DEVELOPMENT OF PULP
11/13/2019 7:16:00 AM
Synopsis
 Introduction to pulp
 Morphology
 Development
 Histological appearance
o Odontoblastic zone
o Cell free zone ( Zone of Weil)
o Cell rich zone
o Pulp core
 Principal cells of pulp
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Odontoblasts
Fibroblasts
Undifferentiated ectomesenchymal cells
Macrophages
o Immunocompetent cells
 Odontoblasts
o Functional stages
 Preodontoblast
 Secretory
 Transitional
 Aged
o Cellular junction changes
 Fibroblasts
o Sites present
o Function
o Histological appearance in young and old age
 Undifferentiated ectomesenchymal cells
o Areas present
o Function
o Histological appearance in young and old age
 Dental pulp stem cells
o Site
o Importance
 Inflammatory cells
o Macrophages
o B and T lymphocytes
o Dendritic cells
 Matrix and ground substance
o Composition
 Collagen fibers- types 1 & 3
 Ground substance
o Variation of collagen concentration within tooth
o Composition of ground substance
 GAG
 Glycoprotein
 Water
o Function of ground substance
 Vasculature and lymphatic supply of pulp
o Vasculature
 Site variation
 Pericytes
 Variation in afferent and efferent vasculature
o Lymphatic supply
 Innervation of dentin-pulp complex
o Path of innervation
o Innervation in the crown
o Innervation in the root
o Structural composition of nerve bundles
 Dentin sensitivity
o Mechanisms of dentin sensitivity
 Direct neural stimulation/innervation
 Odontoblast receptor theory
 Hydrodynamic theory
 Pulp stones
o True or false pulp stone
o Attached or free pulp stone
 Age changes of pulp
 Pulp in forensic odontology
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11/13/2019 7:16:00 AM
INTRODUCTION ( Tencate and Berkovitz)
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The pulp is a soft connective tissue that supports the dentin. It is
contained within the pulp chamber and root canals of the tooth.
At the apical constriction it becomes continuous with the PDL.
Most active during development and eruption of tooth, but productive
throughout life.
It can respond to stimuli to a certain limit such as caries, trauma,
tooth movement and restorative dentin by the production of tertiary
dentin.
MORPHOLOGY (sir book)
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Shape of pulp is similar to that of the tooth. Has a coronal and
radicular portion.
The extension of coronal pulp to the cusps is called pulp horns, that
are present one on each cusp.
The radicular portion is called root canal and more than one root canal
may be present in a single root.
The lateral branches of these canals are called accessory canals.
The volume of pulp in a tooth is about 0.02 cc and total volume of all
teeth is 0.38 cc.
DEVELOPMENT OF PULP
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Occurs in 3 stages.
 In bud stage, the dental lamina protrudes into the adjacent
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ectomesenchyme. The pulp in this stage is characterized by
presence of densely packed cells comprising mostly of
fibroblasts.
In cap stage , these cells further differentiate to form the enamel
organ and dental papilla respectively. Earlier the vessels are
situated centrally and with growth blood vessels also develop
peripherally .
In the last bell stage, morphodifferentiation and
histodifferentiation of pulp occurs wherein the dental papilla
differentiated to form pulp . This process is initiated when the
Odontoblasts form dentin around the central region at which the
central dental papilla forms the pulp.
HISTOLOGICAL APPEARANCE
 Odontoblastic zone in the periphery
 Cell free zone – Zone of Weil
 Cell rich zone- composed of fibroblasts, undifferentiated
mesenchymal cells, macrophages and Immunocompetent cells
and pulpal stem cells.
 Pulp core- blood vessels and nerves of the pulp
PRINICIPLE CELLS OF PULP
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Odontoblasts
o Most abundant and prominent cell of the pulp
o Number of Odontoblasts corresponds to number of dentinal
tubules.
o They are larger and columnar in the crown while they are
smaller and cuboidal in the root. Along the apical region,
the cells are even more flattened
o Can be classified based on their function.
 The active synthetic phase
 Elongated cells composed of basal nucleus ,a
basophilic cytoplasm and a prominent golgi
apparatus
 Numerous vesicles and prominent organelles
are seen on the dentinal side of the cell while
mitochondria are dispersed throughout the cell
body
Nucleus contains numerous nucleoli and dense
peripherally displaced chromatin
 Secretory granules present within the
Odontoblasts are released via the
Odontoblastic processes.
