Journal of Dentistry xxx (xxxx) xxx–xxx

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Journal of Dentistry
journal homepage: www.elsevier.com/locate/jdent

Changes in the radicular pulp-dentine complex in healthy intact teeth and in

response to deep caries or restorations: A histological and
histobacteriological study
⁎
Domenico Ricuccia, , Simona Loghina, Li-na Niub,c, Franklin R. Tayc
a
Private Practice, Cetraro, Italy
b
School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, PR China
c
Department of Endodontics, The Dental College of Georgia, Augusta University, Augusta, GA, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Introduction: The present study reported the histological events that occurred in the radicular pulp of human
Apoptosis mature teeth in the presence of medium/deep untreated caries lesions, and those teeth with restorations or direct
Dental caries pulp capping, with particular emphasis on the morphology of the canal wall dentine and the odontoblast layer.
Odontoblasts Methods: Sixty-two teeth with medium/deep caries lesions, extensive restorations or after application of a direct
Radicular dentine
pulp capping procedure were obtained from 57 subjects. Fourteen intact mature teeth served as controls. Stained
Radicular pulp
serial sections were examined for the pulp conditions of the coronal pulp. The teeth were classified as those with
Tertiary dentine
pulpal inflammation, or those with healed pulps. Histological changes that occurred in the roots at the pulp-
dentine junction were investigated in detail.
Results: All teeth (100%) in the experimental group showed pathologic changes in the radicular pulp, with
varying amounts of tertiary dentine on the canal walls and absence of odontoblasts. These changes were
identified from different portions of the canal wall surface. Non-adherent calcifications in the pulp tissue were
observed in more than half of the specimens. Changes that deviate from classically-perceived histological re-
lationships of the pulp-dentine complex were also observed in the radicular pulps of 33.7% of the control teeth.
Conclusion: When challenged by bacteria and bacterial by-products invading dentinal tubules, odontoblasts in
the radicular pulp may undergo cell death, possibly by apoptosis. This phenomenon may be caused by pro-
gressive root-ward diffusion of bacterial by-products, cytokines or reactive oxygen species through the pulp
connective tissue.
Clinical significance: Although the vitality of the dental pulp in teeth with deep dentinal caries may be main-
tained with direct pulp capping or pulpotomy, the repair tissue that is formed resembles mineralised fibrous
connective tissues more than true tubular dentine.

1. Introduction by recruitment and proliferation of pulp-derived stem/progenitor cells

Odontoblasts are long-lived, terminally-differentiated post-mitotic
cells that are not replaced during the lifetime of an individual [1]. After
secreting primary dentine during the period of active tooth develop-
ment, these cells continue to deposit secondary dentine as a slower rate
throughout life in the absence of pathophysiological insults that chal-
lenge their integrity. Traditionally it has been held that reactionary
dentine is produced in the presence of mild injurious challenges by the
original surviving odontoblasts as well as genetically-committed
daughter cells that reside in the subodontoblastic cell-rich zone [2]. In
the presence of more severe challenges, reparative dentine is produced
that differentiate into mineralised dentine matrix-producing odonto-
blast-like cells [3]. A recent histological study challenged the generally-
accepted view that stem cells are recruited to produce a new generation
of odontoblast-like cells that produce reparative dentine [4]. In that
study, the authors demonstrated that under deep caries lesions and
following capping of pulp exposures, an amorphous, atubular calcified
tissue was deposited with no evidence of morphologically identifiable
odontoblast-like cells. The stem/progenitor cells may not have received
adequate epigenetic signals, such as the lack of cross-talk with cells
derived from the ectoderm [5]. Hence, after their full differentiation,
the secondary odontoblasts possess a phenotype that differs from the

⁎
Corresponding author at: Piazza Calvario, 7, 87022 Cetraro, CS, Italy.
E-mail address: dricucci@libero.it (D. Ricucci).

