Organogenesis:

Osteogenesis

Kuliah S2
then We made out of the Mudghah
bones and clothed the bones with
flesh; then We developed out of it
another creature: so blessed be
Allah the Best creator!
(Surah Al-Mu'minun, 23: Ayat 12-14)
 Terms (examples)
chondro refers to cartilage
chondrocyte
endochondral
perichondrium
osteo refers to bone
osteogenesis
osteocyte
periostium
blast refers to precursor cell or one that
produces something
osteoblast
cyte refers to cell
osteocyte
Major derivatives of the embryonic germ layers
 Skeletal System(2)
The skeletal system consists mainly of bone and cartilageprovides
supporting framework for muscle
Bone is specialised connective composed ofcells, organic matrix and
inorganic matrix.
Bone is formed by process of oestogenesis
Cell typesosteoblasts, osteocytes and osteoclasts participate in
oestogenesis.
The organic matrix consists of type I collagen and amorphous ground
substance containing proteoglycans forms about one-third of bone mass.
Two-thirds of bone is mineralised matrix of calcium phosphate in form of
hydroxyapatite crystals.
Bone has range of physical properties giving high degree of flexibility,
undergoes continual replacement and remodelling.
Ossification = Osteogenesis
 Osteoprogenitor Cells
Derived from embryonic mesenchymal
cells and retain their ability to undergo
mitosis
Located in the inner cellular layer of
the periosteum, lining haversian
canals, and in the endosteum
Have the potential to differentiate into
osteoblasts.
 ADULT DERIVATIVES OF THE TYPES OF MESODERM

MESODERM INTERMEDIATE MESODERM

TYPE STRUCTURE DERIVATIVE
Head Head muscle, skull
cartilage
Chorda- Notochord
Limb muscles
Axial skeleton
Paraxial Somites Trunk muscles
ICM EPIBLAST MESODERM
Dermis
Intermediate Parts of kidney and
reproductive tract

Limb skeleton
Lateral Heart
Body cavity dividers

Blood cells
Amnion, Chorion
Yolk sac,
Allantois
 SOMITOGENESIS -3
Migration from differentiated regions of the somite give rise to dermis,
musculature and axial skeleton

SOMITE 9 OF 12 SOMITE CHICK (33 h) Vertebra/axial skeleton and

tendon
Neural tube Dermatome (dorsolateral) Dermis
Myotome (Ventral) Muscles of back
Sclerotome (Ventromedial)
Shoulder muscle
Pronephric tubule
(see later)

Limb muscle
Splanchnopleure
Muscles of body wall
Aorta
Somatopleure

5. Differentiationcommitted to specific cell lineage within each region.e.g.

myotomemuscles of back(close to neural tube), abaxial muscles of body
wall(farthest from neural tube).
 DERIVATIVES OF SOMITES(4)

Paraxial mesoderm Somitogenesis

Somites
Ventromedial region

Dermomyotome
Dorsolateral Ventral region Sclerotome
region
Myotome
Dermatome Chondrocytes
Skeletogenic
Myogenic cells cells

Dermis and supporting Cartilage

tissues Myoblasts
Tendon
Red arrows show Ossification
Regulation by Axial and appendicular into bone
genes Skeletal muscles
 Anatomical Classification of Bones
Bones are classified by their basic shape
Flat bones
Sutural bones

Irregular bones

Long bones

Short bones
Sesamoid bones
 Osteogenesis:The development of bones(1)
Calcified organic matrix
Mesoderm
Mesenchymal cells

