Journal of Dentistry 100 (2020) 103430

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

Pulp and dentine responses to selective caries excavation: A histological and T

histobacteriological human study
Domenico Ricuccia,**, José F. Siqueira Jr.b,c, Isabela N. Rôçasb,c, Mariusz Lipskid, Amal Shibane,
Franklin R. Tayf,*
a
Private Practice, Cetraro, Italy
b
Department of Endodontics, Faculty of Dentistry, Grande Rio University (UNIGRANRIO), Rio de Janeiro, RJ, Brazil
c
Department of Endodontics and Dental Research, Iguaçu University (UNIG), Nova Iguaçu, RJ, Brazil
d
Department of Preclinical Conservative Dentistry and Preclinical Endodontics, Pomeranian Medical University, Szczecin, Poland
e
Department of Restorative Dental Science, Faculty of Dentistry, King Khalid University, Abha, Saudi Arabia
f
Department of Endodontics, The Dental College of Georgia, Augusta University, Augusta, GA, USA

ARTICLE INFO ABSTRACT

Keywords: Objective: The present study investigated the histobacteriological condition of human carious dentine, and the
Bacteria histological response of dental pulps after selective caries excavation to firm dentine and cavity restoration with
Caries adhesive procedures.
Dentine Methods: Twelve vital teeth with medium/deep occlusal caries from 12 patients were scheduled for extraction.
Pulpal response
The patients gave consent to have caries removed selectively and the cavity restored with adhesive procedures
Selective excavation
prior to extraction. Caries excavation was achieved using burs and sharp hand excavators until “leathery” or
“firm” dentine was encountered. After extraction, the teeth were completely-demineralised, processed for light
microscopy, serial-sectioned and stained with haematoxylin and eosin staining for histological examination of
dentine characteristics and pulpal responses. Additional sections were stained with Taylor-modified Brown and
Brenn technique for histobacteriological examination of bacteria infiltration of the dentinal tubules and dental
pulp.
Results: The 12 teeth showed varying degrees of tertiary dentine formation. Chronic inflammatory cell infiltrates
were identified in the pulp of all specimens and appeared as scattered inflammatory cells or exiguous localised
accumulations. Capillaries were heavily congested with erythrocytes and polymorphonuclear leukocytes. A large
amount of stainable bacteria was observed in the dentine subjacent to the cavity floor in all specimens.
Conclusions: The present study demonstrated that “leathery” or “firm” carious dentine is infected. The remnant
bacteria in the dentine provoked subclinical pulpal inflammation over the entire evaluation period. The presence
of potentially-arrested caries does not necessarily mean that bacterial infection is absent or under control.
Clinical significance: Knowledge on the pulpal response to active caries and the inflammatory responses asso­
ciated with bacteria ingress into dentine is paramount in helping clinicians make an informed, rational choice
based on biologically-robust principles.

1. Introduction
Dental caries is a destructive disease of the oral cavity. If left un­
treated, it may cause irreversible damages to the dentine substrate,
dental pulp and the tooth periapex. Controversies exist in the literature
concerning the role and impact of dentine and pulpal reactions that
occur during caries attack. Many authors considered caries a progres­
sively destructive process [1–5]. Others opined that caries is a
productive process that creates tertiary dentine to protect the dental
pulp from invasion by noxious agents [6,7]. Treatment recommenda­
tions based upon these disparate views differ considerably. Whereas the
first group mandated total removal of soft caries in one or more ap­
pointments, the other group advocated leaving carious dentine beneath
the restoration.
The idea of leaving carious dentine on top of the pulp dated back to
the seminal work of Pierre Fauchard (1678–1761), a French physician

⁎
Corresponding author.
⁎⁎
Corresponding author at: PiazzaCalvario 7, 87022 Cetraro, Italy.
E-mail addresses: dricucci@libero.it (D. Ricucci), ftay@augusta.edu (F.R. Tay).

