xCompact bone (dense bone, cortical bone): Compact bone is ivorylike and is
dense in texture without cavities. It is the shell of many bones, surrounding the
trabecular bone in the center. It consists mainly of Haversian systems or
secondary osteons

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xSponge bone (trabecular bone, cancellous bone): Sponge bone is spongelike
with numerous cavities and is located within the medullary cavity. It consists of
extensively connected bony trabeculae, oriented along the lines of stress.
5) Based on matrix arrangement, bone tissue can be classified as follows:
x Lamellar bone (secondary bone tissue/Bundle bone): Lamellar bone is mature
bone with collagen fibers arranged in lamellae. In sponge bone, lamellae are

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arranged parallel to each other, whereas in compact bone, they are
concentrically organized around a vascular canal, termed a Haversian canal.
x Woven bone (primary bone tissue): Woven bone is immature bone with
collagen fibers arranged in irregular random arrays, containing smaller amounts
of mineral substance and a higher proportion of osteocytes than lamellar bone.
Woven bone is temporary and eventually is converted to lamellar bone. Woven
bone is pathologic tissue in adults, except in a few places, such as areas near
sutures of the flat bones of the skull, tooth sockets), and the insertion site of
some tendons

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x Composite bone: Formed by the deposition of lamellar bone within a woven
bone lattice- Cancellous Compaction .It Is the predominant osseous tissue for
stabilizing during early phases of retention or post operative healing
6) Based on maturity, bone tissue can be classified as follows:
x Immature bone (primary bone tissue): Immature bone is woven bone.
x Mature bone (secondary bone tissue): Mature bone characteristically is
lamellar bone. Almost all bones in adults are lamellar bones.
7) Based on developmental origin, bones can be classified as follows:
x Intramembranous bone (mesenchymal bone): Intramembranous bone develops
from direct transformation of condensed mesenchyme. Flat bones are formed in
this way.
x Intracartilaginous bone (cartilage bone, endochondral bone): Intracartilaginous
bone forms by replacing a reformed cartilage model. Long bones are formed in
this way.
8) Paired and Unpaired bones
Paired Cranial Bones:
x Parietals - the two parietal bones each have a superior and inferior temporal
line, to which the temporal muscle is attached.
x Temporals - the two Temporal bones (near the temples) each have two major
portions, the squamous (flat) portion, and the petrosal portion.

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Unpaired Cranial Bones:
x Frontal - roughly, the forehead and upper part of the eye orbit.
x Occipital - the flat, concave base, which rests upon the first vertebrae. The
occipital bone has a hole, the Foramen Magnum , through which the blood
vessels and nerves of the spine connect with the base of the brain.
x Sphenoid - difficult to describe; winged, with many fissures and protusions. It
roughly leads from the sinuses to the eyes.
x Ethmoid - also hard to describe; cannot be seen from every angle of the skull.
Like the sphenoid, it is located in the the mid-sagittal plane and helps connect
the cranial skeleton to the facial skeleton. It consists of various plates and
paired projections. The most superior projection is the Crista Galli , (or "cocks
comb," owing to its appearance), which helps divides the left and right frontal
lobes of the brain.
Paired Facial Bones:
x Lacrimals - these two are the smallest and most fragile of the facial bones,
forming the front of the side wall of each eye orbit. Basically rectangular with
two surfaces and four borders. Each of the four borders articulate with the
bones that surround them.
x Nasals - two small rectangular bones which form the bridge of the nose above
the nasal cavity
x Zygomatics - the cheekbones, running from the maxilla to the wall of the eye
orbit.

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x Maxillae - the paired upper jaw bones. They are nearly hollow, each with a
large cavity called a maxillary sinus.
x Palatines - wing-shaped. They assist in forming the rear of the hard palate and
part of the nasal cavity.
x Inferior Nasal Conchae - small and complicated. These conchae are thin,
porous, and fragile. They are elongated and curled in on themselves. They lay
horizontally and are attached to the side wall of the nasal cavity. They increase
the surface area inside the cavity and increases the amount of mucus membrane
and olfactory nerve endings exposed to the air.
Unpaired Facial Bones:
x Vomer - forms the nasal septum, creating the left and right nasal passages. The
part of the nose that most often gets broken.
x Mandible - the lower jaw. The formal anatomical name for its tip (the chin) is
the mental protuberence.
x Hyoid - the small U-shaped bone in the front of the throat, under the jaw but
above the larynx. (the "Adam's Apple.")

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MECHANICAL PROPERPERTIES OF BONE1,5
AGE (in years)
Property 10-20
(years)
20-30
(years)
30-40
(years)
40-50
(years)
50-60
(years)
60-70
(years)
70-80
(years)
ULTIMATE STRENGTH (MPa)
Tension 114 123 120 112 93 86 86
Compression - 167 167 161 155 145 -
Bending 151 173 173 162 154 139 139
Torsion - 57 57 52 52 49 49
ULTIMATE STRENGTH (%)
Tension 1.5 1.4 1.4 1.3 1.3 1.3 1.3
Compression - 1.9 1.8 1.8 1.8 1.8 -
Torsion - 2.8 2.8 2.5 2.5 2.7 2.7

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Composition of Bone7,8,9
Bone is a specialized mineralized connective tissue .Aboutx 60%.of its wet weight is
inorganic material, about 25% organic material and about 15% water. By volume,
about 36% is inorganic, 36% is organgic and 28% is water. .The association of
organic and inorganic substances gives bone its hardness and resistance.
Bone
Inorganic Organic
hydroxy apatite 28% 5%
collagen Osteo-calcin
(type I) Sialo-protein
Osteonectin
Osteopontin
Phospo-Protein
Biglycan, Decorin
Growth Factors
Bone specific Protein

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Organic Matrix
Collagen is defined as a molecule composed of three polypeptide chains termed
D chains which associate into a triple helical molecule. Bone consists predominantly
of type I collagen with traces of type III, V & XI collagen. Type I collagen comprising
about 90% of bone matrix is a complex molecule that consists of a heterotrimer of two
pro - D1(I) and pro – α2(I) polypeptide chains. These peptide chains are structurally
similar but genetically distinct. Type I procollagen is characterized by the repeated
triplet, Gly – x – y where glycine is frequently followed by a proline & hydroxy
proline. The post translational modifications of Type I collagen specific for bone
include hydroxylation of some proline & lysine residues and glycosylation of
hydroxylysyl residues to form galactosyl - hydroxy lysyl residues. Interstitial collagen
is composed of these rod shaped molecules that associate both end to end and laterally
in a quarter stagerred fashion to form fibrils.
Transmission electron micrographs of individual mineralized collagen fibrils show
that hydroxyapatite crystals are located mainly within the fibrils at the level of the gap

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regions. The plate-shaped crystals are observed to be more or less uniformly stacked
across the fibril diameter.Collagen fibers are synthesized from osteoblasts,
polymerizing tropocollagen extracellularly & becoming progressively more cross
linked as they mature. The formation of collagen cross-links is attributable to the
presence of two aldehyde-containing amino acids which react with other amino acids
in collagen to generate difunctional, trifunctional, and tetrafunctional cross-links. A
necessary prerequisite for the development of these cross-links is that the collagen
molecules be assembled in the naturally occurring fibrous polymer. Once this
condition is met, cross-linking occurs in a spontaneous, progressive fashion. The
chemical structures of the cross-links dictate that very precise intermolecular
alignments must occur in the collagen polymer. This seems to be a function of each
specific collagen because the relative abundance of the different cross-links varies
markedly, depending upon the tissue of origin of the collagen.In primary bone they
form a complex interwoven meshwork (non-lamellar or woven bone) which is later
replaced by the regular arrays of nearly parallel collagen fibers (lamellar bone).
Collagen fibers from the periosteum are incorporated in cortical bone (extrinsic or
sharpey’s fibers) , anchoring this fibrocellular layer at its surface.
Non-collagenous proteins
In total 10% of organic phase is made of a variety of non collagenous proteins
including proteoglycans, glyco-proteins, J - carboxy glutamic acid containing proteins
and proteolipids.

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Proteoglycan & Glycoproteins
Osteonectins
Also known as SPARC (secreted protein, acidic and rich in cysteine)This acidic
glycoprotein is highly enriched in bone matrix and is synthesized by osteoblasts, skin
fibroblasts, tendon cells and odontoblasts. Its function is unknown. It is highly cross
linked and binds strongly to type I collagen and hydoxy apatite and may have a
function in acalcium –mediated organization of extracellular matrices..
RGD containing proteins (Arg – Gly – ASP) Fibronectin, Thrombospondin,
Osteopontin, Bone Sialo Protein as they contain specific amino acid sequence
arginie-glycine-aspartic acid.The bone matrix contains four proteins that contain the
amino acid sequence RGD (Arg – Gly – ASP) which binds to cell surface receptors
thereby mediating cell attachment.
Fibronectin is produced by osteoblastic cells and mediates cell attachment and
spreading of bone cells.
Thrombospondin an endogenous product mediates only adhesion of cells but
not spreading.
Osteopontin is produced by certain bone cells & mono nuclear cells and
mediates cell attachment. It is similar to bone sialoprotein, that is expressed in
differentiating bone cells. Osteopontin binds to the osteoclast integrin receptor and

20
leads to activation of the phospholipase C pathway in osteoclasts and increases in
intracellular calcium.
Bone sialoprotein is found only in osteoblasts and osteocytes.
The other bone sialo protein, Bone acidic glycoprotein (B A G) is similar to B S
P and osteopontin. Their functions are not clear and they more likely helps to increase
bone formation. These cell attachment proteins in bone can maintain osteoclasts or
other bone cells in particular location.
γ-Carboxy Glutamic Acid (Gla) – containing protein.
Matrix Gla Protein and Osteocalcin are two proteins bearing modified amino
acid, J-carboxy glutamic acid (gla) generated by Vitamin K dependent enzymes.
Osteocalcin appears late in bone development . Osteocalcin is regulated by 1,25 –
dihydroxy Vitamin D3. Osteocalcin is produced by osteoblasts and osteocytes and is
used as a marker of osteoblast activity in clinical states. It is postulated that it could
retard mineralisation. It also acts as chemo attractant for osteoclast progenitors
attracting them towards bone surfaces.
Proteoglycan
They are composed of a central protein core to which glycosoaminoglycans are
attached. Decorin and biglycan comprise < 10% of the noncollagenous proteins in
bone, but this decreases with maturation of bone.A third small proteoglycan
(chondroitin sulfate proteoglycan) has been found entirely associated with mineral

