You are on page 1of 182



Leader in continuing dental education
Craniofacial development
Facial development in the embryo involves the
origin of the facial mesenchyme which arises from
neural crest cells.

Unusually, they disrupt the ectodermal-
mesodermal junction and migrate into the
underlying tissue as ectomesenchymal cells.
Among the derivatives of the cephalic neural crest
cells are the maxilla, mandible, zygomatic, nasal
bones, and bones of the cranial vault.

This process is presumed to be under the control
of genes known as homeobox genes (1984), which
endow neural crest cells (NCC) with a positional
identity, which mediates aspects of craniofacial
morphogenesis and patterning.
Role of Homeobox genes
genes which are highly conserved throughout
evolution of diverse organisms

fundamental in evolution of the specialised body
parts of many animal species
master genes of the head and face controlling
patterning, induction, programmed cell death, and
epithelial mesenchymal interaction during
development of the craniofacial complex.

Those of particular interest in craniofacial
development include the Hox group, Msx1 and
Msx2 (muscle segment), Dlx (distalless), Otx
(orthodontical), Gsc (goosecoid), and Shh (sonic
Proteins encoded by these homeobox genes are
transcription factors, which can switch genes on
and off by activating or repressing gene
expression, and therefore control other genes
producing a co-ordinated cascade of molecular
events which, in turn, control patterning and
morphogenesis .
(Thesleff, 1995).
At a cellular level this control is expressed through
two main groups of regulatory proteins, the growth
factor family and the steroid/ thyroid/retinoic acid
super family (Evans, 1988), that regulate growth
(Johnston and Bronsky, 1995).

These regulatory molecules in the mesenchyme
are fibroblast growth factor (FGF), epidermal
growth factor (EGF), transforming growth factor
alpha (TGF ), transforming growth factor beta
(TGF ), and bone morphogenetic proteins (BMPs)
MSX genes
Homologous to drosophila msh (muscle segment

Murine msx family msx1, msx2, msx3

Msx1 & 2 transcriptina; repressors in cellular

Along with dlx genes (activator)- mutually modulate
their own transcriptional activities
Mice studies
Msx1- targeted disruption loss of palatal shelves
& maxillary bones , slight shortening of maxilla &/or
mandible, no tooth development beyond bud stage

Msx2-(less well defined role in lip & palate
development) deficiency-skull ossification defects
& persistent calvarial foramen; mutation-
craniofacial malformation- mandibular hypoplasia,
cleft palate medial facial palate.
DLX genes
Homologous to drosophila distal-less (Dll gene)

At least 6 members in mice- dlx1,2, 3, 5,6 &7
encode transcription factors involved in orofacial
patterning from neural crest cells
Dlx 1 & 2-development of maxi arch (development
of palatine & pterygoid bones of palate)

Dlx 1, 2, 3, 5, 6 development of mandible
LHX genes
(lim homeobox genes)

Tissue patterning/specification & differentiation of
different cell types

Lhx 6 & 7- expressed prior to initiation of tooth
formation in oral & odontogenic mesenchyme of
maxi & mandi processes (mice)
Lhx 8(L3)-differentially expressed in maxilla,
mandible & ventral forebrain; continuous
expression in mesenchyme during diff stages of
palatogenesis- candidate gene for isolated
nonsyndromic form of cleft palate
PRRX gene
Paired related homeobox

Homologous to drosophila paired & gooseberry
genes & to mouse pax3, 6 & 7 genes.

2 best studied prrx genes-
prrx1(mhox/phox1/prx1/K2) & 2(S8/prx2)-similar
expressions in cranium, branchial arches, body
wall & limbs
Prrx 1- detected in most of the ectoderm including
brain precursors- inactivation (mice)-
microcephaly, low set ears, pointed snout, cleft
palate & mild mandibular hypoplasia.

Prrx 2 no expression in developing brain
inactivation is compensated by prrx 1 functionality
Involved in the final stages of formation of
craniofacial structures like the ear, nose & mouth.

Disruption lower mandible & associated
musculature including tongue, nasal cavity, nasal
pits, malleus & external auditory meatus,
malformation of various bones of base of skull
RYK gene
Related to tyrosine kinases (receptor proteins).

Rugulate diverse cellular functions-mitogenesis,
differentiation & morphogenesis
Disruption (mice study Halford 2000) some limb
abnormalities with slightly smaller & more rounded
cranial vault, shorter snout premaxillary/maxillary
hypoplasia(flattened midface) & reduced mandible
with clefting of palate.
FUNCTIONS angiogenesis, wound healing,
embryonic development & malignant
transformation, regulate cell proliferation,
differentiation & migration in different tissues

At least 7 members are expressed in the
developing face Fgf 1,2,4,5,8,9 & 12
Receptors FGFRs fgfrs 1,2 & 3- expressed in
development of face & later associated with some
regions of chondrogenesis

FGFR activating mutations embryonic lethality &
limb & craniofacial abnormalities such as CP &
reduced maxillary bone (Apert, Crouzon
2 RECEPTORS pdgfr-alpha & pdgfr-beta

Pdgfs & Pdgfrs regulatory roles in development of
CNS, vascular system, in maintenance of tissue
homeostasis & in wound healing along with imp
role in palate development

Homozygous knockout of Pdgfra midline defects
& underdevelopment of face with absence of some
facial bones (Soriano 1997)
TGF ALPHA & BETA-contribute to facial
development, especially palate formation.

Tgf A some human studies indicate a positive
association between tgfa & CL with/without
CP(Ardinger 1989)

Rather than a necessary & sufficient determinant ,
it has been postulated that tgfa acts as a modifier
TGF Beta superfamily
Tgf beta 1,2,3,4 & 5 and more distantly related
bone morphogenetic proteins

Tgh beta 1,2, 3-expressed in early embryogenesis
& are associated later with some regions of
skeletal development
Depletion of tgf beta 3(Brunet 1995)-prevents in
vitro fusion of palatal shelves.

Critical role of tgf beta2& 3 in molecular control of
orofacial clefting (Lidral, 1998, Sanford 1997)
Necessary for normal craniofacial development.