https://doi.org/10.1016/j.jdent.2020.103430
Received 21 May 2020; Received in revised form 7 July 2020; Accepted 11 July 2020
Available online 13 July 2020
0300-5712/ Published by Elsevier Ltd.
D. Ricucci, et al.
who was credited as the “father of contemporary dentistry”. While
Fauchard was acclaimed for his fearless proposition to the peers of his
era that the tooth worm theory was not adept at explaining the cause of
dental decay, his rationale for leaving caries was based on his appre­
hension of exposing “nerves” in a tooth that rendered treatment out­
come worse than the original disease [8]. Realising that the cause of
dental decay was sugar, he recommended using human urine (viz am­
monia) for treatment of early caries, although the regime was met with
harsh resistance by physicians and patients.
Dr. Maury Massler made the distinction between active and arrested
carious lesions [6,9]. Whereas dentinal tubules in active caries are
open, those present in arrested lesions are occluded in the calciotrau­
matic zone, the boundary between secondary dentine and tertiary
dentine in arrested caries. According to Massler, this zone could not be
penetrated by irritants derived from the oral cavity. Dr. Margaret Byers
and colleague observed in rat teeth that sclerotic tertiary dentine is
impermeable to relatively large fluoro-gold nanoparticles, and that the
tertiary dentine forms an effective barrier against the penetration of
toxic materials into the pulp [10]. The existence of a superficial layer of
caries-affected dentine was reinforced by Dr. Takao Fusayama, who
stated that “when caries penetrates dentine, softening is always the
deepest, discolouration is the next and bacterial invasion is the last”
[11]. Even though bacteria remain in the softened dentine during
partial caries excavation, these organisms are entrapped within a
“stressful environment”, being secluded from nutrients derived from the
oral environment and the dental pulp by tubular sclerosis and re­
parative dentine [12].
Since their inception, some of the aforementioned claims have been
critically examined. Permeability of the calciotraumatic line and ter­
tiary dentine in chronic or arrested carious lesions have been identified
in histological studies; penetration of bacteria, stains, silver nitrate or
radiographic markers have been observed in vivo [13]. Bacteria have
been routinely observed in “leathery” dentine [14] and in specimens
taken from the hard carious dentine [5,14–17].
A clinical approach based on the basic pathological tenet that bac­
teria left in the proximity of a wound are detrimental for tissue healing
has prevailed among many clinicians. Surveys conducted in different
countries indicate that the majority of dentists prefer a non-selective
approach for caries removal [18–22]. Among the dental specialties,
endodontists are more inclined toward complete caries excavation,
compared to general practitioners and paediatric dentists [21]. Never­
theless, the notion of non-selective caries removal has been aggressively
challenged in recent years, with inadvertent pulpal exposure considered
as a highly-unfavourable prognostic factor [12,23–26]. Because of the
perceived metabolic changes in bacteria that persist in caries-demi­
neralised dentine [27], a different clinical approach, referred to as se­
lective caries removal or indirect pulp capping, has been advocated
[12,27,28]. The technique consists of removing only the superficial
layers of soft dentine, and leaving carious dentine that is firm and
leathery over the pulpal surface; the soft dentine along the enam­
odentinal junction is removed in a non-selective manner with low-speed
burs until the underlying dentine is hard on probing [12,28].
The International Caries Consensus Collaboration, a group of 21
cariology experts from 12 countries, assembled in Leuven Belgium in
2015 to discuss strategies on the management of carious lesions [29].
The group reached a consensus that complete excavation of carious
tissue to hard dentine is over-treatment and favoured selective, step­
wise removal of carious tissue [30]. More recently, the European So­
ciety of Endodontology published a position statement that further re­
inforces selective removal of carious dentine in teeth with reversible