The transitional phase (seen with electron
microscope)
 It is a narrower cell with nucleus displaced
from basal extremity and has a condensed
chromatin.
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The
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The number of ER are reduced and autophagic
vacuoles are present
resting phase
Under light microscope seen as a stubby cell,
with little cytoplasm and a more
hematoxyphilic nucleus.
They are usually crowded and the nucleus is
more apically situated, and below very few
cytoplasmic organelles are seen.
Supra- nuclear region has no organelles but
present with lipid filled vacuoles.
 Secretory granules are scarce or absent.
o The Odontoblasts attach to adjacent cells by gap junctions,
tight junctions and desmosomes.
o Gap junctions usually occur on lateral surface of
Odontoblasts and at base of the cell for connection with
the pulpal fibroblasts.
o Odontoblastic processes
 Begins at the neck of the cell as they enter the
predentin layer.
 They are devoid of organelles but are composed of
multiple microtubules and pits along its length.
 Coated vesicles and pits are also seen within the
process which show pinocytotic activity ( uptake of
material from external environment by the formation
of the vesicle around them which is later suspended
in the cytoplasm.
o The basic structural organization of the Odontoblastic
process within the dentinal tubule is as follows:
 Each dentinal tubule is occupied by an Odontoblastic
process
 Each tubule is lined by peri- tubular dentin
 A fluid called the dentinal fluid circulated between
the tubule and the process.
 Some of the studies suggest that the possible
content of the dentinal tubule is proteoglycans,
tenascin, fibronectin, glycoprotein and transferrin.
Further studies are needed to evaluate the contents
and their possible actions.
RESEARCH RELEVANCE FOR ODONTOBLAST
Sclerostin inhibits odontogenic differentiation of human pulp‐derived
odontoblast‐like cells under mechanical stress
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Sclerotic dentin is a natural self‐protective barrier beneath non‐
carious cervical lesions (NCCLs), which are mainly induced by
mechanical stress. Sclerostin is a mechanosensory protein and
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serves as an inhibitor of dentinogenesis. However, its function on
mechanotransduction in dentine–pulp complex has not been
elucidated yet
Numerous studies have determined that mechanical stress could
activate the osteogenic function of osteocytes to promote bone
formation (Rochefort, Pallu, & Benhamou, 2010). Dentin
resembles bone in mesenchymal origin, composition and
especially forming mechanism, and odontoblasts have a similar
mechanosensory ability to osteocytes enabling cell processes to
sense external stimuli (Magloire, Couble, Thivichon‐Prince,
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Maurin, & Bleicher, 2009). Accordingly, it could be concluded
that odontoblasts should be responsible for the formation of
reactive dentin under mechanical stress. Sclerostin is a
mechanosensory protein encoded by the SOST gene, known for
its inhibitory effect on osteogenesis (Tu et al., 2012)
Recent studies reported that sclerostin was expressed in the
secretory odontoblasts of fetal mouse tooth germ (Naka &
Yokose, 2011), and sclerostin deficiency could hasten the
formation of reparative dentin after pulp injury (Collignon et al.,
2017). Our previous study also found that sclerostin could impair
odontoblastic differentiation potential of human dental pulp cells
(hDPCs) in vitro (Ou, Zhou, Liang, & Wang, 2018)
In this study it was concluded that MS downregulates sclerostin
expression via the ERK1/2 and proteasome signaling pathways to
promote odontogenic differentiation of hOBs through the STAT3
signaling pathway. It can therefore be inferred that under
mechanical stress, sclerostin inhibition promotes reactive dentin
formation by enhancing odontogenic differentiation of
odontoblasts, which might be one of potential forming
mechanisms of sclerotic dentin beneath NCCLs
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Fibroblasts
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o Most abundant cells in the pulp.
o Occur in larger numbers in the coronal portion of the pulp
and lead to formation of the cell rich zone.
o Main function is to form and maintain the extracellular
matrix of the pulp that is primarily made up of collagen
and ground substance.
o Based on their functional state
 In early stages, where they actively produce the
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matrix, they are more rounded, with plump
cytoplasm with abundant cellular organelles that
effectively help produce the matrix
At later stages, with age, the need for matrix
production is reduced and thus they resume a
spindle shape, with lesser organelles and a dense
nuclei.
o Also capable of the collagen they produced and thus
considered to play a role in the turnover of the pulpal
matrix.
