ANATOMY HISTOLOGY:

STRUCTURES
OF BONE AND CARTILAGE

Wong Kah Hui

wkahhui@um.edu.my
Department of Anatomy,
Faculty of Medicine,
Universiti Malaya
Learning Outcomes

Students must be able to describe:

1. Types of cartilage – hyaline cartilage, elastic carilage and fibrocartilage

2. Bone matrix composition – collagen, non-collagenous protein, mineral
3. Bone structure – bone terminology
4. Bone cells – osteoprogenitor cells, bone-lining cells, osteoblasts, osteocytes, osteoclasts
5. Bone turnover / bone remodelling – roles of osteoblasts and osteoclasts in resorption,
formation, coupling
6. Bone classification – compact vs spongy bones, and immature vs mature bones
 CARTILAGE
Develops from mesenchymal cells.
Consists of Cells + Extracellular matrix

Extracellular matrix = connective tissue fibres + ground substance + high H2O content
Collagen fibres / Proteoglycans
Elastic fibres Glycoproteins

Characteristic:

• Avascular
• Receives nutrients and elimates metabolic waste by diffusion through extracellular matrix (highly hydrated)
• Cells – chondrocytes and chondroblasts – synthesise extracellular matrix
• Surrounded by perichondrium (dense irregular CT) – type I collagen fibres (except articular cartilage and
fibrocartilage – X perichondrium)
• Provides firm structural support for soft tissues
• Semirigid tissue and act as shock absorber
• Allows flexibility without distortion
• Resilient (elastic, flexible) to compression (highly hydrated ECM)
• Exhibits tensile strength (ultimate strength) → Fibrocartilage
Mesenchyme

Embryonic mesenchyme differentiates into various connective tissues of the body, muscle, vascular and
urogenital systems, serous membranes of body cavities.

Mesenchymal tissue from a developing fetus.

Cytoplasmic processes give the cell a tapering or spindle
appearance. Extracellular component contains a sparse
arrangement of reticular fibres and abundant ground
substance.
Three types of cartilage
(based on amount and types of connective tissue fibres in extracellular matrix):

i. Hyaline cartilage
ii. Elastic cartilage
iii. Fibrocartilage

Hyaline cartilage

• Most common in the body.

• Contains type II collagen fibres in the matrix.
• Provides a model for the developing skeleton of the fetus.
In early fetal development, hyaline cartilage is the precursor of bones that develop by endochondral
ossification.
• In developing bones of young individuals, hyaline cartilage present in epiphyseal plates for bones to grow
in length.
As the individual grows, the cartilage model is gradually replaced with bone by a process called
endochondral ossification.
• In adults, most of the hyaline cartilage model is replaced with bone, except on the articular surfaces of
bones, costal cartilages of rib cage, cartilages of external nose and septal cartilage of nasal cavity, larynx,
rings of trachea and plates in bronchi.
Proximal end of a growing long bone. Ephiphyseal growth plate (a disc of
hyaline cartilage) separates the epiphysis from the funnel-shaped diaphysis.
Articular cartilage on the surface of epiphysis contributes to the synovial joint
and is composed of hyaline cartilage. The cartilage of ephiphyseal plate
disappears when length-wise growth of the bone is completed, but the articular
cartilage remains throughout the life. The spaces within the bone are occupied
by marrow.
 Hyaline cartilage and surrounding structures: Trachea, H&E
• Perichondrium surrounds the cartilage:
(i) Inner cellular (chondrogenic) layer that give rise to new cartilage cells.
(ii) Outer fibrous layer
• In the middle of the cartilage plate – cells are large and spherical.
Chondrocytes in lacunae appear singly / in isogenous groups,
• Toward the periphery – cells are flattened = differentiating chondroblasts.
• Interterritorial matrix (matrix between chondrocytes) stains lighter.
• Territorial matrix around the lacunae stains darker.

Outer layer

Watery secretions
Lubricating mucous
Delivers secretions to tracheal lumen.
 Cells and matrix of mature hyaline cartilage, H&E

• Matrix shows ovoid spaces called lacunae with mature cartilage cells (chondrocytes).
• Chondrocytes fill the lacunae, are seen either singly/in isogenous groups.
• Each chondrocyte has a granular cytoplasm and a nucleus.
• During histologic preparation, chondrocytes shrink and the lacunae appear as clear spaces.
• Matrix is homogenous and basophilic.

More basophilic, darker than

the interterritorial matrix,
adjacent to chondrocytes

Matrix between chondrocytes,

lighter than the territorial matrix
 Hyaline cartilage: Developing bone, H&E

• A section through a developing bone shows a portion of the hyaline cartilage and its homogenous matrix.
• Located within the matrix – mature hyaline cartilage cells (chondrocytes) in their lacunae.
• Perichondrium surrounds the hyaline cartilage – outer fibrous layer and inner chondrogenic layer
• Chondrogenic layer is on the inner surface of the perichondrium.
• Central cells in the cartilage appear as rounded chondrocytes.
• Peripheral cells are more flattened and appear as chondroblasts.

