Histology of Bone and

Bone Formation
Richard R. Shivers, Ph.D.
Professor Emeritus (Zoology)
University of Western Ontario,
London, Ontario Canada

Professor (Neurosurgery)
Shandong University
Jinan, P.R. China

Dr. Richard R. Shivers 2005

 Objectives
Discuss the concept of bone as a tissue
as contrasted to bone as an organ.
To compare intramembranous with
endochondral bone development.
Discuss details of the structure and
functions of osteoclasts, osteoblasts,
and osteocytes in bone formation,
maintenance and resorption.

Bone and Bone Formation 2

 Objectives
Outline the correct chronological sequence
of cellular events in bone repair.
Compare the following: periosteum vs
perichondrium; bone growth in width vs
bone growth in length; interstitial vs
appositional growth; bone matrix vs
cartilage matrix.
Outline the ultrastructural features of bone
and its cells. Compare bone ultrastructure
with cartilage fine structure.
Bone and Bone Formation 3
 Bone-General
Connective tissue with mineralized matrix
Support and protection
Mineralized Matrix
Serves as storage site for calcium &
phosphate
calcium phosphate in form of
hydroxyapatite crystals
consists of Type I collagen
and ground substance
with proteoglycans,
non-collagenous glycoproteins

Bone and Bone Formation 4

 Bone-General cont
Matrix contains spaces called
Lacunae;
each houses an OSTEOCYTE
Osteocyte sends numerous processes
into little canals called CANALICULI
canaliculi run through mineralized matrix
connect adjacent lacunae
allow contact between cell processes of
neighboring osteocytes
Processes of osteocytes communicate
via gap junctions
Bone and Bone Formation 5
 Bone-General cont
Four types of cells in bone:
Osteoprogenitor cells-
give rise to osteoblast
Osteoblasts-
secrete extracellular matrix of bone
Osteocyte
once blasts are surrounded by bone matrix
they become osteocytes
Osteoclasts
bone resorbing cells located on bone surface

Bone and Bone Formation 6

 Bone Tissue
Consists of bone matrix & cells
Other connective tissues
including:
hemopoietic tissue
Hyaline cartilage
Fat tissue
Blood vessels
Nerves

Bone and Bone Formation 7

 Bone Tissue-
Classification
compact (dense)
compact found in
outer region
spongy
(cancellous)
spongy situated
on the inside
forming meshwork
Meshwork filled
with bone marrow
and blood vessels

Bone and Bone Formation 8

Bone and Bone Formation 9
Bone and Bone Formation 10
Bone and Bone Formation 11
 Bone Tissue-Classification
cont
Shaft is almost all dense bone
Only small amount of spongy
bone faces medullary cavity
At epiphysis the reverse is true:
made up mostly of spongy bone
with a thin dense outer layer

Bone and Bone Formation 12

Bone and Bone Formation 13
Covered with
periosteum;
sheath of dense
connective tissue
contains
osteoprogenitor
cells
Outer fibrous
layer
collagen fibers
arranged parallel
to surface
Inner cellular
layer-
with progenitor
cells
Inner layer in
inactive bone
called periosteal
 Where ligaments and
tendons attach:
collagen fibers extend
directly at an angle into
bone tissue,
where they are
continuous with bone
collagen fibers.
Fibers called SHARPEYS
FIBERS
Articular Surfaces
Contacts of bone
covered with hyaline
cartilage
ARTICULAR CARTILAGE
this cartilage not covered
with PERICHONDRIUM

Bone and Bone Formation 15

Bone and Bone Formation 16
 Bone Cavities
Lined by ENDOSTEUM
layer of connective tissue
cells with
OSTEOPROGENITOR cells
Found in:
long bones
and trabeculae of spongy
bone
one cell thick
flattened cells
look like fibroblasts
can differentiate into
osteoblasts
called ENDOSTEAL CELLS

Bone and Bone Formation 17

 Adult Bone-Structure
Composed of structural
units called
HAVERSIAN SYSTEMS
(OSTEONS)
cylindrical units of
compact bone
consist of concentric
lamellae of matrix
around a central canal;
the osteonal canal or
HAVERSIAN CANAL
contains vascular and
nerve supply of the
osteon