https://doi.org/10.1016/j.jdent.2018.04.007
Received 14 September 2017; Received in revised form 8 April 2018; Accepted 11 April 2018
0300-5712/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Ricucci, D., Journal of Dentistry (2018), https://doi.org/10.1016/j.jdent.2018.04.007
D. Ricucci et al.
primary odontoblasts [6]. Although the newly-formed tertiary dentine-
producing cells may express several genes that are present in the pri-
mary odontoblasts [7], these cells do not exhibit the typical elongated
polarized shape, and do not produce a tubular matrix that resembles the
morphological characteristics of primary, secondary or reactionary
dentine [8].
Histological investigations of the pulp response to medium and deep
caries were almost exclusively performed on caries-affected dentine and
the adjacent tissues in the pulp chamber [9–15]. Detailed morphologic
changes in the dentine structure had been reported, including the for-
mation of reactionary or reparative tertiary dentine, uniformity of the
predentine and the odontoblast layer, changes in the sub-odontoblastic
areas, as well as the accumulation of inflammatory cells in the vicinity
of the pulp chamber where the dentinal tubules were affected by caries
[4,9–14,16–19]. It is generally assumed that pulp reactions only occur
in the pulp subjacent to the carious lesion and that no morphological
changes occur in the radicular pulp.
Contrary to these findings, a recent study identified that histo-
pathological and histobacteriological changes occurred in the radicular
pulp of immature teeth affected by deep caries [20]. These changes
were identified in areas that were remote from the site of infection in
the coronal dentine. To date, information is lacking on the changes that
may occur in the radicular pulp of mature human teeth. Accordingly,
the objective of the present study was to examine the histological
events that occurred in the radicular pulp of human mature teeth in the
presence of medium/deep untreated caries lesions, and those teeth with
restorations or direct pulp capping, with particular emphasis on the
morphology of the canal wall dentine and the odontoblast layer.
Knowledge of the radicular pulp tissue response in these conditions is
important in understanding and predicting the healing mechanisms of
treatment procedures targeted at maintaining the vitality of all or part
of the dental pulp.
2. Materials and methods
The materials for the present study consisted of 62 mature human
teeth with caries or amalgam/resin composite restorations, or teeth that
had been subjected to direct pulp capping (48 molars, 11 premolars, 3
canines). Those teeth were obtained from a single dental practice from
57 patients (34 females, 23 males) aged between 16 and 75 (mean age
40.1 years). From this pool, 40 teeth had untreated carious lesions, 18
teeth had amalgam or resin composite restorations and 4 teeth had
received direct pulp capping with calcium hydroxide paste (calcium
hydroxide powder mixed with distilled water).
The carious lesions and the restorations were clinically classified as
shallow, medium or deep, as determined from periapical radiographs
[21]. Shallow caries/restorations were those involving less than one-
quarter of the dentine thickness. Medium caries/restorations were those
involving approximately one-quarter to three-quarters of the dentine
thickness. Deep caries/restorations were those involving more than
three quarters of the dentin thickness. Only teeth with medium and
deep caries or restorations were included in the present study. Teeth
with periodontal pockets exceeding 5 mm were excluded. The teeth
were clinically categorised as having “normal pulp”, “reversible pul-
pitis” or “irreversible pulpitis” based on pre-established criteria [15].
The teeth were extracted for prosthetic or orthodontic reasons, or be-
cause of the patientś desire in not having the tooth treated.
Fourteen intact mature human teeth (two premolars extracted for
orthodontic reasons and 12 third molars extracted for pericoronitis)
obtained from 10 patients (six females, four males) aged between 13
and 37 (mean age 23.6 years), were used as controls. The study was
approved by the institutional review board for the use of extracted teeth
in research. Written consent was received from all patients (or from
their legal guardians in case of minors) for histologic analysis.
Immediately after extraction the following approaches were un-
dertaken to facilitate proper fixation of the pulp tissues and correct
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Journal of Dentistry xxx (xxxx) xxx–xxx
orientation of the specimen in the paraffin block. The teeth were shaved
under magnification with high speed diamond burs using water spray
on the mesiodistal or the buccolingual plane until one or two pulp horns
were encountered. In some maxillary and mandibular molars, the roots
were separated 2 to 3 mm apically to the root canal orifices before
shaving the crowns. The teeth were immersed in 10% neutral buffered
formalin for 48 h. Demineralization was performed using an aqueous
solution consisting of a mixture of 22.5% (v/v) formic acid and 10%
(w/v) sodium citrate for 3 to 4 weeks, with the end point of deminer-
alization determined radiographically. All specimens were subse-
quently washed in running tap water for 48 h, dehydrated in ascending
grades of ethanol, cleared in xylene, infiltrated and embedded in par-
affin (melting point 56 °C) according to standard procedures of tissue
processing. With the microtome set at 4–5 μm, meticulous longitudinal
serial sections were taken of the whole tooth, for those teeth that were
processed in one piece, until the pulp tissue was exhausted. For some of
the roots that were separated from their crowns, longitudinal serial
sections were taken, until no more radicular pulp tissues were en-
countered. Cross sections were prepared for the rest of the roots.
Approximately 200 sections were cut at the transition from the coronal
to middle third, and another 200 sections at the transition from the
middle to apical third of each root. Every fifth section was stained with
haematoxylin and eosin for screening purpose and evaluation of the
reactions. Those sections were used to locate the areas with the most
severe reactions. Based on this initial evaluation, all slides adjacent to
the location with the most severe reactions were stained. In addition, a
modified Brown and Brenn technique for staining bacteria [22] was
used for the selected slides. Cover slips were then placed on the slides,
and the sections were examined using a light microscope. The worst
histologic condition observed was recorded for each pulp. Two eva-
luators examined the sections independently. In the event that dis-
agreement occurred, it was resolved by involving the third evaluator.
Irrespective of the clinical diagnosis, the teeth were histologically
classified into four categories according to a slight modification of
previously-reported criteria [15]:
a) Reversible histological pulpal changes. This group included specimens
with evidence of moderate or severe chronic inflammation confined
to the coronal pulp. Lymphocytes and plasma cells were identified in
varying concentrations beneath the deepest areas of caries pene-
tration. No coagulation or liquefaction necrosis was evident.
Bacteria were seen in large numbers in the carious dentine, but were
absent from the pulpal tissues.
b) Irreversible histological pulpal changes. At least one area (even if it was
very small) of the coronal pulp tissue had undergone liquefaction or
coagulation necrosis (i.e. pulpal abscess), surrounded by masses of
live and dead polymorphonuclear neutrophils (PMNs). Bacterial
aggregations/biofilms were observed colonising the necrotic pulp
tissues or the adjacent dentine walls. Peripherally, concentrations of
chronic inflammatory cells (lymphocytes, plasma cells and macro-
phages) formed a dense halo around the central zones of the abscess.
c) Normal, intact pulp. Pulps with no changes in the dentin/predentine/
odontoblast complex in the crown. Dentinal tubules could be seen
running parallel to each other through the dentine and predentine,
with no reduction in numbers. There was no reduction of the
odontoblast layer or reduction in the odontoblast cell size. Tertiary
dentine or other calcifications were also absent. In addition, there
were no inflammatory cell accumulations or dilated vessels, and no
bacteria.
d) Healed pulp. Pulps with no evident accumulation of inflammatory
cells, but deviations from normal histologic findings were present.
That is, a variable amount of tubular/atubular tertiary dentine, or
free/adherent pulp stones could be identified in the pulp chamber.
Scattered inflammatory cells were present, together with hyper-
aemic vessels, fibrosis, and a reduced odontoblastic layer.
D. Ricucci et al.
In addition to these features which were related only to the coronal
portion of the tooth, the following aspects, related to the radicular
portion, were investigated and recorded in the present investigation:
1. Changes in the regularity of the odontoblast layer on the root canal
walls;
2. Tertiary dentine formation on the root canal walls;
3. Isolated, non-adherent calcifications in the radicular pulpal tissues.
Because quantification of the surface of the root canal wall involved
in these changes was impossible using serial sections, these features
were recorded qualitatively as being present or absent. Accordingly, no
statistical analysis was performed in the present study.
3. Results
3.1. Control teeth
Descriptive statistics of the control teeth are included in Table S1.
Only the dental pulps of the control intact teeth could be classified as
normal, intact pulps. These pulps contained uninflamed connective
tissues in the pulp chamber, with abundant cells and neurovascular
bundles, and a peripheral palisading layer of odontoblasts (Fig. 1). The
predentine was of uniform thickness and the course of the dentine tu-
bules could be easily identified, with no interruption through dentine
and predentine (Fig. 1). In the radicular pulp, the same uniformity in
the interrelationship between dentine, predentine and the odontoblast
layer could only be observed in 9 of the 14 (64.3%) of the control teeth.
In the 9 teeth in which there was no disruption in the interrelationship
between the dentine, predentine and the odontoblast layer, the only
identifiable difference was in the shape of odontoblasts. The odonto-
blasts were mostly cuboidal or spindle-shaped in the apical direction
(Fig. 1D–E). No interruption or other disturbances could be seen in the
course of the dentinal tubules from all serial sections derived from the
same tooth (Fig. 1F). There was no sign of tertiary dentine formation
along the canal walls (Fig. 1F).
In the remaining 5 teeth, deviation from classically-described den-
tine morphology could be observed (Figs. 2 and 3). These deviations
were minimal in general and consisted of restricted areas in which the
canal walls appeared to be devoid of odontoblasts, despite the presence
of dentinal tubules (Fig. 2C–F). In 4 of those 5 teeth with loss of
odontoblasts, atubular tertiary dentine could be identified along the
canal walls (Fig. 3B, D). In 3 of those 5 teeth, small, non-adherent pulp
stones that were completely segregated from the predentine could be
identified in the vicinity of the neurovascular bundles (Fig. 1E, G).
3.2. Experimental teeth
Irrespective of whether the teeth were untreated or treated, all teeth
from the experimental group, including those that had received direct
pulp capping, showed changes in the morphology of the radicular pulp-
dentine complex to varying extents. These morphologic changes, which
included the loss of odontoblasts and the presence of tertiary dentine,
were observed irrespective of the overall clinical diagnosis of reversal
or irreversible pulpitis or histological manifestation of reversible/irre-
versible pulpal morphologic features or those characteristic of a healed
dental pulp (Tables S2–S4).
Of the 31 teeth histologically that were presented with irreversible
pulpal changes, non-adherent calcifications were observed in the radi-
cular pulps of 21 teeth (67.7%). These calcified masses were present in
10 of the 18 teeth diagnosed with reversible pulpitis (55.5%), and in 7
of the 13 teeth diagnosed with healed pulps (53.8%) (Tables S2–S4).
3.2.1. Teeth with irreversible pulpal changes
The most severe and diffuse reactions in the radicular pulp/dentine
complex were seen in untreated and treated teeth presented with the
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Journal of Dentistry xxx (xxxx) xxx–xxx
histologic features of irreversible pulpal changes (Figs. 4–6). Irregular
calcified tissues, hereby referred to as tertiary dentine, were present
extensively on the root canal walls. These calcified tissues had reduced,
irregular dentinal tubules or were entirely atubular, and were separated
from the secondary dentine by a dark “calciotraumatic line” (Figs.
4J–M; 5 G–H; 6D–I). The tertiary dentine were variable in thickness,
sometimes featuring scattered lacunae containing cells with projecting
canaliculi that resembled cellular cementum (Fig. 4H–I). In a few cases,
these tissues were discontinuous, being interrupted by secondary den-
tine in close proximity with large multinucleated cells (odontoclasts)
(Fig. 4J–K). The tertiary dentine appeared highly irregular and un-
evenly stained in some areas, and was present in successive layers
(Fig. 6E). On the pulpal side, the tertiary dentine showed relatively
regular, darkly-stained profiles (Fig. 5G–H) and was occasionally pre-
sented with irregular edges (Fig. 6I). In some instances, the pulpal
margins appeared highly eosinophilic, with dense collagen bundles
inserted into the tertiary dentine at varying angles, resembling the
periodontal ligament (Fig. 4L–M). These changes were predominantly
present in the middle and apical thirds of the root (Figs. 4G–M; 5G–K).
Paradoxically, they were often absent in the coronal third of the roots,
where the most severe inflammatory reactions occurred. It was not
infrequent to observe normal relationship among the dentine, pre-
dentine and the odontoblasts in the canal orifice and coronal third of
the roots (Figs. 4E–F; 5C).
No odontoblasts were present on the pulpal side of the affected
areas (Figs. 4G–M; 5G–K; 6E–I); only occasional flat, elongated cells
exhibiting the morphology of fibroblasts could be observed (Figs. 4H–I,
L–M; 5G–H; 6E-I). Polymorphonuclear leukocytes were sometimes seen
infiltrating the cells that approximated this tissue (Fig. 5J–K). Free,
diffuse calcifications were often present at any level of the radicular
pulp (Fig. 5D–F). In some instances, pulp stones embedded in tertiary
dentine could be identified along the canal walls, considerably nar-
rowing the canal lumen (Fig. 6F–G).
3.2.2. Teeth with reversible pulpal changes
Teeth with deep caries or deep restorations and with the histologic
presentation of reversible pulpal changes showed reactionary responses
in the radicular pulp that were similar to those observed in teeth with
irreversible pulpal changes (Fig. 7). That is, more severe histological
changes were identified in the middle and apical third of the roots
(Fig. 7E–P), with minimal morphologic aberrations in the coronal third
of the roots (Fig. 7D) were sometimes observed. Large areas of the canal
walls were covered by layers of atubular tertiary dentine of variable
thickness, with highly irregular margins on the pulpal side of the de-
positions (Fig. 7E–J; M–P). Odontoblasts were always absent from this
atubular calcified tissue. Only a few elongated cells with fibroblast
characteristics were identified (Fig. 7G–J; N–P), Dense collagen bundles
were occasionally inserted at 90° into these calcified tissues, resembling
insertion of Sharpey fibre bundles into the cementum (Fig. 7H–J). De-
position of irregular calcified tissues could also be observed on the walls
of lateral canals, when present (Fig. 7K–L). Diffuse unattached calcifi-
cations, were frequently identified in the radicular pulp (Fig. 7M–P).
Deposition of atubular tertiary dentine and the absence of odonto-
blasts were also observed in all teeth affected by caries of limited ex-
tension (medium caries), although the changes were less prominent. In
some instances, inconsistency in histologic conditions could be identi-
fied between the pulp chamber and the root canals (Fig. 8). That is, the
pulp chamber tissue appeared normal, with dentine and predentine
lined by an unaffected odontoblast layer with the exception of the area
of the mesial pulp horns under the infected carious dentin (Fig. 8G),
where there was reduction of the odontoblast layer and increased
predentine deposition (Fig. 8F). In contrast, the radicular pulp con-
tained atubular calcified tissues along the canal walls, where odonto-
blasts were completely absent (Fig. 8H–R). Cross sections of canals
taken from the mesial roots of mandibular molars revealed similar re-
actionary changes along the canal circumference with the exception of
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 1. Control tooth. (A) Radiograph of intact tooth 28 (upper left third molar) in a 29-year-old woman taken after extraction. (B) Photograph of the extracted tooth
after buccolingual sectioning to facilitate pulpal fixation. (C) Section cut approximately at the centre of the pulp chamber (haematoxylin and eosin, original
magnification ×16). (D) Cross-cut section of the root taken at the level of the line in (A) (original magnification ×25). (E) Detail from the left part of the large canal
(original magnification ×50). (F) High power view from the area of the left wall indicated by the horizontal arrow in (E). Dentinal tubules with a regular,
uninterrupted course traversing dentine and predentine. Normal odontoblast layer. Voids in the odontoblast layer are artefacts (original magnification ×400). (G)
High power view of the area of the pulp indicated by the vertical arrow in (E). A cross-cut vessel with an adjacent calcification (original magnification ×400).