pulpitis [31]. The rationale for selective caries excavation is based on
the results derived from a few short/medium-term randomised clinical
trials [28,32]. These trials were of the consensus that bacteria, once
isolated from their source of nutrition by a restoration of sufficient
integrity, either die or remain dormant, thereby posing minimal risk to
the health of the dentition [27]. The results of those trials were
2
Journal of Dentistry 100 (2020) 103430
buttressed by clinical studies that identified no detrimental pulpal ef­
fects associated with sealing in of bacteria [33]. Adversaries to these
esteemed consensus reports, however, deplored that definitive evidence
is lacking on the fate of the residual micro-organisms in the retained
carious dentine. This group enunciated the necessity to simultaneously
perform microbiological studies to scrutinise the viability of bacteria
that remain in carious dentine, and histologic studies to examine the
responses of the underlying pulp [30]. To resolve this issue, the present
study strived to investigate the histobacteriological condition of human
carious dentine, and the histological response of dental pulps after se­
lective caries excavation to firm dentine and cavity restoration with
adhesive procedures. The hypothesis tested was that selective excava­
tion arrests carious progression and prevents infection of dental pulp
without creating adverse pulpal reactions.
2. Materials and methods
The present study included 12 carious teeth from 12 human subjects
(8 females, 4 males). Their ages ranged from 18 to 62 years (mean 35
years). Four intact third molars (two maxillary and two mandibular)
were used as the control. The carious teeth were scheduled to be ex­
tracted for prosthetic or orthodontic reasons. Only teeth meeting the
following criteria were selected:
a) presence of an exclusive occlusal caries lesion with intact inter­
proximal surfaces, as evidenced from periapical and bitewing
radiographs;
b) caries lesion infiltrating 1/2 of the occluso-gingival dentine thick­
ness as evidenced from radiographs (designated as medium caries),
or 2/3 or more of the dentine thickness (designated as deep caries);
c) absence of spontaneous pain in the dental history; the tooth was
asymptomatic and diagnosed as reversible pulpitis based on clinical
examination, thermal/electric pulp tests, and radiographs;
d) periodontal probing depth not exceeding 5 mm; and
e) the tooth could be isolated with rubber dam.
The study was conducted in accordance with ethical principles,
including the World Medical Association Declaration of Helsinki (ver­
sion 2008). Each patient received thorough explanation on the purpose
of the study, clinical procedures, possible discomfort and complica­
tions. All patients gave written consent. The experimental protocol was
approved by the Institutional Review Board.
2.1. Operative procedures
Periapical and bite-wing radiographs were taken for all teeth prior
to the experimental procedures. The clinical crown of each tooth was
polished for 1 min with a rubber cup and prophylaxis polishing paste.
After anaesthesia and rubber dam isolation, photographs of the occlusal
surface were taken. All clinical procedures were performed using a
magnifying device (EyeMag Pro 4x, Carl Zeiss Meditec Dentistry,
Oberkochen, Germany) or an operating microscope (OPMI PROergo,
Carl Zeiss Meditec AG, Germany).
The enamel cavitation was enlarged with high-speed cylindrical
diamond burs with water spray. Superficial soft dentine was removed
with stainless steel round burs in a low-speed hand piece with water
cooling. Selective caries excavation was achieved using sharp hand
excavators until “leathery” or “firm” dentine was encountered, with
resistance to hand excavation [30]. Soft dentine long the enam­
odentinal junction was removed using low-speed burs, until the dentine
was hard (scratchy) on probing [12,30].
After caries excavation, each cavity was restored with a nanohybrid
resin composite using a 3-step dentine adhesive system. The restora­
tions were finished and polished. Photographs were taken during all
phases of the restorative procedures. Patients were instructed to report
post-operative sensitivity or discomfort. The teeth were extracted after
D. Ricucci, et al. Journal of Dentistry 100 (2020) 103430