Increases matrix metalloproteinases(MMPs)
secretion (downstream signal transduction
Mice studies (Miettinen 1999) egfr knockout-facial
mediolateral defects like narrow & elongated
snouts, underdeveloped lower jaws, CP &
diminished secretion of MMPs in palatal shelf
Major inhibitory neurotransmitter with many critical
functions as an intercellular signaling molecule in
the CNS

Receptors gaba A,B & C. gaba A can be
modulated by steroids, benzodiazepines.
Gaba- capable of promoting survival, differentiation
& migration of embryonic neurons

Mice studies(Miller 1975, Homanics 1997)
Abberations in gaba/gaba A induces clefting of
Growth factors

This collective term originally referred to
substances that promote cell growth.
The genes and mechanisms of our body are
tirelessly operating day in, day out, and are
part of a long interconnected chain of
reactions that make our body work.
There are various factors involved that affect
growth. It is the genetic coding of our bodies
that determine the way we are and how we
work, with the external environment either
emphasizing or inhibiting the effectiveness of
some of these genes.
Growth factors

Growth factors are proteins that bind to
receptors on the cell surface, with the
primary result of activating cellular
proliferation and/or differentiation. Many
growth factors are quite versatile,
stimulating cellular division in numerous
different cell types; while others are
specific to a particular cell-type.
Growth factors comprises of molecules that
function as growth stimulators but also as
growth inhibitors (sometimes referred to as
negative growth factors ), factors that
stimulate cell migration or as chemotactic
agents or inhibit cell migration or invasion of
tumor cells, factors that modulate
differentiated functions of cells, factors
involved in apoptosis , factors involved in
angiogenesis , or factors that promote
survival of cells without influencing growth
and differentiation.
Growth factors are polypeptides that belongs
to a number of families.
Cell surface receptors capture them.
Upon capturing receptor interacts with
membrane and cytoplasmic bound
components to bring about alteration in gene
expression of a cell.
Thus a growth factor is an inductive agent.
A growth factor produced by one cell and
acting on another is described as paracrine
Whereas the process of a cell that recaptures
its own product is known as autocrine
Few growth factors act during
By contrast, the retinoic acid family freely
enters a cell to complex with intracellular
receptors, which eventually affect gene
Both growth factors and the retinoids regulate
the expression of the homeobox genes,
which, in turn, regulate the expression of
growth factors(role of regulatory loops in

Epidermal Growth Factor (EGF)
Platelet-Derived Growth Factor (PDGF)
Fibroblast Growth Factors (FGFs)
Transforming Growth Factors-b (TGFs-b)
Transforming Growth Factor-a (TGF-a)
Erythropoietin (Epo)
Insulin-Like Growth Factor-I (IGF-I)
Insulin-Like Growth Factor-II (IGF-II)
Interleukin-1 (IL-1)
Interleukin-2 (IL-2)
Interleukin-6 (IL-6)
Interleukin-8 (IL-8)
Tumor Necrosis Factor-a (TNF-a)
Tumor Necrosis Factor-b (TNF-b)
Interferon-g (INF-g)
Colony Stimulating Factors (CSFs)
Osteoblasts synthesize and regulate the
deposition and mineralization of the
extracellular matrix of bone. Systemic and
locally active hormones, growth factors, ions,
lipid metabolites and steroids are regulators
of osteoblastic activity and/or differentiation.
Members of the transforming growth factor
beta (TGF-b) family, particularly TGF-b and
the bone morphogenetic proteins (BMPs) are
important to bone homeostasis. These
factors modulate osteoblast proliferation and
differentiation .
Extracellular matrix degrading metallo
enzymes are known collectively as
Tissue inhibitors of
Depend on Zn+ and Ca+ for activity.
Rather than being primarily involved in matrix
degradation MMPs have equally or more
important roles as efficient processing
enzymes of many bioactive mediators such
as cytokines, chemokines, growth factors,
their receptors and specific matrix protein
anchors for these molecules.
Includes a family of molecules which are
small proteins with either paracrine or
endocrine functions which are involved in
local inflammation or immunoregulation.
Within this definition growth factors could be
Cytokines are a unique family of growth
Secreted primarily from leukocytes, cytokines
stimulate both the humoral and cellular
immune responses, as well as the activation
of phagocytic cells.
A large family of cytokines are produced
by various cells of the body.
Many of the lymphokines are also known
as interleukins (ILs), since they are not
only secreted by leukocytes but also able
to affect the cellular responses of
Specifically, interleukins are growth
factors targeted to cells of hematopoietic
The list of identified interleukins grows
continuously with the total number of
individual activities now at 22.
Factor Principal Source Primary
PDGF platelets,
endothelial cells,
proliferation of
tissue, glial
and smooth
muscle cells
two different
chains form
3 distinct
dimer forms;
AA, AB and
Factor Principal Source Primary Activity
EGF submaxillary
gland, Brunners
promotes proliferation of
mesenchymal, glial and
epithelial cells
Factor Principal Source Primary
TGF-a common in
transformed cells
may be
important for
related to
Factor Principal
Primary Activity Comments
FGF wide range
of cells;
protein is
with the
promotes proliferation
of many cells; inhibits
some stem cells;
induces mesoderm to
form in early embryos
at least 19
members, 4
Factor Principal
Primary Activity Comments
NGF promotes neurite
(axites & dendrites)
outgrowth and
neural cell survival
proteins first
identified as
trkB, trkC
Factor Principal
Primary Activity Comments
Erythropoietin kidney promotes
proliferation and
differentiation of
Factor Principal
Primary Activity Comments
TGF-b activated TH1
cells (T-helper)
and natural
killer (NK) cells
production and
class II MHC
promotes wound
healing, inhibits
macrophage and
at least 100
Factor Principal Source Primary
IGF-I primarily liver promotes
of many cell
related to IGF-
II and
proinsulin, also
Factor Principal Source Primary
IGF-II variety of cells promotes
of many cell
primarily of
fetal origin
related to
IGF-I and
Factors modulating growth, chemotactic behavior and/or
functional activities of smooth muscle cells include Activin A ,
Adrenomedullin , aFGF, ANF , Angiogenin , Angiotensin-2 ,
Betacellulin , bFGF , CLAF , ECDGF (endothelial cell-derived
growth factor ), ET (Endothelins ), Factor X , Factor Xa , HB-EGF
, Heart derived inhibitor of vascular cell proliferation , IFN-gamma
, IL1 , LDGF (Leiomyoma-derived growth factor ), MDGF
(macrophage-derived growth factor , monocyte-derived growth
factor ), Oncostatin M , PD-ECGF , PDGF , Prolactin , Protein S ,
SDGF (smooth muscle cell-derived growth factor ), SDMF
(Smooth muscle cell-derived migration factor ), Tachykinins ,
TGF-beta , Thrombospondin .
Factors modulating growth, chemotactic behavior
and/or functional activities of vascular endothelial
cells include AcSDKP , aFGF , ANF , Angiogenin ,
angiomodulin , Angiotropin , AtT20-ECGF , B61 ,
bFGF , bFGF inducing activity , CAM-RF , ChDI ,
Neurothelin , Endostatin , Endothelial cell growth
inhibitor , Endothelial cell-viability maintaining
factor , Epo , FGF-5 , IGF-2 , HBNF , HGF , HUAF
, IFN-gamma , IL1 , K-FGF , LIF , MD-ECI , MECIF
, NPY , Oncostatin M , PD-ECGF , PDGF , PF4 ,
PlGF , Prolactin , TNF-alpha , TNF-beta ,
Transferrin , VEGF .
A mind once
stretched by a
new idea, never
regains its
Bone enormous reservoir
Binding proteins to keep these factors within
bone itself.
Transforming Growth Factors-
(TGFs- )