the buccal and lingual aspects (Fig. 8K–P). In case of oval or circular
canals, the changes were present in variable portions along the canal
circumference, often concentrated in the proximity of neurovascular
bundles (Fig. 8Q–R).
3.2.3. Treated teeth with healed pulp
Teeth with medium and deep restorations with no pulp exposures,
histologically classified as healed pulps, showed uninflamed pulp
chamber tissues, with a band of tertiary dentine of variable thickness
subjacent to the cavity (Fig. 9). The tertiary dentine was atubular and
odontoblasts were absent. Cells with fibroblast characteristics and
abundant collagen fibres could be identified adjacent to the tertiary
dentine (Fig. 9E). No stainable bacteria could be seen in the cavities. In
the middle third of the roots, in some areas the canal walls were lined
by atubular tertiary dentine, and odontoblasts were absent (Fig. 9G–I).
The aforementioned morphologic features co-existed with areas

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Fig. 2. Control tooth. (A) Intact tooth

18 (upper right third molar) of a 29-
year old woman. (B) Buccolingual sec-
tion cut at the centre of the pulp
chamber (haematoxylin and eosin, ori-
ginal magnification ×16). (C)
Longitudinal section encompassing
apical root canal and foramen (original
magnification ×25). (D) Detail from
(C) (original magnification ×50). (E)
High power view of the area of the
apical canal wall indicated by the left
arrow in (D). Normal appearance of
dentine, predentine, and odontoblast
layer (original magnification ×400).
(F) High power view of the area of the
apical canal wall indicated by the right
arrow in (D). Tubular dentine is pre-
sent. Absence of predentine and odon-
toblast layer, in the absence of pulpal
inflammation. Cells adjacent the apical
canal walls do not resemble typical
odontoblasts (original magnification
×400).

exhibiting a normal pulp/dentine complex (i.e. tubular dentine with
predentine and an odontoblast layer) (Fig. 9G, J). This was particularly
evident in cross sections of the root canals (Fig. 9G). Cross sections of
the apical third of the root canals were often devoid of odontoblasts for
the entire canal circumference, with deposition of irregular atubular
calcified tissues (Fig. 9K, right insets). A few fibroblasts were present
subjacent to this calcified tissue, and a large part of the canal lumen was
occupied by neurovascular bundles (Fig. 9K) that contained niduses of
calcification (Fig. 9K, left inset).
3.2.4. Teeth that had undergone direct pulp capping
The four cases previously treated with pulp capping procedures
were all classified as with healed pulps (Table S4). Bacterial staining
did not reveal the presence of bacteria along the cavity walls. The
connective tissues in the pulp chamber were uninflamed, and the ex-
posure sites were repaired by an amorphous, atubular, irregularly-

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Fig. 3. Control tooth. Intact tooth 28 (upper left third molar) of a 21-year-old man. (A) Cross-cut section taken at the middle third of the mesiobuccal (MB) root. A
tiny MB2 canal can be seen adjacent to the MB1 canal (haematoxylin and eosin, original magnification ×25). (B) Detail of the MB1 canal (original magnification
×50). (C) Magnification of the area demarcated by the rectangle in (A). The MB2 canal appears enclosed in calcified tissue formed between two tubular dentine
walls, and is separated by two dark haematoxyphilic lines (original magnification ×100). D) High power view of the area of the canal wall indicated by the arrow in
(B). Tubular dentine ends abruptly into a layer of tertiary dentin with only a few dentinal tubules. No odontoblasts can be seen on the pulpal side. Inflammation is
absent (original magnification ×400).

calcified tissue lined by cells with flat-shaped morphology (Fig. 10C).
Description of the morphological features of calcified tissue repairing
carious pulp exposures is beyond the purpose of the present study. The
radicular dentine/pulp tissue complex exhibited changes that are si-
milar to those observed in the previous tooth categories. That is,
atubular irregular mineralized tissues were present along the canal
walls, and odontoblasts were absent from some areas of the canal cir-
cumference (Fig. 10D–H). In three of the four cases, diffuse calcifica-
tions were absent from the radicular connective tissues (Table S4).
4. Discussion
In the present study, dentine remodelling in the radicular pulp was
identified in only a few of the control intact teeth (28.6%), while si-
milar remodelling in the radicular pulp were observed in all (100%) of
the cases that were clinically diagnosed as irreversible and reversible
pulpitis, in restored teeth with healed pulps, and in teeth following
successful direct pulp capping. Interestingly, morphological aberrations
in these teeth occurred at sites that were far remote from the site of
insult (i.e. the coronal pulp); the most prominent features being the
deposition of atubular tertiary dentine on the root canal walls with
concomitant loss of the cells that formed this repair tissue. Two perti-
nent features could be identified for both groups of teeth. The first is
that a continuous layer of hard tissue-forming cells was frequently
missing from the pulpal side of the atubular tertiary dentine. Absence of
a continuous cell lining was confirmed by examination of serial sec-
tions, with the conclusion that the phenomenon was not caused by
artefacts produced during laboratory processing. Rather, the observa-
tion suggests that cell death occurred after deposition of the tertiary
dentine. The second feature is that when cells were present directly
beneath the tertiary dentine, they appeared flat in shape. Although
immunohistochemical examination had not been performed, those cells
were morphologically different from typical odontoblasts with elon-
gated cell bodies and processes present in the dentinal tubules [23].
The phenomenon in which primary odontoblasts decrease in size
and number, and disappear in certain areas of the pulp has previously
been reported in intact teeth. These changes were seen on the pulpal
floor over the bifurcation or trifurcation of multi-rooted teeth [24].
Changes in the odontoblast layer and formation of tertiary dentine were
also observed in impacted teeth [25]. Out of those 155 impacted teeth,
calcifications were identified in 30 cases and irregular dentine in 19
cases, both in the crown and roots. In addition, the dentinal tubules
appeared irregular when disturbance in dentine formation occurred.
Morphological changes along the dentine-pulp interface on canal
walls of teeth with inflamed pulps were previously reported in im-
mature teeth affected by caries [20]. Similar changes were identified
from the adult teeth examined in the present study. For the coronal pulp
that is directly beneath the site of caries involvement, it is under-
standable that primary odontoblasts are exposed to bacteria and their
by-products [26]. The latter include lipoteichoic acid from the cell wall