Bone and Bone Formation 18

Bone and Bone Formation 19
Bone and Bone Formation 20
Bone and Bone Formation 21
Bone and Bone Formation 22
 CANALICULI
with osteocyte
processes
arranged radially
with respect to
Haversian canal
canaliculi system
opens into
Haversian canal:
serves for
passage of
substances
between
osteocytes and
blood vessels
Bone and Bone Formation 23
 INTERSTITIAL
LAMELLAE
remnants of
previous osteonal
lamellae
located between
osteons
This form of
bone called
LAMELLAR BONE

Bone and Bone Formation 24

Bone and Bone Formation 25
 Long axis of osteon
parallel to long axis
Collagen fibers in
concentric lamellae in
an osteon are laid
down parallel to each
other in any one
lamella
but are in different
directions in adjacent
lamellae
Cut surface resembles
plywood
gives great strength to
bone
Lamellar bone also
found at sites other
than osteons
Bone and Bone Formation 26
 Lamellar Bone
Lamellar bone
compared to
WOVEN BONE
in which collagen
fibers and bundles
are randomly
oriented and loosely
intertwined
Spongy bone may
be either woven or
lamellar
Compact bone is
almost entirely
lamellar

Bone and Bone Formation 27

Bone and Bone Formation 28
 VOLKMANNS
CANALS
(perforating
canals);
channels in lamellar
bone through which
blood vessels and
nerve travel
from periosteal and
endosteal surfaces to
reach the Haversian
Canal
VOLKMANNS
CANALS CONNECT
HAVERSIAN CANALS
TO EACH OTHER
Bone and Bone Formation 29
 Volkmann
Canals
Run at right angle
to Haversian canal
NOT surrounded
by concentric
lamellae of bone
like Haversian
canal

Bone and Bone Formation 31

 Adult Spongy Bone

Very similar to adult

compact bone except
tissue is arranged as
TRABECULAE or
SPICULES
with numerous
interconnecting marrow
spaces of various sizes.
If trabeculae are
sufficiently thick,
they will contain
osteons
The matrix of the bone
is lamellated
Bone and Bone Formation 32
Bone and Bone Formation 33
Adult Bone-Blood Supply
Chiefly from arteries entering
marrow cavity through nutrient
foramina
Greatest # of nutrient foramina are
in epiphyses and diaphyses
Drainage of bone is via veins
leaving through nutrient foramina
or through bone tissue of shaft
and out through periosteum

Bone and Bone Formation 34

Adult Bone-Blood Supply
cont
Blood nourishing bone tissue moves from:
marrow cavity into and through bone tissue
and out via periosteal veins
For nourishment of bone itself,
Volkmanns canals provide major route of entry
into compact bone
Smaller vessels enter Haversian canals
where a single arteriole and venule may be
located
Smaller supply from periosteal vessels
Bone lacks lymphatics

Bone and Bone Formation 35

 Mature and Immature
Bone
Display patterns of lamellar bone
both compact and spongy
Immature bone in fetal skeleton differs
from adult in:
immature bone has no organized lamellated
appearance (NONLAMELLAR)
nonlamellar also called WOVEN or BUNDLE
BONE due to collagen fiber interlacing
Contains more cells per unit area than adult
Cells tend to be randomly arranged
whereas in adult their long axis parallels lamellae

Bone and Bone Formation 36

 Cells of Bone
4 cell types:
Osteoprogenitor cells
Osteoblasts
Osteocytes
Osteoclasts
Except for osteoclasts, the other
cells are differentiated forms of
same basic cell type

Bone and Bone Formation 37

 Osteoprogenitor Cell

Resting cell that

can transform into
osteoblast and
secrete bone
matrix
Found on external
and internal
surfaces of bone

Bone and Bone Formation 38

 Osteoprogenitor Cell
Comprised of:
periosteal cells and
endosteal cells
which line :
marrow cavities,
Haversian Canals
Volkmanns
canals
Can divide &
proliferate