CIF-B ( cartilage inducing factor B =TGF-
beta-2 )
DIF ( differentiation-inhibiting factor )
DSF ( decidual suppressor factor =TGF-beta-
2 )
EIF ( Epstein-Barr virus inducing factor )
EGI (epithelial growth inhibitor )
MDGF ( milk-derived growth factor )
MGF ( milk growth factor )
MGF-a ( milk growth factor =TGF-beta-2 )
MGF-b ( milk growth factor =TGF-beta-1 )
TCGF ( transformed cell growth factor )
TGI ( tissue-derived growth inhibitor )
TIF-1 ( tumor inducing factor-1 ).
Sources of TGFs-
Found predominantly in spleen and bone tissues.
Platelets - milligrams of TGF-beta/ kg.
other tissues - microgram TGF/kg.
Human milk MGF.
Synthesized also by - macrophages(TGF-beta-1 ),
lymphocytes(TGF-beta-1 ), endothelial cells(TGF-beta-
1 ), keratinocytes(TGF-beta-2 ), granulosa cells(TGF-
beta-2 ), chondrocytes (TGF-beta-1 ), glioblastoma
cells(TGF-beta-2 ), leukemia cells(TGF-beta-1 ).
Inducers of TGFs-
secretion can be induced by a no. of different
stimuli including: steroids, retinoids, EGF ,
NGF , activators of lymphocytes, vitamin D3 ,
and IL1 .
Inhibitors of TGFs-
The synthesis of TGF-beta can be inhibited
by: EGF , FGF , dexamethasone, calcium,
retinoids and follicle stimulating hormone .
TGF-beta also influences the expression of
its own gene and this may be important in
Wound healing .
With the extracellular matrix as a complex
with betaglycan and decorin.
Stored in a biologically inactive form.
The exact molecular mechanisms underlying
its release from these reservoirs is unknown.
Five isoforms
TGF-beta-1 , TGF-beta-2 , TGF-beta-3 ,
TGF-beta-4 , TGF-beta-5 .
They are not related to TGF-alpha .
Their amino acid sequences display
homologies on the order of 70-80 percent.
TGF-beta-1 - prevalent form.
The biologically active forms of all isoforms
are disulfide-linked homodimers.
Sometimes hetrodimers.
The isoforms of TGF-beta arise by proteolytic
cleavage of longer precursors.
Isoforms isolated from different species are
evolutionarily closely conserved and have
sequence identities on the order of 98
Mature human, porcine, simian and bovine
TGF-beta-1 are identical and differ from
murine TGF-beta-1 in a single amino acid
Carboxy terminal end and Amino terminal
end of precursor.
Biosynthesis and processing of mature
TGF-beta (dark blue)
Almost all forms of TGF- are released as
biologically inactive forms that are known
also as L-TGF ( latent TGF ).
Latent forms are complexes of TGF-, an
aminoterminal portion of the TGF-beta
precursor, designatedTGF-LAP ( TGF-
latency associated peptide ), and a specific
binding protein, known as LT-BP ( latent TGF
binding protein).
L-TGF - localized at the cell surface by
binding to the mannose-6-phosphate/IGF-2
Biologically active TGF-beta results after
dissociation from the LAP complex.
The nature of the activation mechanism of L-
TGF in vivo is unclear.
Direct cell-to-cell contacts, proteases,
specifically plasmin, transglutaminases
Thrombospondin .
Alpha2M/TGF-beta complexes
The main fraction of the factor in the serum is
covalently attached to one of the acute phase
proteins , Alpha-2-Macroglobulin (Alpha2M).
Alpha2M/TGF-beta complexes are believed
to represent TGF-beta molecules released by
platelets after tissue injuries and destined to
Mutant TGF-
Mutant forms of TGF- have been created.
They form wild-type/mutant heterodimers
deficient in assembly or processing.
Such mutants behave as dominant negative
mutants and are useful in investigation of the
role of TGF- in normal and pathological
The different isoforms of TGF- are encoded
by different genes.
All genes have a length of more than 100 kb
and contain seven exons.
The genes map to different chromosomes.
The TGF-beta-1 gene maps to human chromosome
The TGF-beta-2 gene maps to human chromosome 1q41.
The TGF-beta-3 gene maps to human chromosome
These genes are expressed differentially.
The TGF-beta-3 gene is expressed strongly in embryonic
heart and lung tissues but only marginally in liver, spleen,
and kidney tissues. TGF-beta-1 is expressed strongly in
spleen tissues.
TGF-beta is the prototype of a protein family
known as the TGF-beta superfamily.
This family includes Inhibins , Activin A , MIS
(Mllerian inhibiting substance ), BMP (bone
morphogenetic proteins ), dpp
(decapentaplegic ) and Vg-1 .
An entire family of glycoprotein receptors for
TGF-beta has emerged.
Some of these proteins do not bind TGFbeta-
related factors belonging to the TGF-beta
Type-1 receptors (hematopoietic
progenitor cells) and type-2 receptors.
Individual TGF-b isotypes - varying affinities.
E.g., TGF-beta-1 binds approximately tenfold
better than TGF-beta-2.
Expression of the TGF-beta receptors is
decreased by EGF (Receptor
In endothelial cells the expression of the
TGF-beta receptor is decreased by bFGF .
Almost all types of cells express, type-3
This receptor type is not expressed in primary
epithelial, endothelial, and lymphoid cells .
The type-3 receptor is a proteoglycan
(Betaglycan), binds TGF-beta-1 and TGF-
beta-2 equally well.
Not species-specific.
TGF-beta-2 is the only variant that does not
inhibit the growth of endothelial cells.
Most pronounced differences in the TGF-beta
isoforms is their spatially and temporally
distinct expression of both the mRNAs and
proteins in developing tissues, regenerating
tissues, and in pathologic responses.
TGF-beta is the most potent known growth
inhibitor for normal and transformed epithelial
cells, endothelial cells, fibroblasts, neuronal
cells, lymphoid cells and other hematopoietic
cell types (CFU-S ), hepatocytes, and
Inhibits the proliferation of T-lymphocytes.
Inhibits the growth of natural killer cells in
Deactivates macrophages.
Blocks the antitumor activity mediated in vivo
by IL2 and transferred lymphokine-activated
or tumor infiltrating lymphocytes .
Inhibits the growth of immature hematopoietic
progenitor cells .
In particular growth of CFU-GEMM .
Inhibits megakaryocytopoiesis.
Antagonizes the biological activities of EGF ,
PDGF , aFGF and bFGF .
Latent form of TGF-beta is a strong inhibitor
of erythroleukemia cell lines.
The extent of growth inhibition induced by
TGF-beta depends on the cell type, on the
concentration of TGF-beta, and on the
presence of other factors.
The growth-inhibitory activities of TGF-beta
can be abolished by HGF (hepatocyte growth
factor ).
At concentrations of 1-2 fg/cell - growth inhibition for
smooth muscle cells, fibroblasts, and chondrocytes.
At higher concentrations - stimulation.
This bimodal activity is mediated in part by PDGF .
Low concentrations of TGF-beta - synthesis and
secretion of PDGF.
Higher concentrations lower expression of the
PDGF receptors and hence diminish the biological
effects of PDGF .
Overproduction of TGF-beta-1 by tumor cells
- neovascularization and may help promote
tumor development in vivo.
TGF-beta is an autocrine growth modulator
for malignant gliomas.
It stimulates the growth of fibroblasts and
osteoblasts in vivo and in vitro.
TGF-beta induces the synthesis of bone
matrix proteins in osteoblasts.
Factors that promote bone resorption (IL1 ,
vitamin D3 , parathormone) induce the
synthesis of TGF-beta in bone cells.
While calcitonin, an inhibitor of bone
resorption, reduces the synthesis of TGF-
It suppresses the expression of class II MHC
antigens .
Microglial cells.
TGF-beta stimulates the synthesis of the
major matrix proteins including collagen,
proteoglycans , glycosaminoglycans,
fibronectin , integrins, Thrombospondin ,
osteonectin, osteopontin .
It inhibits degradation mainly by inhibiting the
synthesis of neutral metalloproteinases and
by increasing the synthesis of proteinase
Involved in metastatic processes.
It is responsible for the transformation of
epithelial cells into mesenchymal cells.
Suppressive effects on the immune system .
TGF-beta-1 is the most potent known
chemoattractant for neutrophils .
It may be a potent regulator of Wound
healing and of bone fracture healing.
Local application of TGF-beta has been
shown to accelerate wound repair.
In combination with bone morphogenetic
protein-2 it causes development of
ossification of the posterior longitudinal
ligament of the cervical spine.
The factor may be helpful in the treatment of
traumatic tissue injuries.
Treatment of osteoporosis.
Reverses age- or glucocorticoid-impaired
Wound healing even if given 24 hours before
Bone morphogenetic protein (BMP)
Responsible for osteoinductive activity in
bone matrix.
Water soluble.
Bone morphogenetic protein (BMP)
The cellular and molecular events governing
bone formation in the embryo, healing of a
fractured bone, and induced bone fusion
follow a similar pattern.
Bone is unique of all the tissues.
When injured, it heals by formation of new
The molecular and cellular processes that
lead to the development of the skeletal
structures within the embryo are very similar
to the cascades that occur in the healing
process in an injured bone.
Thus, there is a common theme in the
development of bone from primitive
mesenchymal tissues to a well-structured,
well-organized mature bone.
The ongoing remodeling process in an adult
organism, which is exposed to external
physical and hormonal influences, is also
modulated through a similar molecular
Intracartilaginous process.
Intramembranous process.
Postfracture healing - intracartilaginous
ossification process.
Very high concentration of BMP -
intramembranous route may be taken.
It is unclear what factor(s) direct(s) one
process as opposed to the other in the
embryonic phase or during fracture healing.

