Part 2- Basic dentistry for implants
Chapter 1 The histology of bone
Associate Professor of Maxillofacial Anatomy Section, Graduate School of Medical and
Dental Sciences, Tokyo Medical and Dental University
Tatsuo Terashima
The adult human body is composed of 206 bones that differ in size and shape, each with a structure tosuits its function. Adult bones can largely be divided into the axial skeleton and the appendicularskeleton. The axial skeleton is composed of 23 cranial bones, 6 auditory ossicles, 26 vertebrae, abreastbone and 24 ribs, and the appendicular skeleton consists of the 64 bones of the upper limb and the
62 bones of the lower limb (Fig. 2-1-1-a).
Bones are differentiated into long bones, short bones, flat bones and irregular bones according to theirstructural form. Other unique bones include the pneumatic bones such as the maxilla, which makes up
the maxillary sinus. The long bones constitute the majority of bones in the body as can be seen in both theupper and the lower limbs, and are comparatively long and thin (Fig. 2-1-1-d). They are hollow cylinders
surrounded by thick cortical bone, with a medullary cavity in the interior. The short bones form the smallbones of the hands and feet, the surfaces of which are covered by relatively thin cortical bone and theinteriors of which consist of cancellous bone (Fig. 2-1-1-e). Flat bone forms the calvarium and thebreastbone, which surfaces are covered by relatively thick cortical bone, with cancellous bone in theinterior (Fig. 2-1-1-c). The irregular bones have the complex form seen in the vertebrae and the hip bones,in which the ratio of cancellous to cortical bone differs among the bone types (Fig. 2-1-1-b).

Fig. 2-1-1-a,b,c,d,eThe

classification

of

bones

according

to

their

structural formsa. Human skeletonb. The irregular bone (the vertebra)c. The flat bone (the breastbone)d. The long bone (the thigh bone)e. The short bone (the tarsal bone)1

I. The function of osseous tissue
Osseous tissue makes up 18% of the body’s weight, produces hardness and strength, and functions in theform of a skeleton to provide overall support. It is also essential for organization of the following bodilyfunctions:1) Support: The skeletal structure supports the body, providing points for the attachment of the skeletal
muscles, and giving rise to the overall framework of the body.
2) Protection: Protects the internal organs, brain, spinal cord, heart and lungs.
3) Motor: Motor activities are the result of the collective action of bone and the skeletal muscles.
4) Homeostasis of calcium and phosphate: Osseous tissue stores minerals such as calcium and phosphate
as inorganic compounds, which make the bones strong. In addition, it maintains homeostatic equilibriumby releasing minerals into the blood and storing extra minerals, deposited as calcium compounds.
5) Hemopoiesis: Hematocytes are generated in the red marrow, a hematogenous tissue present in the
medullary cavity of the bone.
The osseous tissues are constructed of matrix that contains a high concentration of calcium salts, withosteocytes, osteoblasts and osteoclasts that are entrapped through their projections. The bones arecontinuously formed throughout the lifetime of the individual; even in adults several percent of the bone
is undergoing active remodeling, so a bone is completely replaced in roughly five years.

II. The structure of osseous tissue
The inner surface of the long bone, referred to as the diaphysis, is composed of compact bones that are
hard and thick. The epiphyses at both ends are cancellous (have spongy architecture). The diaphysis
encloses a hollow medullary cavity that communicates freely with the intratrabecular spaces. The bonemarrow, the site of hematopoiesis, also resides in this space. (Fig. 2-1-2 a,b,c).
The proximal and distal ends of bones are covered with articular cartilages that are made up of hyaline
cartilage, forming a joint. During development of the long bone, epiphyseal cartilages (the epiphyseal
plate) exist in between the diaphysis and epiphysis, and growth occurs in a longitudinal direction fromthis location. In fully-grown adults where growth has ceased, the epiphyseal cartilage becomes ossified
and completely replaced by bone, though remnants of the communication with the marrow cavity ofdiaphyses can be seen, referred to as the epiphyseal line (Table 2-1-3).
The external structure of the cortical bone is surrounded by a fibrous layer (the periosteum), and in its
interior resides the potential for osteogenesis. The periosteum consists of a rich supply of blood vessels
and nerve fibers that enter into the bone structure via Volkmann's canals. The periosteum and corticalbones interact intimately by means of Sharpey's fibers that are well-developed bundles of collagen fibers.
The periosteum of immature bone is thick, with a rich supply of blood vessels for efficient osteogenesis.
However, in elderly individuals, this structure is thin with a poor blood supply, and reduced osteogenic
ability. The interior surface of cortical bone is covered with the endosteum which consists of osteogenetic
cells as with the endosteal layer.
There are red hematopoetic, and yellow adipose types of marrow, depending on the age and site of themarrow. The former type is the active site of hematopoiesis, whereas the latter loses its hematopoietic
function and turns into fatty tissue. Hematopoiesis in the bone marrow starts at the 8th week ofembryonic development, and after birth, becomes the only site of hematopoiesis.
2