RESEARCH RELAVANCE FOR FIBROBLASTS
Human Pulp Fibroblast Implication in Phagocytosis via Complement
Activation
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Pulp fibroblasts constitutively express and secrete the
complement C3b fragment.
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The released C3b fragment is fixed on bacteria and recognized
by its CR1 receptor on the macrophages.
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This opsonization is followed by a stimulation of bacteria
phagocytosis.
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Pulp fibroblasts mediate the process of phagocytosis and play a
significant role in the dental pulp local regulation of
inflammation.
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Undifferentiated mesenchymal cells
o They are cells of the mesenchymal origin present in the
pulp rich zone and the pulp core.
o These cells can give rise to either odontoblasts or
fibroblasts based on the stimuli received
o Histologically they appear as large polyhedral cells with a
pale stained centrally placed nucleus. They have abundant
cytoplasm and peripheral cytoplasmic extension.
o With age, their number decreases, thus reducing the
regenerative potential of the pulp.
RESEARCH RELEVANCE FOR UNDIFFERENTIATED MESENCHYMAL STEM
CELLS
Hypoxia upregulates the expression of the pluripotency markers in the
stem cells from human deciduous teeth
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Cultivation under hypoxia promotes different responses in the
mesenchymal stem cells and it has been producing promising
results for clinical applications. Pulp tissue from deciduous teeth
is a source of stem cells which has a high proliferative potential
but this is usually discarded. This study has evaluated the effects
of hypoxia on proliferation, apoptosis, and the expression of the
pluripotency-related genes of the stem cells from human
exfoliated deciduous teeth (SHED).
No differences in the metabolic activity, the proliferation rate,
and the apoptosis of SHED when cultivated under hypoxia or
normoxia were observed. The expression of the pluripotent
genes was significantly higher after 24 h and 7 days of the cells
that were exposed to hypoxia
Hypoxia culture may help maintain the quiescent state of the
SHED, which could be advantageous for their future clinical
applications.
o Dental pulp stem cells
 These are pluripotent cells that are usually derived
post- natally and under optimum conditions can give
rise to odontoblasts, chondrocytes , neurons and
adipocytes.
 They are a promising tool for tissue regeneration.
 Dental Stem Cells – Sources and Identification
Methods
o Cells derived from dental pulp consist of heterogeneous
population of progenitor cells that are also called
odontoblastoid cells as these cells synthesize and secrete
dentin matrix like the odontoblast cells of dentin
o It has been suggested that odontoblastoids differentiate
from the zone of Hohl cells which is a subodontoblstic cell‐
rich zone
o They express positivity for CD9, CD10, CD13, CD29, CD44,
CD49d, CD59, and CD73. Furthermore, they express
CD90, CD105, CD106, CD146, CD166, STRO‐1, Oct‐4,
Nanog, SSEA‐4, and Vimentin.
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Ageing of stem cells:
o After 120 days, MSCs start losing their proliferative
potential in vitro expansion. Various changes occur in stem
cells during culturing which including. • Gradual decrease
in proliferation index
o • Shortening of telomere • Functional impairment •
Typical Hayflick phenomenon of cellular aging.
o “The Hayflick phenomenon is the number of times a
normal human cell population will divide until cell division
stops. Leonard Hayflick discovered 40 years ago that
cultured normal human cells have limited capacity to
divide, after which they become senescent, a phenomenon
now known as the Hayflick limit
storage of stem cells
o Cryopreservatives are necessary additives to stem cell
concentrates, since they stop cell death by inhibiting the
formation of intra‐ and extracellular crystals. Dimethyl
sulfoxide is the standard cryoprotectant used in
laboratories as it prevents freezing damage to living cells.
Rapid freezing of stem cells prevent the ice formation in or
around the cells and also plays a role in prevention of
dehydration of cells.