observation periods ranging from 1 to 9 months. Thermal and electric a Presence of stainable bacteria in the dentine overlying the pulp:
pulp tests were performed prior to extraction. Periapical radiographs some tubules occasionally colonised by bacteria (+); limited
and intraoral photographs of the occlusal surface were also taken before segments of dentine with a large number of tubules filled with
extraction. The teeth were extracted as atraumatically as possible, bacteria (++); large segments of dentin heavily filled with bac­
taking care to avoid root fracture. Immediately after extraction, each teria, with areas where dentinal tubular structure was lost (++
tooth was gently washed to remove blood and saliva and then immersed +);
in 10 % neutral buffered formalin. b Capillaries filled with blood in the odontoblastic and sub-odon­
toblastic layers underneath the cavity, indicating hyperaemia and
2.2. Histological procedures circulatory disturbances: yes/no;
c Presence of acute (neutrophilic leukocytes) and/or chronic in­
To facilitate pulp tissue fixation, each tooth root was severed 3 mm flammatory cells: lymphocytes, macrophages, plasma cells, for­
apical to the cementoenamel junction, using a diamond disk with co­ eign body giant cells) in the odontoblast and subodontoblast
pious water cooling. Each tooth was abraded with a high-speed cy­ layers (scattered inflammatory cells (+); mild, localised accu­
lindrical diamond bur with water spray in a mesiodistal or buccolingual mulations of mostly chronic inflammatory cells (++); moderate
direction until one or two pulp horns were exposed. Photographs were accumulations of chronic inflammatory cells (+++); severe ac­
taken of each tooth specimen prior to the successive step. cumulations of acute and chronic inflammatory cells (++++);
The specimens were completely demineralised for 4 weeks in 22.5 d Increase of leukocytes in the blood vessels afferent to the pulp
% (v/v) formic acid and 10 % (w/v) sodium citrate, with the end-point beneath the cavity, which is indicative of chemotaxis: yes/no.
determined radiographically. When the enamel was completely dis­
solved, the resin composite restoration that firmly adhered to the un­
3. Results
derlying dentine was removed with a high-speed round diamond bur
using copious water spray. The procedure was conducted under an
No patient reported post-operative discomfort. All teeth examined
operating microscope to avoid removal of dentine from the cavity floor.
in the present study were asymptomatic during the varying observation
The specimens were rinsed in running tap water for 24 h, dehydrated
periods and responded within normal limits to thermal and electric
using ascending grades of ethanol (50 %–100 %), cleared in xylene, and
pulp tests immediately prior to local anaesthesia for tooth extraction.
embedded in paraffin. Five micrometre-thick serial sections were cut
All teeth were extracted without complications. No specimens were lost
using a microtome until the entire pulp tissue in the chamber was ex­
during histologic processing and the staining protocols achieved op­
hausted. Six to eight sections were collected on each slide. Every fifth
timal results.
slide was stained with haematoxylin and eosin (H&E) for evaluation of
The four control intact teeth showed dental pulps with no changes
pulp changes. These stained sections were used to locate the areas ex­
in the dentine/predentine/odontoblast complex. Dentinal tubules could
hibiting the most severe inflammatory reactions. Additional slides were
be followed running parallel to each other through dentine and pre­
stained as needed. Selected slides were stained using the Taylor-mod­
dentine, with no reduction in numbers. Palisading layers of odonto­
ified Brown and Brenn technique for bacteria identification. The slides
blasts were observed with no reduction in the dimensions of these cells.
were examined using a light microscope (DMLB; Leica Microsystems,
No tertiary dentine or other calcifications were present. The pulp tissues
Wetzlar, Germany) for acquisition of digital photographs.
were free of inflammatory cell accumulations or dilated vessels. Brown
and Brenn staining did not reveal bacteria in the circumpulpal dentine.
2.3. Histological criteria