*
*

Dense irregular
connective tissue * Chondrocyte in lacuna
* Chondroblast
Perichondrium
 Elastic cartilage

• Contains mainly branching elastic fibres within its matrix in addition to

type II collagen fibres.
• Provides flexible support.
• Pinna of external ear, external acoustic meatus, auditory/Eustachian tube
and cartilages of larynx (epiglottis, corniculate and cuneiform cartilages).

Auditory
tube

Parasagittal section of left

side of head and neck
showing location of
respiratory structures
• Numerous elastic fibres (protein elastin) in the cartilage matrix – provide great flexibility and support.
Elastic fibres are distributed as branching and anastomosing fibres of various sizes.
• Density of elastic fibres varies among elastic cartilages and among different areas of the same cartilage.
• Larger chondrocytes in lacunae in the interior of the cartilage plate.
• The smaller and flatter chondrocytes are located peripherally in the inner chondrogenic layer of perichondrium =
differentiating chondroblasts.
• Perichondrium – a layer of dense irregular connective tissue surrounds the cartilage (fibroblasts and small blood
vessels).
Elastic cartilage: Epiglottis, H&E/Verhoeff

*
*

* * *
* *
* * * *
* Chondrocyte in lacuna
* Chondroblast
Perichondrium
 Fibrocartilage

• Combination of dense regular connective tissue and cartilage.

• Large bundles of type I collagen fibres in addition to type II collagen fibres in matrix.
• Intervertebral disks, symphysis pubis, meniscus, temporomandibular joint, sternoclavicular joint, shoulder joint, along the
insertions of tendons and ligaments to the bones.
• Limited distribution in the body.
• Provides tensile strength and resist to both compression and shearing forces in the tissue – to absorb sudden physical
impact.
 Fibrocartilage: Intervertebral disc, H&E

• Matrix is filled with dense collagen fibres that exhibit parallel arrangement.
• Small chondrocytes in lacunae are arranged in rows within the matrix.
(rather than in random / isogenous groups, as seen in hyaline or elastic cartilage).
• All chondrocytes in lacunae are of similar size.
(there is no gradation from larger central chondrocytes to smaller and flatter peripheral cells)
• Perichondrium is absent because fibrocartilage is a transitional area between hyaline cartilage and tendon or ligament
(dense regular CT).
• Collagen fibres may be so dense that the matrix is invisible. Chondrocytes and lacunae appear flattened.
• Collagen fibres within a bundle are parallel, but may course in different directions
 Fibrocartilage: Intervertebral disc, Mallory's trichrome
• Dense composition of the fibrocartilage.
• The chondrocytes are contained within lacunae:
- cytoplasm stains deeply
- appear individually or in rows
- between the layers of dense collagen fibres that course throughout the fibrous portion of the disc
- the surrounding cartilage matrix material is scant and blend into the dense connective tissue.
• Cartilage matrix – light homogeneous area around the cell nest and light staining area between the collagen fibres and
the chondrocytes.

lacunae
 BONE
Develops from mesenchymal cells.
Vascular tissue.
Consists of Cells + Mineralised extracellular matrix

Extracellular matrix = 65% minerals (calcium phosphate in the form of hydroxyapatite crystals)
+
35% organic matter (type I collagen fibres + ground substance/noncollagenous protein)

Bone matrix Multiadhesive proteoglycan

protein/ [bone sialoprotein (BSP-1 [osteopontin] and BSP-2), thrombospondin]
unmineralised Glycoprotein
bone Bone-specific, vitamin K-dependent protein
(osteoid) (osteocalcin, osteonectin, protein S and matrix Gla-protein)
Growth factor and cytokine
Functions Tissue nonspecific alkaline phosphatase
1. Mineralisation of matrix provides support and protection:
• Bones form the strong and rigid endoskeleton to which skeletal muscles are attached to permit movement.
• Bone is heavy and its architecture is optimally arranged to provide maximum strength with minimal weight.
2. Storage site for calcium + phosphorus. Both can be mobilized from matrix and taken by blood as needed to maintain
appropriate levels throughout the body (body’s calcium and phosphate homeostasis) / Homeostatic regulation of blood
calcium levels.
3. Red marrow is the site of hemotopoiesis – blood cell formation.
Structure of a typical long bone
• The diaphysis (shaft) of a long bone in
the adult contains yellow bone marrow
in a large marrow cavity surrounded by
a thick-walled tube of compact bone. A
small amount of spongy bone may line
the inner surface of the compact bone.
• The proximal and distal ends, or
Cancellous/
epiphyses, of the long bone consist
Trabecular/Spo
chiefly of spongy bone with a thin outer
ngy bone
shell of compact bone.
Trabeculae –
• Metaphysis – the expanded or flared thin and
part of the diaphysis nearest the anastomosing
epiphysis spicules of
• Marrow cavity/ medullary cavity – a bone tissue
large cavity filled with bone marrow that
forms the inner portion of the bone.
Epiphysis of an adult long bone.
• Except for the articular surfaces that
Outer portion has a solid structure.
are covered by hyaline (articular) compact/dense bone.
cartilage, the outer surface of the Interior of the bone exhibits a spongy
bone is covered by a fibrous layer of configuration and represents
connective tissue periosteum. spongy/cancellous bone. It consists of
numerous interconnecting trabeculae
separated by labyrinth of interconnecting
marrow spaces.
Parts of growing (young) bones Epiphysis the head of a kitten femur,
H&E /Alcian blue
A typical long bone develops from a preformed
model of hyaline cartilage model in three parts:

Epiphyses
The ends of long bones which ossify from
secondary ossification centres
Epiphyseal growth plate
• A plate of hyaline cartilage that intervenes
between epiphysis and diaphysis of a
growing long bone.
• Proliferation of chondrocytes in epiphyseal
plate → growth in length.
• After fusion of epiphysis with diaphysis, the
bone can no longer grow in length
Metaphysis
• Separates the epiphysis and diaphysis
• At the end of diaphysis towards the
epiphyseal growth plate. The cartilaginous epiphysis E is separated from the diaphysis D by the
• Most actively growing area of a long bone epiphysial growth plate GP. Note the thickening cortical bone C at the
Diaphysis outer aspect of the diaphysis and the network of trabecular of bone in the
• Elongated part of bone between medulla of the diaphysis. SC – centre of secondary ossification in the
metaphyses epiphysial cartilage.
• Develops from primary ossification centre Epiphysial growth plates provide for growth in length of long bones while
accommodating functional stresses in the growing skeleton.
 Vasculature of a long bone
Nutrient foramina are openings in the bone
through which blood vessels pass to reach
the marrow. The greatest numbers of
nutrient foramina are found in the
diaphysis and epiphysis.

Nutrient artery
It enters midshaft through nutrient
foramen, runs obliquely through cortex
and divides into ascending and
descending branches in the medullary
cavity. Each branch subdivides into
parallel vessels entering metaphysis and
form hair-pin loops. These loops
anastomose with epiphyseal, metaphyseal
and periosteal arteries.
It supplies medullary cavity and inner
two-third of compact bone of diaphysis
and metaphysis.
Metaphyseal arteries
• Arise from periosteal arteries that become incorporated into Periosteal arteries
metaphysis as the bone grows in diameter. ▪ Ramify beneath the periosteum
• Supply the ends of long bones. ▪ Enter the bone through Volkmann’s
Epiphyseal arteries derived from arterial anastomosis around the joint canal to supply outer one-third of
and supply the epiphysis compact bone.
 Outer surface of bone

Cellular
periosteum
Periosteum
Fibrous
periosteum

Tendon

• Bones are covered by periosteum (a sheath of dense fibrous connective tissue containing osteoprogenitor cells), EXCEPT
in areas where they articulate with another bone (articulating surface is covered by cartilage).
• The periosteum that covers an actively growing bone consists of:
▪ an outer fibrous layer – dense irregular connective tissue
▪ an inner cellular layer that contains the osteoprogenitor cells.
• The fibrous layer is the main component of the periosteum if active bone formation is not in progress on the bone surface
or the inner layer is not well defined.
• Periosteal cells (relatively few cells) are capable of undergoing division and becoming osteoblasts under appropriate
stimulus.
Periosteum, H&E

The outer surface of most bone is covered by a layer of condensed

fibrous tissue, the periosteum P, which contains cells capable of
converting into osteoprogenitor cells and osteoblasts. When no new
bone is being formed on the bone surface, these cells are
insignificant flattened cells with spindle-shaped nuclei but, when
there is active new bone formation at the periosteal surface, these
cells proliferate and increase in size to become osteoblasts.

a. Inactive periosteum with barely detectable inactive

osteoprogenitor cells Op and mature formed bone containing
established osteocytes Oc.