The main types of bone are classified into cortical bone (substantia compacta) (Fig. 2-1-2-c), andcancellous bone (trabeculae) (Fig. 2-1-2-b). The quantitative ratios of these two bony tissues are
dependent on the functions of the bone, and vary within the bone at different parts.
Cortical bone displays substantial hardness, is thickened particularly at sites of compression, andlamination is seen here in large mammals. Cancellous bone exists in the medullary cavity in its spongy
form (trabeculae), forming a lattice network that connects each component. The surface area covered bycancellous bone is extremely large. The trabeculae of cancellous bones are ordered in such a way that they
can resist external forces.
The intercellular matrix of bone is embedded with numerous collagenous fibers in the ground material.
The spaces between these fibers are filled with non-collagenous, organic and inorganic mineralsubstances that include hydroxyapatite crystals as the main constituent, as well as a small quantity ofcarbonic salts.

Fig.2-1-2-a,b,c

Fig.2-1-3

The structure of the long bone

The epiphysial line which is seen

CB: cortical bone (compact bone)during the growth period in the long

BMC: medullary cavity

bone (epiphysial cartilage is present in

SB: cancellous bone (sponge bone)an organism).

A. Bone tissue structure
A large pore known as the nutrient foramen exists close to the center of the diaphysis of the long bone, for
supply of nutrients to the osseous tissues. One or two main diaphyseal nutrient arteries enter the shaft
obliquely through this pore of the compact bone after which they divide into branches in theintramedullary cavity. The blood vessels that are arranged parallel to the major axis of the bone are
known as the Haversian canals, and the blood vessels arranged in a perpendicular direction to the
Haversian canals, and supply the bone surface (periosteum), are known as Volkmann's canals. Haversianand Volkmann's canals are interconnected, together with the blood vessels of the bone interior (bonemarrow). Therefore, it can be seen that there are two types of developed blood vessel systems that reside3

in the compact bones (Fig. 2-1-4-a, b).
The Haversian canal is a vessel with a diameter of 20 to 100 µm, which consists of blood capillaries or finearteries and veins in the center, and loose connective tissue in the surrounding area. The canals placed at
the center, the lamellae of the bones, are 5 µm thickness, and are arranged in a concentric manner similar
to annual rings of a tree, forming a Haversian system or osteon (bone unit) that is 200 µm in diameter(Fig. 2-1-5-a). The Haversian system is regarded as the fundamental functional unit of compact bone, andconsists of lacunae situated between lamellae. Osteocytes make contact with the cytoplasmic processes of
their counterparts via a network of small canals or canaliculi, and it is through this network that the cells
are oxygenated and nutrients are supplied from the Haversian canal (Fig. 2-1-5-b, Fig. 2-1-6-a).
Haversian canals run in the same direction as the blood vessels, and also branch and anastomose, or form
a cul-de-sac in a similar manner. In contrast, the Volkmann’s canal does not have a sheath of lamellae.
The boundary of a Haversian system or osteon is the cement line, and between adjoining osteons, angularintervals are present that are occupied by interstitial lamellae. An interstitial lamella is incomplete anddoes not contain a Haversian canal, the bone tissue of which is under continuous generation andabsorption; the remnants ofdestroyed osteons therefore exist as interstitial lamellae in between

newly

generated osteons (Fig.2-1-5-a).
No Haversian system is present in either the outermost or innermost (marrow) layers of compact bones.
Instead, these layers are covered by a few to ten parallel layers of lamellae, referred to as the external
and internal basic lamellae, respectively (Fig. 2-1-4-a). These structures are formed by the accumulationof osteoblasts on both the periosteal and endosteal layers. The collagenous fibrils inside both lamellae run
in parallel, though in the Haversian system the direction is arranged diagonally in relation to the majoraxis. The adjacent collagenous fibrils are orientated so that they cross each other at a 90º angle.
The fibrils that run parallel with the long axis contribute to the tensile and compressive strength of theosteon, whereas those that are horizontal contribute to elasticity.
The trabecular bones, in contrast, do not consist of blood vessels or Haversian systems, and therefore rely
on canalicular diffusion of oxygen and nutrients directly from the adjacent marrow. The lamellae areorientated towards the direction of the applied force, and are continuously formed and arranged by thecoordinated action of osteoclasts and osteoblasts in such way that the bone can withstand the applicationof external forces.

Fig.2-1-4-a,b
The ground section of the bonea. A number of Haversian
systems can be seen in the
ground longitudinal section of
the long bone.
b.

Haversian

canal

runs

longitudinally at the center ofthe Haversian system.