The following features were evaluated during histological ex­
amination (Table 1):
1 Changes predominantly attributable to the pre-existing caries lesion
2) Presence/absence of irregular tertiary dentine on the pulpal side
where dentinal tubules affected by the caries lesion ended in the
pulp;
3) Reduction in the odontoblast layer.
4 Changes attributable exclusively to persisting dentinal infection
3.1. Tertiary dentine
In the experimental group, all 12 teeth showed layers of tertiary
dentine with varying thickness (Table 1). Tertiary dentine was clearly
demarcated from the adjacent unaffected secondary dentine (Figs. 1
H–I; 2 G–H; 3 K; 4 F–G, I and K). The transition from secondary to
tertiary dentine was characterized by a dark calciotraumatic line in H&
E-stained or Brown and Brenn-stained sections (Figs. 1–4). The tertiary
dentine contained a reduced number of tubules; the tubules were
sparsely-distributed and ran an irregular course. On the pulpal side, the

Table 1
Demographics of the specimens and information on their pulpal histology and bacteria status.
Case Sex Age Tooth Clinical Obser- Post- Pulp Tertiary Reduction of Stainable Inflammatory cells Capillaries PMNs in
number evaluation of vation opertaive response to dentine odontoblast bacteria in filled afferent
caries period symptoms tests prior to layer dentine vessels
extraction

1 F 43 32 Medium 9 m No Normal Yes Yes +++ ++ Yes Yes

2 F 45 32 Medium 1 m No Normal Yes Yes +++ + Yes Yes
3 F 42 32 Medium 3 m No Normal Yes Yes ++ + Yes Yes
4 F 34 1 Medium 3 m No Normal Yes Yes +++ + Yes Yes
5 F 18 32 Medium 1 m No Normal Yes Yes ++ ++ Yes Yes
6 F 62 17 Medium 1 m No Normal Yes Yes ++ + Yes Yes
7 M 29 1 Deep 1 m No Normal Yes Yes +++ + Yes Yes
8 M 19 1 Medium 8 m No Normal Yes Yes ++ + Yes Yes
9 F 26 17 Medium 6 m No Normal Yes Yes ++ + Yes Yes
10 M 38 1 Deep 2 m No Normal Yes Yes +++ ++ Yes Yes
11 F 32 1 Medium 2 m No Normal Yes Yes +++ ++ Yes Yes
12 M 37 16 Medium 5 m No Normal Yes Yes +++ ++ Yes Yes

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D. Ricucci, et al. Journal of Dentistry 100 (2020) 103430

Fig. 1. Medium caries (Case 1, Table 1) (A) Pre-opera­

tive radiograph. (B) Pre-operative occlusal view. (C)
Photograph taken after non-selective caries excavation,
conditioning and bonding procedures. (D) Photograph
taken after restoration and polishing. (E) Post-operative
radiograph. (F) 9-month follow-up radiograph. (G)
Photograph taken after preparation of a mesiodistal
sectioning plane to allow pulp fixation. (H) Section cut
in the buccal portion of the pulp chamber, not encom­
passing the canal orifices (haematoxylin and eosin (H&
E), original magnification ×16). (I) Detail of the mesial
portion of the pulp chamber. Accumulation of cells with
dilated vessels can be observed (original magnifications
×50). (J) High magnificaton view from the centre of the
cellular concentration in (I). Mononuclear inflammatory
cells and Russel bodies (original magnifications ×400;
inset ×1000). (K) Dentine overlying the mesial portion
of the pulp chamber (Taylor’s modified Brown & Brenn,
original magnification ×16). (L) Semi-high magnifica­
tion of the area of the superficial dentine demarcated by
the rectangle in (K). The dentine is lined occlusally by a
red fuchsine-stained line, representing the hybrid layer.
Severe dentinal infection can be identified below (ori­
ginal magnification ×50). (M) High magnification view
of the rectangular area in (L). Lateral branches of the
dentinal tubules are filled with bacteria. A vertical
pocket engulfed with bacteria is observed on the right
(original magnification ×400). (For interpretation of
the references to colour in this figure legend, the reader
is referred to the web version of this article).

original odontoblast palisade layer in the affected areas was reduced in 3.2. Inflammatory cells
thickness. The odontoblasts sometimes appeared as a single layer of
flattened cells with small cell volume (Figs. 2G and H; 4 G). Inflammatory cells were identified in the pulp connective tissue in
all specimens. These appeared as scattered inflammatory cells (7 cases)

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D. Ricucci, et al. Journal of Dentistry 100 (2020) 103430