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PROCEDURE TO CULTURING DENTAL PULP STEM CELLS
TYPES OF STEM CELL ISOLATION
o The following materials are needed for the isolation and
culturing of DSCs.
o α-Minimal essential medium (α-MEM) supplemented with
3� concentrated antibiotic/antimycotic (1x concentration
for each component is 100 units/mL penicillin G, 100
μg/mL streptomycin, and 0.25 μg/mL Fungizone).
o Tissue digestion solution: 3 mg/mL collagenase type I and
4 mg/ mL dispase. Prepare fresh to get the best result, or
store in �80 �C in aliquots.
o BASIC CELL CULTURE MEDIUM FOR DPSC:
o α-MEM (up to 500 mL volume) supplemented with:
o (a) Fetal bovine serum (FBS) (10–20% of the total volume
of α- MEM)
o (b) 100 μM L-ascorbic acid
o (c) 2 mM L-glutamine
o (d) 100 units/mL Pen-Strep/0.25 μg/mL Fungizone
solution
o TO DETACH CELLS FOR PASSAGING
o Trypsin-EDTA (porcine trypsin, 0.25%, EDTA, 2.2 mM, in
PBS)
o CRYOPRESERVATION MEDIUM
o 90% FBS 10% Dimethyl sulfoxide (DMSO)
RESEARCH RELEVANCE FOR DENTAL PULP STEM CELLS
A comparative in vitro study of the osteogenic and adipogenic potential of
human dental pulp stem cells, gingival fibroblasts and foreskin fibroblasts
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Fibroblasts are highly accessible and might represent a viable
alternative to dental stem cells. Thus they investigated and
compared the in vitro differentiation potential of human dental
pulp stem cells (hDPSCs), gingival fibroblasts (hGFs) and
foreskin fibroblasts (hFFs).
These cell populations were cultured in osteogenic and
adipogenic differentiation media, followed by Alizarin Red S and
Oil Red O staining to visualize cytodifferentiation. Quantitative
Real-Time Polymerase Chain Reaction (qRT-PCR) was performed
to assess the expression of markers specific for stem cells
While fibroblasts are more prone towards adipogenic
differentiation, hDPSCs exhibit a higher osteogenic potential.
These results indicate that although fibroblasts possess a certain
mineralization capability, hDPSCs represent the most appropriate
cell population for regenerative purposes involving bone and
dental tissues.
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Inflammatory cells
o Macrophages are mainly seen in the pulp core. They are
usually large oval or elongated cells that exhibit a dark
stained nucleus. They can also be seen in their resting
state ( termed as histiocytes or periocytes) they have
varies morphological features.
 They aim at elimination of dead cells that help in the
turnover of fibroblasts in the pulp
o Usually T- lymphocytes are seen in the pulp and their
number increase when there is an injury to the pulp
o Mast cells are also present, but difficult to demonstrate in
the pulp as their lost during processing for histology due to
their fragility.
o Dendritic antigen- presenting cells
 They are derived from the bone marrow
 Found around the Odontoblastic layer in nonerupting teeth and below the Odontoblastic layer in
the erupted teeth.
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They capture and present foreign antigens to the Tcells and aid in T-lymphocyte division and
differentiation and also take part in immunosurveillance.
They increase in number in case of carious teeth.
RESEARCH RELEVANCE FOR INFLAMMATORY CELLS
Pulp capping materials modulate the balance between inflammation and
regeneration
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The interrelations between inflammation and regeneration are of
particular significance within the dental pulp tissue inextensible
environment
pulp capacity to respond to insults by initiating an inflammatory
reaction and dentin pulp regeneration.
the pulp has been shown to possess an inherent antiinflammatory potential and a high regeneration capacity in all
teeth and at all ages.
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Extracellular matrix
o Contains 75% water and 25% organic material by weight
o The main functions of the matrix are:
 Stabilizing the structure of the pulp tissue
 Controlling the development, migration, division,
shape and function of cells within it .
 Act as a medium of transport of nutrients from the
vasculature to the cells and of metabolites from the
cells to the vasculature.
o They are composed of
 Collagen fibers (Type I & III)
 Type I (60%) and type III (40%) present
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always. Even with age, the number might
reduce but the proportion of the two types
remains stable.