21
crystals. Biglycan is more prominent in developing bone and has been mineralized to
pericellular areas.It can bind TGF-β and extracellular matrix macromolecules,
including collagen and thereby regulate fibrillogenesis. Decorin binds mainly within
the gap region of collagen fibrils and decorates the fibril surface. The primary
calcification in bones is reported to follow the removal of decorin and fusion of
collagen fibrils.The other proteoglycans are biglycan, versican and serum proteins.
Lysyl oxidase and tyrosine rich acidic matrix proteins (TRAMP) are
components of demineralized bone and dentin matrix. Lysyl oxidase is a critical
enzyme for collagen crosslinking. TRAMP, also known as dermatopontin, binds
decorin and TGF-β and together these proteins regulate the cellular response to TGF-
β.
Other protein constituents
Growth Factors
Many growth regulatory factors that influence cell proliferation and/ or
differentiation is found in bone. They include transforming growth factor E - I, TGF E
- II, bone morphogenetic proteins, platelet derived growth factor, fibroblast growth
factors and insulin like growth factors.
Bone morphogenetic proteins are members of the extended transforming growth factor
E (TGF - E ) family and are synthesized by bone cells locally. They help in regulation
of normal bone remodeling.

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Inorganic Component
The inorganic component of bone consists of calcium hydroxy apatite which is
represented as Ca10(PO4)6(OH)2. The unit cell of the apatite has the shape of a rhombic
prism when stacked together, these prisms form the lattice of a crystal. A layer of
water called the hydration shell, exists around each crystallite, thereby there are 3
surfaces to an apatite crystal – the crystal interior, the crystal surface and the hydration
shell, all of which are available for the exchange of ions.
Thus the major ions are calcium, phosphate, hydroxyl, carbonate. Less numerous ions
are citrate, magnesium, sodium , potassium, fluoride, chloride, iron, zinc, copper,
aluminium, lead, strontium, silicon , boron,carbonate and lead. The percentage of
calcium in bones is 99% and phosphate is 85%. The relative ratio of calcium to
phosphorus can vary markedly under different nutritional conditions, the Ca/P ratio on
a weight basis varying between 1.3 and 2.0

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ANATOMY OF BONE1,2,5
Epiphysis
In long bones, the epiphysis is the region between the growth plate and the
expanded end of bone, covered by articular cartilage. An epiphysis in a skeletally
mature person consists of abundant trabecular bone and a thin shell of cortical bone.
While an epiphysis is present at each end of long limb bones, it is found at only one
end of metacarpals (proximal first and distal second through the fifth metacarpals),
metatarsals (proximal first and distal second through fifth metatarsals), phalanges
(proximal ends), clavicles, and ribs.
The epiphysis is the location of secondary ossification centers during
development. The structure of the epiphysis is more complex in bones that are fused

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from more than one part during development. Examples include proximal and distal
ends of the humerus, femur, and vertebrae. For instance, the proximal end of the
humerus is developed from 3 separate ossification centers, which later coalesce to
form a single epiphyseal mass. In the proximal humeral epiphysis, one of the centers
forms the articular surface, and the other two become the greater and lesser
tuberosities. Carpal bones, tarsal bones, and the patella are also called epiphysioid
bone and are developmentally equivalent to the epiphyses of long bones.
Knowledge of the location of the epiphysis and its equivalents in various bones
aids in recognition of the origin of bone lesions and further facilitates the diagnostic
consideration, as some bone tumors such as chondroblastoma have a strong
predilection for the epiphysis or epiphysioid bones.
Metaphysis
The metaphysis is the junctional region between the growth plate and the
diaphysis. The metaphysis contains abundant trabecular bone, but the cortical bone
thins here relative to the diaphysis. This region is a common site for many primary
bone tumors and similar lesions. The relative predilection of osteosarcoma for the
metaphyseal region of long bones in children has been attributed to the rapid bone
turnover due to extensive bone remodeling during growth spurts.

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Diaphysis
The diaphysis is the shaft and the region between metaphyses, composed
mainly of compact cortical bone. The medullary canal contains marrow and a small
amount of trabecular bone.
Physis (epiphyseal plate, growth plate)
The physis is the region separating the epiphysis from the metaphysis. It is the
zone of endochondral ossification in an actively growing bone or the epiphyseal scar
in a fully-grown bone.
Bone Surface markings
Depressions and openings that allow blood vessels and nerves to pass:
Foramen:
Round or oval opening through a bone through which blood vessels nevers or
ligaments pass. Eg optic foramen of the sphenoid bone.( foramen= hole)
Fissure:
Narrow, slitlike opening betweenadjacent parts of bone through which blood
vessels or nerves pass. Eg Superior orbital fissure
Fossa:
Shallow, basin like depression in a bone, often serving as an articular surface.
(Foramen=trench)Eg Coronoid fossa of the humerous.

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Meatus:
Canal-like or tube like opening.(Meatus= passageway) Eg external auditory
meatus of the temporal bone.
Sulcus:
Furrow along bone surface that accommodates a blood vessel, nerve or tendon.
(Sulcus =groove).Eg intertubercular sulcus of the humerous.
Sinus:
Cavity within a bone, filled with air and lined with mucous membrane.
Processes: Projections or outgrowths on bone that form joints or attachment points for
connective tissue, such as ligaments and tendons.
Process that form joints:
Condyle:
Rounded articular projection or round protuberance at the end of a
bone.(Condyle= knuckle) Eg lateral condyle of the femur.
Head:
Rounded articular projection.supported on the neck (constricted portion) of a
bone. Eg head of the Femur.

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Facet:
Smooth, nearly flat articular surface.Eg Superior articular surface of the
vertebra
Processes that form attachment points for connective tissue:
Crest:
Narrow, usually prominent, ridge of bone. Eg Illiac crest of the hip bone.
Epicondyle:
Raised area on or above a condyle Eg Medial condyle of the femur.
Line:
Narrow ridge of bone that is less prominent than a crest. Eg Linea aspera of the
femur.
Spinous process:
Sharp, slender, often pointed projection eg spinous process of a vertebra.
Trochanter:
Very large, blunt, irregularly shaped process.Eg greater trochanter of femur.

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Tubercle:
Small rounded process.(tuber=knob) eg greater tubercle of the humerous.
Tuberosity:
Large rounded projection that may be roughened eg ischial tuberosity of the hip
bone.
Marrow Cavity:
The marrow not only fills up the cylindrical cavities in the bodies of the long
bones, but also occupies the spaces of the cancellous tissue and extends into the larger
bony canals (Haversian canals) which contain the bloodvessels. It differs in
composition in different bones. In the bodies of the long bones the marrow is of a
yellow color, and contains, in 100 parts, 96 of fat, 1 of areolar tissue and vessels, and 3
of fluid with extractive matter; it consists of a basis of connective tissue supporting
numerous bloodvessels and cells, most of which are fat cells but some are “marrow
cells,” such as occur in the red marrow to be immediately described. In the flat and
short bones, in the articular ends of the long bones, in the bodies of the vertebræ, in
the cranial diploë, and in the sternum and ribs the marrow is of a red color, and
contains, in 100 parts, 75 of water, and 25 of solid matter consisting of cell-globulin,
nucleoprotein, extractives, salts, and only a small proportion of fat. The red marrow
consists of a small quantity of connective tissue, bloodvessels, and numerous cells,
some few of which are fat cells, but the great majority are roundish nucleated cells,

29
the true “marrow cells” of Kölliker. These marrow cells proper, or myelocytes,
resemble in appearance lymphoid corpuscles, and like them are ameboid; they
generally have a hyaline protoplasm, though some show granules either oxyphil or
basophil in reaction. A number of eosinophil cells are also present. Among the
marrow cells may be seen smaller cells, which possess a slightly pinkish hue; these
are the erythroblasts or normoblasts, from which the red corpuscles of the adult are
derived, and which may be regarded as descendants of the nucleated colored
corpuscles of the embryo. Giant cells (myeloplaxes, osteoclasts), large,
multinucleated, protoplasmic masses, are also to be found in both sorts of adult
marrow, but more particularly in red marrow. They were believed by Kölliker to be
concerned in the absorption of bone matrix, and hence the name which he gave to
them—osteoclasts. They excavate in the bone small shallow pits or cavities, which are
named Howship’s foveolæ, and in these they are found lying.