Fig. 2. Medium caries (Case 2, Table 1) (A) Pre-operative

radiograph. (B) Pre-operative occlusal view. (C) The cavity after
“selective” caries excavation. (D) Photograph taken at comple­
tion of the restoration. (E) 1-month follow-up radiograph, taken
just before extraction. (F) Preparation of a mesiodistal sec­
tioning plane. Adaptation of the resin composite restoration is
optimal. (G) Section cut approximately at the centre of the pulp
chamber. Note the large band of tertiary dentine and the pulp
stones. Inset shows high magnification of the area of the mesial
wall indicated by the arrow, with a dilated capillary in the
subodontoblastic zone (H&E, original magnification ×16; inset
×400). (H) Detail from the pulp chamber roof, with the tertiary
dentine (TD) separated from the secondary dentine (SD) by the
so-called calciotraumatic line. Note the reduced odontoblast
layer (original magnification ×50). (I) Distal root canal orifice
showing an arteriole in which a number of polymorphonulear
leukocytes can be observed (original magnification ×100; inset
×400). (J) Section proximal to that in (G) (Taylor’s modified
Brown & Brenn, original magnification ×16). (K) Magnification
of the area demarcated by the rectangle in (J). Deep colonisa­
tion of the dentinal tubules by bacteria (original magnification
×100). (L) High magnificaiton view of the rectangular area in
(K) (original magnification ×400).

and mild accumulations (5 teeth). Moderate or severe inflammation known as Russel bodies (Fig. 1J).
was not observed in any of the 12 specimens. The inflammatory cells
were predominantly lymphocytes and plasma cells (Fig. 1H–J), some of
which exhibited occasional eosinophilic inclusions in their cytoplasm

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D. Ricucci, et al. Journal of Dentistry 100 (2020) 103430

Fig. 3. Medium caries (Case 4, Table 1) (A) Pre-operative radiograph.

(B) The cavity after “selective” caries excavation. (C) Photograph
taken after completion of restoration. (D) 3-month follow-up radio­
graph, just before extraction. (E) A buccolingual sectioning plane was
prepared. Optimal adaptation of the restorative material. (F) Section
taken approximately at the centre of the pulp chamber. Overview. (H&
E, original magnification ×16). (GeH) Other section. Progressive
magnifications from the mesiobuccal pulp horn. A hyperaemic vessel
with several polymorphonuclear leukocytes in its lumen (original
magnifications ×100 and ×400). (IeJ) Progressive magnifications
from the palatal pulp horn. Hyperaemic vessels in the sub­
odontoblastic area (original magnifications ×100 and ×400). (K)
Section proximal to that in (F). The encircled area, magnified in the
inset, shows a thin band of tertiary dentine (Taylor’s modified Brown
& Brenn, original magnification ×16; inset ×400). (L) Detail of the
cavity floor with underlying dentine, which is heavily infected (ori­
ginal magnification ×50). (M) High magnification view of the area of
the cavity floor demarcated by the lower rectangle in (L) showing the
hybrid layer (red-stained) and the subjacent infected dentine (original
magnification ×400). (N) High magnification view of the upper rec­
tangular area in (L). Dentinal tubules clogged with bacteria (original
magnification ×400). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this
article).

3.3. Capillaries sections or longitudinal sections. These capillaries were heavily filled
with erythrocytes in the subodontoblastic region (Table 1; Figs. 2 G; 3
The dental pulp in all the 12 specimens showed varying numbers of I–J).
capillaries. Depending on their course, the capillaries were cut in cross

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D. Ricucci, et al. Journal of Dentistry 100 (2020) 103430

