1. MOTIVATION LETTER
Embryonic development depends on the precise temporal and spatial
interactions between various cells and extracellular molecules. Tooth development
is a sequential process of reciprocal epithelial-mesenchymal interactions that
involves intricate modulation of complex signaling pathways [1].
Morphologically, tooth formation commences with a structure of dental
epithelial thickening known as dental lamina, which subsequently proliferates and
invaginates into underlying mesenchyme. At the same time, signals from thickened
epithelium induce condensation of mesenchymal cells, which is known as dental
follicle. The condensed mesenchyme then guides further epithelial invagination
and convolution to progress the enamel organ through the sequential bud, cap, and
bell-shaped stages of tooth morphogenesis. During these stages, distinct anatomical
and functional parts of the tooth form, and the basic shape of tooth crown is
established [2].

2. FIGURE – 1 DEVELOPMENTAL STAGES OF TOOTH.
A schematic frontal view of an embryo head at embryonic day (ED) 11.5 is shown with a
dashed box to indicate the site where the lower (mandibular) molars will form. The tooth germ is
formed from the oral epithelium and Neural crest derived mesenchyme. At the bell stage of
development, the ameloblasts and odontoblasts form in adjacent layers at the site of interaction
between the epithelium and mesenchyme. These layers produce the enamel and dentin of the fully
formed tooth[2].

3. According to Sharpe, 1995, that the patterning of tooth shape and position
appears to be controlled by different homeobox genes expressed in mesenchyme.
The homeobox is a small conserved region of DNA that was originally identified
in Drosophila. The products of these homeobox regions are DNA-binding proteins
that regulate transcription, and in effect may hierarchically control gene expression
in morphogenesis. In mammals, homeobox genes are known as Hox genes, which
are important for somatic patterning and development [3,4,5,6].
Morphogens are extracellularly secreted signals governing morphogenesis
during epithelial-mesenchymal interactions by up regulating various genes. The
morphogenetic signaling networks include the five major classes of evolutionarily
conserved genes: bone morphogenetic proteins (BMPs), fibroblast growth factors
(FGFs), wingless and inter- related proteins (Wnts), Hedgehog proteins (Hhs), and
tumor necrotic factor (TNF) superfamilies. These families exhibit redundant and
reiterative signaling, each with distinct temporal and spatial expression during
initiation, patterning formation and morphogenesis, and cytodifferentiation. How
these conserved signals regulate the development of the mammalian tooth need to
be explored [7,8,9 10].

4. Figure -2 Signaling pathways and molecules critical for tooth development.
Teeth form from oral epithelium (green) and underlying mesenchyme (blue) and
interactions between these tissues communication are BMP (bone morphogenetic
protein), WNT, SHH (sonic hedgehog) and FGF regulate development. The most
important signal molecules mediating this (fibroblast growth factor). They regulate
the expression of important transcription factors indicated in boxes. Loss of
function of many of these genes arrests the process of tooth development in
genetically modified mice, and their mutations cause tooth agenesis in humans.
Bone morphogenic proteins (BMPs):

5. BMPs regulate most aspects of embryonic development and they are used
repeatedly during the morphogenesis of various organs. The identification of
signaling centres, i.e. enamel knots, in the developing tooth has greatly advanced
the understanding of the interactions involved in tooth development. During the
initiation of tooth development, epithelial signals induce mesenchymal factors that
then reciprocally act on the dental epithelium to form the signaling center, also
called dental placode [11,12,13].
BMP family members are sequentially and repeatedly involved in
embryonic tooth development. Six different Bmps (Bmp2 to Bmp7) are co-
expressed temporally and spatially. Ten BMP members [Bmp2, Bmp4, Bmp6,
Bmp7, Bmp8, Growth/differentiation factor (Gdf) 1, Gdf5, Gdf6, Gdf7, Gdf11, and
glial cell line- derived neurotrophic factor (GDNF)] were cloned from rat incisor
pulp. The interactions between epithelium and mesenchyme are important in tooth
development [14,15,16].
BMP signaling has multiple roles in the initiation of tooth development,
including regulating the distance between adjacent secondary enamel knots to
control the positioning of cusps, and in root development. Bmp2, Bmp4, and Bmp7
signals expressed in the enamel knot influence both epithelial and mesenchymal
cells and are responsible for the maintenance of the enamel knot and the
subsequent morphogenesis of epithelium.
Repression of BMP signaling by Noggin, which is a wide-range inhibitor of