Bone and Bone Formation 39

 In mature bone
surfaces covered
by flat cells
called BONE
LINING CELLS
little cytoplasm
form incomplete
lining over bone;
when in contact
have gap
junctions
May provide
nutrition to
nearby bone cells
Bone and Bone Formation 40
 Bone Cells-
Osteoprogenitor cell
Derived
cont
from mesenchyme
Can differentiate into
adipose cells
chondroblasts
fibroblasts
Thus play important role in:
bone repair
formation of new connective tissue
and cartilage
Bone and Bone Formation 41
 Bone Cells-Osteoblast

Resembles
fibroblast and
chondroblast
Recognized as
cuboidal or
polygonal cells
organized into
single layer next to
forming bone

Bone and Bone Formation 42

 Basophilic
cytoplasm
large Golgi
osteoblasts
connected via gap
junctions
can divide

Bone and Bone Formation 43

 OSTEOBLAST
Calcification process
initiated by
osteoblastic
secretion into matrix
of:
membrane-limited
MATRIX VESICLES
small 50-250nm
Rich in alkaline
phosphatase

Bone and Bone Formation 44

 OSTEOCYTE

Mature bone cell

surrounded by matrix
is a differentiated
osteoblast
can make matrix and
reabsorb it
Occupies a space-
LACUNA
Nucleus is often only
structure seen
often shrunken due
to preparation

Bone and Bone Formation 45

 Extend
cytoplasmic
processes
through fine
tunnels or
CANALICULI in
matrix
to contact
processes of
other osteocytes
via gap junctions,
Canaliculi not
seen in H&E
evident in
ground
preparations
Bone and Bone Formation 46
 Osteocytes

Homeostasis of blood calcium

Death of osteocyte results in bone
resorption by osteoclasts
followed by repair or remodelling by
osteoblasts
EM shows variation in functional state of
osteocytes
Osteocytes can modify surrounding matrix
through syntheses and absorption

Bone and Bone Formation 48

 Bone Cells-
Large multinucleated cell
function to resorb bone Osteoclast
rest directly on bone
surface
create cavity beneath
osteoclast
called HOWSHIPS LACUNA
cell is conspicuous due
to:
size
marked acidophilia
strong histochemical
reaction for acid
phosphatase

Bone and Bone Formation 50

 Portion of cell directly
in contact with bone
has 2 parts:
CENTRAL REGION
with many plasma
membrane infoldings
microvillous-like
structures
called RUFFLED
BORDER
EM shows hydroxy
apatite crystals in
ruffled border
processes

Bone and Bone Formation 51

 CLEAR ZONE
ring-like perimeter
of cytoplasm-
demarcates limits
of bone being
resorbed
Abundant
microfilaments

Bone and Bone Formation 52

 lysosomes arise
from Golgi of
osteoclast
released into clefts
of extracellular
space
between ruffled
border processes
Example of
lysosomal
hydrolases
functioning
outside the cell

Bone and Bone Formation 54

 Parathormone has
dramatic effect on
osteoclast activity
Calcitonin
from parafollicular
cells of thyroid
counterbalances
parathormone
Osteoclasts arise
from monocytes
Closely related to
other macrophages

Bone and Bone Formation 55

 Bone Formation
ENDOCHONDRALor
INTRAMEMBRANOUS
Distinction rests on whether a
cartilage model serves as a
precursor to bone
or whether bone is formed by a
simpler method
Terms refer ONLY to mechanism by
which a bone is initially formed

Bone and Bone Formation 56

 Bone Formation
Endochondral Ossification
Bones of extremities
those parts of the axial skeleton
that bear weight (ie vertebrae)
continuedgrowth involves
formation of endochondral bone
and histogenesis of
intramembranous bone

Bone and Bone Formation 57

 Bone Formation cont
intramembrane ossification
flat bones of skull and face
the mandible and clavicle
Due to remodeling, initial bone is
replaced.
Replacement is established on
preexisting bone by appositional
growth and is identical in both
cases

Bone and Bone Formation 58

 Bone formed by
Intramembranou
differentiation of s Ossification
mesenchymal cells
into osteoblasts
Begins at 8th week
of gestation
Mesenchymal cells
migrate to site
where bone is to
be formed
aggregate to
form a blastema
this is
intramembranou
s bone formation
Mesenchyme
changes into
osteoblasts
Bone and Bone Formation 59
Bone and Bone Formation 60
 Newly formed bone
matrix
appears as small,
irregularly shaped
spicules and
trabeculae
As calcification
proceeds
cytoplasmic
processes of
osteocytes are
surounded by
matrix
and are thus
within canaliculi