Osteoprogenitor cells

Osteoblasts: bone forming cells

Osteocytes: mature bone cells

Haversian Canals: surround blood

Osteoclasts: a cell that breaks down vessels & nerve cells
bone
Osteogenesis:The development of bones(4)
Two major methods of
osteogenesis:
1.Intramembranous ossification,
no cartilagenous stage
Mesenchymal(neural crest)
condense to form
osteoblastform osteoid
matrixcacified/osteocytes(e.g flat
bones of the skull.
2. Endochondral ossification.
Mesenchymal cellscartilage
ossification into bone(e.g.long bones).
 Bone Cells
In addition to Osteocytes, the mature bone
cells found within the matrix of bone tissue,
there are three other cells that are associated
with bones
Osteoprogenitor cells
Osteoblasts
Osteoclasts
 Bone Cells
Osteoprogenitor cells
Derived from mesenchymal cells
Can undergo mitosis
Mature into osteoblasts
 Bone Cells
Osteoblasts
Responsible for osteogenesis create bone
tissue
Mature into osteocytes
 Bone Cells
Osteoclasts
Derived from embryological WBCs
Secrete enzymes for osteolysis
resorb/break down bone tissue
Necessary for calcium homeostasis
 Under certain conditions of low
oxygen tension, these cells may
differentiate into chondrogenic
[cartilage forming]cells
Most active during the period of
intense bone growth
 OSTEOBLASTS
synthesize the organic matrix of bone
possess receptors for parathyroid hormone.
derived from osteoprogenitor cells
located on the surface of the bone in a
sheet-like arrangement of cuboidal to
columnar cells
osteoblasts exocytose their secretory
products, each cell surrounds itself with the
bone matrix it has just produced
when this occurs, the imprisoned cell is
referred to as an osteocyte, and the space
it occupies is known as alacuna
Osteocyte
 Most of the bone matrix becomes
calcified
Osteoblasts as well as osteocytes are
always separated from the calcified
substance by a thin, noncalcified
layer known as
theosteoid(uncalcified bone
matrix).
 Pre-natal Ossification
Embryonic skeleton:
fashioned from fibrous membranes or
cartilage to accommodate mitosis.
2 types of pre-natal ossification (bone formation)

1. Intramembranous 2. Endochondral
Bone develops from Bone develops from
fibrous membrane hyaline cartilage
Forms bones of skull Forms all bones below
and clavicle (all flat base of skull
bones) Begins 2nd month of
Begins at 8 weeks of dvlpmt
development
 Intramembranous ossification

Begins with osteoblast differentiation

Dermal bones produced
Begins at ossification center
Intramembranous ossification

(osteoid is the organic part)

 Intramembranous Ossification
(prenatal)
Mesenchymal cells create
fibrous CT framework for
ossification

Some mesenchymal cells

differentiate into
osteoblasts in an
ossification center

Osteoblasts secrete bone matrix, osteoid

Intramembranous Ossification
(prenatal)

Mineralization and
calcification of osteoid

Trapped osteoblasts become

osteocytes
Intramembranous Ossification
(prenatal)

Osteoid accumulates in
between embryonic
blood vessels, creating
trabeculae of woven
bone.

Mesenchyme on bone
face condense and
differentiate into
periosteum
Intramembranous Ossification
(prenatal)

A bone collar of thickly woven

osteoid forms around
trabeculae and ossifies into
compact bone

Spongy bone (diplo)

cavities made up of
trabeculae fill with
red marrow created
from vessels
(vascular tissue)
overview
Figure 6.7 Intramembranous
Ossification

Figure 6.7
 Endochondral ossification

Cartilage model gradually replaced by bone

at metaphysis
Increasing bone length
Timing of epiphyseal closure differs
Appositional growth increases bone diameter
 Endochondral ossification
Modeled in hyaline cartilage, called cartilage
model
Gradually replaced by bone: begins late in second
month of development
Perichondrium is invaded by vessels and
becomes periosteum
Osteoblasts in periosteum lay down collar of bone
around diaphysis

See next slide

(5)

Fig.2
 Osteogenesis: Enchondral ossification(6)
Fig.3

The sclerotome cell can

become a chondrocyte
characterised by Sox9
or an osteocyte (osterix
transcription factor).
Chrondrocyte secrete
inhibitor factor that
repress the bone
pathway.

Pre-osteoblast &
Osteoblast
 Endochondral ossification
Calcification in center of diaphysis
Primary ossification centers
Secondary ossification in epiphyses
Epiphyseal growth plates close at end of
adolescence
Diaphysis and epiphysis fuse
No more bone lengthening

See next slide

 Endochondral Ossification
Begins in the second month of
development
Uses hyaline cartilage bones as models
for bone construction
Requires breakdown of hyaline cartilage
prior to ossification
 Stages of Endochondral
Ossification
Formation of bone collar
Cavitation of the hyaline cartilage
Invasion of internal cavities by the periosteal
bud, and spongy bone formation
Formation of the medullary cavity;
appearance of secondary ossification
centers in the epiphyses
Ossification of the epiphyses, with hyaline
cartilage remaining only in the epiphyseal
plates
Endochondral ossification

Stages 1-3 during fetal week 9 through 9th

month Stage 5 is process
Stage 4 is just of long bone growth
before birth during childhood &
adolescence
Endochondral Ossification
Bone collar formed around
diaphysis by osteoblasts
located on inner side of
periosteum
Endochondral Ossification
Cartilage in primary
ossification center
calcifies, then the cells die
and cavities form
(cavitates)

Bone collar provides stability

during cavitation

Cartilage elsewhere continues

to elongate
Endochondral Ossification

Periosteal bud (lymph,

blood vessels, nerves, red
marrow, osteoblasts and
osteoclasts) enters cavity
and builds spongy bone
Endochondral Ossification
Secondary Ossification
Center forms in epiphysis

Osteoclasts dissolve
spongy bone to create
medullary cavity
Endochondral Ossification
Hyaline only remains on
epiphyseal surface (articular
cartilage) and at diaphysis and
epiphysis junction, to form the
epiphyseal plates.