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Fig. 4. Irreversible histological pulpal changes. (A) Tooth 47 (lower right second molar) with secondary distal caries in a 51-year old man. (B) Photograph of the
extracted tooth after sectioning for pulpal fixation. (C) Section cut approximately at the centre of the pulp chamber (haematoxylin and eosin, original magnification
×2). (D) Distal part of the pulp chamber. A micro-abscess is present adjacent to the distal caries (Taylor’s modified Brown and Brenn stain, original magnification
×25). Inset. High power view of the area of the abscess indicated by the arrow. Bacterial aggregations surrounded by erythrocytes and inflammatory cells (original
magnification ×400). Inflammatory cells are absent beyond the coronal pulp. (E) Area of the coronal part of the distal canal indicated by the upper arrow in (C)
(original magnification ×50). (F) High power view from the left canal wall. Predentine is present, lined by a single layer of flattened cells, some of which can be seen
with a cell process extending into a dentinal tubule (original magnification ×400, inset ×1000). (G) Magnification of the area of the distal canal indicated by the
middle arrow in (C) (original magnification ×50). (H) Magnification of the area of the distal wall indicated by the left arrow in (G). Tissue closely resembling cellular
cementum is present. No odontoblasts are present (original magnification ×400). (I) High power view of the upper portion in (H). Cellular lacunae with canaliculi,
typical of cellular cementum (original magnification 1000). (J–K) Progressive magnifications of the area of the mesial wall indicated by the right arrow in (G).
Dentine is lined by acellular cementum, which is interrupted by multinucleated resorbing cells (odontoclasts), initiating resorption of dentine (original magnification
×400 and ×1000). (L–M) Progressive magnifications of the left wall of the apical third indicated by the lower arrow in (C). Connective tissue with thick collagen
bundles, closely resembling periodontal ligament, is present. Only fibroblast-like cells can be observed on the pulpal side. Typical odontoblasts cannot be identified
(original magnification ×400 and ×1000).

of gram-positive bacteria [27] and lipopolysaccharides from the cell
wall of gram-negative bacteria [28], both of which adversely affect the
secretory function of the odontoblasts [29,30]. Likewise, during cavity
preparation and air-drying of the exposed dentine, odontoblasts may
also be irreversibly injured by heat generated during dentine removal
[31], or by aspiration of their cell bodies into the dentinal tubules [32].
However, it is not readily apparent why similar changes also occurred
in the parts of the radicular pulp in all the symptomatic/restored teeth
examined.
Senescence of inherent and recruited cells within the dental pulp
may occur by different means, including necrosis, apoptosis or other
recently reported non-apoptotic pathways such as necroptosis, pyr-
optosis or nemosis [33–35]. The pathway responsible for the death of
the cells that approximate the tertiary dentine in the control teeth and
the symptomatic/restored teeth is likely to be apoptosis. Despite the
presence of inflammation in the coronal pulp in the carious teeth, sites
where odontoblasts were absent in the radicular parts of the dental pulp
were completely devoid of inflammatory cells. Apoptosis is a highly-
regulated, evolutionary-conserved form of programmed cell death that
plays a fundamental role in body tissue development and homeostasis
[36] as well as in diseases [37]. This form of cell death is frequently
identified from odontoblasts during tooth development, ageing and in
responses to inflammation and dental procedures. Apoptosis may be
initiated via the intrinsic or the extrinsic pathway. Both pathways in-
duce cell death by activating initiator caspases, which then activate
executioner caspases and subsequently kill the cell by indiscriminate
protein degradation. In the intrinsic (mitochondrial) pathway, the cell
kills itself upon sensing stress produced by various intrinsic lethal sti-
muli. Release of cytochrome C into the cytosol via mitochondrial outer
membrane permeabilization activates the caspase cascade and results in
programmed cell death. In the extrinsic (death receptor) pathway, the
cell kills itself after receipt of signal ligands derived from immune cells
via transmembrane death receptors, which, in turn, activate the caspase
cascade [38].
Using the transferase-mediated, dUTP-biotin nick end labelling
(TUNEL) assay for examination of DNA fragmentation as well as other
methods, apoptosis of the primary odontoblasts has been reported as
part of the ageing process in uninflamed dental pulps of healthy, virgin
teeth in rodents [39–42] and humans [39,43–46]. Such a process is
analogous to the process of removal of vestigial appendages and su-
perfluous cells in other parts of the body. As primary dentine is secreted
toward the pulp, the odontoblasts become crowded and assume a pa-
lisade relationship; some of these odontoblasts have to be eliminated by
apoptosis to resolve the crowding problem. Using a transgenic mouse

7
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 5. Irreversible histological pulpal changes. (A) Tooth

36 (lower left first molar) with deep caries and the clinical
diagnosis of irreversible pulpitis in a 16-year old woman.
Section cut through the distal canal (haematoxylin and
eosin, original magnification ×2). (B) Section cut through
the carious perforation of the pulp chamber. Bacteria colo-
nise the necrotic tissue in the mesial pulp horn. The tissue
above the mesial canal orifice is severely inflamed (Taylor’s
modified Brown and Brenn stain, original magnification
×16; inset ×100). (C) Middle magnification of the area of
the distal canal indicated by the right upper arrow in (A).
Moderate concentration of chronic inflammatory cells.
Predentine and an odontoblast layer can be recognized on
both sides (original magnification× 100). Inflammation is
absent apical to this region. (D) Middle magnification of the
area of the distal canal indicated by the right intermediate
arrow in (A). Inflammatory cells are absent (original mag-
nification ×100). (E) Higher magnification of the area de-
marcated by the rectangle in (D). Several elongated foci of
calcification can be seen in the pulp tissue. A nerve structure
is present on the right (original magnification ×400). (F)
High magnification of the lower calcified body demarcated
by the rectangle in (E). Numerous fibroblast-like cells are in
close contact with the body. Myelinated nerve fibres are
present on the right (original magnification ×1000). (G)
Middle magnification of the area of the distal canal in-
dicated by the right lower arrow in (A). Predentine and
odontoblasts are absent. The tertiary dentine (TD) is sepa-
rated from the secondary dentine (SD) by a calciotraumatic
line. Although hyperaemia is present, the tissue does not
contain chronic inflammatory cells (original magnification
×100). (H) High magnification of the rectangular area in
(G). The tertiary dentine contains only a few irregular
dentinal tubules. Fibroblasts and collagen fibres are present
on the pulpal side (original magnification ×400). (I) Middle
magnification of the area of the mesial canal at the same
level of the left arrow in (A). No odontoblast layer can be
seen on both canal walls (original magnification ×100).
(J–K) Progressive magnifications of the area of the left canal
wall indicated by the arrow in (I). Elongated cells with large
nuclei are in contact with the atubular tertiary dentine
(probably fibroblasts). The area is infiltrated by several
polymorphonuclear leukocytes (original magnification
×400 and ×1000).