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SUTURES:
A suture (sutur=seam) is a fibrous joint composed of a thin layer of dense
fibrous connective tissue that unites bones of the skull.eg suture between the parietal
and frontal bones. The irregular, interlocking edges of the suture give them strength
and decrease their chance of fracturing.Because the suture is immovable, it is
classified as synarthrosis.
Some suture, although present during childhood,are replaced by bone in the adult.
Such a suture is called as synostosis in which there is complete fusion of the bone
across the suture line. Eg frontal suture between the left and right sides of thr frontal
bone that begins to fuse during infancy.
Cranial sutures:
Norma Verticalis
Sagittal suture,Coronal suture,Lambdoid suture,Metopic suture
Norma Occipitalis
Occipitomastoid suture,Parietomastoid suture
Norma Frontalis
Internasal Frontonasal, Naso-maxillary, Lacrimo-maxillary, Frontomaxillary,
Inermaxillary, Zygomaticomaxiilary,Zygomaticofrontal

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Norma Lateralis
Zygomatico temporal, Squamomastoid

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Periosteum
The periosteum is composed of an inner cambium layer immediately adjacent to
the bone surface and an outer dense fibrous layer. The cambium layer consists of
osteoprogenitor cells, which are flat and spindle shaped and are capable of
differentiating into osteoblasts and forming bones in response to various stimulations
.The collagen fibers in the outer layer are contiguous with the joint capsule, ligament,
and tendons. The periosteum is thicker and is more loosely attached to the cortex in
children but is thinner and more adherent in adults. The periosteum completely covers
a bone except in the region of the articular cartilage and at sites of muscle
attachments. It is somewhat anchored to the cortex by Sharpey fibers that penetrate
into the bone. The periosteum carries a dense network of blood, lymphatic vessels,
and predominantly sensory nerves for maintenance of the bone structure.
Different patterns of periosteal stimulation result in different patterns of periosteal
bone formation. Continual insult results in streams of periosteal bone perpendicular to
the bone surface, resulting in a hair-on-end appearance on radiographs eg Sickle cell
anemia, Thalassemia. Intermittent periosteal stimulation results in multiple partially
separated streams of periosteal bone parallel to the bone surface, giving an onion skin
appearance on radiographs.eg Ewing’s Sarcoma. As opposed to osseous tissue,
periosteum has nociceptors nerve endings, making it very sensitive to
manipulation.The periosteum has an osteogenic role. In the adult, the osteogenic role
is demonstrated during fracture repair. In addition, periosteum is becoming

33
increasingly attractive for the treatment of certain clinical problems: cleft palate
repair, treatment of severely comminuted fractures ,pseudoarthrosis of the tibia and
for the repair of tracheal defects.
The functions are follows:
x Provides attachment to muscles, tendons and ligament
x Nourishes the underlying bone with the help of blood vessels.
x Helps in bone formation during growth period
x Repair of fractures because of the presence of osteoprogenitor cells
x Prevents overgrowth of bone by acting as a limiting membrane
Mucoperiosteum:
Mucoperiosteum is a compound structure consisting of mucous membrane and
of periosteum. In regions such as the gingiva and parts of the hard palate, oral mucosa
is attached directly to the periosteum of underlying bone, with no intervening
submucosa. This arrangement is called a mucoperiosteum and provides a firm
inelastic attachment.

34
INDICATIONS:
x Areas with irregular bony contours, deep craters and other defects
x Pockets on teeth for which a complete removal of root irritant is not possible
x In cases of furcations involvement
x Intrabony pockets on distal to last molars, frequently associated with
mucogingival problems
x Persistent inflammation in areas of moderate to deep periodontal pockets
Endosteum
The endosteum is composed of osteoprogenitor cells and only a small amount
of connective tissue, covering the surface of bone trabeculae and the medullary
surface of cortical bone and Haversian canals. It serves as one of the functional
surfaces for bone remodeling.
MICROSCOPIC STRUCTURE OF BONE:
BONE CELLS

35
Osteoprogenitor cells:1,7,8,9
Are derived from the pleuripotent stromal stem cells present in the bone
marrow & other connective tissues which can proliferate & differentiate into
osteoblasts before bone formation. They are mesenchymal in origin. In
intramembranous bone they aggregate & undergo proliferation before differentiating
into osteoblasts while in endochondral bone formation.,similar cells migrate with the
ingrowth of blood vessels from the perichondrium into areas of degenerating cartilage
& differentiate into osteoclasts.
There are two types or stages of osteoprogenitor cells, one totally committed to
bone formation (committed osteoprogenitor cells),found associated with bone & the
other, (inducible osteoprogenitor cells) widely present in connective tissue,& probably
able to differentiate into various connective tissue cells depending on the nature of
inducer.

36
Osteoblast1,7,8,9
Any cell that forms bone whether during growth or remodeling or during
fracture healing is an osteoblast.
Osteoblasts are large non dividing cells with a rounded to polygonal shape with an
eccentrically placed nucleus. Cytoplasm is deeply basophilic and exhibits a distinct
negative Golgi image. The cytoplasmic processes are in contact with one another and
also the processes of osteocytes in the lacunae beneath them. Gap junctions do form
between adjacent cells, preosteoblasts to osteoblasts, osteoblasts to osteocytes and
osteocytes to osteocytes.Preosteoblasts and Osteoblasts exhibit high levels of alkaline
phosphatase on the outer surface of their plasma membranes.
Differentiation of Osteoblasts
Mesenchymal stem cells differentiate into osteoblasts when they are
exposed to bone morphogenic proteins (BMP). BMPs are part of the
transforming growth factor (TGF) superfamily.

37
Regulation of osteoblast differentiation10
Bone morphogenic protein
They regulate osteoblast and chondrocyte differentiation during skeletal
development
Smads
SMAD's (Small Mothers Against Decapentaplegic) are a class of proteins that
modulate the activity of transforming growth factor beta ligands.Smad transcription
factors are substrates of the activated type I receptor kinases in the cytoplasm. The
phosphorylated Smad proteins move into the nucleus, bind to the regulatory regions of
target genes, and regulate their transcription. Thus, Smad proteins are key molecules
in the transduction of signals from the cell membrane to the nucleus. There are three
classes of SMAD:
x The receptor-regulated Smads(R-SMAD) which include SMAD1, SMAD2,
SMAD3, SMAD5 and SMAD9
x The common-mediator Smad(co-SMAD) which include only SMAD4,
x The antagonistic or inhibitory Smads(I-SMAD) which include SMAD6 and
SMAD7.

38
Runx2 and Osterix
Runx2 interacts tightly with BMP signaling through Smads in osteoblast
differentiation.Osterix acts downstream of Runx2 during bone development
Factors affecting bone resorption and formation, directly and indirectly9
Systemic hormones:
PTH, 1,25(OH)2D3, Calcitonin, Sex steroids, Glucocorticoids, Growth hormone,
Thyroid hormone.
Cytokines growth factors
Prostaglandins,Interleukin 1, Tumor necrosis factor, Interferon Y, Insulin-like
growth factors, Macrophage colony stimulating factor, Epidermal growth factor,
Transforming growth factor, Bone morphogenic proteins, Platelet-derived growth
factor, Fibroblast growth factor, Vasoactive intestinal peptide, PTH-related peptide,
Osteoprotegerin ligand, Osteoprotegerin,Calcitonin gene- related peptide.
Miscellaneous agents
Immobilisation, weightlessness, Stress/exercise, Protons, Calcium, Phosphate,
Fluoride, Bisphosphonates, Alcohol/tobacco.

39
Functions of osteoblasts
x Osteoblasts are responsible for production of the proteins of bone matrix type I
and IV collagen and other non collagenous proteins like osteocalcin,
osteopontin, bone sialoprotein and osteonectin.
x Osteoblasts secrete the growth factors which are stored in bone matrix such as
transforming growth factor E, bone morphogenetic protein, platelet – derived
growth factor and the insulin – like growth factor.
x Osteoblasts mineralize newly formed bone matrix which maybe mediated in
part by sub-cellular particles known as matrix vesciles enriched in alkaline
phosphatase which are generated from the osteoblast cytoplasm. Osteoblasts
also produce phospholipids and proteoglycans which may be important in the
mineralization process.
x Osteoblast may be required for normal bone resorption to occur. Under
physiologic conditions that support resorption, the osteoblasts are stimulated by
lymphokines to produce interleukin-6 which in turn stimulate the osteoblast to
produce proteolytic enzymes which prepare the bone surface for osteoclastic
resorption. Functional lifespan of osteoblasts may range from 3 – 4 months to
1-5 years with an average of about 5 – 6 months.
x Osteoblasts has a controlling influence in activating the bone-resorbing cells,
the osteoclasts.Itis the source of factors involved in this process (colony-
stimulating factors,prostaglandins, osteoprotegerin ligand).

40
x Osteoblasts contains receptors for parathyroid hormone and regulates the
osteoclastic response to this hormone.
Lining Cells1,2
Lining Cells are remnants of osteoblasts that previously laid down bone matrix.
The cells have thin flat nuclear profiles. Cytoplasmic organelles are few and these
cells retain their gap junctions with osteocytes creating a network that functions to
control mineral homeostasis and ensure bone vitality. It also manages bone
maintenance by forming a bone membrane that controls ion fluxes into and out of
bone and by secreting additional phosphoproteins and glycoproteins.
Osteocytes1,7,8,9
Osteocytes constitute the major cell type of mature bone, lyingscattered within
its matrix, but interconnected by numerous cellular extensions to form a complex
cellular network. They are derived from osteoblasts which have reduced or ceased
matrix formation and become enclosed in matrix, but retain contact with each other
and with cells at the surfaces of bone (osteoblasts and bone lining cells) throughout
their lifespan.
Mature, relatively inactive osteocytes possess a cell body which has the shape
of a three-axis ellipsoid, the longer axis(about 25 μm) parallel to the surrounding body
lamella and its shortest axis perpendicular to the plane of the lamella. The cytoplasm
is faintly basophilic and contains few organelles. Numerous fine processes emerge

41
from the cell body and branch a number of times to form an extensive tree. Such
processes contain bundles of microfilaments and some smooth endoplasmic reticulum.
At their distal tips they contact the processes of adjacent cells (other osteocytes and at
surfaces, osteoblasts and bone lining cells)
Embryonic (woven) bone and repair bone have more osteocytes than does
lamellar bone. After their formation the osteocytes become reduced in size and the
space in the matrix occupied by an osteocyte is called the osteocytic lacuna.
Functions of osteocytes
Their normal functions are not clearly known. Functions may include :
x Maintenance of bone matrix – Osteocytes possess enough organelles to
continue producing relatively small amounts of matrix constituents throughout
life.