Secondary Ossification
Center does NOT calcify.
Spongy bone retained.
 Growing taller
throughout childhood!
Figure 6.10 Appositional Bone
Growth

Figure 6.10a
Figure 6.10 Appositional Bone
Growth

Figure 6.10b
 Growing Taller!
(A closer look at the epiphyseal plate)

Lots of activity!
rapidly mitotic cartilage, lengthening bone; chondrocytes
form columns

enlarging size of chondrocytes (hypertrophy)

matrix of cartilage calcifies and cells die forming spiky

tips

spiky calcified cartilage reshapes into spongy bone, converted

into medullary cavity or compact bone later as bone grows.
 Zone of resting cartilage
Zones of Growth in
anchors growth plate to bone Epiphyseal Plate
Zone of proliferating cartilage
rapid cell division (stacked
coins)
Zone of hypertrophic cartilage
cells enlarged & remain in
columns
Zone of calcified cartilage
thin zone, cells mostly dead
since matrix calcified
osteoclasts removing matrix
osteoblasts & capillaries move
in to create bone over calcified
cartilage
 Growth at epiphyseal plates

Zones of epiphyseal plates

Zone of Resting Cartilage

Zone of Proliferating Cartilage
Zone of Hypertrophic Cartilage
Zone of Calcified Cartilage
 Growth at epiphyseal plates

Zones of epiphyseal plates

Zone of Resting Cartilage

Zone of Proliferating Cartilage
Zone of Hypertrophic Cartilage
Zone of Calcified Cartilage
 Growth at epiphyseal plates

Zones of epiphyseal plates

Zone of Resting Cartilage

Zone of Proliferating Cartilage
Zone of Hypertrophic Cartilage
Zone of Calcified Cartilage
 Growth at epiphyseal plates

Zones of epiphyseal plates

Zone of Resting Cartilage

Zone of Proliferating Cartilage
Zone of Hypertrophic Cartilage
Zone of Calcified Cartilage
 Regulation of development of the Somite
Wnt Shh
musculoskeletal system(3).
Myotome induced by Wnt
+ (Notochord
& neural
genes Myotome
(muscle)
-Plate)

Sclerotome formation is BMP

regulated by fibroblast growth Fgf Sclerotome
factors(Fgf) from myotome and
sonic hedgehog(Shh) gene
secreted by notochord and
neural tube Sox
Shh inhibits bone morphogenetic
Scleraxis
protein(BMP) gene in part that Cartilage
forms tendon. Bone
Sox stimulates formation of
cartilage, but inhibits scleraxis Red arrows
gene(sclerotome forms tendon). show Tendon
Cartilage cells ossifies into bone. Regulation by
 Bone is Dynamic!
Bone is constantly remodeling and recycling

Coupled process between:

1. Bone deposition (by osteoblasts)
2. Bone destruction/resorption (by osteoclasts)
5-7% of bone mass recycled weekly
All spongy bone replaced every 3-4 years.
All compact bone replaced every 10 years.

Prevents mineral salts from crystallizing; protecting against

brittle bones and fractures
 When does lengthening stop?
End of adolescence - lengthening stops
Chondrocytes stop mitosis.
Plate thins out and replaced by bone
Diaphysis and epiphysis fuse to be one bone
Epiphyseal plate closure (18 yr old females, 21 yr
old males)
Thickening of bone continuous throughout
life
 Bone growth regulated by
hormones
Human Growth Hormone (HGH): from pituitary gland in
brain promotes epiphyseal plate activity
Thyroid hormones: regulate HGH for proper bone
proportions
Puberty: Testosterone or Estrogen cause adolescent
growth spurt and skeletal differences between the sexes:
Wider shoulders, larger bones, narrow pelvis in men
Wider hips, smaller upper body in women
Excesses in any hormones can cause abnormal skeletal
growth
Yao Defen, gigantess currently in
Ex. gigantism or dwarfism treatment for pituitary tumor in China.
Robert Wadlow, worlds tallest man 8 ft 7 ft 7 inches 396 lbs
11 inches
 Bone Remodeling
While bone is getting longer, the epiphysis
has to continually be reshaped to maintain
proportions
Involves:
Dissolving/destroying bone
New bone growth