Stages of bone healing and remodeling
GF and cytokines involved in generation
of new bone
Senn(1889)-Decalcified ox bone promotes
healing of osteomyelitic defects.
Lexer(1908)-Necrotic bone tissue released
stimulating factors that affect osteoblasts.
Polettini(1922)-Substance released from graft
tissue resulted in differentiation of fibroblasts
into bone and cartilage forming cells.
Leriche(1928)-Ca materials contained in the
graft tissue were the agents inducing new
bone formation.
Levander(1934)-crude alcohol extracts of
bone induce bone formation in muscle.
Sharrard and Collin(1961)-EDTA decalcified
allograft induced spinal fusion in children.
Urist(1965)-acid-decalcified bone induced
ectopic bone in rat model. He coined the term
"bone morphogenetic protein" or "osteogenic
protein" .
Reddi and Sampath(1983)-crude but
reproducible bioassay for BMP; bone matrix
when dissociated from BMP ineffective in
bone induction; reconstituted matrix effective.
Johnson(1992)-first clinical study; purified human
BMP successful clinically.
Creative biomolecules and genetic institute(1990s)-
simultaneous gene sequencing for various BMPs
and related patent dispute.
Stryker Corp., Medtronic Sofamor Danek(2002)-FDA
approval of OP-1 (BMP-7) for long bone defects and
BMP-2 in a collagen carrier within a cage for anterior
lumbar interbody fusions.
Classification of BMP
Bone morphogenic proteins are members of TGF
The BMP subfamily comprises more than 10
proteins, and newer ones are being discovered.
Several structural homologies between BMPs and
TGF growth factors.
The amino acid sequence of BMPs is considered to
be as old as 600 million years.
Because of this conservation, human
recombinant BMPs are highly effective in
lower life forms, including fruit flies.
BMPs are synthesized as precursor proteins.
The mature portion of the protein is located at
the carboxy terminal of the precursor
It is the only morphogen of all known growth factors
that has the ability to transform connective tissue
cell into osteoprogenitor cells.
Thus, it is not only a mitogen but can be a
morphogen as well.
All other growth factors such as TGF, insulin-like
growth factor, fibroblast growth factor, PDGF, and
vascular endothelial growth factor all induce
multiplication of cells but do not transform one cell
type into the other.
Signaling Mechanism of BMP
BMP receptors - Type I and Type II
serine/threonine kinase proteins.
The binding of the ligand to the Types I and II
serine/threonine kinase transmembrane
receptors results in the activation of the
signaling cascade.
Type II receptor kinase phosphorylates the
Type I receptor.
Signaling Mechanism of BMP
Type I receptor phosphorylates the
intracytoplasmic signaling molecules Smads
1, 5, and 8.
Smads 1, 5, and 8 bind to Smad 4.
Translocate into the cell nucleus.
Activation of transcriptional factors for the
early BMP response genes.
Normal bone contains approximately 0.002
mg of BMP per kilogram of pulverized bone.
At a fracture site, presumably the BMP is
released at a higher concentration.
The concentration required for ideal induced
bone bridging in osseous defects depends on
several factors.
- state of the organism in the evolutionary
- type of defect.
Bone induced under the influence of BMP
matures faster than natural healing of the
Other uses
Brain protective agent.
kidneys are their primary source in the
human adult. In chronic renal disease levels
of BMP are lower. systemic administration of
BMP may restore some of the renal
Local application for dialysis patient in
Named because of their osteoinductive
Role in embryonic and post embryonic
Signaling molecules in no. of tissues.
Implicated in mesodermal patterning,
neurogenesis and organogenesis.
BMP signaling pathways other growth
factors hormonal signaling pathways.
Cross talk between them must be evaluated
to avoid side effects of BMP based therapies.
Mutations perturbing functions of BMP genes:
BMP-5 gene mutation short ear abnormal
growth & patterning of skeletal structures and
diminished repair of bone fracture.
GDF-5 gene mutation brachypod
phenotype in mice & in autosomal recessive
syndromes Hunter Thompson
chondrodysplasia in humans shortening of
appendicular skeleton and loss or abnormal
development of some joints.
BMP-2 & BMP-4 knock out mice die early in
embryonic development, long before
development of skeleton, because of defects
in gastrulation.
BMP-7 knock out eye and kidney defects,
only mild skeletal defects.
Several extracellular proteins regulate
activities of BMP.
Noggin (BMP-4 & BMP-2)
Follistatin (BMP-4 & BMP-7)
Protein chordin