model to induce overexpression of the anti-apoptotic protein “B-cell wild-type mice [47]. Because the dental pulp volume slowly decreases
lymphoma-2” (Bcl-2), primary dentinogenesis was found to be impaired due to the continuous deposition of secondary dentine, the number of
when apoptosis was inhibited, resulting in thinner and less dense odontoblasts has to be further eliminated to accommodate for the vo-
dentine deposition when compared with was observed for age-matched lume reduction. The massive reduction in odontoblast numbers at this

8
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

(caption on next page)

stage may result in the reorganization of the primary odontoblasts into the regeneration potential of the dental pulp is reduced with age.
a single layer along the pulpal surface of the predentine [43]. Because The aforementioned studies all involved apoptosis of the primary
programmed cell death involves both the primary odontoblasts and odontoblasts that lined the secondary dentine. This phenomenon is
odontoblast lineage-committed subodontoblastic daughter cells [45], different than what was observed in the present study, in that apoptosis

9
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 6. Irreversible histological pulpal changes. (A) Radiograph of tooth 38 (lower left third molar) with deep caries in a 20-year-old woman. (B) Section cut
approximately at the centre of the pulp chamber (haematoxylin and eosin, original magnification ×2). (C) Section proximal to that in (B). The pulp chamber contains
a micro-abscess; bacterial penetration can be identified in the mesial pulp horn (Taylor’s modified Brown & Brenn, original magnification ×16). Chronic in-
flammatory cells are present beneath the micro-abscess. The rest of the pulp chamber is devoid of inflammatory cells. (D) Area of the mesial canal indicated by the
arrow in (B). An odontoblast layer is present on the mesial canal wall, but is absent on the distal wall (original magnification ×100). (E) High magnification of the
distal canal wall in (D). Tubular secondary dentine (SD) is separated from the atubular tertiary dentine (TD) by a calciotraumatic line. Odontoblasts are absent. Cells
lining the tertiary dentine are fibroblast-like cells (original magnification ×400; inset ×1000). (F) Area of the distal canal indicated by the right arrow in (B). A pulp
stone embedded in the wall causes narrowing of the canal lumen (original magnification ×25). (G) Middle magnification from (F). No predentine and odontoblast
layer are present beneath the pulp stone (original magnification ×100). (H) Detail of the area demarcated by the rectangle in (F). Odontoblasts are present along the
right canal wall but are absent on the left wall (original magnification ×100). (I) High magnification of the left area in (H) shows atubular tertiary dentine and
absence of odontoblasts (original magnification ×400).

Fig. 7. Reversible histological pulpal changes. (A) Tooth 15 (upper right second premolar) with deep caries in a 52-year old woman. (B) Longitudinal section cut
approximately at the centre of the pulp chamber (haematoxylin and eosin, original magnification ×2). (C) Detail of the pulp chamber. Bacteria can be seen between
the secondary dentine and the large band of tertiary dentine. No bacteria are identified in the pulp chamber. Severe inflammation is present in the subjacent pulp
tissue (Taylor’s modified Brown & Brenn, original magnification ×100). (D) Magnification of the area of the orifice indicated by the arrow in (B). Dentine,
predentine, and odontoblast layer exhibit normal characteristics. No inflammatory cells are present (original magnification ×100). (E) Middle third of the root. The
section contains a lateral canal (original magnification ×16). (F) Magnification of the area demarcated by the rectangle in (E). Although dentine and predentine can
be observed in their normal relationship on the right canal wall, tertiary dentine (TD) is present on the left canal wall, overlying the secondary dentine (SD). The two
tissues are separated by a calciotraumatic line (original magnification ×100). (G) High magnification of the area demarcated by the rectangle in (F). The tertiary
dentin (TD) contains only a few dentinal tubules. No odontoblasts can be seen (original magnification ×400). (H) High magnification of the area of the left canal wall
indicated by the arrow in (E). The tertiary dentine tapers off apically. Irregular calcified bodies and collagen bundles can be seen on the pulpal side. Odontoblasts are
absent (original magnification ×400). (I–J) Very high magnification views from (H). The calcified bodies are in close contact with fibroblasts (original magnification
×1000). (K) Area where the lateral canal joins the main canal (original magnification ×100). (L) High magnification shows amorphous calcified structure that is
formed on the walls of the lateral canal (original magnification ×400; inset ×1000). (M) Apical part of the root (original magnification ×16). (N) High magni-
fication of the rectangular area in (M). Diffuse calcifications can be identified in the pulp tissue (original magnification ×100). (O) High magnification of the area of
the canal wall indicated by the left arrow in (N). Dentinal tubules in the secondary dentine end abruptly in an irregularly-calcified tertiary dentine. No odontoblasts
can be seen on the pulpal side (original magnification ×400; inset ×1000). (P) Very high magnification of the area indicated by the right arrow in (N), with
calcifications in the pulp tissue and the atubular calcified tissue on the root canal wall. The latter is devoid of odontoblasts. No chronic inflammatory cells can be
identified (original magnification ×400).