b. Active periosteum with new bone being formed by active

periosteal osteoblasts Ob, some of which are being
incorporated into newly formed bone to become osteocytes Oc.
 Bone cavities
Bone cavities are lined by The marrow cavity and the spaces in spongy bone
endosteum, a layer of connective contain bone marrow
tissue cells that contains
osteoprogenitor cells. Red bone marrow consists of blood cells in
different stages of development and a network of
• A lining tissue of compact bone reticular cells and fibers that serve as a supporting
facing the marrow cavity and framework for the developing blood cells and
the trabeculae of spongy bone vessels. As an individual grows, the amount of red
within the cavity marrow does not increase proportionately with
• Only one cell layer-thick and bone growth.
consists of osteoprogenitor
Compact bone
cells that can differentiate into In later stages of growth and in adults, when the
osteoblasts (bone matrix- rate of blood cell formation has diminished, the
secreting cells) and endosteal tissue in the marrow cavity consists mostly of fat
cell (bone-lining cells). cells; it is then called yellow marrow. In response
• Osteoprogenitor cells and to appropriate stimuli, such as extreme blood loss,
endosteal cells – flattened in yellow marrow can revert to red marrow. In the
shape with elongated nuclei adult, red marrow is normally restricted to the
and indistinguishable spaces of spongy bone in a few locations such as
cytoplasmic features. the sternum and the iliac crest.

Trabecular bone
 Cells of bone tissue

Osteoprogenitor cells

• Derived from the mesenchymal stem cells in the bone marrow

• Osteoprogenitor cells are resting cells that can differentiate into osteoblasts
• Location: external and internal surfaces of bones, microvasculature supplying bone, resemble the periosteal cells that
form the innermost layer of the periosteum and the endosteal cells that line the marrow cavities, Haversian canals and
Volkmann’s canals.
• In growing bones, the cells appear as flattened or squamous cells with lightly staining, elongate or ovoid nuclei and
inconspicuous acidophilic or slightly basophilic cytoplasm.

Bone-lining cells

• Derived from osteoblasts

• Cover the bone that is not remodelling
• Appear as a layer of flat cells with attenuated cytoplasm and lack of organelles beyond the perinuclear region
• Maintenance and nutritional support of the osteocytes embedded in the underlying bone matrix
• Regulation of movement of calcium and phosphate into and out of the bone
• Location: lining the external bone surfaces – (i) periosteal cells, lining the internal bone surfaces – (ii) endosteal cells
 Schematic drawing of cells associated with bone

• All cells except osteoclasts originate from the mesenchymal stem cells. MSCs differentiate into osteoprogenitor cells,
osteoblasts, followed by osteocytes and bone-lining cells.
• Periosteal cells – bone-lining cells on external bone surfaces
• Endosteal cells – bone-lining cells on internal bone surfaces
• Osteoclasts originate from hematopoietic progenitor cells which differentiate into bone-resorbing cells.
❖ Osteoprogenitor cells and bone-lining cells have a similar microscopic appearance and non-distinguishable from each
other.
 Osteoblasts
• Osteoblasts are the differentiated bone-forming cell that secrete and deposit type I collagen (which constitutes 90% of the
protein in bone) and bone matrix proteins, which constitute the initial unmineralised bone, or osteoid
• Responsible for the calcification of bone matrix through the secretion of membrane-limited matrix vesicles rich in alkaline
phosphatase.
• When inactive, osteoblasts appear as narrow, attenuated, spindle-shaped cells lying on the bone surface.

Active osteoblasts and osteoid

Osteoblasts actively deposit new osteoid on a bone surface. When active, the
osteoblasts Ob are large, broad, spindle-shaped or cuboidal cells with abundant
basophilic cytoplasm.
H&E The tissue has been decalcified before sectioning and staining, so the distinction
between mineralised bone and the newly formed unmineralised osteoid cannot be seen.
When new bone formation is nearly over, osteoblasts become spindle-shaped again and
virtually undetectable. Only the long, narrow nucleus being visible histologically. A few
cells are being incorporated in the newly formed bone as osteocytes Oc

Undecalcified resin section, Goldner trichrome stain

Tissue has not been decalcified
The mineralised bone (blue) can easily be distinguished from the new osteoid (red)
which is being produced by the row of cuboidal osteoblasts. There is always a short
delay between osteoid production and its mineralisation.