Astacin family of metalloproteases cleaves
Platelet-Derived Growth Factor
MDGF ( monocyte-derived growth factor )
ODGF ( osteosarcoma-derived growth factor )
stored in the alpha granules of platelets(PDGF-
released after cell activation of platelets for example
by thrombin .
Unstimulated cells of osteoblastic lineage
Other cells - macrophages, endothelial cells,
fibroblasts, glial cells, astrocytes, myoblasts,
smooth muscle cells, and a number of tumor
cell lines.
Synthesis of PDGF can be induced by IL1 ,
IL6 , TNF-alpha , TGF-beta and EGF .
Platelet-Derived Growth Factor
PDGF is composed of two distinct
polypeptide chains, A and B, that form
homodimers (AA or BB) or heterodimers
3 isoforms.
PDGF receptors have intrinsic tyrosine
kinase activity.
PDGF-BB binds to receptor activates
extracellular signal regulated kinase 1&2 cellular
proliferation by accelerating cell recycle & inducing
quiescent cells into the proliferation portion of the
cell cycle.
This effect is mediated by protein kinase B, a serine-
threonine protein kinase.
TGFb1 inhibits receptor autophosphorylation
neutralizes mitogenic effect of PDGF.
Proliferative responses to PDGF action are
exerted on many mesenchymal cell types.
Two related receptors, called PDGFR alpha
or PDGFR beta.
PDGF is not released into the circulation.
The biological half-life is less than two
minutes after intravenous administration.
In the adult organism PDGF is involved in
Wound healing processes.
The dimeric form of PDGF is mainly
mitogenic for cells of mesenchymal origin
while monomeric forms of PDGF are mainly
Disruption of PDGF signaling perinatal
lethality > 50%.
At low concentrations PDGF is a
chemoattractant for fibroblasts.
PDGF is also chemotactic and activating for
monocytes and neutrophils.
PDGF (alone and in combination) may be
useful in promoting bone formation.
Promotes fracture healing.
Doesnt provides entire osteogenic properties
Osteoblasts can specifically bind and
proliferate in response to PDGF.
Enhanced proliferation of both osteoblasts
and osteoclasts.
In tissue culture, PDGF alone has not yet
been proved to be osteoinductive in vivo.
In osteosarcoma positive feedback loop.
Platelet gel.
PDGF is chemotactic for both alkaline
phosphatase positive and negative cells.
It may so contribute to recruitment of bone
cells during remodeling and repair.
Used in implants and periodontal therapies.
With or without IGF-I.
Fibroblast Growth Factors (FGFs)
19 distinct members
FGF1 (acidic-FGF, aFGF) and FGF2 (basic-
Studies of human disorders & gene knock-out
studies in mice show the prominent role for
FGFs is in the development of the skeletal
system in mammals.
Best sources of aFGF is brain tissue.