42
x Release of calcium ions – osteocytes may have the capacity to transfer calcium
ions from bone mineral to the blood plasma.
x Osteocytes may play a role in sensing strain resulting from mechanical force
applied to the skeleton during mechanical usage or they could act as part of a
transducer mechanism that converts changes in the strain environment into
organized bone cell work. Plays the role of mechanoreceptor of bone.
Osteoclasts1,7,8,9
Osteoclasts are multi nucleated giant cells which resorb bone. They range from 20 Pm
to over 100 Pm and contain 2–50 nuclei. They occupy shallow pits called ‘Howship’s
lacunae’ on flat bone surfaces, and they are present in the leading edge of cutting
cones in haversian bone. Features seen on light microscopy include a foamy
acidophilic cytoplasm, a striated or brush border appearance at the site of attachment
to the bone due to the projecting free collagen fibrils and a positive staining for
tartarate - resistant acid phosphatase. The part of an osteoclast that is directly
responsible for carrying out bone resorption is a transitory and highly motile structure
called its ruffled border seen on electron microscopy. Encircling the periphery of the
ruffled border is the clear zone a ring shaped region devoid of organelles. This region
is also called the podosome or filamentous zone where the ruffled border is sealed to
the bone surface. This seal apparently localizes the highly acidic micro environment,
which is conducive to resorption of bone. Farthest away from bone lies the basal
region of the cell containing multiple nuclei, golgi saccules, numerous mitochondria,

43
some secretary vesicles and lysosomes. Their lifespan is uncertain, though it may be
as long as 7 weeks.
Origin and Cell Lineage1,7,11
They come from mononuclear precursors i.e. blood monocytes. The proposed
model for the formation of osteoclast is that the colony stimulating factors (CSFs)
stimulate the proliferation and differentiation of the granulocyte – macrophage
committed progenitor cells (CFU-GM). The CFU–GM stimulated by CSF to form
pro-monocytes which are immature non-adherent progenitors of mono nuclear
phagocytes and osteoclasts. The pro-monocyte proliferate and differentiate along the
macrophage pathway or along the osteoclast pathway. The first osteoclast to form the
early pre-osteoclasts proliferates and circulate in blood. They contain non specific

44
esterase and not tartarate resistant. The early pre-osteoclast gives rise to a late pre-
osteoclast regulated by 1,25 – dihydroxy vitamin D, PTH. Then the late pre-osteoclast
attaches to bone, expresses osteoclast specific antigens and then fuses with other cells
to form a multinucleated osteoclast.
Differentiation of Osteoclasts10
The molecule that inhibits osteoclastogenesis is known by two different
names, OPG (osteopotegerin) and OCIF (osteoclastogenesis inhibiting factor).
OPG is secreted by osteoblasts and functions to block the formation of
osteoclasts as well as bone resorption.

45
Regulation of osteoclast differentiation
TNF receptor-ligand family members
Osteoblasts/stromal cells regulate osteoclast differentiation and function
through TNF receptor –ligand family members.
RANKL–RANK interaction
Activation of NF-jB and JNK through the RANK mediated signaling system
appears to be involved in the differentiation and activation of osteoclasts.
Inflammatory cytokines
Interleukin-1 directly stimulates osteoclast function through the IL-1 type 1
receptor expressed by osteoclasts. LPS and some inflammatory cytokines such as
TNFa and IL-1 are directly involved in osteoclast differentiation and function through
a mechanism independent of RANKL–RANK interaction.
RECEPTOR ACTIVATION
Osteoclasts also express integrin receptors including the vitronectin receptor
which plays an important role in the adhesion of osteoclasts to bone
surface. Peptides containing the RGD motif have been shown to inhibit
osteoclast-mediated bone resorption in vitro and prevent osteoporosis in vivo

46
Osteoclastogenesis5
PTH stimulates bone resorption by osteoclasts, but it does so indirectly.
Receptors for PTH are located on osteoblasts, which then signal to bone marrow-
derived osteoclast precursors to stimulate their fusion, differentiation and activation.
Osteoclast precursors express a cell-surface receptor known as RANK (Receptor
Activator of Nuclear factor-Kappa B). Osteoblasts express RANKL (RANK Ligand)
on the extracellular surface of their plasma membrane.
When they are stimulated by PTH, osteoblasts up-regulate expression of
RANKL, which binds to RANK, activating signaling pathways that promote
osteoclast differentiation and survival. Osteoblasts also express a secreted factor
called osteoprotegerin. As its name implies, osteoprotegerin "protects bone" by
preventing bone resorption. Osteoprotegerin works as a decoy receptor for RANKL: it
binds RANKL and therefore prevents binding to RANK and stimulation of
osteoclastogenesis. The ratio of osteoprotegerin:RANKL produced by osteoblasts will
determine the extent of bone resorption.

47
Interrelationship between osteoblasts and osteoclasts:5
There is close relationshiop between bone deposition and bone
resorption.During the growing phase of a child, the amount of deposition exceeds that
of resorption, giving an increase in bone mass. During the adult phase, the amount of
bone deposition is equivalent to that of bone resorption and bone masses more or less
constant. In old age, the amount of bone deposition is generally less than that of bone
resorption and there is overall decrease in bone mass.In postmenopausal women
particularly this loss may be sufficient to lead to clinical condition of osteoporosis.
Many of the factors that result in bone resorption are known to have no direct
effect on osteoclasts, but act indirectly through osteoblasts. Most of the receptors to
bioactive molecules that cause bone resorption are present on osteoblasts.There are
several mechanisms whereby osteoblasts might promote bone resorption:
x By the local release of substances such as cytokines and growth factors
(macrophage colony-stimulating factor, osteoprotegrein and interleukins),
osteoblasts could stimulate the production of osteoclasts.
x By releasing enzymes (such as MMPs) to degrade the unminerialised osteoid
layer covering forming bone, osteoblasts could help expose mineralized matrix
on which osteoclasts could attach and commence resorption.
x By bioactive molecules present within bone (cytokines, BMPs, TGF-β) that
could be activated as a result of osteoclastic bone resorption and subsequently
have an effect on remodelling.

48
Reversal lines mark the position where bone activity changes from resorption to
deposition. Such lines are darkly stained and irregular in outline, being composed of a
series of concavities that were once the sites of the resorptive Howship’s lacunae.
They may be seen to contain the enzyme acid phosphatase.
BONE MATRIX
Haversian system 1,7,8
The primary structural unit of compact bone is the Haversian system. Each
Haversian system is a long, often bifurcated, cylinder parallel to the long axis of bone,
formed by successive deposition of 4-20 (average 6) concentric layers of lamellae.

49
Collagen fibers are parallel to each other within each lamella but are oriented
perpendicularly to those in the neighboring lamellae. Such an arrangement can be
highlighted as alternating bright and dark layers in polarized microscopy.
Lamellar deposition starts from the periphery, so that younger lamellae are
closer to the center of the system, and the younger systems have larger canals.
Between the lamellae are lacunae containing the cell bodies and canaliculi holding the
cytoplasmic processes of osteocytes.
In the center of each Haversian system is a Haversian canal, which is lined by
endosteum and contains a neurovascular bundle and loose connective tissue.
The Haversian canals connect with each other by transverse or oblique
Volkmann canals, communicating with the marrow cavity and the periosteum to
provide channels for the neurovascular system. Volkmann canals are not surrounded
by concentric lamellae; rather, they perforate the lamellae. They contain blood vessels,
nerves and lymphatics and connect haversian canals with the medullary cavity and the
surface of the bone

50
Interstitial lamellae
Interstitial lamellae are incomplete or fragmented osteons located between the
secondary osteons. They represent the remnant osteons left from partial resorption of
old osteon during bone remodeling.
The mixture of interstitial lamellae and complete osteons produces a mosaic
pattern. Thus, the age of the bone can be deduced from the proportion of interstitial
lamellae and intact osteons. Younger bone has more complete osteons and less
interstitial lamellae in between the osteons.

51
Circumferential lamellae
Circumferential lamellae are circular lamellae lining the external surface of the
cortex adjacent to the periosteum and lining the inner surface of the cortex next to the
endosteum. There are more outer than inner circumferential lamellae.
Bony trabeculae
Bony trabeculae are seen as a system of plates, rods, arches and struts traversing
the medullary cavity and attached to the cortex endosteum. The internal surface of the
bone is covered by a single layer of bone cells, the endosteum which physically
separates the bone surface from the bone marrow within.

52
Bone Marrow
The term bone marrow is usually restricted to the soft red or yellow tissue
occupying the macroscopically visible cavities in a fresh bone. It is essentially a frame
work of reticular tissue (reticulum cells & fibres) supporting blood vessels especially
venous sinusoids and either colonies of developing blood cells or large fat cells.
Reticulum cells readily turn into osteoprogenitor cells.
Bone marrow is considered one of the most valuable diagnostic tools to
evaluate hematologic disorders. Indications have included the diagnosis, staging, and
therapeutic monitoring for lymphoproliferative disorders such as chronic lymphocytic
leukemia (CLL), Hodgkin and Non-Hodgkin lymphoma, hairy cell leukemia,

53
myeloproliferative
disorders,andmultiplemyeloma. Furthermore, evaluationof cytopenia, thrombocytosis,
leukocytosis, anemia, and iron status can be performed. The bone marrow analysis has
also been used to evaluate nonhematologic, conditions. For example, in the
investigation for fever of unknown origin (FUO), specifically in those patients with
(AIDS), the marrow may reveal the presence of microorganisms, such as tuberculosis,
Mycobacterium avium intracellulare (MAI) infections, histoplasmosis, leishmaniasis,
and other disseminated fungal infections. Furthermore, the diagnosis of storage
diseases (eg. Niemann-Pick disease and Gaucher disease), as well as the assessment
for metastatic carcinoma and granulomatous diseases (eg, sarcoidosis) can be
performed.
In a bone marrow biopsy, a sample of solid bone marrow material is taken. A
bone marrow aspiration is usually done at the same time as a biopsy. In an aspiration,
a sample of the liquid portion of your marrow is withdrawn. Together, a bone marrow
biopsy and aspiration are often called a bone marrow exam. "Dry tap" is a term used
to describe failure to obtain bone marrow on attempted marrow aspirations. Extensive
marrow fibrosis and hypercellularity have been proposed as mechanisms to account
for the inability to withdraw marrow by aspiration.
HISTOGENESIS OF BONE:1,2,7,9
The bone is of mesodermal origin. The process of bone formation is called
OSSIFICATION. Formation of most bones is preceded by the formation of a