• When completely surrounded by
osteoid or bone matrix, the
osteoblast is referred to as an
osteocyte (mature bone cell).
The cells are metabolically
active, multifunctional and
respond to mechanical forces
applied to the bone.
• The process of transformation
from osteoblast to osteocyte ~3
days. During this time,
osteoblast produces a large
amount of ECM, reduces cell
volume by ~70%, decreases size
and number of organelles, and
develops long cell processes
that radiate from its cell body.
• Each osteocyte develops ~50
processes. After bone matrix
mineralisation, each osteocyte
occupies a lacuna, that conforms
to the shape of the cell.
Cytoplasmic processes are
enclosed by the canaliculi
within the matrix.
Osteocytes
Osteocyte lacunae with extensive
network of canaliculi, SEM
Resin-embedded sample of bone from a
4-month-old mouse shows a network of
canaliculi interconnecting three osteocyte
lacunae (OL) and endosteal cells. Resin
fills the osteocyte lacunae, canaliculi,
osteoid and bone marrow spaces but
does not penetrate mineralised bone
matrix. Phosphoric acid removes the
mineral, leaving behind a resin cast. Bone
marrow cells (BM), endosteum (EOS)
A growing bone spicule, Mallory-Azan
An osteoblast is
surrounded by its
osteoid product –
osteocyte
Osteocytes are embedded within bone matrix of
spicule (dark blue). Osteoblasts are aligned on right
side of spicule. These cells are metabolically active,
laying down the unmineralised bone matrix (osteoid).
Osteoid (thin, light-blue-stained layer, uncalcified
matrix material produced by osteoblasts) appears
between the osteoblasts and calcified bone spicule.
Inactive osteoblasts on the left side of spicule –
flattened nuclei and attenuated cytoplasm.
 Osteoclasts
• Derived from An osteoclast on bone spicule,
Osteocyte
granulocyte/monocyt Mallory-Azan
e progenitor (GMP)
cells that give rise to
granulocyte and Cytoplasm of
Bone marrow
inactive bone-
monocyte cell
lining cells on the
lineages.
nongrowing
• Osteoclasts are surface
Osteoclasts
large, multinucleated Trabecular bone
cells found at sites
where bone is being
removed.

• Rest directly on the bone tissue where resorption is taking place. As a result of
osteoclast activity, a shallow or resorption bay (Howship’s lacuna) can be observed in
the bone directly under the osteoclast. The cell is visible because of its large size and
marked acidophilia.

A spicule made of calcified cartilage (light blue) and a covering of bone tissue (dark
blue). An osteoclast on the left side of spicule has resorbed bone tissue and lies in a
depression (Howship’s lacuna). The light band between the osteoclast and the bone
spicule corresponds to the ruffled border of the osteoclast. Bone is deposited on the
opposite side of the spicule, as evidenced by the presence of osteoblasts on this
surface and newly formed osteocytes just below the surface of the spicule.

H&E
Undecalcified resin section,
Goldner trichrome stain
Osteoclasts
Micrographs (a) and (b) are taken from bone showing excessive
osteoclastic activity due to the effects of Paget’s disease. The disease is of
unknown cause in which there is random and excessive osteoclastic
erosion of bone occurring in waves, followed by increased osteoblastic
activity attempting to replace eroded bone. However, the new osteoid and
bone formation does not always occur where bone has previously been
eroded, so the architecture of the bone is grossly distorted (usually with
woven bone) and the bone is structurally weak.
Micrograph (b) shows uncoordinated new osteoid formation by a row of
cuboidal osteoblasts.
Resorption of bone is performed by large multinucleated cells called
osteoclasts O, which are often seen lying in depressions resorbed from the
bone surface called Howship lacunae H. The aspect of the osteoclast in
apposition to bone is characterised by fine microvilli which form a ruffled
border – secretes several organic acids to dissolve mineral component,
while lysosomal proteolytic enzymes are employed to destroy the organic
osteoid matrix.
Osteoclastic resorption contributes to bone remodelling in response to
growth or due to changing mechanical stresses upon the skeleton.
Osteoclasts also participate in the long-term maintenance of blood calcium
homeostasis by their response to parathyroid hormone and calcitonin.
 Bone turnover
• Once peak bone mass has been achieved by mid 30’s, net bone mass remains relatively constant throughout early adult
life until age 50.
• Bone remains metabolically active throughout life. It constantly remodels its mineral storage along the lines of mechanical
stress and repairs itself following fracture.
• Bone turnover / bone remodelling is characterised by formation of new bone by osteoblasts and resorption of old bone
by osteoclasts, a process that takes place on bone surfaces.
• In the normal adult skeleton, bone formation and resorption processes are balanced in a state called coupling, therefore
bone formation equals net bone resorption.
• In bone resorption, dissolution of bone mineral and catabolism of bone matrix by osteoclasts results in the formation of
resorptive cavity and release of bone matrix components. In bone formation, osteoblasts synthesise bone matrix, which
fills in the resorptive cavity and undergoes mineralisation. If there is uncoupling of this process, with either excessive
osteoclastic resorption or defective osteoblastic function, then there is a net loss of bone = OSTEOPOROSIS

SEM of trabecular bone.