4

Fig.2-1-5-a.b

Fig.2-1-6-a,b

The structures of the Haversian system

The organized structure of the Haversian systema. A ground section featuring the lamella ofa. Bone corpuscle consists of the bone lacuna andbone in the Haversian system.

the bone canalicule (high-powered image of theb. Bone corpuscles exist between the lamellas

Schmorl’s stain).

of the bone (Schmorl’s stain)

b. Bony tissues exists inside of the bone corpuscle[high-powered image of the hematoxylin andeosin (HE) stain]

B. The cellular components of osseous tissues
1. Osteocytes
Osteocytes occupy a space called a lacuna which can be found distributed in bony tissue. Typically, thelacuna originates from the two layers of epiphyseal plates, however, it can also be seen within one layer(Fig. 2-1-6-b). Osteocytes form as the osteoblasts become embedded in the matrix that they have secreted,within the lacunae. Osteocytes have a structure that is characterized by numerous dendritic projections,and communicate with the adjacent cells or osteoblasts via gap junctions, forming a cellular network
within the osseous tissues. This network system allows efficient communication, transmitting
information from the osteoblasts on the bone surface, and relaying nutrients and oxygen into the depthsof the bone. Furthermore, this system also has an essential role in the rapid mobilization of calcium thathas been released from cellular osteolysis resulting from the coordinated actions of osteocytes andosteoblasts.
The morphological characteristics of osteocytes vary with their distance from the exterior of the bone, andthey are classified depending on their morphological properties into: 1) generative osteocytes, 2)
resorbable osteocytes, and 3) degenerative osteocytes.
The generative osteocytes are also known as juvenile osteocytes and are localized close to the surface ofthe bone where osteogenesis is at its most active; these cells have the ability to generate matrix. They
occupy a large volume within the lacunae, react to mechanical stimuli and transmit this information tothe osteoblasts and osteoclasts. The resorbable osteocytes, found in the depths of the bone matrices, aresmall, and have little ability to develop organelles. The degenerative osteocytes are found deeper in thebone matrices and thought to be the cells in which processes such as pyknosis, accumulation of lipid
droplets, vacuolization or clasmatosis occur, resulting in denaturation and necrosis of the cells.
52. Osteoblasts
These large cells, 20 to 30 µm in diameter, proliferate and differentiate to become osteocytes and arearranged on the surfaces of bone matrices. The greater part of the organic constituents of the bone matrix
is synthesized and secreted by the osteoblasts. In addition, these cells also play essential roles incalcification, formation of matrix vesicles, and induction of differentiation of osteoclasts. Osteoblasts
mature to become osteocytes, embedded in the matrix that they have secreted; a fraction of the cells
remain as quiescent osteoblasts — flattened epithelioid cells on the surface (Fig. 2-1-7-a,b).
The active osteoblasts that undergo osteogenesis often assume a cubic or ovoid shape, and the presence ofrough endoplasmic reticulum (rER), a Golgi apparatus and mitochondria can be observed. The rER of
osteoblasts that are undergoing active proliferation shows an expanded structure, and the Golgi complex
consists of the layers of the Golgi body, alongside the vesicles, and the Golgi vacuole, forming a developedarea of Golgi (Fig. 2-1-7-c,d).

Fig.2-1-7-a,b,c,d
The histology of osteoblasts (HE staining)a. Osteoblasts in their resting phase of mitosisb. Osteoblasts in their resting phase of mitosis(high powered image). It exists on the surfaceof the bone in a uniform layer, without thepresence of a large number of osteoids.
c. Osteoblasts in the developmental stages
d. Osteoblasts in the developmental stages(high powered image). Exists in either therectangular or egg shape, undergoing active
secretion, with the presence of developed
golgi apparatus. The osteoblasts exist incontact with the thick osteoids.
The bone matrix does not calcify immediately after it is secreted by osteoblasts, but exists as an
uncalcified structure called osteoid. The osteoblasts are in constant interaction with the osteoid, anddetermine the thickness of the layers of the osteoid depending on the activation state of the osteoblast.
During the active stages, it can be 5 to 10 µm thick, however, it can be scarce or non-existent in areas
adjacent to quiescent osteoblasts.
Osteoblasts synthesize and secrete organic matrix, i.e. type I collagen, or glycoproteins such as
osteocalcin, bone sialoproteins, osteopontin and osteonectin. The majority of the collagenous proteins andglycoproteins show affinity for calcium and are thought to control the calcification process. Osteoblastsalso secrete cytokines such as the IGF-I, IGF-II, TGF-β and BMP and store them in the matrix.
In the surrounding structures and the circulating blood near active osteocytes that are undergoing matrix
secretion, a raised concentration of alkaline phosphatase can be detected.
Matrix calcification occurs once matrix formation has finished. At the initial stages of calcification, matrix
vesicles of 0.03 to 1 µm in diameter surrounded by unit membrane can be detected in the extracellular6