10
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 8. Reversible histological pulpal changes. (A) Radiograph of tooth 48 (lower right third molar) in a 58-yr-old man. The radiograph shows occlusal caries and
calculus on the mesial aspect. The tooth was asymptomatic and the pulp responded normal to sensitivity tests (thermal and electric pulp testing). (B) Radiograph of
the extracted tooth. (C) Occlusal view. (D) The tooth was sectioned along the mesiodistal plane to facilitate pulpal fixation. The photograph shows that caries involves
only superficial dentine. (E) Section cut approximately at the centre of the pulp chamber. This overview shows normal aspect of the pulp chamber with minimal
changes in the mesial pulp horn (Taylor’s modified Brown & Brenn, original magnification ×16). (F) Middle magnification of the mesial pulp horn. Thickness of the
predentine is slightly increased with reduction of the odontoblast layer (original magnification ×100). (G) Overlying carious dentine with dentinal tubules colonised
by bacteria (original magnification ×100). (H) Orifice and coronal portion of the mesial canal (haematoxylin and eosin, original magnification ×16). (I)
Magnification of the rectangular area in (H). No predentine can be seen at this level (original magnification ×100). (J) High magnification of the rectangular area in
(I), showing atubular tertiary dentine and absence of odontoblasts. Foci of calcification in the pulp tissue. Chronic inflammatory cells are completely absent (original
magnification ×400). (K) Cross-cut section from the mesial canal taken approximately at the level of the right line in (B). Only one flat canal is present in the mesial
root (original magnification ×16). (L–M) Medium magnification of the buccal and the lingual portion of the canal, respectively. Normal predentine and odontoblast
layer can be seen only on the extreme portions of the canal (original magnification ×100). (N) The central portion of the canal in (K) showing absence of
odontoblasts (original magnification ×100). (O) High magnification from the upper canal wall in (N). The secondary dentine is covered by an haematoxyphilic layer,
without odontoblasts (original magnification ×400). (P) High magnification from the lower canal wall in (N), showing atubular dentine and the absence of
odontoblasts (original magnification ×400). (Q) Cross-cut section from the distal canal taken at approximately the level of the left line in (B). Predentine and the
odontoblast layer can be observed only for about two thirds of the canal circumference (original magnification ×100). (R) High magnification of the area indicated
by the arrow in (Q). Atubular tertiary dentine is deposited over the tubular secondary dentine. The tertiary dentine is in close contact with a neurovascular bundle.
No odontoblasts can be identified along the pulpal side of the tertiary dentine (original magnification ×400).

purportedly occurred in cells that formed tertiary dentine in the roots of
some of the intact teeth. Although formation of tertiary dentine in in-
tact teeth has been perceived as a manifestation of ageing [48–50], 3
out of the 4 teeth that had tertiary dentine deposited in the root canals
were contributed by young individuals in their early twenties (Table
S1). Hence, apoptosis of tertiary dentine forming cells at distant root
locations have to be confirmed with TUNEL assay in our future work. If
the formation of tertiary dentine is perceived as a repair response to
injury [4,51], the sites where tertiary dentine was formed in the roots of
intact, uninflamed teeth are likely to be mineralised scars produced in
response to previous injuries to those roots. These injuries may be
caused by excessive masticatory stresses [52], trauma [53] or ortho-
dontic movement [54]. Persistence of these injuries may trigger apop-
tosis of the cells that produce tertiary dentine, in the absence of in-
flammation. For example, prolonged orthodontic traction has been
associated with decreased expression of Bcl-2 in odontoblasts, resulting
in their apoptosis [55,56]. At the cellular level, these physical stresses
may be translated into intrinsic lethal stimuli such as cytokine depri-
vation, DNA damage, endoplasmic reticulum stress, hypoxia (ischemia)
and metabolic stress [57], that induce apoptosis of the tertiary dentine-
forming cells via the intrinsic apoptotic pathway.
Whereas dentine remodelling in the roots was identified in only a
few of the control intact teeth, similar manifestations were seen in all
the symptomatic/restored teeth, with practically no inflammation
identified in those specific sites. It is not known whether the mechanism
responsible for these morphological changes in the radicular pulp are
different from those that trigger the aberrations in the coronal part of
the pulp chamber. Unlike the coronal caries process, it is the odonto-
blast cell body and not the odontoblast process that is directly chal-
lenged by noxious stimuli. Both lipoteichoic acid and lipopolysacchar-
ides produced by cariogenic bacteria have the potential to induce
apoptosis in dental pulp cells [58,59]. Ischemia produced in the pulp
[60] and nitric oxide produced by inflammatory cells recruited to the
infected site [61] may also trigger apoptosis of the primary odonto-
blasts or odontoblast-like cells beneath the caries-infected coronal
dentine. Unlike apoptosis of odontoblasts in uninflamed intact teeth
that only involve the intrinsic apoptotic pathway, both the intrinsic and
the extrinsic apoptotic pathways are activated in dental caries [62].
This may result in more wide-spread apoptosis in the pulps of carious
teeth compared with non-carious teeth. Pro-inflammatory mediators
are upregulated in the odontoblast layer by carious bacteria to mediate
the host inflammatory response to caries [63,64]. Some of these upre-
gulated molecules, such as tumour necrosis factor α (TNF-α) may ac-
tivate TNF-α-related apoptosis-inducing ligand (TRAIL) death receptors

11
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 9. Tooth with histological features of a healed dental pulp. (A) Radiograph of tooth 45 (lower right second premolar) in a 58-yr-old man. The radiograph shows a
deep occlusal restoration, with calculus deposition and periodontal bone loss. The pulp responded normally to sensitivity tests (thermal and electric pulp testing). (B)
Radiograph of the extracted tooth taken in the mesiodistal direction. (C) Occlusal view. (D) The tooth was ground along the buccolingual plane to facilitate pulpal
fixation. The photograph shows that the deepest part of the cavity had been covered with a white direct pulp capping agent (partly dissolved) and a layer of glass-
ionomer cement. The cavity was restored with resin composite. (E) Section cut approximately at the centre of the pulp chamber. The overview shows that the pulp is
uninflamed and a large band of tertiary dentine is present below the cavity. The tertiary dentine is atubular and lined by fibroblast-like cells with ample collagen
fibres. Odontoblasts are absent along the pulpal side of the tertiary dentine (haematoxylin and eosin, original magnification ×16; inset ×400). (F) Cross-cut section
taken at the level of the upper line in (B). An intact layer of cementum with periodontal ligament fragments can be seen along the mesial and distal side of the
extracted tooth (original magnification ×16). (G) Detail of the canal. Normal predentine and an odontoblast layer can be discerned only on the buccal and lingual
sides (original magnification ×50). (H) High magnification taken from the area of the distal side of the canal indicated by the upper arrow in (G) showing deposition
of irregular atubular tertiary dentine and the absence of odontoblasts (original magnification ×400). (I) High magnification taken from the area of the distal side of
the canal indicated by the lower arrow in (G), showing irregular atubular tertiary dentine lined by fibroblast-like cells only. Collagen fibres are abundant in the pulp
(original magnification ×400). (J) High magnification taken from the area of the lingual side of the canal indicated by the right arrow in (G). Dentinal tubules run
parallel to each other uninterruptedly from the dentine and predentine. A palisade layer of odontoblasts is evident (original magnification ×400). (K) Cross-cut
section taken at the level of the lower line in (B). Atubular tertiary dentine without odontoblasts can be seen along the entire canal circumference. The bulk of the
dental pulp is occupied by a neurovascular bundle containing a nidus of early calcification (original magnification ×100; right insets ×400; left inse ×1000).