a. A tissue section of a vertebral
body of a healthy individual.
b. A tissue section of a vertebral
body of an elderly woman
showing extensive signs of
osteoporosis.
 Endochondral ossification
• Long bones, vertebrae, pelvis and bones of the base of skull
• A small model of long bone is formed in solid hyaline cartilage. It undergoes appositional growth to form an elongated
dumbbell-shaped mass of cartilage consisting of a diaphysis and future epiphyses, surrounded by perichondrium.
• Within the shaft of the cartilage model, the chondrocytes enlarge greatly, resorbing the surrounding cartilage → slender,
perforated trabeculae of cartilaginous matrix. This cartilage matrix then becomes calcified and the chondrocytes
degenerate, leaving large interconnecting spaces.
• The perichondrium of the shaft develops osteogenic potential and assumes the role of periosteum, laying down a thin
layer of bone around the surface of the shaft.
• After degeneration of the chondrocytes, primitive mesenchymal cells and blood vessels invade the spaces left within
the shaft. Primitive mesenchymal cells differentiate into osteoblasts and blood-forming cells of the bone marrow. The
osteoblasts form a layer of cells on the surface of the calcified remnants of the cartilage matrix and commence the
formation of irregular woven bone.
• A large site of primary ossification in the shaft separates the ends of the original cartilage model. The cartilaginous
ends continue to grow in diameter, undergo regressive changes and ossification → the developing bone now consists of
an elongated diaphysial shaft with a semilunar cartilage epiphysis at each end.
• The interface between the shaft and each epiphysis constitutes an epiphysial growth plate. The cartilage proliferates
continuously, resulting in progressive elongation of the bone. At its diaphysial aspect, the chondrocytes mature and die, the
degenerating zone of cartilage being replaced by bone → bony diaphysis lengthens and the growth plates are pushed
further apart.
• On reaching maturity, growth plates are replaced by bone, causing fusion of diaphysis and epiphyses. In the centre of
mass of cartilage of each developing epiphysis, regressive changes, bone formation and appositional growth of
cartilage over the whole external surface of epiphysis occur. This conversion of central epiphysial cartilage to bone is
known as secondary ossification. A thin zone of hyaline cartilage always remains at the surface as articular cartilage.
• Under functional stresses, the calcified cartilage remnants and the surrounding irregular woven bone are
completely remodelled →the bone ultimately consists of a compact outer layer with a central medulla of cancellous bone.
Epiphyseal growth plate
H&E/Alcian blue
Epiphysial growth plate GP shows a distinct progression of morphological changes
between the epiphysial cartilage E and the newly forming bone B of the diaphysis.
SC, secondary ossification centre within the epiphysis. The Alcian blue
counterstains ground substance of cartilage. V, blood vessels passing into the
secondary ossification centre of the epiphysis via cartilage canals.
Epiphyseal growth plate
H&E/Alcian blue

• Zone of reserve cartilage R. A typical hyaline cartilage,

chondrocytes arranged in small clusters surrounded by a
large amount of moderately stained matrix.
• Zone of proliferation P. The clusters of cartilage cells
undergo mitotic divisions to form columns of chondrocytes
separated by strongly stained matrix.
• Zone of maturation M. Cell division has ceased and
chondrocytes increase in size.
• Zone of hypertrophy and calcification H. The
chondrocytes become greatly enlarged and vacuolated, and
the matrix becomes calcified.
• Zone of cartilage degeneration D. The chondrocytes
degenerate and lacunae of the calcified matrix are invaded
by osteogenic cells (Op cells) and capillaries from the marrow
cavity of diaphysis.
• Zone of ossification O. The osteogenic cells differentiate
into osteoblasts which assemble on the surface of the
spicules (thin plates) of calcified cartilage matrix
commencing bone formation. This transitional zone is
known as the metaphysis. Mineralised bone (blue), new
osteoid (red)
 Epiphyseal growth plate

Produces platelets
 Formation of secondary ossification centre and epiphyseal plate in long bones

covers articulating ends of future bone

between the secondary ossification

centre and developing shaft of the
bone.
 Limb of a 10-day old mouse, epiphyseal growth plate, Mallory-Azan
Zone of reserve cartilage R, Zone of proliferation P, Zone of maturation
M, Zone of hypertrophy and calcification H, Zone of cartilage
degeneration D, Zone of ossification O.
Secondary ossification centre

M H O
P D
P R
M
H
D

O D
 Endochondral bone formation, epiphysis of long bone,
human, H&E
BM, bone marrow CC, calcified cartilage EB, endochondral bone Ob,
osteoblast Oc, osteocyte Ocl, osteoclast T, trabeculae ZCC, zone of
calcified cartilage ZH, zone of hypertrophy ZP, zone of proliferation ZR,
zone of resorption ZRC, zone of reserve cartilage
ruffled border of osteoclast