54
cartilaginous model which is subsequently replaced by bone. This kind of ossification
is called ENDOCHONDRAL OSSIFICATION and the bones formed are called
CARTILAGE BONES.
Bone formation can take place in the mesenchymal blastema of some bones like
the bones of the skull cap. This is called INTRAMEMBRANOUS OSSIFICATION.
The 2 main forms of ossification are:
x Intramembranous ossification
x Endochondral ossification
Bone forms only by appositional deposition of matrix on the surface of a
preformed tissue. Woven bone is formed initially and is later converted to lamellar
bone by subsequent remodeling.
INTRA MEMBRANOUS OSSIFICATION:
Intramembranous ossification is the formation of bone directly on or within
fibrous connective tissue membranes formed by condensed mesenchymal cells. Such
bones form directly from mesenchyme without first going through a cartilage stage.It
begins approximately towards the end of second month of gestation. The process
involves the following steps:

55
Formation of bone matrix within the fibrous membrane:
At the site where a bone will develop, there is initially loose mesenchyme,
which appears as widely separated, pale-staining, stellate cells with interconnecting
cytoplasmic processes. Then a center of osteogenesis develop in association with
capillaries that grow into the mesenchyme. The mesenchymal cells proliferate and
condense into compact nodules.Some these cells develop into capillaries.The
mesenchymal cells in the center become round and basophilic with thick
interconnecting processes. These cells differentiate into osteoblasts. These cells
secrete the organic matrix. Once surrounded by bone matrix, these are called
osteocytes. The matrix soon begins to calcify. The osteocytes obtain nutrients and
oxygen by diffusion along bone canaliculi. The organic matrix is also formed around
their interconnecting processes. The first small of newly formed bone matrix is an
irregular spicule.
Formation of woven bone
The bony spicules gradually lengthen into longer anastomosing structures called
trabeculae.The trabeculae extend in radial pattern. These trabeculae extend the local
blood vessels. This early membranebone is termed as woven bone.External to woven
bone, there is condensation of vascular mesenchyme called the periosteum.At this
stage, few mesenchymal cells remain undifferentiated.But, before these cells
disappear, they leave a layer of flat cells called as osteogenic cells or trabeculae which
do not have osteoblasts.In richly vascular areas, these osteogenic cells give rise to

56
osteoblasts that form the bone matrix. In areas,with no capillary blood supply, they
from chondroblasts which lay down cartilage.
Appositional growth mechanism and formation of compact bone plates:
Osteoblasts and osteogenic cells cover the spicules and trabeculae of
bone.These osteogenic cells proliferate in a richly vascularised environment and give
rise to osteoblasts that deposit new layers of bone matrix on preexisting bone surface.
They are always in a superficial position repeating the process again and again. This is
appositional growth, which results in build up of bone tissue one layer at a time.Every
generation of osteoblasts produce their own canaliculi. Hence, all the new osteocytes
remain linked throughout canaliculi to bone surface above and to osteocytes below.
As the trabeculae increase in width due to appositional growth, neighbouring
capillaries are incorporated to provide nutrition to osteocytes in deeper layers.New
bone is deposited on some surfaces and resorbed at other sites leading to remodelling
of trabeculae. This remodelling maintains shape and size of bone throughout life.
Continued appositional growth and remodelling of trabeculae converts
cancellous bone to compact bone. Cancellous bone is in the central part of the bone as
the trabeculae do not increase in size.The vascular tissue in cancellous bone
differentiate into red marrow.

57
Formation of osteon:
As cancellous bone gets converted into compact bone, a number of narrow
channels are formed lined by osteogenic cells.These cells enclose vessels that were
present in soft tissue spaces of cancellous network.The consecutive lamellae of bone
added to the bony walls of spaces in cancellous bone, which is called osteon or
Haversian system.These osteons are called as primitive osteons as they are short,
compared to those in long bones.
The mechanism of intramembrasnous ossification involves bone morphogenetic
proteins and activation of transcription factor called cbfa1. BMP activate cbfa1 gene
in mesenchymal cells.The cbfa1 transcription factor transforms mesenchymal cells
into osteoblasts.It is believed that the proteins activate the genes for osteocalcin,
osteopontin and other bone specific extracellular matrix proteins.

59
ENDOCHONDRAL OSSIFICATION:
Endochondral ossification is the formation of bone within hyaline cartilage. In
this ossification process, mesenchymal cells are transformed into chondroblasts,
which initially produce a hyaline cartilage ‘model’ of the bone. Subsequently,
osteoblasts gradually replace the cartilage with bone.
Formation of cartilaginous model:
This process begins late in the second month of development At the site where
bone is going to form, mesenchymal cells crowd together in the shape of the future
bone. The mesenchymal cells differentiate into chondroblasts that produce a cartilage
matrix, hence the model consists of hyaline cartilage.In addition, a membrane called
perichondrium develops around the cartilage model consisting of outer fibrous layer
and inner chondrogenic layer. No osteoblasts are produced by the cells in the
chondrogenic layer, because differentiation is taking place in an avascular
environment. Fibroblasts in the fibrous layer produce collagen and a dense fibrous
covering is formed.
Growth of the cartilaginous model:
Growth of the cartilage model is by interstitial and appositional growth.
Increase in length is by interstitial growth due to repeated division of chondrocytes,
along with production of additional matrix by the daughter cells.Widening of the
model is due to further addition of matrix to its periphery by new chondroblasts,

60
derived from chondrogenic layer of the perichondrium.This is called appositional
growth. As the differentiation of cartilage cells moves towards the metaphysis, the
cells organize into longitudinal columns which are subdivided into three zones:
Zone of proliferation: The cells are small and flat, and constitute a source of new
cells.
Zone of hypertrophy and maturation: This is the broadest zone.The chondrocytes
hypertrophy, and in the early stages secrete Type II collagen. As hypertrophy
proceeds, proteoglycans are secreted.The increased cell size and cell secretion, lead to
an increase in the size of the cartilaginous model. As the chondrocytes reach the
maximum size, they secrete type X collagen and noncollagenous proteins.
Subsequently, there is partial breakdown of proteoglycans, creating a matrix
environment receptive for mineral deposition.
Zone of provisional mineralization: Matrix mineralization begins in the zone of
mineralization by formation of matrix vesicles. These membrane bound vesicles bud
off from the cell and form independent units in the longitudinal septa of the cartilage.
Formation of bone collar:
The capillaries grow into the perichondrium that surrounds midsection of the
model. The cells in the inner layer of the perichondrium differentiate into osteoblasts
in a vascular environment and form a thin collar of bone matrix around the midregion
of the model. At this stage, perichondrium is reffered to as periosteum as the

61
differentiation of cells from the inner layer of the perichondrium is giving rise to bone.
Vascularisation of the middle of the cartilage occurs, and chondroclasts resorb most of
the mineralized cartilage matrix. The bone collar holds together the shaft, which has
been weakened by disintegration of the cartilage. Hence, more space is created for
vascular ingrowth.
Formation of periosteal bud:
Periosteal capillaries accompanied by osteogenic cells invade the calcified
cartilage in the middle of the model and supply its interior.The osteogenic cells and
the vessels comprise a structure called the periosteal bud. The periosteal capillaries
grow into the cartilage model and initiate the development of a primary ossification
center. Osteogenic cells in the periosteakl bud give rise to osteoblasts that deposit
bone matrix on the residual calcified cartlage. This results in the formation of
cancellous bone that has remanents of calcified cartilage. This is the mixed spicule.
Formation of medullary cavity:
As the primary ossifications centre enlarges, spreading proximally and distally,
osteoclasts break down the newly formed spongy bone and open up a medullary
cavity in the center of the shaft. Hematopoietic stem cells enter the medullary cavity
giving rise to myeloid tissue.
The two ends of the developing bone are at this stage still composed entirely of
cartilage. The midsection of the bone becomes the diaphysis and the cartilaginous

62
ends of bone become the epiphysis.Hence, the primary center of ossification is the
diaphyseal center of ossification.
Formation of secondary ossification center:
A birth, most of the long bones have a bony diaphysis surrounding remanents of
spongy bone, widening medullary cavity, and two cartilaginous epiphysis. Shortly
befor or after birth, secondary ossification centers appear in one or both epiphysis.
Initially chondrocytes in the middle of the epiphysys hypertrophy and mature, and the
matrix partitions between their lacunae calcify.
Periosteal buds carry mesenchymal cells and blood vessels and here spongy
bone I sretained and no medullary cavity forms in the epiphysis. The ossification
spreads from secondary center in all directions. Eventually, the cartilage in the middle
of epiphysis gradually gets replaced by cancellous bone. When secondary ossification
is complete hyaline cartilage remains at two places-on the epiphyseal surface as
articular surface and at the junction of the diaphysis and epiphyseal plates.this plate
continues to form new cartilage, which is replaced by bone, a process that increases
the length of the bone. Long bones have one or two secondary ossification centers.
Short bones have one ossification center. The union of primary and secondary center
is called epiphyseal line.