Bone and Bone Formation 61

 More cells of
periosteum
proliferate
become
progenitor cells
Progenitor cells
in contact with
spicules
become
osteoblasts;
add matrix
etc

Bone and Bone Formation 62

Bone and Bone Formation 63
Bone and Bone Formation 64
 APPOSITIONAL
GROWTH:
Spicules enlarge
and are joined into
trabeculae
progenitor cells
maintain population
of bone forming
cells
by mitosis
New osteoblasts lay
down matrix in
successive layers
Called WOVEN BONE
Spaces occupied by:
marrow
& vessels
Bone and Bone Formation 65
 Begins with
proliferation and
Endochondral
aggregation of
mesenchyme cells
Ossification
these differentiate
into chondroblasts
Produce cartilage
matrix
Hyaline cartilage
produced acquires
general shape of
bone that will be
formed
Cartilage model of
bone
Grows by
interstitial and
appositional growth

Bone and Bone Formation 66

Bone and Bone Formation 67
Bone and Bone Formation 68
 Increase in length
interstitial growth
Increase in width
large by addition of
cartilage matrix
from new
chondrocytes
differentiated from
chondrogenic layer
of perichondrium
appositional growth
First sign of
ossification
appearance of cuff of
bone around model

Bone and Bone Formation 69

 For cuff,
perichondrium stops
making chondroblasts
and makes
osteoblasts
Now called
periosteum
inner layer makes
osteoblasts
Thin layer of bone
formed around
cartilage model
called PERIOSTEAL
BONE
as opposed to
membrane bone
Name due to method
of development

Bone and Bone Formation 70

Bone and Bone Formation 71
Bone and Bone Formation 72
 After collar
established
chondrocytes
become hypertrophic
As chondrocytes
enlarge
matrix is
compressed
forming thin plates
between
chondrocytes
Surrounding matrix
calcifies
not to be confused
with bone
calcification

Bone and Bone Formation 73

 Calcified cartilage
matrix inhibits
diffusion of nutrients
causes death of
chondrocytes in model
When chondrocytes die
matrix breaks down,
neighboring lacunae
become confluent to
produce a large cavity
One or several blood
vessels grow through
thin diaphyseal bony
collar to vascularize
the cavity

Bone and Bone Formation 76

 Endochondral Ossification
cont
Periosteal cells migrate into cavity
along with blood vessels
Periosteal cells become progenitor cells
Other primitive cells leave circulation to
become marrow
Some calcified cartilage remains as
spicules
Osterprogenitor cells contact cartilage
spicules
they become OSTEOBLASTS

Bone and Bone Formation 77

 Osteoblasts begin to
lay down bone
(osteoid) on spicule
framework
Bone formed in this
manner is
ENDOCHONDRAL bone
Combined cartilage and
bone spicules called
MIXED SPICULES
Recognized by staining
characteristics:
calcified cartilage is
basophilic,
bone is eosinophilic;
remains until cartilage is
resorbed

Bone and Bone Formation 78

Bone and Bone Formation 80
 Growth of
Begins in second
trimester
Endochondral
12th week of gestation Bone
til early adult
Growth of long
bones depends on
presence of
EPIPHYSEAL
CARTILAGE
throughout growth
period
Diaphyseal marrow
cavity enlarges,
distinct zonation seen
at either end of cavity

Bone and Bone Formation 81

 Zones of epiphyseal
cartilage beginning
most distally are:
ZONE OF RESERVE
CARTILAGE:
no cellular proliferation
or active matrix
production
ZONE OF
PROLIFERATION:
cartilage cells undergo
division
and are organized in
distinct columns.
Cells are larger than in
reserve zone
actively making matrix
Bone and Bone Formation 84
 ZONE OF
HYPERTROPHY:
greatly enlarged
cells
clear cytoplasm
due to
accumulated
glycogen
matrix
compressed into
bands between
columns of
chondrocytes
Bone and Bone Formation 85
Bone and Bone Formation 86
Bone and Bone Formation 87
 ZONE OF CALCIFIED
CARTILAGE:
enlarged cells begin to
degenerate
matrix calcifies
ZONE OF RESORPTION:
zone nearest diaphysis.
cartilage in contact with
connective tissue of
marrow cavity.
Small blood vessels and
connective tissue invade
region occupied by dying
chondrocytes
Form spearheads leaving
calcified cartilage as
longitudinal spicules