matrix (ECM). It is in these vesicles that initiation of apatite crystal formation occurs. Matrix vesicles
that show enzymatic activity with the presence of alkaline phosphatase and ATPase, and have a highlipid concentration, bud off from the osteoblast cell surface into the newly formed osteoid. The alkaline
phosphatase acts to remove the pyrophosphoric acid that is an inhibitor of calcification, and increases thephosphoric acid concentration locally. The phospholipid contained in the vesicle, which shows strong
affinity for calcium, is highly adapted to concentrating the calcium in the vesicle as the source of
calcification. The apatite crystals grow, and eventually extend externally, and playing a role in the
induction of calcification of collagenous fibrils.
3. Osteoclasts
These are large (over 50 µm in diameter), polymorphic cells that consist of up to 100 different nuclei. The
presence of projections results in an irregular structure, and the osteoclast crawls around, the way a slug
crawls, causing resorption and thus modeling and remodeling bone. It is now known that the osteoclasts
are derived from fusion of the precursor forms of monocytes or macrophages. They can be found lying on
the bone surface inside resorption bays called Howship’s lacunae or found lying free, away from the bone
surface, or wandering with their pseudopodia extended. Their size and structure is extremely diverse (Fig.
2-1-8-a).
The cytoplasm of the osteoclasts is typically acidophilic and stains red with hematoxylin and eosin, with abrush-like structure adjacent to the bone (Fig. 2-1-8-1). Microscopically, the height of the cells adjacent tobone is often 1.5 to 2 µm, with complex invaginations on the plasma membrane that sometimes containcalcareous structures. A vast number of vesicles of various sizes fill the cytoplasm, alongside large
numbers of mitochondria, rER, and free ribosomes, as well as lysosomes that are involved in protein
degradation, and the Golgi apparatus located in the area surrounding the nucleus.
In terms of enzymatic histochemistry, osteoclasts display a strong tartrate-resistant acid phosphatase(TRAP) activity, which has been adopted as a marker of osteoclast activity (Fig. 2-1-8-c).

Fig.2-1-8-a,b,c
The histology of osteoclastsa. Osteoclasts with multiple nuclei exist in the resorption pit, present on the bone surface (HE staining).
b. The high-powered image of the osteoclast. Brush border exists next to the bone.
c. Osteoclasts that display high tartrate-resistant acid phosphatase (TRAP) activity

The osteoclast forms a belt-like isotropic band when they adhere to the cytoplasm, and it is through thisstructure that it interacts with bone. A ruffled border exists in the interior where the active bone7

resorption takes place.
Resorption by the osteoclast can be considered to occur by two different means: the absorption of theminerals that constitute the matrix, and the decomposition of the organic components. For mineraldissolution, an acidic condition is required in the resorption bay. Mechanistically, protons produced bycarbonic anhydrase (Type II) located in the cytoplasm are actively transported into the resorption bay by
proton ATPase (a proton pump) localized on the ruffled ridge of the plasma membrane. This maintains
strong acidity with a pH of around 3–4, necessary for the dissolution of hydroxyapatite (Fig. 2-1-9).
Dissolution of organic material is promoted by lysozymes, mainly cathepsin K and matrix
metalloprotease 9 (MMP-9), both of which digest Type I collagen, the main constituent of the bone matrix
in the resorption cavity.

Fig. 2-1-9

PumpchannelThe mechanism of the bone resorption by the

α‐channel

osteoclast(Taken from Biology of the bone - in Japanese).

Nuclei

Lysosome

Secretory granuleActin ring

Low pHenvironment

Bone matrix

Protease

III. Growth of bone
There are intramembranous and endochondral processes of ossification. Intramembranous ossification is
the formation of bony tissues within the primitive mesenchyme. This process is applied to therudimentary formation of cranial bones (except in some parts of the cranial base) and clavicles. In
endochondral ossification, small cartilages are formed, then osseous tissues grow and replace thecartilages absorbed and eliminated as they grow. This type of issification is seen with the bones of otherparts of the body, including the bones of the cranial base, the torso and the limbs.

A. Intramembranous ossification
The mesenchymal progenitor cells, which are in contact with each other through their projections,differentiate into osteoblasts upon invasion of blood vessels into the center of ossification. Osteogenesis isinitiated with the secretion of collagenous fibrils and bone matrix by the osteoblast in the proximal
regions of the bone. Calcification of the matrix occurs in association with the extracellular matrix vesicles,
and the embedded osteoblast is converted to an osteocyte. The cancellous bone that was formed first is
replaced by bone tissue that proliferates from the center out to the surroundings (Fig. 2-1-10-b). The point8

where ossification starts is referred to as the primary center of ossification, and the bones formed in thismanner are called membranous bones.