6. BMP, results in the transformation of tooth shape from incisor to molar. Moreover,
tooth development gets arrested at the bud stage in Bmp deficient mice, indicating
that BMP signaling is essential for tooth development [17].
BMP 4 functions to regulate tooth organ shape by controlling cell cycle
progression and apoptosis. Mild inhibition of BMP signaling in K14-Noggin mice
results in severe crown and root defects, suggesting that BMP signaling is essential
for tooth hard tissue formation and root development [18].
Clinical application of the morphogenic signals for the
tissue engineering in Endodontics:
Tissue engineering is an emerging multi disciplinary field that applies
the principles of engineering and life sciences for the development of
biological substitutes that can restore, maintain, or improve tissue function
(Langer & Vacnati, 1993) [19]. Regenerative endodontic procedures can be
defined as biologically based procedures, designed to predictably replace
damaged, diseased, or missing structures, including dentin and root structures
as well as cells of the pulp dentin complex with live viable tissues preferably
of the same origin that restore the normal physiologic functions of the pulp
dentin complex (Murray et al, 2007) [20].
Regenerative endodontics comprises of research in:

7. 1. Adult stem cells.
2. Growth factors (morphogens)
3. Organ tissue culture.
4. Tissue engineering materials.
` The ability of both young and old teeth to respond to injury by
induction of reparative dentinogenesis suggests that a small population of
competent progenitor pulp stem cells may exist within the dental pulp
throughout life. The regeneration of dental tissue relies on the ability of stem
cells to produce extracellular matrix proteins encountered in the dental pulp
tissue [21].
Growth factors:
Bone morphogenic proteins (BMPs) are important growth factors
required in tooth development and regeneration. Recombinant BMP-2, -4, -7
induces formation of reparative dentin in vivo. According to Lovschall et al,
2001, the application of recombinant human insulin like growth factor-1
together with collagen has been found to induce complete dentin bridging and
tubular dentin formation This indicates the potential of adding growth factors
before pulp capping or incorporating them into restorative and endodontic
materials to stimulate dentin and pulp regeneration [22].

8. According to Kim et al, 2010, FGF2 plays a role not only as a
differentiation inducing factor in the injury repair process of pulpal tissue but
also as a positive regulator of chemokine expression, which may help in
tissue engineering and pulp regeneration using Human DPSCs. However, the
fate of odotoblastic or osteoblastic differentiation, effective local delivery for
FGF2 interaction of chemotactic and odontogenic factor limitations need to
be explored [23].
Recombinant human insulin-like growth factor-I with collagen
membrane induces complete dentin bridging and tubular dentin formation. It
is still unclear what regulates the abrupt transition of stem/progenitor cells
from quiescent to active state in terms of proliferation, migration,
differentiation, and matrix secretion following pulp tissue injury. The
molecular control mechanisms underlying morphogen release requires to be
elucidated for therapeutic uses in regenerative endodontics [24].
The innervation of the pulp has a critical role in the homeostasis of the
dental pulp. The denervation of pulp resulted in rapid necrosis of the exposed
pulp. Sensory nerves help in the invasion of immune and inflammatory cells
into sites of injury. Reinnervation of pulp resulted in deposition of dentin.
Schwann cells appear to release neurotrophic growth factors and play a role
in recruitment of sensory and sympathetic nerves during reinnervation [25].

9. Thus, the pulpal nerve fibers contribute to angiogenesis, extravasation
of immune cells and regulate inflammation to minimize initial damage,
maintain pulp tissue, and strengthen pulpal defense mechanisms. The
members of the BMP family have pronounced effects on neurogenesis.
BMPs can be used for regenerative pulpal therapy and dentinogenesis [25].
Hence the exploration of knowledge regarding morphogens will aid in
the better understanding of tooth development and its underlying
developmental malformations. This knowledge can be utilized for the gene
therapy of various treatment protocols in tissue engineering.
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