64
BONE DYNAMICS:13
The dynamics of bone involve three different processes:
x Growth
x Modelling
x Remodelling
BONE GROWTH:1,14,15
Bone growth can be of two tyes:
x Appositional = bone growth on pre-existing bone surface
x Interstitial = bone growth via new cartilage formation within pre-existing
cartilage mass

65
Long Bone Growth:
WIDTH --> Appositional (bone)
LENGTH --> Interstitial (cartilage)
During childhood, bones throughout the body grow in thickness by appositional
growth, & long bones lengthen by the addition of bone material the epiphyseal plate.
Bones stop growing in length at about age 25, although they may continue to thicken.
GROWTH IN LENGTH:
The epiphyseal growth plate is made up of three tissue types: the cartilage
component divided into distinct zones, the bony tissue of the metaphysis and the
fibrous tissue that surrounds the growth plate. The cartilage matrix is primarily
composed of collagens and proteoglycans. These macromolecules play a critical role
in the development and maintenance of a variety of functions including tissue
strength, architecture, and cell to cell interactions. Type II collagen is the most
abundant of the collagens in the growth plate, and since it is found almost exclusively
in cartilage it is a specific phenotype marker for chondrocytes. Type II collagen is
composed of three identical chains that are wound into the characteristic triple helix of
the collagen molecule. Type II collagen molecules form banded fibers seen with the
electron microscope and are therefore classified as fiber forming (class I) collagen. In
the developing limb and in models of endochondral ossification, type II collagen
synthesis can be correlated with chondrogenesis.Type II procollagen may be

66
expressed in two forms, IIA or IIB, due to differential splicing of recently transcribed
RNA. In embryonic human vertebral column, type IIB mRNA expression is correlated
with cartilage matrix synthesis, whereas IIA is expressed in pre-chondrocytes, the
cells surrounding the cartilage. Type XI collagen, also a class I collagen, is present in
cartilage matrix and is integrated into the interior of type II collagen fibrils. Its
function is not known. Type IX collagen is also found in cartilage, but is not a fiber
forming collagen since it will not form supramolecular aggregates alone. Type IX is
associated with the exterior of the type II collagen molecules and, since it has a single
glycosaminoglycan side chain, it is also a proteoglycan.
Type X collagen is a short chain, non-fibril forming collagen with a restricted
tissue distribution within the hypertrophic calcifying region of growth plates in fetal
and developing bone, where it makes up 45% of total collagen . It has been proposed
that type X collagen may play a role in regulating mineralisation of cartilage
calcification, however, this remains to be proven.
The other main structural component of cartilage is proteoglycan.
Proteoglycans are proteins with one or more attached glycosaminoglycan side chains,
e.g. chondroitin sulphate, heparan sulphate, dermatan sulphate. These sulphated side
chains occupy approximately two thirds of the C terminus region of the molecule,
while the other third, the carbohydrate-rich portion, binds to hyaluronic acid.. The
main proteoglycan of cartilage is aggrecan, a large proteoglycan composed of
approximately 90% chondroitin sulphate chains. Aggrecan is found as multi-

67
molecular aggregates composed of many proteoglycan monomers (up to 100) bound
to hyaluronan. A small link protein helps to stabilize the aggregate. Synthesis of
aggrecan is another specific marker of the chondrocyte phenotype.
Another important matrix component is the enzyme alkaline phosphatase
(ALP). ALP is abundant in matrix vesicles and on the plasma membrane of the
maturing chondrocytes, and is required in the calcification process although the
precise mechanism of action remains unclear Growth plate chondrocytes are organised
into different zones with each cell population being part of a different stage of
maturation in the endochondral sequence,.
ZONE OF RESTING CARTILAGE;1,16,17
The resting zone lies immediately adjacent to the secondary bony epiphysis.
Various terms have been applied to this zone, including resting zone, zone of small-
size cartilage cells, and germinal zone. They appear to store lipid and other materials
and perhaps are held in reserve for later nutritional requirements. The cells in this
zone are spherical, exist singly or in pairs, are relatively few when compared with the
number of cells in other zones, and are separated from each other by more
extracellular matrix than are cells in any other zone. Electron microscopy reveals
these cells to contain abundant endoplasmic reticulum, a clear indication that they are
actively synthesizing protein. They contain more lipid bodies and vacuoles than do
cells in other zones but contain less glucose-6-phosphate dehydrogenase, lactic
dehydrogenase, malic dehydrogenase, and phosphoglucoisomerase. The zone also

68
contains the lowest amount of alkaline and acid phosphatase, total and inorganic
phosphate, calcium, chloride, potassium, and magnesium. The matrix in the reserve
zone contains less lipid, glycosaminoglycan, protein polysaccharide, moisture, and ash
than the matrix in any other zone. It exhibits less incorporation of radiosulfur (35S)
than any other zone and also shows less Iysozyme activity than the other zones. It
contains the highest content of hydroxyproline of any zone in the plate.Collagen
fibrils in the matrix exhibit random distribution and orientation. Matrix vesicles are
also seen in the matrix, but they are fewer than in other zones. The matrix shows a
positive histochemical reaction for the presence of a neutral mucopolysaccharide or an
aggregated proteoglycan.
ZONE OF PROLIFERATING CARTILAGE;1,16,17
The spherical, single or paired chondrocytes in the reserve zone give way to
flattened chondrocytes in the proliferative zone. They are aligned in longitudinal
columns with the long axis of the cells perpendicular to the long axis of the bone.
The zone of proliferation contains the highest content of hexosamine,inorganic
pyrophosphate, and sodium, chloride, and potassium. It also has the highest level of
Iysozyme activity.
The chondrocytes in the proliferative zone are, with few exceptions, the only
cells in the cartilage portion of the growth plate that divide. The top cell of each
column is the true "mother" cartilage cell for each column, and it is the beginning or
the top of the proliferating zone that is the true germinal layer of the growth plate.

69
Longitudinal growth in the growth plate is equal to the rate of production of new
chondrocytes at the top of the proliferating zone multiplied by the maximum size of
the chondrocytes at the bottom of the hypertrophic zone.
The matrix of the proliferating zone contains collagen fibrils, distributed at
random, and matrix vesicles, confined mostly to the longitudinal septa. The matrix
shows a positive histochemical reaction for a neutral mucopolysaccharide or an
aggregated proteoglycan.
Thus the function of the proliferative zone is twofold: matrix production and
cellular proliferation. The combination of these two functions equals linear or
longitudinal growth. It is a paradox that while this chondrogenesis or cartilage growth
is solely responsible for the increase in linear growth of the long bone, the cartilage
portion of the plate itself does not increase in length. This, of course, is due to the
vascular invasion that occurs from the metaphysis with the resultant removal of
chondrocytes at the bottom of the hypertrophic zone, events that, in the normal growth
plate, exquisitely balance the rate of cartilage production.
ZONE OF HYPERTROPHIC CARTILAGE:1,16,17
The flattened chondrocytes in the proliferative zone become spherical and
greatly enlarged in the hypertrophic zone. These changes in cell morphology are quite
abrupt, and one can usually determine the end of the proliferative zone and the
beginning of the hypertrophic zone within an accuracy of one to two cells. By the time

70
the average chondrocyte reaches the bottom of the hypertrophic zone, it has enlarged
some five times over what its size was in the proliferative zone.
On light microscopy, the chondrocytes in the hypertrophic zone appear
vacuolated. Toward the bottom of the zone, such vacuolation becomes extensive,
nuclear fragmentation occurs, and the cells appear nonviable. At the very bottom of
each cell column the lacunae appear empty and are devoid of any cellular content.
On electron microscopy the chondrocytes in the top half of the hypertrophic
zone appear normal and contain the full complement of cytoplasmic components.
However, in the bottom half of the zone, the cytoplasm contains holes that occupy
over 58% of the total cytoplasmic column. Obviously, it is holes and not vacuoles that
account for the "vacuolation" seen on light microscopy. Electron microscopy also
shows that glycogen is abundant in the chondrocytes in the top half of the zone,
diminishes rapidly in the middle of the zone, and disappears completely from the cells
in the bottom portion of the zone. The last cell at the base of each cell column is
clearly nonviable and shows extensive fragmentation of the cell membrane and the
nuclear envelope with loss of all cytoplasmic components except a few mitochondria
and scattered remnants of endoplasmic reticulum. Clearly, the ultimate fate of the
hypertrophic chondrocyte is death.
ZONE OF CALCIFIED MATRIX :1,16,17
Matrix calcification occurs in longitudinal septae between the columns of
chondrocytes, and this calcified matrix becomes the scaffolding for bone deposition in

71
the metaphysis. The hypertrophic zone contains the highest levels of alkaline
phosphatase. The traditional view was that these cells were metabolically very
inactive, and that increasing vacuolation indicated death by hypoxia. However, these
cells are clearly actively involved in the synthesis of type X and type II collagen.
Improvements in techniques of growth plate fixation that retain chondrocyte
morphology have led to the proposal that a terminal chondrocyte spends most of its
life as a fully viable cell indistinguishable from hypertrophic chondrocytes positioned
further proximally in the growth plate. The cells then die by apoptosis, a distinct
biological form of cell death, lasting approximately 18% of a terminal chondrocyte's
life span .Apoptosis may be triggered by the metaphyseal vasculature beyond the last
intact cartilage septum
ZONE OF JUNCTION OF GROWTH PLATE WITH METAPHYSIS:1,17
The region where the transition from cartilage to bone occurs. Chondrocyte
lysis is evident from empty lacunae invaded by vascular endothelial loops. The
vascular region of calcified cartilage is the primary spongiosum, upon which
osteoblasts lay down unmineralised bone, the osteoid. Metaphyseal bone formation is
associated with type I procollagen mRNA expression in the empty lacunae, osteoid,
bone and perichondrium .Type I collagen, a marker of the osteoblast phenotype, is
immunolocalised to the same areas, while types II and X collagen have restricted
immunolocalisation to calcified cartilage trabecular remnants within spongy
bone.Newly formed woven metaphyseal bone is gradually replaced by lamellar bone

72
following osteoclastic degradation of bony matrix and chondroclastic removal of
remaining cartilage trabeculae. At the same time external reshaping of the bone is
brought about by surface osteoclastic bone resorption and appositional bone formation
by periosteally derived osteoblasts.
FIBROUS AND FIBROCARTILAGINOUS COMPONENTS1
Encircling the typical long-bone growth plate at its periphery are a wedge-
shaped groove of cells, termed the ossification groove, and a ring or band of fibrous
tissue and bone, termed the perichondrial ring. Ranvier, the first to describe these
structures, concentrated his study on the cells in the groove.
The ossification groove contains round to oval cells that, on light microscopy,
seem to flow from the groove into the cartilage at the level of the beginning of the

73
reserve zone. .The function of the groove of Ranvier is to contribute chondrocytes to
the growth plate for the growth in diameter, or latitudinal growth, of the plate.
Three groups of cells were identified in the ossification groove: a group of
densely packed cells that seemed to be progenitor cells for osteoblasts that form the
bony band in the perichondrial ring; a group of undifferentiated cells and fibroblasts
that contribute to appositional chondrogenesis and, hence, growth in width of the
growth plate; and fibroblasts amid sheets of collagen that cover the groove and firmly
anchor it to the perichondrium of the hyaline cartilage above the growth plate.
The perichondrial ring is a dense fibrous band that encircles the growth plate at
the bone-cartilage junction and in which collagen fibers run vertically, obliquely, and
circumferentially. It is continuous at one end with the group of fibroblasts and
collagen fibers in the ossification groove and at the other end with the periosteum and
subperiosteal bone of the metaphysis
Hence the function of the ossification groove is to provide chondrocytes for the
growth in width of the growth plate, and the function of the perichondrial ring is to act
as a limiting membrane that provides mechanical support to the growth plate.
The activity of the epiphyseal plate is the only way that the diaphysis can
increase in length. As a bone grows, chondrocytes proliferate on the epiphyseal side of
the plate. New chondrocytes cover older ones, which are then destroyed by the
process of calcification. Thus the cartilage Is thus replaced by bone on the diaphyseal
side of the plate .In this way thickness remains constant but increases in length.