Bone spicule, Mallory-Azan

Bone has been deposited on calcified

cartilage spicules. The bone spicules
have already grown to create an
anastomosing trabeculae.
Light blue – remnants of calcified
cartilage
Dark blue – bone tissue

an osteoclast is aligned near the

surface of spicule, where remodelling is
about to be initiated
 Intramembranous ossification
• The first evidence of intramembranous ossification appears in the 8th week
of human gestation within embryonic connective tissue (mesenchyme) →
flat bones of skull, facial bones, maxilla, mandible and clavicle.
• Intramembranous bone formation occurs within membranes of condensed
primitive mesenchymal tissue. Mesenchymal cells differentiate into
osteoblasts which begin synthesis and secretion of osteoid at multiple
centres of ossification. This is followed by mineralisation of osteoid.
• As osteoid is laid down, osteoblasts are trapped in lacunae to become
osteocytes and their fine cytoplasmic extensions shrink to form the fine
processes contained within the canaliculi.
• Osteoprogenitor cells at the surface of centres of ossification undergo
mitotic division to produce further osteoblasts which then lay down more
bone. This progressive bone formation results in the eventual fusion of
adjacent ossification centres to form bone which is spongy in gross
appearance.
• The collagen fibres of developing bone are randomly arranged in interlacing
bundles, giving rise to the term woven bone. The woven bone then
undergoes progressive remodelling into lamellar bone by osteoclastic
resorption and osteoblastic deposition to form mature compact in the
periphery and spongy bone in the centre. The primitive mesenchyme
remaining in the network of developing bone differentiates into bone marrow.
Developing skull vault of a cat fetus, H&E
Spicules of woven bone B are separated by primitive mesenchymal tissue. Condensed primitive mesenchyme M
delineates the outer margin of the developing bone. It will eventually develop into the periosteum.
 Intramembranous ossification: Intramembranous ossification:
Developing mandible, Mallory-Azan Developing mandible, H&E

• Osteoprogenitor cells in periosteum differentiate into

osteoblasts and form bony trabeculae that surrounds
primitive marrow cavities – mesenchyme and blood
vessels. Os
• Osteoblasts are located along the developing bone
trabeculae and deposit bone matrix.
• Osteoid (newly synthesised bone matrix) – margin of
trabeculae.
• Osteocytes in lacunae are trapped within the bony
trabeculae.
• Formation of new bone is not a continuous process. Osteoid
and osteoblasts are not present in inactive ossification areas.

Primitive
Os

Trabeculae of bone are being formed by osteoblasts lining

their surface
Osteocytes trapped in lacunae and primordial osteons
(Os) begin to form. Osteonic canals contain blood capillaries.
 (Decalcified) bone formation: Primitive bone marrow and development of osteons in a compact bone, H&E

around the central canals, and the

margin of innermost bone lamella

eroded shallow depressions (Howship lacunae).

The cells continue to resorb and remodel the
bone as it forms.

hematopoeisis in
progress
 Bone classification
Structural classification: Compact bone, Cancellous / trabecular/spongy bone
Microscopic classification: Immature bone and mature bone

Featurement Immature bone Mature bone

(non-lamellar bone / woven bone) (lamellar bone)
Initial formation Skeleton of a developing fetus, more rapid In adults, less rapid
[In adults: alveolar sockets of oral cavity,
insertion of tendons to bones and during bone
remodelling]
Arrangement of Interlacing arrangement Cylindrical units: osteon / Haversian systems that consist of
collagen fibre Does not display an organised lamellated concentric lamellae that surround a central canal
appearance (contains the vascular and nerve supply of the osteon)
Cells per unit area More Less
Arrangement of cell Random Long axes in the same direction as lamellae
Amount of ground More Less
substance
Mineralisation Not heavily mineralized during initial formation Prolonged secondary mineralisation
Perforating No • Run at right angles to long axis of the osteons and of
(Volkmann’s) canals the bone, Not surrounded by concentric lamellae
Others • Compact bone
• Spongy bone: trabeculae (spicules), interconnecting
marrow spaces, lamellated matrix
Microradiograph of the cross-section of a bone shows various degrees of mineralisation in different osteons

• The secondary mineralisation of mature bone is evident in

microradiographs of ground sections that show younger Haversian
* systems to be less mineralised than older Haversian systems
• The degree of mineralisation is reflected by the shade of light and dark
in the microradiograph.
i. Light areas represent the highly mineralised tissue that deflects the
* X-rays and prevents them from striking the photographic film.
ii. Dark areas contain less mineral and are less effective in deflecting
* the X-rays.
• * Mature compact bone is actively replacing immature bone, which is
seen on the periosteal (upper) surface.
• * Interstitial lamellae of the older bone are very light.
• * Some (newly formed) osteons appear very dark.
• * Haversian canals containing soft tissues appear black.
*
Immature (woven) bone does not
display an organised lamellar
appearance because of
interlacing arrangement of the
collagen fibres. The cells tend to be
randomly arranged.
The cells in mature compact
Mature spongy bone represents a
bone are organised in a circular
meshwork of trabeculae (thin,
the fashion that reflects the lamellar
anastomosing spicules of bone tissue).
structure of the Haversian
The spaces within the meshwork are
system. Resorption canals in
continuous and, in a living bone, are
mature bone are lined by
occupied by bone marrow.
osteoclasts (in cutting cones) and
have their long axes oriented in
the same direction as the
Haversian canals.
a. Decalcified immature bone, H&E b. Decalcified mature bone, H&E
a. The immature bone has
more cells, and the
matrix is not layered in
osteonal arrays.
b. Mature bone has fewer
osteocytes per unit area
IL
than immature bone.
The mature bone has
several osteons O with
concentric lamellae.
Haversian canals
contain blood vessels,
nerve and connective
tissue. Osteocytes
undergo considerable
shrinkage during routine IL
slide preparation,
revealing empty lacunae
with a small nucleus
attached to their walls. IL
interstitial lamellae
between neighboring
osteons.
Bone remodelling and repair, an irregular spicule of woven bone (WB) from a fetus, H&E