in the odontoblasts to activate the extrinsic apoptotic pathway [38,62].
Progressive diffusion of pro-inflammatory mediators through the pulpal
connective tissues may induce injury in the roots of those teeth and
stimulate repair by tertiary dentine and apoptosis of the tertiary den-
tine-forming cells in the manner previously described for the intact
teeth.
Similar to dentinal caries, cavity preparation also causes damage to
the odontoblasts by inducing apoptosis [65]. Two waves of apoptosis
are induced in odontoblasts after cavity preparation, and the apoptotic
cells had to be eliminated by phagocytosis prior to the initiation of
reparative dentinogenesis [66]. Larger cavity preparations are more
prone to apoptosis induction during pulp wound healing than smaller
preparations [67]. Although heat stress induces death signals that
trigger apoptosis [68], dental pulp cells are relatively resistant to heat
stress due to upregulation of heat shock proteins [69,70]. During re-
storation of carious teeth, signalling molecules such as bone morpho-
genetic protein and transforming growth factor-beta1 (TGF-β1) are
released from bacterial acid, inorganic acid or acidic resin monomer-
demineralised dentine [71,72] or highly-alkaline pulp capping agents
[73,74], These cytokines are capable of inducing apoptosis of dental
pulp cells [75–77]. Incompletely-polymerised resin monomers derived
from adhesives and resin composites, such as HEMA, 4-META/MMA-
TBB, TEGDMA and Bis-GMA, are cytotoxic and induce apoptosis in
human dental pulp cells via the formation of reactive oxygen species
[75,78–81]. We speculate that diffusion of apoptosis-inducing signal-
ling biomolecules and reactive oxygen species through the pulpal
connective tissues into the root canal system may be responsible for the
more profuse dentine remodelling seen in the roots of restored carious
teeth as well as in healed teeth that had undergone successful direct
pulp capping. Nevertheless, it is inexplicable why only some parts of the
roots were remodelled instead of the entire root, and why only a seg-
ment of the cross sections of the root was affected instead of entire
circumference.
With respect to the observation of flat-shaped cells that approxi-
mated the pulpal side of the radicular tertiary dentine, there are two
possibilities. On one hand, one may conjecture that these cells are fi-
broblasts. Although fibroblasts secrete extracellular collagen matrices,
they do not usually participate in the mineralisation of those matrices.
Nevertheless, fibroblasts participate in ectopic soft tissue calcification
when they are appropriately stimulated [82]. When exposed to elastin
degradation products (derived from blood vessel walls) and TGF-β1, rat
dermal fibroblasts developed gene and protein profiles with osteogenic
characteristics, contributing to vascular calcification [83]. The osteo-
genic characteristics of fibroblasts induced by the two aforementioned

12
D. Ricucci et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 10. Post-direct pulp capping. (A) Tooth 37 (lower left second molar) in a 16-yr-old woman. Exposure of the distal pulp horn occurred during excavation of the
deep occlusal caries. The pulp was capped with calcium hydroxide paste and the tooth was restored with resin composite. (B) After 19 months, fracture of the distal
wall occurred in the tooth up to bone level. A radiograph showed normal periapical conditions and the pulp responded normally to sensitivity tests. The tooth was not
restorable because of the fracture and was subsequently extracted. (C) Section cut approximately at the centre of the pulp chamber. The overview shows extensive
tertiary dentine deposition and uninflamed pulpal tissues (haematoxylin and eosin, original magnification ×16). (D) Cross section of the mesial root taken at
approximately the level of the left line in (B) (original magnification ×16). (E-F) Middle magnifications of the lingual and buccal canals. Odontoblasts are absent
from a large portion of the canal circumference (original magnification ×100; insets ×400). (G) Cross section of the distal root taken at approximately the level of
the right line in (B) (original magnification ×16). (H) Middle magnification reveals that normal appearing predentine, dentine and odontoblasts can be identified
from only two-thirds of the canal circumference (original magnification ×100).

components were further amplified in the presence of high glucose
(simulating diabetes mellitus) [84,85]. Both elastin degradation pro-
ducts and TGF-β1 are likely to be present in coronally-inflamed pulps of
partially-demineralised carious dentine, in teeth restored by acid-
etching, or in exposed pulps that have undergone direct pulp capping.
On the other hand, it is also possible that those flat-shaped cells
represent undifferentiated dental pulp stem cells, These cells may have
migrated to the injured site but without undergoing osteogenic differ-
entiation. Mesenchymal stem cells derived from dental tissues possess
fibroblast-like morphology prior to their differentiation into phenotype-
committed, specialised cells [86]. The cytokine TNF-α is present in
abundance in coronally-inflamed dental pulps; osteogenic differentia-
tion of dental pulp stem cells is suppressed in the presence of high doses
of TNF-α [87,88]. One of the limitations of the present study is that
immunohistochemical staining had not been performed on the histo-
logical sections. Without identifying the characteristics of the tertiary
dentine forming cells or dentine-specific proteins associated with those
cells, it is inappropriate to over-speculate on the nature of those cells
based on morphological observation at the light microscopy level alone.
This should be contemplated in future studies.
5. Conclusion
Within the limits of the present histological and histopathological
study, it may be concluded that any tooth treated for caries will exhibit
pulpal morphological changes in response to the previous pathology for
life. Atubular tertiary dentine and loss of the original number of
odontoblasts remain permanently in that pulp as a permanent scar. The

13
D. Ricucci et al.
radicular pulp appears to be affected by this reduction earlier and more
severely than the coronal pulp. When challenged by bacteria and bac-
terial by-products invading dentinal tubules, odontoblasts in the radi-
cular pulp may undergo cell death, possibly by apoptosis. This phe-
nomenon may be caused by root-ward diffusion of bacterial by-
products, pro-inflammatory mediators, cytokines or reactive oxygen
species through the pulp connective tissue. From a clinical perspective,
although the vitality of the dental pulp may be maintained with direct
pulp capping or pulpotomy, the repair tissue that is formed resembles
mineralised fibrous scar tissue instead of true tubular dentine.
Conflicts of interest
None.
Acknowledgements
The authors had no financial support for the conduct of the research
and/or preparation of the article.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.jdent.2018.04.007.
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