74
Between the ages of 18 & 25, the epiphyseal plates close, they stop dividing &
bone replaces cartilage. It fades leaving a bony feature called the epiphyseal line.
GROWTH IN THICKNESS:1,4
Bone can grow in thickness by appositional growth. At the bone surface,
periosteal cells differentiate into osteoblasts, which stores collagen fibers & other
organic molecules that form bone matrix.The osteoblasts become surrounded by
matrix & develop into osteocytes. This process form bone ridges on either side of a
periosteal blood vessel. The ridges slowly enlarge & create a groove on the periosteal
blood vessel.Eventually, the ridges fold together & fuse, & groove becomes a tunnel
that encloses the blood vessel. The former periosteum now becomes the endosteum
that encloses the blood vessel.Bone deposition by osteoblasts from the endosteum
forms, new concentric lamellae. The formation of addition concentric lamellae
proceeds inward towards the periosteal blood vessel. In this way, the tunnel fills in, &

75
a new osteon is created.As a osteon is forming, osteoblasts under the periosteum
deposit new outer circumferential lamellae, further increasing the thickness of the
bone .as the additional periosteal blood vessel becomes enclosed further growth
process continues. As a new bone tissue is being deposited on the outer surface of
bone, the bone tissue lining the medullary cavity is destroyed by the osteoclasts in the
endosteum .In this way the medullary cavity enlarges as the bone increases in
diameter
FACTORS AFFECTING BONE GROWTH:
The regulation of postnatal somatic growth is complex. Genetic, nutritional
factors and hormones exert regulatory functions
Calcium-Regulating Hormones
Three calcium-regulating hormones play an important role in producing healthy
bone: 1) parathyroid hormone or PTH, which maintains the level of calcium and
stimulates both resorption and formation of bone; 2) calcitriol, the hormone derived
from vitamin D, which stimulates the intestines to absorb enough calcium and
phosphorus and also affects bone directly; and 3) calcitonin, which inhibits bone
breakdown and mayprotect against excessively high levels of calcium in the blood.
Parathyroid hormone or PTH
PTH is produced by four small glands adjacent to the thyroid gland. These
glands precisely control the level of calcium in the blood. They are sensitive to small

76
changes in calcium concentration so that when calcium concentration decreases even
slightly the secretion of PTH increases. PTH acts on the kidney to conserve calcium
and to stimulate calcitriol production, which increases intestinal absorption of
calcium. PTH also acts on the bone to increase movement of calcium from bone to
blood. Excessive production of PTH, usually due to a small tumor of the parathyroid
glands, is called hyperparathyroidism and can lead to bone loss. PTH stimulates bone
formation as well as resorption. In recent years a second hormone related to PTH was
identified called parathyroid hormone related protein (PTHrP). This hormone
normally regulates cartilage and bone development in the fetus, but it can be over-
produced by individuals who have certain types of cancer. PTHrP then acts like PTH,
causing excessive bone breakdown and abnormally high blood calcium levels, called
hypercalcemia of malignancy.
Calcitriol
Calcitriol is the hormone produced from vitamin D. Calcitriol, also called 1,25
dihydroxy vitamin D, is formed from vitamin D by enzymes in the liver and kidney.
Calcitriol acts on many different tissues, but its most important action is to increase
intestinal absorption of calcium and phosphorus, thus supplying minerals for the
skeleton. Vitamin D should not technically be called a vitamin, since it is not an
essential food element and can be made in the skin through the action of ultra violet
light from the sun on cholesterol. Vitamin D deficiency leads to a disease of defective
mineralization, called rickets in children and osteomalacia in adults. These conditions

77
can result in bone pain, bowing and deformities of the legs, and fractures. Treatment
with vitamin D can restore calcium supplies and reduce bone loss.
Calcitonin
Calcitonin is a third calcium-regulating hormone produced by cells of the
thyroid gland, although by different cells than those that produce thyroid hormones.
Calcitonin can block bone breakdown by inactivating osteoclasts, but this effect may
be relatively transient in adult humans. Calcitonin may be more important for
maintaining bone development and normal blood calcium levels in early life. Excesses
or deficiencies of calcitonin in adults do not cause problems in maintaining blood
calcium concentration or the strength of the bone. However, calcitonin can be used as
a drug for treating bone disease.
Sex Hormones
Along with calcium-regulating hormones, sex hormones are also extremely
important in regulating the growth of the skeleton and maintaining the mass and
strength of bone. The female hormone estrogen and the male hormone testosterone
both have effects on bone in men and women. The estrogen produced in children and
early in puberty can increase bone growth. The high concentration that occurs at the
end of puberty has a special effect—that is, to stop further growth in height by closing
the cartilage plates at the ends of long bone that previously had allowed the bones to
grow in length. Estrogen acts on both osteoclasts and osteoblasts to inhibit bone
breakdown at all stages in life. Estrogen may also stimulate bone formation. The

78
marked decrease in estrogen at menopause is associated with rapid bone loss.
Testosterone is important for skeletal growth both because of its direct effects on bone
and its ability to stimulate muscle growth, which puts greater stress on the bone and
thus increases bone formation. Testosterone is also a source of estrogen in the body; it
is converted into estrogen in fat cells. This estrogen is important for the bones of men
as well as women. In fact, older men have higher levels of circulating estrogen than do
postmenopausal women.
Growth hormone
Is an important regulator of skeletal growth. It acts by stimulating the
production of another hormone called insulin-like growth factor-1 (IGF-1), which is
produced in large amounts in the liver and released into circulation. IGF-1 is also
produced locally in other tissues, particularly in bone, also under the control of growth
hormone. The growth hormone may also directly affect the bone—that is, not through
IGF-1. Growth hormone is essential for growth and it accelerates skeletal growth at
puberty. Decreased production of growth hormone and IGF- 1 with age may be
responsible for the inability of older individuals to form bone rapidly or to replace
bone lost by resorption. The growth hormone/IGF-1 system stimulates both the bone-
resorbing and bone-forming cells, but the dominant effect is on bone formation, thus
resulting in an increase in bone mass.

79
Thyroid hormones
Increase the energy production of all body cells, including bone cells. They
increase the rates of both bone formation and resorption. Deficiency of thyroid
hormone can impair growth in children, while excessive amounts of thyroid hormone
can cause too much bone breakdown and weaken the skeleton . The pituitary hormone
that controls the thyroid gland, thyrotropin or TSH, may also have direct effects on
bone.
Cortisol
Cortisol, the major hormone of the adrenal gland, is a critical regulator of
metabolism and is important to the body’s ability to respond to stress and injury. It has
complex effects on the skeleton .Small amounts are necessary for normal bone
development, but large amounts block bone growth. They can cause bone loss due
both to decreased bone formation and to increased bone breakdown, both of which
lead to a high risk of fracture.There are other circulating hormones that affect the
skeleton as well. Insulin is important for bone growth, and the response to other
factors that stimulate bone growth is impaired in individuals with insulin deficiency
.A recently discovered hormone from fat cells, leptin, has also been shown to have
effects on bone.

80
Local factors in regulation of growth1,18
Local factors are necessary for intercellular communication and include
cytokines and growth factors. A cytokine can be defined as a soluble low molecular
weight cell product that affects the activity of other local cells in a paracrine manner;
they may act on their cell of origin by an autocrine mechanism, or via release into the
circulation may affect cells at a distant site, behaving as classic endocrine agents. In
hard tissues another mechanism of control exists, where locally produced growth
factors, or those in the circulation, are incorporated into mineralized matrix and are
released during matrix dissolution by osteoclasts or chondroclasts. The term cytokine
is now generally used to include molecules that were originally defined as growth
factors, e.g., the insulin-like growth factors (IGFs), the transforming growth factors
(TGF alpha and TGF beta), platelet-derived growth factor (PDGF), and fibroblast
growth factors (FGFs)
Local mediators in skeletal tissues
Factor Expression of mRNA or protein in bone
and cartilage cells
Growth factors
Insulin-like growth factors (IGF-I & II) Osteoblasts (OB) & chondrocytes (C)
Transforming growth factors (TGFbs 1-3) OB & C
Fibroblast growth factors acidic and basic
(aFGF & bFGF)
OB & C
Platelet derived growth factor (PDGF) OB
Bone morphogenetic proteins BMPs 1-7 OB
Interleukins (IL)

81
IL-1 b OB & C
IL-3 (Multi CSF) OB
IL-4
IL-6 OB & C
IL-8 OB & C
Tumour necrosis factors
TNFa OB
TNFb
Interferons
IFNg
Colony stimulating factors
GM-CSF OB & C
M-CSF OB & C
Others
Prostaglandins OB & C
PTH-RP OB & C
CGRP
Insulin-like growth factors (IGF-I & IGF-II)
Of the growth factors, those with the most potent effects on growing skeletal
tissue are the IGFs, previously known as somatomedins. IGFs are synthesized in the
liver and circulate bound to carrier proteins .The major factors regulating IGF
concentrations in serum are growth hormone, nutritional intake and thyroid hormones,
the latter being necessary for growth hormone secretion. The traditional view was that
growth hormone acted indirectly on the growth plate via IGF-I, a potent mitogen for
growth plate chondrocytes. However, there is increasing evidence that growth
hormone has direct effects on the growth plate .In addition to having effects on the