Fig. 2-1-10-a,b
The histology of the intramembranous ossification (HE staining)a. The initiation source of osteogenesis
The osteoblast differentiates in the undifferentiated mesenchymal tissue, calcification occurs in the bonematrix, and initiating the osteogenesis process.
b. The new bone matrix is formed around the formed bone substance and the bone tissues spread in suchmanner.

B. Endochondral ossification
Bone can sometimes be referred to as replacement bone as it is completed by the replacement of cartilage,
formed at the period of fetal development, by osseous tissue. Hyaline cartilage, which is covered by
perichondrium, forms a miniature template. The differentiation of osteoblasts close to the center of theenchondrium of the cartilage follows alongside the development of sheathing bone that surrounds thediaphysis (Fig. 2-1-11-a). Simultaneously, changes occur in the middle of the diaphysis (i.e. the primarycenter of ossification). Hypertrophy (substantial enlargement) of chondrocytes, compression of nuclei,
cytoplasmic vacuolation and calcification of cartilage matrix all occur. The blood vessels invade thecalcified cartilage from the periosteum via the connective tissue (Fig. 2-1-11-b). The chondroclasts andmacrophages that are transported from the invading blood vessels remove the chondrocytes and calcified
matrix, forming a large cavity — the primary bone marrow (primitive medullary cavity) (Fig. 2-1-11-c). At
the same time, cancellous bone is formed from the differentiated osteoblasts.
In the cartilaginous regions of the epiphysis, the differentiated and generated chondrocytes migrate
towards the diaphysis, and through longitudinal arrangement of the chondrocytes, cartilage columns areconstructed. Next, the chondrocytes situated on the diaphyseal side of this ordered arrangement
metamorphose, and matrix becomes calcified (Fig. 2-1-11-d). Chondroclasts and macrophages infiltrate
this area, remove the denatured chondrocytes and digest the matrix, and form a tunnel in the area
between the rows of chondrocytes. The sides of this tunnel are lined with osteoblasts that initiate theconstruction of skeletal tissue (Fig. 2-1-12-a). The columns of chondrocytes become denatured, are then9

resorbed, and gradually become replaced by osseous tissue. Newly differentiated chondrocytes arecontinuously added to the line of chondrocytes, but in order to achieve progressive longitudinal growth,
the epiphyseal plate migrates in a mesiodistal direction for effective longitudinal extension (Fig.2-1-12-b).
This process only ceases upon termination of growth of the bone (i.e. the person reaches their maximum
height); until then the cartilaginous column exists as the epiphyseal cartilage.
A center of ossification also becomes apparent in the epiphyseal region, and the cartilage in this area
becomes denatured and resorbed, at the same time becoming increasingly bony. The layers of articularcartilage involve in the formation of the articular cartilage.
Fig. 2-1-11-a,b,c,d
The histology of the enchondral ossification (HE stain)
a. The vaginate bone which surrounds the diaphysis of the cartilage is formed
close to the central part of the cartilage (arrow).
b. The cartilage at the center of the diaphysis becomes thicker, the calcification(★) of the cartilage occurs, and the blood vessel with connective tissue invades
the calcified cartilage (arrow).
c. The chondrocyte and the calcified cartilage matrix are absorbed and theprimitive bone marrow is formed in the diaphysis (arrow).
d. Longitudinal columnar (palisades) layer is formed in the cartilage situatedclosely to the epiphysis, followed by the cartilage becoming denatured on thediaphysis side, and the matrix becomes calcified (indicated by the arrow).
PB: the primitive bone marrow

Fig. 2-1-12-a,b
The osteogenesis at the epiphysial cartilage (HE stain)a. In the epiphysial cartilage (CL), the osteoclast becomes aligned on the surface of calcified cartilage
matrix that becomes absorbed, forming bone tissues (arrow).
BM: bone marrowb. The cartilage (CL) lined in columnar form becomes denatured and absorbed, and becomes rearranged
as the bone tissues.
SB: Bone marrow
10IV. Growth of the bone
A. Longitudinal growth
The longitudinal growth of the long bones is mediated by the epiphyseal cartilage. The chondrocytes thatmake up the epiphyseal chondrocyte columns exhibit characteristic longitudinal structures, sequentiallytermed the resting zone, the proliferative zone, the hypertrophic zone and the zone of calcified cartilage(Fig. 2-1-13-a).
The chondrocytes in the resting layer constitute a set of undifferentiated cells, with reduced ability todevelop organelles and a thin layer of intercellular matrix. The cells in the palisade layer are flattened
and are packed very closely, and often undergo cell division. The cellular organelles show gradualdevelopment, accompanied by accumulation of glycogen granules, and the appearance of mitochondrial
granules. The cartilaginous lacuna is filled with a proteoglycan-rich matrix that shows metachromasia,
and in the mediastinal matrix, both fibers and proteoglycan accumulate simultaneously. The cells in thehypertrophic zone gradually increase in size, with the development of cellular organelles. Although cell
division does not occur, the cells differentiate and swell. The cells in the zone of calcified cartilage are
replaced by means of enchondral ossification, initiated by calcification of matrix vesicles. The cancellous
bone that is formed by the addition of bone matrix by the osteoblast to the cartilaginous trabeculae iscalled “primary cancellous bone”. This bone forms the metaphysis (Fig. 2-1-13-b).
Remodeling replaces the primary cancellous bone with mature, secondary cancellous bone. In a manner
similar to longitudinal extension of bone, the diaphyseal side of the trabecula is resorbed by osteoclasts ina manner that is proportional to the speed of osteogenesis. As a consequence, the width of the cancellous
bone in the metaphysis is kept constant.