• The surfaces of the spicule exhibit osteoblastic proliferation and

activity Ob, and in the process of being resorbed by osteoclasts
Oc.
• WB is also the first bone to be laid down during the repair of a
fracture. At the fracture site, a blood clot initially forms, and
replaced by highly vascular collagenous tissue (granulation
tissue) which becomes progressively more fibrous.
• Mesenchymal cells then differentiate into chondroblasts and
progressively replace this fibrous granulation tissue with hyaline
cartilage. This firm but flexible bridge is known as temporary
callus that is strengthened by deposition of calcium salts within the
cartilage matrix.
• Osteoprogenitor cells in the endosteum and periosteum are
activated and lay down a meshwork of woven bone within and
around the temporary callus → bony callus.
• Bony union is achieved when the fracture site is completely
bridged by woven bone. Under the influence of functional stress,
the bony callus is then remodelled to form mature lamellar bone.
Fracture callus, H&E

Fracture callus C around

the site of a rib fracture.
This mass of healing
tissue stabilises the
broken ends of the bone
B, initiating bone repair.
There is new bone
formation within the
callus followed by
organisation into mature
lamellar bone. Striated
muscle M is present at
the lower border of the
bone.
 Decalcified cancellous bone: Sternum, H&E • Slender bony trabeculae that
concentric lamellae ramify, anastomose and enclose
marrow cavities with blood
vessels + hematopoietic tissue.
• Endosteum (a thin layer of inner
Inferiorly – parallel lamellae
cells) – lines the bony trabeculae
concentric lamellae
in marrow cavities.
• Osteoprogenitor cells in the
periosteum and endosteum give
rise to osteoblasts.
• Formed bone matrix contains
osteocytes in lacunae.
parallel lamellae • Large, multinucleated
osteoclasts erode part of the
bone and lie in the eroded
depressions (Howship lacunae).

• Trabecular
network
• Lamellar
architecture
•
Osteocytes
are embedded
in an orderly
manner in
bone matrix.
 Compact bone, H&E (Dried) Compact bone: Osteon

PC

HC

*
• Central (Haversian) canal (centre of the osteon) is surrounded by
An osteocyte in lacuna concentric lamellae.
Canaliculi (transparent tiny canals) • Interstitial lamellae are found between adjacent osteons.
*
HC Haversian canal • Lacunae (almond-shaped structures between the lamellae) house
PC Perforating canal osteocytes in living bone.
An osteon • Tiny canaliculi (cytoplasmic extensions of osteocytes) radiate from
individual lacuna to adjacent lacunae and form a system of
communicating canaliculi throughout the bone matrix and within the
central canal.
• Osteocytes communicate with each other and blood vessels in the
central canals.
• Cement line - outer boundary
 (Dry) compact bone: Ground
• The bone was ground to a thin section to show empty canals for blood vessels, lacunae for osteocytes and connecting canaliculi.
• Structural units of a compact bone matrix - osteons / Haversian systems
• Each osteon consists of layers of concentric lamellae arranged around a central canal.
• Lamellae are thin plates of bone that contain osteocytes
• Radiating from each lacuna in all directions are tiny canals (canaliculi). Canaliculi penetrate lamellae, anastomose with canaliculi
from other lacunae and form a network of communicating channels with other osteocytes. Canaliculi also open directly into central
canal and marrow cavities.
• Interstitial lamellae are small irregular areas between osteons (represents eroded or remodelled osteons).
• External circumferential lamellae form the external wall of compact bone (beneath periosteum) and run parallel to each other and
to the long axis of bone.
• Internal circumferential lamellae line the endosteum along the marrow cavity.
= Anastomoses between
central canals
 (Dried) compact bone: Ground
• Central canals are surrounded by lamellae with lacunae and
radiating canaliculi.
• Perforating (Volksmann’s) canals extend in either a transverse
or oblique direction, join the central canals of osteons with
marrow cavity and do not have concentric lamellae. The canals
penetrate through lamellae.

vertical tube that

shows branching

CL circumferential lamellae, O osteon, HC Haversian canal,

IL interstitial lamellae, L lacuna, VC Volksmann’s canal

THANK YOU
REFERENCES REFERENCES