Histologically, seen as single fibers dispersed
throughout the pulp in young pulp . But with
age, as the number of collagen increases, they
organize themselves into bundles.
 They are concentrated more in the apical
portion of the pulp.
Non fibrous matrix
 Glycosaminoglycan (GAGs) are mainly for
movement of water and ions. They act as a
reservoir for holding growth factors and other
bioactive molecules. The adult pulp is mainly
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composed of hyaluronic acid while chondroitin
sulphate is the main component in developing
pulp.
Proteoglycans are a diverse group of molecules
that contribute to the bulk of the matrix. Some
help in binding the various components of the
tissue and limit the permeability of the matrix.
Glycoproteins such as fibronectin and tenascin
are abundantly seen near the Odontoblastic
layer and believed to contribute in the
development of dentin. Other glycoprotein are
selectins ( guide leukocytes in diapedisis),
integrins ( anchor cells to matrix).
VASCULATURE AND LYMPHATICS OF PULP
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They enter through the apical foramen and move longitudinally
toward the coronal region. Below the odontoblastic layer, they
form a meshwork of blood vessels which is believed to provide
the nutrition for development of dentin.
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The venules leave the pulp through the apical foramen as well.
Their wall is similar to that of the arterioles but they have a
thinner wall with intermittent muscle layer that results in a larger
lumen.
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Lymphatic vessels arise as small, blind, thin walled vessels in the
coronal region of pulp. They can be distinguished from the blood
vessels by the lack of blood cells in the lumina and the
discontinuous vessel wall.
INNERVATION OF PULP
o The nerves enter the pulp through the apical foramina and the
accessory canal along with the blood vessels to form the
neurovascular bundle.
o They are primarily afferent sensory fibers of the trigeminal nerve
and the sympathetic branches from the superior cervical
ganglion.
o As they move coronally they branch out to form an extensive
plexus of nerves in the cell free zone of Weil called the “ subodontoblastic plexus of Rashkow”
o There is no counterpart for the same in the radicular portion.
Instead, they are given off branches by the ascending trunk as
they move towards the coronal region.
o Histologically, the bundles have myelinated and unmyelinated
fibers where more unmyelinated fibers are seen in the coronal
aspect of the pulp.
o Mostly they terminate in the sub-odontoblastic layer but some
may pass through the odontoblasts and pierce into the dentinal
tubules.
RESEARCH RELAVANCE ON NEUROVASCULATIRY OF DENTAL PULP
Establishment of tooth blood supply and innervation is developmentally
regulated and takes place through differential patterning processes
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The aim of this study was to describe in detail the developmental
time‐course and localization of blood vessels during early tooth
formation and to compare that to innervation, as well as to
address the putative role of vascular endothelial growth factor
(VEGF), which is an essential regulator of vasculature
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development, in this process.
The localization of blood vessels and neurites was compared
using double immunofluorescence staining on sections at
consecutive stages of the embryonic (E) and postnatal (PN)
mandibular first molar tooth germ (E11‐PN7)
VEGF showed developmentally regulated epithelial and
mesenchymal mRNA expression domains including the enamel
knot signaling centers that correlated with the growth and
navigation of the blood vessels expressing Vegfr2 and VEGFR2 to
the dental papilla and enamel organ
Developing blood vessels were present in the jaw mesenchyme
including the presumptive dental mesenchyme before the
appearance of the epithelial dental placode and dental neurites.
Similarly, formation of a blood vessel plexus around the bud
stage tooth germ and ingrowth of vessels into dental papilla at
E14 preceded ingrowth of neurites.
pioneer blood vessels in the dental papilla started to receive
smooth muscle coverage at the early embryonic bell stage.
Establishment and patterning of the blood vessels and nerves
during tooth formation are developmentally regulated, stepwise
processes that likely involve differential patterning mechanisms.
Development of tooth vascular supply is proposed to be
regulated by local, tooth‐specific regulation by epithelial–
mesenchymal tissue interactions and involving tooth target
expressed VEGF signaling.