more specific details on how this happens later

overview
 Bone remodeling
Osteoclasts
Bone resorption
Osteoblasts
Bone deposition
Triggers
Hormonal: parathyroid hormone
Mechanical stress
Osteocytes are transformed osteoblasts
 Bone Resorption
Osteoclasts are related to macrophages:
secrete lysosomal enzymes and HCl acid
Move along surface of bone, dissolving grooves
into bone with acid and enzymes
Dissolved material passed through osteoclasts
and into bloodstream for reuse by the body
 OSTEOCLASTS
Multinucleated cells originating
from granulocyte-macrophage
progenitors
Play a role in bone resorption
Occupy shallow depressions,
calledHowship's lacunae,that
identify regions of bone resorption.
Osteoclast A Bone-Degrading
Cell
A giant cell with many nuclei
Crawls along bone surfaces
Breaks down bone tissue
Secretes
concentrated
hydrochloric acid
Lysosomal
enzymes are
released
Figure 6.13a
 Bone Resorption
Accomplished by osteoclasts
Resorption bays grooves formed by
osteoclasts as they break down bone matrix
Resorption involves osteoclast secretion of:
Lysosomal enzymes that digest organic matrix
Acids that convert calcium salts into soluble forms
Dissolved matrix is transcytosed across the
osteoclasts cell where it is secreted into the
interstitial fluid and then into the blood
 Bone Deposition
Thin band of osteoid (unmineralized bone)
laid down by osteoblasts, located on inner
surface of periosteum and endosteum.
Mineral salts (Ca2+ and Pi) are precipitated
out of blood plasma and deposited
amongst the osteoid fibers
Requires proper Ca2+ and Phosphate ion
concentration
Vitamin D, C, A, and protein from diet
(Poor nutrition will negatively affect bone health)
 Bone is a reservoir for Calcium
Constant supply of Ca2+ in the blood stream
needed for:
Transmission of nerve impulses
Muscle contraction
Blood coagulation
Cell division
A narrow range of 9-11 mg Ca/100 ml blood
maintained at all times.
Bone remodeling = key in maintaining proper
blood calcium levels
 Factors regulating bone growth
Vitamin D: increases calcium from gut
Parathyroid hormone (PTH): increases blood
calcium (some of this comes out of bone)
Calcitonin: decreases blood calcium
(opposes PTH)
Growth hormone & thyroid hormone:
modulate bone growth
Sex hormones: growth spurt at adolescense
and closure of epiphyses
 Bone Remodeling
Constant osteoblast & osteoclast activity
throughout life
Causes:
Ca+2 levels in blood
If too low, parathyroid hormone activates
osteoclasts
If higher than needed, calcitonin (a hormone)
activates osteoblasts
Pull of gravity & muscles
Activates osteoblasts
If not active, activates osteoclasts - atrophy
 Positive or negative
feedback?????

Ah, yes, its

NEGATIVE!
Diferensiasi Regulasi
Molecular View
 Fractures
Reduction realignment of bone ends
Remember granulation tissue? During
which step does it occur?
New capillaries, phagocytes
Tetrapod Limb
anterior
ventral
proximal distal
dorsal
posterior
Limb Bud Formation
Establishment of Limb Fields
Hox Gene Expression in Trunk During
Limb Bud Development

Fig 23.21 Analysis of Biological Development 2 nd ed, Kalthoff

FGF10 Induces FGF8

supernumerary limbs from implanting

FGF10 beads
 Positioning of the Limb Field
Forelimb position lies near anterior
expression boundary of hox b8, b9, c6, c9, &
d9
FGF8 expression in intermediate meso + hox
genes induces FGF10 in the lateral plate
meso
 Positioning of the Limb Field
Wnts in the lateral plate maintain FGF
expression in regions of hoxd9 expression
Between the fore & hindlimb buds, the
developing kidney (mesonephros) inhibits
FGF expression
Exogenous FGF can induce supernumerary
limbs
Forelimb vs Hindlimb
Tbx5 Forelimb vs
Hindlimb
Correlates
with Tbx5 &
Tbx4
Tbx4
Expression
Respectively
 Tbx4 Gain of Function Study