Bone and Bone Formation 88

Bone and Bone Formation 89
Bone and Bone Formation 90
 In XS cartilage is
honeycomb
invading vessels etc
Bone deposition occurs
on cartilage spicules
same as for ossification
centers
Cartilage is resorbed
leaving spongy bone.
spongy bone reorganizes
through:
osteoclastic activity
and addition of new bone
tissue
After birth, secondary
ossification center
occurs in upper
epiphysis.

Bone and Bone Formation 91

Bone and Bone Formation 92
 cartilage cells
hypertrophy and
degenerate
calcification of
matrix occurs
blood vessels and
periosteum invade
create a new
marrow cavity.
similar ossification
center (also a
secondary center)
develops at other
end of bone

Bone and Bone Formation 93

 Only cartilage
remaining from
original model is
articular cartilage
end ends of bone
and a transverse disc-
the EPIPHYSEAL PLATE
separates
epiphyseal and
diaphyseal cavities
cartilage of epiphyseal
plate responsible for
maintaining growth
To keep shape,
external and internal
remodeling must occur
as bone grows in
length.

Bone and Bone Formation 94

 Proliferative zone of
cartilage plate
makes cartilage on
which bone is later
laid down
Thickness of
epiphyseal plate
is constant during
growth
Amt of new cartilage
= amt of resorbed
Resorbed cartilage is
replaced by spongy
bone

Bone and Bone Formation 95

 Actual lengthening
of bone occurs:
when new cartilage
matrix is produced
at epiphyseal plate
pushes epiphysis
away from diaphysis
Increase in width
(diameter) occurs :
when appositional
growth of new bone
occurs between
cortical lamellae
and periosteum

Bone and Bone Formation 96

Bone and Bone Formation 97
 Bone Remodeling
Consistsof preferential
resorption of bone in some areas
and deposition in other areas

Bone and Bone Formation 98

Bone and Bone Formation 99
 Cessation of Bone
Growth
When maximal growth is
achieved:
proliferation of new cartilage
ceases
cartilage is finally replaced by bone
epiphyseal and diaphyseal cavities
become one
Epiphyseal cavity closes
epiphyseal plate is removed
Bone and Bone Formation 100
 Development of the
Haversian System
Osteons develop in preexisting
compact bone originating from:
A) fetal spongy bone
B) adult compact bone
C) may be older compact bone
consisting of osteons and
interstitial lamellae

Bone and Bone Formation 101

 BONE REMODELLING
UNIT:
osteoclast resorption
and osteoblast
synthesis
tunnel is bored
through compact bone
by osteoclasts
blood vessels and
surrounding connective
tissue occupy the tunnel
new bone deposition
immediately begins on
tunnel wall

Bone and Bone Formation 102

 Osteoclasts cut canal
200 um dia
diameter of future
Haversian system
(osteon)
Osteoblasts deposit
matrix on walls of of
canal in successive
lamellae
as each lamella is
deposited from
periphery inward
canal achieves small
diameter of adult
osteonal canal
Process where new
osteons are formed
called INTERNAL
REMODELING

Bone and Bone Formation 103

 Compact Adult Bone
Haversian Systems
Haversian systems of varying age
and size
Younger Haversian systems
not completely mineralized
Dynamic internal remodeling of
bone:
absorption balances deposition
in aged, resorption often exceeds
deposition
=OSTEOPOROSIS

Bone and Bone Formation 104

 Mineralization
Mineralization is an extracellular
event
Occurs in extracellular matrices of
bone, cartilage, dentin, cementum and
enamel
All except enamel contain collagen
fibrils,and matrix
calcification occurs within and around
collagen
Biologic
mineralization is a cell-
regulated event
Bone and Bone Formation 105
 Bone Repair
CALLUS:
dense connective
tissue and newly
formed cartilage
covers bone at
site of fracture
callus always
forms
helps stabilize
and bind
fractured bones