Fig. 2-1-13-a,b
The structure of the epiphysisal cartilage
a. Epiphyseal chondrocyte columns consist of layers of resting cells (RC), proliferating cells (PC),hypertrophic cells (HC) and calcified cells (CC).
b: In the layer of calcified cells, the calcification occurs to the cartilages and the blood capillary invasion(CP), and the primary cancellous bone is formed by the addition of the bone matrix (OS) by the osteoblast(CB) on the surface of the cartilaginous trabecula (CC).

B. Increase in the diameter
The diaphysis of the long bone increases in thickness due to subperiosteal ossification. Thickening of theperiosteal collar occurs at the center of the diaphysis, and the trabecular meshwork increases as woven
11

bone forms at the endosteal side. Once the diaphysis reaches a certain thickness, concurrent actions ofosteoclasts and osteoblasts define the thickness of the long bone. The coordinated actions ofosteclast-mediated resorption of the trabeculae in the endosteum, and addition of bone by osteoblasts
result in an increase in the volume of the bone marrow, retaining the distance between the exterior andthe endosteum as the width of the long bone increases.

V. Vascular and nervous supply of bone
A. Blood vessels
Two main vascular systems are distributed within bone. One of the systems arises when numerous thinarteries invade and occupy the Haversian canals from the arterial plexus of the periosteum throughVolkmann's canals. The other nourishes the bone marrow via nutrient foramina that enter the medullary
cavity, also supplying oxygen and nutrition to the trabeculae of the cancellous bone on the surface. The
thin vessels that feed parts of the compact bony interior are sourced from the inner medulla, passing via
the Volkmann’s canals (Fig. 2-1-14).
The artery that supplies the bone marrow of the epiphysis is an arterial branch that arises from thesystemic circulation in a manner that is independent of the vessels of the diaphysis and metaphysis, andis referred to as the epiphyseal artery. This artery pierces the periosteum of the bone where cartilage isabsent, and forms a capillary network. This network has no connections with those of metaphysis, andthe blood vessels are underdeveloped in comparison to those of the metaphysis.

Epiphyseal Fig.2-1-14
arteryThe vasculature of the bone
Metaphyseal (Taken from “Cell and Tissue Biology”）
arteryArterial plexusof periosteum
NutrientarteryB. Nerves
The abundant, predominantly medullated sensory nerves (some are non-medullated) are distributed in
the periosteum and the joint capsule, and participate in detection of pain and proprioception.

They also

penetrate the bone marrow through the nutrient foramina.

VI. Remodeling and modeling of bone
The skeleton of the human enlarges as growth and development occur. The bone tissues continuously
undergo processes of resorption and deposition throughout life, becoming replaced with new bony tissue.
12

These processes are referred to as modeling and remodeling, and describe the restructuring of the bone
due to the resorptive actions of osteoclasts, and deposition by osteoblasts. The restructuring of corticalbone and cancellous trabeculae that accompanies growth and development is called modeling, whereas
the restructuring that does not involve alterations in size or form is known as remodeling.
In remodeling, the woven bone that is primarily constructed is converted gradually into cortical bone byenlargement of the trabeculae, and deposition of bone within the perivascular cavities. This process ofosteogenesis that proceeds concentrically, with blood vessels at the center, forms the primary osteon. As
the cortical bone constructed by this process is resorbed by osteoclasts that differentiate in theperivascular regions, a secondary osteon forms.