Tbx4
expressing
retrovirus
infected
embryo
 Proximal Distal Limb Axis
AER
overlying ectoderm
responsible for maintaining outgrowth of limb
Progress zone
underlying mesoderm
region of cell division
maintained in active mitotic state by AER
AER Apical Ectodermal
Ridge
AER Necessary for Limb Bud
Outgrowth

Removal of AER at various times prematurely truncates limb

AER Necessary for Limb Bud
Outgrowth
Reimplantation of younger AER into
older bud causes duplication of distal
structures

Reimplantation of older AER into

younger bud causes loss of proximal
structures
Interactions of
AER and PZ
 FGF8 in the AER
3 day embryo limb
FGF in ectoderm AER buds
 Hox Code of the Limb

Proximal Distal

Hoxa & d = forelimb; Hoxc & d = hindlimb

Mammalian Hox Genes
Hox Genes Required for Proper
Limb Development

wild-type

Hoxa-11/Hoxd-11 human with synpolydactyly resulting

double knockout from homozygous HOXD-13 null
mutation
Proximal-Distal Structures
Anterior-Posterior Limb Axis

Zone of Polarizing Activity

ZPA
Classic Experiments: Retinoic Acid
Induced Limb Duplication

Implanting RA soaked beads caused

duplication of zeugopod/autopod
structures
Depletion of RA reduces the normal
structures
Shh is Expressed in ZPA
Demonstration that Sonic
Hedgehog is Primary ZPA
Component
Dorsal-Ventral Gene Patterning in Limb
Bud

Fig 23.23 Analysis of Biological Development 2 nd ed, Kalthoff

Dorsal-Ventral Limb Axis
1. Fgf-8 induces Shh
2. Shh & Fgf-8 induce Fgf-4
3. All these induce Lmx-1
4. Wnt-7a in the ectoderm
represses Lmx-1 and
induces Shh
Lmx

BMPs
Molecular Interactions in Limb
Bud
Dorsal-to-Ventral Transformations

WT mouse autopod

Wnt7a-/- mouse autopod

Homology of Process: A-P Axis
Homology of Process: D-V
 The Syndetome: Tendon Formation
Tendon joins bone to muscle. The last row of sclerotome is induced
by the overlying myotome to differentiate into those connectors.
 Malformations(1)
Bone
Osteogenetic defectsinherited characterised by extreme fragility of bones, long
bones prone to fracture
Vertebral defects
--spinal bifida occulta/block vertebrae fusion of 2 or more
adjacent vertebrae and hemivertebrae.
--hemivertebraeonly one half develops, condition confined to thoracolumbar region
results from failure of sclerotome differentiation on one side of developing vertebral
Body
--cervical vertebraeabnormal segmentation of caudal occipital and cervical
sclerotomes result in atlantoaxial malformations
--abnormal curvature of vertebral column;
Lordosisabnormal ventral curvature
Kyphosisabnormal dorsal curvature.
--short-spined dogs results from compaction and fusion of thoracic and lumbar
vertebrae
--Stenosis of vertebral foramen constricts the spinal cord and neurological defects.
Rib defectsassociated with abnormalities of vertebral column or sternum
Sternal defects
--.incomplete fusion of paired sternal bbones during morphogenesis
--associated with ectopic heart.
Limb defects
 Malformation of Limb Development(2)
Autonomy of development produces
many abnormalities.
Limb reductionsinvolve loss of
specific parts,e.g.
1. Ameliacomplete absence of limb
2. Ectromeliapartial or complete
absence of parts, e.g.carpal ectromelia.
3.Micromelialimb reduced in size.

Limb duplications.
1. Polydactylyextra digits
2. Whole or partial limbs
Limb and joint deformities.
Arthrogryposiscrooked limb, heredity
in animals.
Deficiency in gene expression
e.g.Hox and BMP.

Polydactyly
 References
1. Carlson, B. M., Foundations of Embryology (6 th.Edition) 1996.
McGraw-Hill inc. London. Page 393 - 424

2. Gilbert, S.F., Developmental Biology (8th. Edition) 2006. Sinauer Associates

Inc. Sunderland, Massachuetts. USA. Page 505 -527

3. McGeady, T.A., Quinn, P.J., Fitzpatrick, E.S., & Rayan, M.T., (2006).
Veterinary Embryology. Page 184 -203

4. Noden, D.M., DeLaHunta, A., The Embryology of Domestic Animals.

1985, Williams & Wilkins. London. Page196 -206