The mechanism underlying the release of
aFGF is unknown.
Almost all tissues of mesodermal and
neuroectodermal origin.
Also in tumors derived from these tissues.
Endothelial cells.
potent inducers of mesodermal differentiation
in early embryos.
Specific cell-surface receptors.
4 distinct receptor types identified as FGFR1
- FGFR4.
Receptors has intrinsic tyrosine kinase
activity .
autophosphorylation of the receptor is the
immediate response to FGF binding.
FGFs also bind to cell-surface heparan-sulfated
proteoglycans with low affinity
The FGF receptors are widely expressed in
developing bone.
Mutations in the FGFR genes-autosomal dominant
disorders of bone growth e.g.
FGFR3 is predominantly expressed in quiescent
chondrocytes & it restricts chondrocyte proliferation
and differentiation.
bFGF stimulates the growth of fibroblasts,
myoblasts, osteoblasts, endothelial cells,
chondrocytes, and many other cell types.
bFGF is not only a mitogen for chondrocytes
but also inhibits their terminal differentiation.
Animals experiments with bFGF - promotes
endosteal, but not periosteal, bone formation.
bFGF may thus be a potential agent for
treatment of osteoporosis which may
increase bone mass without causing outward
deformation of the skeletal bones.
Craniosynostosis syndromes have been
shown to result from mutations in FGFR1,
FGFR2 and FGFR3.
Sometimes the same mutation can cause two
or more different craniosynostosis
Syndrome Phenotypes
FGFR1 Pfeiffer broad first digits,
FGFR2 Apert mid-face hypoplasia,
fusion of digits
FGFR2 Beare-
mid-face hypoplasia,
corrugated skin
Syndrome Phenotypes
FGFR2 Crouzon mid-face hypoplasia
FGFR2 Jackson-
mid-face hypoplasia, foot
FGFR2 Pfeiffer same as for FGFR1
Syndrome Phenotypes
FGFR3 Crouzon mid-face hypoplasia,
acanthosis nigricans
FGFR3 Non-syndromatic
digit defects, hearing
Insulin-Like Growth Factor
IGFs are single chain peptides.
2 isoforms (IGF-I and IGF-II).
40-50% homology with insulin. Still all 3 have
unique binding site to their receptors.
IGF also has general activity (metabolic &
growth promoting) in many tissue types.
Mainly liver.

IGF responsible for fetal and postnatal
growth and development in general.
IGF-I receptor gene deleted mice died at birth
putatively due to poor muscular development.
IGF-I: pre + postnatal development.
IGF-II: prenatal stages only.
IGF-I/ IGF-II ratio increases with age in many
Role in skeletal development and skeletal mass
maintenance and development of teeth.
Insulin-Like Growth Factor-I (IGF-I)
Called somatomedin C(considered as
circulating mediator of growth hormone).
Primary protein involved in responses of cells
to growth hormone (GH)
IGF-I is produced in response to GH and then
induces subsequent cellular activities,
particularly on bone growth.
IGF-I has autocrine and paracrine activities in
addition to endocrine activities on bone.
Family of transmembrane IGF-I(tyrosine kinase),
IGF-II(mannose-6-phosphate receptor) & insulin
Receptor has intrinsic tyrosine kinase activity.
Plays role in general growth and maintenance of
body skeleton.
Insulin-Like Growth Factor-II (IGF-II)
Exclusively expressed in embryonic and
neonatal tissues.
Following birth, the level of detectable IGF-II
protein falls significantly.
The IGF-II receptor is identical to the
mannose-6-phosphate receptor.
Osteoblast aging is associated with impaired
production of the stimulatory components of
the IGF-system, that may contribute to age-
related decline in osteoblast functions.

Of all IFG binding proteins, IGFBP-5 is
abundant in bone matrix.
IGF-I & II are potent survival factors for
fibroblasts, hematopoetic cells, cardiac
muscle cells & pancreatic beta cell.
IGF-I has anti apoptotic activity in these cell
types and in certain tumors.
Autocrine loop tumor promoting effect.
IGF-I has chemotactic effect on osteoblasts
in a dose dependent manner. IGF-II effects
only at lowest conc.
It promotes expression of bone specific
protein e.g. bone sialoprotein, and
In vivo, systemic application of IGF-I rapidly
activated bone turnover increase in serum
osteocalcin, increased collagen marker of
bone formation, and an increased urinary
ratio of Ca/creatinine.
In inflammatory tissue (e.g. fracture repair)
IL1 increases IGF-I production.
IGF-I activity can be suppressed by NSAIDs
e.g. indomethacin.
Taken from the AJO-DO on
CD-ROM (Copyright 1997
AJO-DO), Volume 1993
Aug (121 - 131): Heritability
of skeletodental
relationships - King, Harris,
and Tolley, Fig. 6.
Fig. 6. The epigenetic
landscape (redrawn from
Taken from the AJO-DO on CD-ROM
(Copyright 1997 AJO-DO), Volume
1981 Oct (366 - 375): Genetics,
epigenetics, and causation - Moss, Fig.
Fig. 5. This figure shows how boundaries
might be analogously demonstrated to
operate in the epigenetic regulation. If the
ball at the top of the figure is taken to
represent a cell, or a tissue, capable of
rolling down the convoluted surface
shown, then the developmental pathway
will be regulated by the convex ridges of
that inclined surface. These epigenetic
boundaries will effectively prevent the
ball from passing into adjacent grooves,
and thus determine the developmental
pathway. (Adapted from Waddington:
New Patterns in Genetics and
Development, New York, 1962, Columbia
University Press.)
The ball may be considered, for example, as a simple, totipotential cell with its complete,
genomically encoded information regulatory of the full range of species-specific polypeptide
synthesis. The subsequent history of this cell and its descendants is a function of which
developmental pathway (or "Chreod") it moves along. Some initial epigenetic factor or
process determines the initial path selected, at which time portions of the genome become,
respectively, repressed and derepressed, so that initial cytodifferentiation occurs. This new
state, or epigenetic environment, keeps the vital material moving along this particular
pathway until another bifurcation point occurs (Fig. 5). Once again, the instantaneous
epigenetic state regulates this decision, and a "catastrophic" event occurs; that is, a new
structually higher-order state or path is evolved. These pathways become "deeper" (have
higher "walls") as they progressively become hierarchically more complex. This represents,
in such a model, the fact that there is an increasing ability to withstand lateral, homeostatic
perturbations during "movement'' along the landscape. This movement is termed
homeorrhesis. In this model, the selection of pathways is not genomically but epigenetically
regulated. Yet the genomic information must be present to permit synthetic activity by the
cell and is one type of intrinsic, necessary information needed for ontogenesis to occur.
The constantly added epigenetic information is the other type of necessary causation
required. There is no reason for conflict between the genomic and epigenetic hypotheses
of ontogenetic regulation when it is perceived that they are interdependent, yet different,
categories of necessary causes and that only their unity provides the sufficient condition for
growth and development to occur.74