82
growth plate chondrocytes, locally synthesized and circulating IGFs retained in bone
matrix are important in the regulation of bone remodelling. Osteoblasts synthesize
IGFs; with human bone cells producing more IGF-II relative to IGF-I, and in human
bone matrix IGF-II is present in 10-15-fold greater concentrations than IGF-I. Both
IGF-I and IGF-II stimulate osteoblast and chondrocyte proliferation, induce
differentiation in osteoblasts and maintain the chondrocyte phenotype .Some of the
anabolic effects of PTH and oestrogen on bone may be effected by alterations in the
local synthesis of IGFs. Local concentrations of IGFs will also be regulated by
osteoblastic synthesis of binding proteins (IGFBPs), IGFBPs synthesis itself being
altered by growth hormone and oestradiol
Transforming growth factors (TGFs)
TGFs have diverse effects on growth and differentiation in normal and
neoplastic cell types. Most important in skeletal tissue are members of the TGF-β
gene family which includes the activins, inhibins, mullerian inhibiting substance, bone
morphogenetic proteins (BMPs), the drosophila decapentaplegic gene complex
product (dpp), and products of the mammalian Vgr gene. At least three isoforms of
TGF- β have been isolated in mammalian tissues (TGF-β). There is considerable
sequence identity and shared biological effects between these isoforms. TGF- β is
produced by several cell types, with bone matrix one of the most abundant sources of
both TGF- β1 and TGF- β2. Regulation of TGF- β, like that of many cytokines, occurs
not only at a transcriptional or translational level; it is secreted and stored in a latent

83
form that requires activation to become functional. Considerable evidence exists
supporting a role for TGF- β in morphogenesis, in the regulation of endochondral
ossification and in bone remodelling .High levels of TGF- β messenger RNA are
expressed in the growth plate of fetal human long bones. . TGF-β regulates the
synthesis of collagen by growth plate chondrocytes; increasing the synthesis of type I
relative to type II collagen,it may therefore control mineralisation by regulation of
hypertrophic chondrocyte differentiation. The effects of TGF- β on endochondral
ossification may be to stimulate growth in the undifferentiated cell, with different
effects on the terminally differentiated chondrocyte. TGF- β has a role to play in
regulation of bone remodelling, having effects on the proliferation and differentiation
of osteoblastic cells. TGF- β inhibits interleukin-1 and 1,25(OH)2D3 induced bone
resorption and the formation of multinucleated osteoclast-like cells in a human
marrow culture system. These diverse effects of TGF- β on bone cells have led to the
hypothesis that TGF- β may have a role in the coupling of bone formation to bone
resorption. One proposed mechanism is that during bone resorption latent TBF- β is
released from bone matrix and activated (possibly by the low pH and/or proteases), to
act locally on bone cells.
Bone morphogenetic proteins (BMPs)
This large family of proteins has aroused considerable interest in the bone cell
field, since the discovery that the implantation of demineralised matrix at
subcutaneous or intramuscular sites leads to bone formation. The factors in bone

84
matrix responsible for this induction of bone formation were named the bone
morphogenetic proteins (BMPs). There are now known to be 7 members of this family
(BMPs 1-7); all except BMP1 are members of the TGF- β family. BMP1 has been
classed as a novel regulatory protein. Chromosome mapping has shown that the
BMP2A and BMP3 genes map to conserved regions between mouse and human,
while the BMP1 gene does not.BMPs are the only molecules so far discovered capable
of independently inducing endochondral ossification in vivo. TGF- β1 and TGF- β2
enhance the osteoinductive properties of BMPs; however, injection of TGF- βs on
their own leads to extensive fibrous tissue formation only Recombinant forms of
BMP2 and BMP4 induce ectopic bone formation, and BMP2 will heal cortical bone
defects by an endochondral process BMP2 stimulates the growth and differentiation
of growth plate chondrocytes in vitro, and results in the development of the osteoblast
phenotype in a rat pluripotential cell line Osteoblasts have been shown to have high
affinity binding proteins for BMP on the cell surface). Indirect lines of evidence
demonstrate that BMPs have a critical role in bone development. Firstly, the protein
encoded by the decapentaplegic locus (dpp) in Drosophila is a member of the TGF- β
family member with 75% sequence homology to BMP2, suggesting a common
ancestral gene. Developmental anomalies produced by mutations of the dpp gene are
similar to patterns of disease expression in fibrodysplasia ossificans progressive, a
developmental disorder characterised by deformations of the hands and feet and
heterotopic chondrogenesis. In addition, the chromosomal locations of the BMP genes
overlap with the loci for several disorders of cartilage and bone formation. More direct

85
evidence is provided by a recent study which demonstrated that BMP2, together with
fibroblast growth factor-4, is important in regulating limb growth in the mouse
embryo.
Fibroblast growth factor (FGF)
FGF is a heparin binding peptide that exists in two forms, acidic and basic, with
55% sequence homology between the two.FGFs are potent mitogens for osteoblasts,
chondrocytes and endothelial cells, and stimulate proliferation of mesenchymal cells
in the developing limb that leads to limb outgrowth. FGF receptors are expressed in
limb mesenchyme as is mRNA for FGF-4.There is increasing evidence that basic FGF
(bFGF) is also important at later stages of bone growth, bFGF interacts with two
classes of binding sites on bovine growth plate chondrocytes: a high-affinity bFGF
receptor and a low-affinity heparin-like binding site FGFs are not secreted proteins
since a leader sequence is lacking, so they may only be released from their cell of
origin after membrane disruption. In this way FGF released from the degenerating
chondrocyte may act as a mitogen for metaphyseal vessels (since FGF is a potent
angiogenic factor) and cells of the osteoclast lineage.). During bone remodelling, FGF
synthesised by osteoblasts and stored in bone matrix may be released following
osteoclastic bone resorption. Activated FGF may then be important in stimulating
bone formation by increasing the number of osteoblastic precursor cells. bFGF has no
effect on osteoblast differentiation.

86
Platelet-derived growth factor (PDGF)
PDGF, a dimeric 30kDa peptide, was initially isolated from human platelets and
is known to exist in both homo- and heterodimeric forms . PDGF has been found in
bone matrix extracts and is secreted by human osteosarcoma cells and untransformed
rat osteoblasts. However, its synthesis by normal human osteoblasts or chondrocytes
has not been reported. The PDGF located in bone matrix may be sequestered from the
systemic circulation. PDGF is mitogenic for osteoblasts, fibroblasts and periosteal
cells, although it is possible some of these effects may be mediated by IGF-I, since
PDGF increases IGF-I synthesis in mesenchymal cells.PDGF may play a role in bone
development and growth. Both homodimeric forms of PDGF bind and increase DNA
synthesis in growth plate chondrocytes, having an additive effect with IGF-1. In the
stunted child, where disease may be a significant contributing factor, cytokine effects
on the bone growth plate may be of particular importance. For example, in post-
menopausal osteoporosis, cytokine production by circulating cells may be altered, and
this mechanism is believed to be important in the uncoupling of bone formation from
resorption characteristic of this disease.
Tumor necrosis factors (TNF)
Alpha and beta forms of TNF exist and, although there is only 28% sequence
identity, they share the same receptors, and their range of biological activities
overlaps, with many similar functions to IL-1. A second form of the TNF receptor
exists that binds to circulating TNF, and is shed after cleavage of the extracellular

87
TNF cell surface receptor. TNFa is produced by most cell types, including osteoblasts,
in response to a range of non-specific signals .TNFb is only induced by specific
antigens and has only been shown to be synthesized by activated T cells. In skeletal
tissues, TNFs stimulate bone and cartilage resorption and cell division . Since TNFa
induces neo-vascularisation in vivo, it may work with other local factors, including
FGF and TGFa to stimulate vascular invasion of the growth plate.
Interleukin 1 (IL-1)
IL-1 exists in two 17 kDa forms, alpha and beta, that have a similar spectrum of
biological activity but little sequence homology. IL-1 was originally isolated from
cells of the monocyte series but has subsequently been shown to be expressed by most
cell types, including human osteoblasts. The range of biological effects of IL-1 is
extensive, with activities previously attributed to leucocyte endogenous mediator
(LEM), mononuclear cell factor (MCF), osteoclast activating factor (OAF) and
catabolin now known to be those of IL-1 .The first cell surface receptor to be
identified for IL-1 was found to be a member of the immunoglobulin superfamily.
There is evidence that there may be a soluble form of IL-1 receptor. The most potent
inducer of IL-1 synthesis is endotoxin, but it is also induced by a number of other
cytokines and in an autocrine manner by IL-1. IL-1b stimulates bone, and increases
the proliferation of osteoblast cells and the production of other cytokines by
osteoblasts. IL-1 mRNA has been localised in the calcified cartilage zone of growth
plate, and together with BMP enhances ectopic bone formation , and cartilage

88
formation .Since IL-1 suppresses cell proliferation and proteoglycan synthesis in
chondrocytes, and decreases types II and IX collagen synthesis, it may suppress the
cartilage phenotype in the hypertrophic zone that precedes the onset of mineralisation
. Local synthesis of IL-1 and TNFa may also be important in the remodelling of
matrix in the metaphysis via stimulation of the synthesis of proteinase enzymes by
bone and cartilage cells.
IL-6 is a 23-28 kDa protein produced by many cell types including fibroblasts,
bone and cartilage cells as well as monocytes. Synthesis in osteoblastic cells is
stimulated by a range of factors including IL-1 and PTH. The considerable overlap in
the biological activities of IL-6 and IL-1 has led to the suggestion that IL-6 mediates
some of the actions of IL-1.. Direct effects have been demonstrated in osteosarcoma
cells, although it has not been shown to affect cell growth or differentiation in primary
cultures of human osteoblast.IL-6 may mediate some of the effects of oestrogen on
bone.
IL-8, or neutrophil activating factor (NAF), is an inflammatory mediator
produced by a wide variety of cell types. IL-8 is a potent attractant for neutrophils and
may have an important role to play in diseases such as rheumatoid arthritis and
osteoarthritis. Other members of the IL-8 supergene family may also have effects

Structure and Classification of Bone

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