3D-Imaging of Whole Neuronal and Vascular Networks of the Human
Dental Pulp via CLARITY and Light Sheet Microscopy
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Direct visualization of the spatial relationships of the dental pulp
tissue at the whole-organ has remained challenging. CLARITY
(Clear Lipid-exchanged Acrylamide Tissue hYdrogel) is a tissue
clearing method that has enabled successful 3-dimensional (3D)
imaging of intact tissues with high-resolution and preserved
anatomic structures.
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Dental pulps from sound teeth were CLARITY-cleared,
immunostained for PGP9.5 and CD31, as markers for peripheral
neurons and blood vessels, respectively, and imaged with light
sheet microscopy.
Innervation comprised 40% of the dental pulp volume and the
vasculature another 40%. Marked innervation morphological
differences between uni- and multiradicular teeth were found,
also distinct neurovascular interplays. Quantification of the
neural and vascular structures distribution, diameter and area
showed that blood vessels in the capillary size range was twice
as high as that of nerve fibers.
This represents an outstanding tool to study the molecular and
structural intricacies of whole dental tissues in the context of
disease and treatment methods.
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PULP STONES
 they are discrete calcified masses that have calcium-phosphorus
ratios comparable to that of dentin.
 They can be singular or multiple in any tooth and are more
common at the orifice of the pulp chamber or within the root
canal
 Histologically, they usually consist of concentric layers of
mineralized tissue formed by surface accretion around blood
thrombi, dying or dead cells, or collagen fibers.
 At times, a pulp stone may contain tubules and be surrounded
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by cells resembling odontoblasts. Such stones are rare and, if
seen, occur close to the apex of the tooth. Such stones are
referred to as true pulp stones .
Stones having no cells associated with them are called false pup
stones.
They can also be classified as free and attached pulp stone
o Attached pulp stone is when union occurs between the pulp
stone and the dentin wall, or if secondary dentin deposition
surrounds the stone .
o Free pulp stone is when the stone is completely surrounded
by soft tissue.
They decrease the total number of cells and affect the efficient
root canal debridement for treatment.
AGE CHANGES OF PULP
 With increased age, the total volume of pulp chamber and root
canal is reduced. This reduces the vascular supply of the tooth
and thus bring about the other changes in pulp.
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Total number of cells reduce from 20 yrs. and becomes about
half by 70 years of age.
The collagen appears to be organized as bundles with age.
Loss and degeneration of myelinated and unmyelinated axons
that correlate with reduced sensitivity in the pulp .
Increased dead tracts and sclerotic dentin is also seen with age
Occurrence of areas of dystrophic calcification, especially in the
pulp core
Reduced potential for repair occurs with age.
PULP AND FORENSIC ODONTOLOGY
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Age estimation is a crucial concern in establishing the
distinctiveness of an individual as the development of human
dentition follows a consistent developmental sequence of teeth
starting from 4 months in utero until the emergence of third
molars, i.e. second to third decade of life
Odontoblasts and sub-odontogenic cells undergo apoptotic cell
death by apoptotic cell markers such as bcl-2.[8] As age
advances decrease in length, cytoplasmic organelles with
reduced capacity of synthetic and secretion are observed in
odontoblasts.
Barr bodies are intensely stained chromatin material present in
nucleus of female somatic cells which plays a pivotal role in
gender identification in individuals. The study of Barr bodies
provides valuable information and sufficient evidence in remains
of burnt and mummified bodies
F-bodies are present in Y chromosomes and can be used in sex
determination. Numerous studies have been carried out to
identify F‐bodies from pulpal tissue. The most efficient and
reliable method to determine gender is by fluorescent staining of
Y chromosome in healthy pulps
The sex-determining region Y (SRY) gene extracted from pulp
DNA can be used for gender determination in forensic samples.
The short (p) arm of the Y chromosomes at position 11.3 harbors
SRY gene
Reduction in the length of chromosomes can be seen during
aging process. The end of human chromosomes and senescenceassociated distension of satellites are formed by telomeres.
During the aging processes of many cells and tissues, shortening
of telomere occurs. Thus, estimating telomere shortening of
extracted DNA from pulp is a valuable method in determining
age at the time of death
As pulp is highly vascularized, most undoubtedly, blood group
antigens are present in the pulp. Many studies conducted
concluded that the dental pulp tissue harbors stable blood group
antigen and can be used for forensic analysis
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