(1) all of the extrinsic (extraorganismal) factors impinging on vital
structures, including importantly mechanical loadings and electroelectric
states and (2) all of the intrinsic (intraorganismal) biophysical,
biomechanical, biochemical, and bioelectric microenvironmental events
occurring on, in, and between individual cells, extracellular materials,
and cells and extracellular substances.

As previously noted,99 epigenetic factors include (1) all of the extrinsic,
extraorganismal, macroenvironmental factors impinging on vital
structures (for example, food, light, temperature), including mechanical
loadings and electromagnetic fields, and (2) all of the intrinsic,
intraorganismal, biophysical, biomechanical, biochemical, and
bioelectric microenvironmental events occuring on, in, and between
individual cells, extracellular materials, and cells and extracellular
AJO-DO:1993 Mar - Sandy, Farndale, and
Simplified diagram depicting
interactions of molecules at focal
contacts. The model depicts how the
cytoskeleton is linked through the
membrane glycoprotein integrin to the
extracellular matrix. Many of the
extracellular matrix proteins which are
responsible for cell adhesion contain
common peptide sequences as cell
recognition sites. These sites are
recognized by integrins that are a
family of glycoproteins, which span the
cell membrane from the cytoplasm to
the extracellular matrix. Integrin does
not bind directly to microfilament
structures, such as actin, but is
dependent on associated proteins for
this function. Integrin binds to
fibronectin in the extracellular matrix
and to talin on the cytoplasmic
surface. Actin and vinculin then bind to
this talin-integrin complex.
From macro to - micro
From macro to - micro
Cell basic living, structural & functional unit of
Chromosome highly coiled & folded DNA molecule
combined with protein molecules, present in the
nucleus of cell
Gene-The unit of heredity: one or more nucleic acid
sequences incorporating information necessary for
the generation of a particular peptide or RNA
(AJODO-1997 : MOSS - Meier AE, editor. A is for . .
. gene. Sci Med 1996;3:72.)
These are located on the chromosomes
Apoptosis physiological cell death occuring
in normal tissues/in diseased organs, not
associated with inflammatory reactions

Chemotaxis process of migration of cells
towards an attractant

Ontogenesis - growth and development of the
3 possibilities :
Hypertrophy increase in the size of cells
Hyperplasia increase in the number of cells
Secretion of extra cellular material
Atrophy diminished size and number of cells due
to extreme failure of development
Interstitial growth occurring at all points within the
tissue; hyperplasia primarily & hypertrophy
secondarily, with/without secretion of ECM
Resolving synthesis (AJODO-1997 :
Morphogenesis is regulated (controlled, caused) by
the activity of both genomic and epigenetic
processes and mechanisms. Both are necessary
causes; neither alone are sufficient causes; and only
their integrated activities provides the necessary and
sufficient causes of growth and development.
Genomic factors are considered as intrinsic and
prior causes; epigenetic factors are considered as
extrinsic and proximate causes.

It is probable that ontogeny involves nonlinear
processes and is not fully predictable; that is, growth
and development, to a significant extent, exhibit
both random behaviors and frequent perturbations.
Resolving synthesis - Complexity and self-
The highly ordered morphological properties of adult
complex biological systems (for example, functional
matrices and skeletal units) result from the operation
of a series of spontaneous and self-organized
ontogenetic processes and mechanisms.

Environmental factors play a decisive role in all
ontogenetic processes. But it is the organism itself
that, as an integrated system, dictates the nature of
each and every developmental response . . . the living
organism self-organizes on the basis of its own
internal structuring, in continuous interaction with the
environment in which it finds itself.
Mechanotransduction (AJODO-1997 :
Occurs in single bone cells

All vital cells are "irritable" or perturbed by and
respond to alterations in their external environment.
Mechanosensing processes enable a cell to sense
and to respond to extrinsic loadings by using the
processes of mechanoreception and of

The former transmits an extracellular physical
stimulus into a receptor cell; the latter transduces or
transforms the stimulus's energetic and/or
informational content into an intracellular signal
There are several mechanotransductive
processes, for example, mechanoelectrical and
mechanochemical. Whichever are used, bone
adaptation requires the subsequent
intercellular transmission of the transduced

When an appropriate stimulus parameter
exceeds threshold values, the loaded tissue
responds by the triad of bone cell adaptation
processes..trio of possible osteoblastic
responses to loading (deposition, resorption, or
maintenance of bone tissue)
Osseous mechanotransduction is unique in four ways:

(1) Most other mechanosensory cells are
cytologically specialized, but bone cells are not;
(2) one bone-loading stimulus can evoke three
adaptational responses, whereas nonosseous
processes generally evoke one;
(3) osseous signal transmission is aneural, whereas
all other mechanosensational signals use some
afferent neural pathways and,
(4) the evoked bone adaptational responses are
confined within each "bone organ" independently,
e.g., within a femur, so there is no necessary
"interbone" or organismal involvement.
1. Electromechanical
As in most cells, the osteocytic plasma membrane
contains voltage-activated ion channels, and
transmembrane ion flow may be a significant
osseous mechanotransductive process.