Primary osteons thus become fully replaced bysecondary osteons; the osteons of cortical bones are continually replaced by this remodeling process. Thissame process is observed in the trabeculae of cancellous bone, and after osteoclasts absorb the bonematrix, bone is formed by osteoblasts. Through this remodeling mechanism, degenerating parts of bone
are renewed, permitting retention of the ability to withstand external forces applied to the body, as wellas preserving the ability to automatically regulate the calcium concentration in bone and blood. By using
these mechanisms to replace older parts with new tissues, bone provides the ability to adapt to newenvironments.
The details of how bony tissue remodels and bones develop, the factors involved in the remodeling process,including the osteoblast and osteoclast, as well as osteocyte cell division and the functional control of thisprocess are all described below.
A. Reconstruction of the Haversian system by remodeling
The renewal of compact bones is conducted by reconstruction of Haversian systems. The osteoclast
appears in the connective tissue of the Haversian canal, and causes bone resorption that is independentof the structure of the previous Haversian system, forming new cavities (Fig.2-1-15-a). These cavities arecharacteristically cylindrical, with the osteoclast at the front, and as bone is resorbed the cavity deepens
due to the position of the osteoclast. Osteoblasts differentiate on peripheral surfaces of the cavity wall,
and add further layers through osteogenesis, progressively narrowing the vascular channels (Fig.
2-1-15-b). This is how a new Haversian system forms, replacing the old, and restructuring the bone (Fig.
2-1-15-c). The remnants of old Haversian systems can be seen in the form of interstitial lamellae.

Fig.2-1-15-a,b,c
13

The reconstruction of the Haversian system by the remodeling of the bonea. Old osteon
The osteoclast appears in the Haversian system, and the bone resorption occurs independently of thestructure of the Haversian system and forming new cavity.
b. The start of the osteogenesis
The osteogenesis is initiated by the differentiation of the osteoblast on the surface of the resorption pit,
lamella is added, narrowing down the internal circumference.
c. The new osteon and the interstitial lamella
The new Haversian system is generated, and the bone reconstructed.
(Taken from “The science of new bones”).

The remodeling of the skeleton of a grown man occurs in the following order: activation, resorption,
reversal, formation and quiescence; it is however thought that the majority of cells on the surface(approximately 80%) usually exist in a quiescent state (Fig. 2-1-16).

Fig.2-1-16
The remodeling cycle
The process of the osteogenesis occurs in the order of the activation phase (I), the resorption phase (II),the reversal phase (III), the formation phase (IV) and quiescent phase (V).
(Taken from “Cell and Tissue Biology”)
The activation phase (I) begins when an osteoclast recognizes and adheres to the surface of the bonematrix. In this phase, the resting osteoblasts surround the surface of the bone matrix, calcification
reaches the bone surface, and osteoid can barely be seen. The bone surface becomes exposed in theactivation phase by degradation of the sparse collagen fibers that lie beneath a layer of quiescenceosteoblasts, as MMP-13 (matrix metalloproteinase 13) is secreted by these osteoblasts. The exposure of
the surface caused by removal of the degraded fibers promotes the adhesion of pre-osteoclasts to the bonematrix surface.
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In the resorption phase (II), once the pre-osteoclasts adhere to the bone surface, several pre-osteoclasts
fuse to form multinucleated osteoclasts that actively resorb bone.
In the reversal phase (III), upon termination of resorption, the osteoclasts retreat, and become replacedby mononuclear macrophages that absorb remaining components such as the matrix. Prior to a process
called coupling, the transition between the reversal and the formation phases, the mesenchymal stem
cells that occupy the pit differentiate into osteoblasts and proliferate.
In the formation phase (IV), the osteoblasts secrete bone matrix and form osteoid. The osteoid becomes
calcified after a lag of few days, and this process progresses with hydroxyapatite deposition. The interface
between the old and the newly formed bone is separated by the cement line. A small fraction of theosteoblasts becomes integrated into the bone matrix, while the majority remains on the surface as flat,
quiescent osteoblasts.
The quiescent phase (V) describes a reduction in the bone-forming activity of osteoblasts. A flat, quiescentlayer of osteoblasts forms, with bone formation ceasing as calcification spreads to the surface of the bone.