Bone and Bone Formation 106

 Formation of new
bone progresses to
fracture site until:
new bone forms a
sheath over the
FIBROCARTILAGENOUS
CALLUS
cartilagenous callus
replaced by bone;
BONY CALLUS
spongy bone
develops and is
replaced by
compact bone

Bone and Bone Formation 107

 Acknowledgements
Color Atlas of Histology, Leslie P. Gartner &
James L. Hiatt - 3rd ed. 2000, Lippincott,
Williams & Wilkins
Basic Histology, Luiz Junqueira & Jose
Carneiro 10th ed. 2003, Lange Medical
Books McGraw-Hill
Freeze-Etch Histology, Lelio Orci & Allain
Perrelet 1975, Springer-Verlag Berlin
Heidelberg
Fine Structure of Cells and Tissues, Keith R.
Porter & Mary A. Bonneville 4th ed.
1973,Lea & Febiger
Bone and Bone Formation 108
 Glossary
acidophilia bone calcification
acidophilic bone deposition
aggregation bone remodeling
alkaline unit
phosphatase border process
appositional growth bundle bone
articular cartilage calcification
axial skeleton calcitonin
blastema callus

Bone and Bone Formation 109

 Glossary cont.
calvarium cleft
canaliculi collagen fiber
cancellous collar of bone
cell-regulated column of cells
event compact bone
cementum compression of
chondroblasts matrix
chondrocyte concentric
lamellae
clavicle
confluent
Bone and Bone Formation 110
 Glossary cont.
conspicuous dentin
cortical lamellae diaphyseal
counterbalance marrow cavity
cuff
diaphysis
cylindrical
differentition
cytoplasmic
diffusion
process enamel
demarcate endochondral
dense bone
endosteal surface

Bone and Bone Formation 111

 Glossary cont.
endosteum flared
eosin gap junction
epiphiyseal plate gestation
epiphyseal cartilage ground bone
exocytosis ground substance
extracellular matrix Haversian canal
fibroblasts Haversian system
fibrocartilage hematoxylin

Bone and Bone Formation 112

 Glossary cont.
hemopoietic hydroxyapatite
tissue crystals
histochemical hypertrophic
reaction inhibition
histiocytes interlacing
histogenesis collagen
internal
homeostasis remodeling
Howships lacuna interstitial
hyaline cartilage lamellae
hydrolase intramembranous

Bone and Bone Formation 113

 Glossary cont.
lacuna macrophage
lamella mandible
lamellar bone marrow cavity
Langhans cell matrix
ligament
matrix
calcification
long axis matrix vesicle
long bone medullary cavity
lysosome mesenchymal cells
mesenchyme
meshwork

Bone and Bone Formation 114

 Glossary cont.
metacarpal odontoblast
metaphysis organic
micron component
mixed spicules orthodontic
monocyte correction
morphology ossification
neutrophils osteoblast
nm-nanometer osteoccytic
nutrient foramina osteocytes

Bone and Bone Formation 115

 Glossary cont.
osteoclast perforating
osteocyte pericellular space
osteoid perichondrium
osteon perimeter
osteoporosis periosteal bone
osteoprogenitor cell periosteal cells
parafollicular cells periosteum
parathormone phagocytic
PAS+ granules

Bone and Bone Formation 116

 Glossary cont.
plasmalemma quiescence
plate-like radial
plywood randomly
polygonal oriented
preferential red bone marrow
resorption remodeling
progenitor reserve cartilage
proliferation resorption
proteoglycans resorptive

Bone and Bone Formation 117

 Glossary cont.
osteocytes skull cap
reticular cell spicule
spongy bone
ruffled border
sternum
secondary center successive lamellae
for bone tendon
formation tibia
Sharpeys fibers
single layer nest

Bone and Bone Formation 118

 Glossary cont.
trabeculae
vesicle
Volkmanns canal
woven bone
yellow bone
marrow
zonation

Bone and Bone Formation 119