Stretch-activated channels - Several types of
deformation may occur in strained bone tissue. One
of these involves the plasma membrane stretch-
activated (S-A) ion channels, a structure found in
bone cells, in many other cell types and significantly
in fibroblasts. When activated in strained osteocytes,
they permit passage of a certain sized ion or set of
ions, including K+, Ca2+, Na+. Such ionic flow may,
in turn, initiate intracellular electrical events.
2. Electrokinetic
Bound and unbound electric charges exist in
bone tissue, many associated with the bone
fluid(s) in the several osseous spaces or
compartments. Electrical effects in fluid-filled
bone are not piezoelectric, but rather of
electrokinetic, that is, streaming potential
(SP) origin. The SP is a measure of the
strain-generated potential (SGP) of
convected electric charges in the fluid flow of
deformed bone. The usually observed SPG
of 2 mV can initiate both osteogenesis and
osteocytic action potentials.
3. Electric field strength
Bone responds to exogenous electrical fields.
Although the extrinsic electrical parameter is
unclear, field strength may play an important
role. A significant parallel exists between the
parameters of these exogenous electrical
fields and the endogenous fields produced by
muscle activity.
Gap junctions &
Connected cellular network (CCN)
Gap junctions are regions on the lateral surfaces of
cells where the gap between the adjoining plasma
membranes is reduced from 20 nm to 2nm in width.
Pits/holes may be present in these regions, permeable
to small tracer particles

All bone cells, except osteoclasts, are extensively
interconnected by gap junctions that form an osseous
CCN. In these junctions, connexin is the major protein.
Each osteocyte, enclosed within its mineralized
lacuna, has many (n = 80) cytoplasmic (canalicular)
processes, 15 mm long and arrayed three-
dimensionally, that interconnect with similar processes
of up to 12 neighboring cells. These processes lie
within mineralized bone matrix channels (canaliculi).
Gap junctions are found where the plasma
membranes of a pair of markedly overlapping
canalicular processes meet. Gap junctions also
connect superficial osteocytes to periosteal and
endosteal osteoblasts. All osteoblasts are similarly
interconnected laterally. Vertically, gap junctions
connect periosteal osteoblasts with preosteoblastic
cells, and these, in turn, are similarly interconnected.

In addition to permitting the intercellular
transmission of ions and small molecules, gap
junctions exhibit both electrical and fluorescent dye
transmission. Gap junctions are electrical synapses,
in contradistinction to interneuronal, chemical
synapses, and, significantly, they permit bidirectional
signal traffic, e.g., biochemical, ionic.
Mechanotransductively activated bone cells, e.g.,
osteocytes, can initiate membrane action potentials
capable of transmission through interconnecting gap

The CCNs show oscillation, i.e.,reciprocal signaling
(feedback) between layers. This attribute enables
them to adjustively self-organize.

Gap junctions, permitting bidirectional flow of
information, are the cytological basis for the
oscillatory behavior of a CCN.
A family of membrane integral proteins that
span the cell membrane from the cytoplasm
to the extracellular matrix.
Many of the extracellular matrix proteins
which are responsible for cell adhesion
contain common peptide sequences as cell
recognition sites, these sites are recognized
by integrins.
Changes in cell shape produce a range of
effects mediated by integrins and the
cytoskeleton, which may be important in
transducing mechanical deformation into a
meaningful biologic response.
A family of glycoproteins. These are
connected extracellularly with the
macromolecular collagen of the organic
matrix and intracellularly with the
cytoskekeletal actin. The molecules of the
latter, in turn, are connected to the nuclear

These aid in transmitting signals from the
ECM directly to the intranuclear genome.
This informational transfer between cells and
ECM is dynamic, reciprocal, and continuous.
Osteoblasts (AJO-DO:1993 Mar - Sandy,
Farndale, and Meikle)
They are now recognized as the cells that
control both the resorptive and the formative
phases of the remodeling cycle, and receptor
studies have shown them to be the target
cells for resorptive agents in bone.

The osteoblast is perceived as a pivotal cell,
controlling many of the responses of bone to
stimulation with hormones and mechanical
All somatic cells commonly share approximately
5000 different polypeptide chains, each specific cell
type is characterized only by approximately 100
specific proteins. And it is claimed that "these
quantitative (protein) differences are related to
differences in cell size, shape and internal

The encoding of the DNA exists in two families; the
vastly preponderant "housekeeping" genes and the
nonabundant "structural" genes. The former regulate
the normal molecular synthesis of agents involved in
(1) the common energetic (metabolic, respiratory)
activities of all cells and, (2) the specific activities of
special cell types (e.g., neurons, osteoblasts,
ameloblasts etc.)
Prenatal craniofacial development is controlled by
two interrelated, temporally sequential, processes:
(1) initial regulatory (homeobox) gene activity and
(2) subsequent activity of two regulatory molecular
groups: growth factor families and
steroid/thyroid/retinoic acid super-family. For
example, "homeobox genes coordinate the
development of complex craniofacial structures" and
in "both normal and abnormal development, much of
the regulation of the development of virtually all of
the skeletal and connective tissue of the face is
dependent on a cascade of overlapping activity of
homeobox genes.
It is claimed that regulatory molecules can (1)
"alter the manner in which homeobox genes
coordinate cell migration and subsequent cell
interactions that regulate growth" and (2) be
involved in the "genetic variations causing, or
contributing to, the abnormal development of
relatively common craniofacial malformations .
. . perhaps modifying Hox gene activity."

Thank you

For more details please visit