B. Differentiation of the osteoclast
It has been proved that the osteoclasts originate from hematopoietic cells of the monocyte-macrophagecell line. In studies that used myeloma cell cultures, active Vitamin D3 and PTH (parathyroid hormone)promoted the formation of osteoclast-like cells; calcitonin inhibits the formation process that is promoted
by Vitamin D3, and prostaglandin E2 does not promote formation of osteoclast-like cells. The
multinucleated cells derived in this study had numerous calcitonin receptors, showed strong
tartrate-resistant acid phosphatase activity, and had a very distinct ruffled border that formed resorption
pits in the underlying bone, confirmatory morphologic characteristics of osteoclasts.
Such osteoclast-like cells were found to occupy regions adjacent to alkaline phosphatase-positive cells; it
was therefore concluded that direct contact between osteoblasts and mesenchymal stem cells was
essential for the formation of osteoclast-like cells; these osteoblasts are thought to act as feeder cells by
producing inducers of osteoclast differentiation.
The inducing factors for osteoclast differentiation were identified by the following means. In a strain of
osteopetrotic mice with a mutation in the MSF-gene (op/op mouse), the observed lack of osteoclasts intheir bony tissues was hypothesized to be due to an abnormality in the process of osteoclastdifferentiation. When M-CSF was administered to op/op mice, generation of osteoclasts was induced, withsubsequent formation of a bone marrow cavity. This indicates that M-CSF plays a role in osteoclastdifferentiation.
In addition, from previous observations that confirmed the importance for osteoclast differentiation of
direct interaction between the osteoblast and mesenchymal stem cells, it was hypothesized that an
osteoclast differentiation factor (ODF) exists. An inhibitor of osteoclast differentiation, osteoprotegerin(OPG), was then discovered - this completely inhibited osteoclast differentiation, and was also an effective
inhibitor of physiological bone resorption.
Next a ligand for OPG called RANKL was identified, which acted as “ODF”. The specific interaction
between OPG and RANKL, inhibition of binding of RANKL to its receptor, RANK, resulted in inhibitionof osteoclast differentiation. Therefore, it is now believed that the RANKL - RANK interaction has an
essential role in the physiological differentiation of osteoclasts.
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Resorption of bone tissue by osteoclasts is conducted by secretion of acids and lysosomal enzymes into aspace sealed by an isotropic band. The factors that control osteoclast functions were found to include TypeII carbonic anhydrase, for acid production; and vacuolar ATPase, for acid secretion. Degradation ofproteins in the bone matrix is mediated by cathepsin K, which digests collagen fibers. Activation of
osteoclasts is induced by their adherence to bony tissue, and it is thought that integrins play animportant role in both recognition and subsequent adherence. Current research in this area aims to
uncover the intracellular signal transduction pathways, and the whole picture is becoming clearer.

C. Differentiation of the osteoblast
The osteoblast becomes flattened and the number of organelles becomes reduced once it has passed its
activation phase. The majority of these cells in direct contact with the calcified bone surface have reduced
ability to synthesize bone matrix. The extracellular matrix protein, osteopontin, localizes in some cellularborders. The quiescent cells secrete MMP-1 that digests and removes the collagen fibers in the space
beneath the bone surface, and also plays a role in conditioning the surface microenvironment of the bone,preceding osteoclast induction and adhesion. The critical importance of these cells in the differentiation
and activation of osteoclasts has thus become clear. Further, it is now known that osteoblasts in theresting phase are reactivated by various stimuli, and are converted into an activated form that readily
synthesizes bone matrix.
Osteoblasts and osteoclasts are in contact with each other through projections. It has been suggested that
their functions are synchronized and linked by gap junctions that play essential roles in intercellularsignal conduction from osteoblasts, and in bone formation. The adhesive components that mediate theinteraction between osteoblasts and bone matrix are thought to be collagen, fibronectin in the matrix, andintegrinβ-1 on the osteoblast. These types of cell-to-matrix adhesion have been indicated to be essential
for the signal transduction pathways, and the signal transduction mechanisms that are transmitted
through the cytoskeleton are essential for osteoblast differentiation and functional expression. Thesefindings have led to further clarification of cellular communication, broadening our understanding of thesignal transduction pathways.
The osteoblast, which plays a crucial role in the formation of bone, is derived from a common precursor ofseveral cells including chondrocytes, myoblasts and adipocytes. Osteoblast differentiation was thought tobe mediated by bone morphogenic proteins (BMP) and Wnt signals. However, recent findings haveindicated involvement of a transcription factor, Runx2, and that this is the critical factor required for thedifferentiation of osteoblasts. Furthermore, another transcription factor, osterix (Osx), has been identified
that induces both osteoblast differentiation and osteocalcin expression. Extensive investigations into thisprocess have found that osteoblasts express genes for type I collagen, alkaline phosphatase andosteocalcin. It has also been found that expression of these genes differs, depending on various phases ofdifferentiation that have been identified. Regarding the genes that control osteoblast differentiation,
there are however still unknown factors that are required for us to establish a complete picture.

References
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1)

Fujita T, Fujita H. Standard histology Generalities, Igaku-Shoin Ltd. (in Japanese)

2)

Ohno T, Kurosawa M, Takahashi K, Hosoya Y. Tortra Principles of anatomy and physiology. Maruzen

Co., Ltd. (in Japanese)
3) Cell and Tissue Biology, A Textbook of Histology. Leon Weiss U&S
4)

Suda T, Kurosawa M, Takahashi E, Tanaka S, Nakamura H, Mori S. Bone biology. Ishiyaku Pub, Inc.

(in Japanese)
5)

Noda M. Biology of the bone. Yodosha Co., Ltd. (in Japanese)17