Fig. 4. Deep caries (Case 7, Table 1) (A) Pre-operative

radiograph. (B) The cavity after “selective” caries excava­
tion. (C) Post-operative radiograph. (D) 1-month follow-up
radiograph, just before extraction. (E) A buccolingual sec­
tioning plane was prepared. (F) Section cut from the mesial
third of the pulp chamber. A thick band of tertiary dentine is
present (H&E, original magnification ×16). (G) Magnifica­
tion of the area demarcated by the rectangle in (F). Tertiary
dentine is lined pulpally by a single layer of flattened cells.
In the subjacent pulp, hyperaemic vessels with poly­
morphonuclear leukocytes in their lumen (original magni­
fication ×100; inset ×400). (H) High magnification view of
the vessel indicated by the right arrow in (G), with nu­
merous polymorphonuclear leukocytes in its lumen (original
magnification ×400). (I) Section taken approximately at the
centre of the pulp chamber revealing heavy colonisation of
the dentine left after “selective” excavation, up to tertiary
dentine (Taylor’s modified Brown & Brenn, original magni­
fication ×16). (J) Magnification of the area overlying ter­
tiary dentine. Vertical and horizontal pockets with loss of
tubular structure, packed with thick bacterial biofilms (ori­
ginal magnification ×100). (K) High magnification view of
the area demarcated by the upper rectangle in (I). Bacteria
have reached the calciotraumatic line, penetrating the ter­
tiary dentine in some areas (original magnification ×400).
(L) High magnification view of the area demarcated by the
lower rectangle in (I). Heavy bacterial infection (original
magnification ×400).

3.4. Leukocytes in the vessels experimental specimens (Table 1). Polymorphonuclear leukocytes were
also seen in the arterioles entering the pulp chamber, at the level of root
Examination of serial sections revealed increased numbers of poly­ canal orifices (Fig. 2I), in dilated vessels of the subodontoblastic layer in
morphonuclear leukocytes in the afferent pulpal vessels in all the pulp horns (Fig. 3G–H), or in pulpal vessels subjacent to the tertiary

7
D. Ricucci, et al.
dentine (Fig. 4G–H).
3.5. Bacteria in dentine
Taylor-modified Brown and Brenn staining revealed a reddish
fuchsine-stained line along the superficial dentine (Figs. 1K–L; 2 J;
3 K–M). This stained line represents the hybrid layer created by the
adhesive procedures. A large amount of stainable bacteria was observed
in the dentine subjacent to the cavity floor in all specimens (Table 1).
The dentinal tubules were heavily colonised by bacteria to a consider­
able depth (Figs. 1K–M; 2 J–L; 3 K–N; 4 I–L). High magnification
images showed that the lateral branches of the dentinal tubules were
also colonised (Figs. 1 M; 4 L). In the cases that were clinically-eval­
uated as deep caries, bacteria had reached the transition between sec­
ondary and tertiary dentine (Fig. 4 I), and had penetrated the super­
ficial portion of tertiary dentine (Fig. 4 K). Bacterial colonisation was
particularly severe in some areas, forming longitudinal or horizontal
pockets within the secondary dentine. These pockets were clogged with
bacterial accumulations, in which the dentinal tubules appeared to
have widened and coalesced, with loss of tubular structure (Figs. 1 M; 4
I–J, L).
4. Discussion
The recommendation for selective caries excavation is largely based
on the fear of pulpal exposure [23]. This is considered by many to be an
unfavourable prognostic factor in pulpal healing [26,30,31,34,35].
Because pulpal exposure causes irreversible damage to the odonto­
blastic palisade layer and kills many primary odontoblasts [26], selec­
tive caries removal has also been advocated to avoid stressing the pulp
and to promote pulpal health [30]. Some clinicians also favoured this
procedure as a means of evading the high cost of invasive root canal
treatment, which requires expensive equipment, supplies and highly-
trained dental health personnel. These factors limit access to dental care
in countries where financial, human and structural resources are scanty
[36]. According to these authors, root canal treatment should be
avoided whenever possible, by replacing complete caries excavation
with indirect pulp treatment [12].
A potential limitation of the present work is the small number of
teeth examined. With respect to this issue, readers have to candidly
consider that it is extremely difficult to obtain clinically-asymptomatic,
restored carious teeth for histological examination. Despite this lim­
itation, extensive stainable bacteria were universally demonstrated in
all specimens. These stained bacteria were found in clinically “firm”
dentine left by selective caries excavation. Bacteria were observed at
considerable depths away from the excavated cavity surfaces, within
the dentinal tubules and their lateral branches, and in the tertiary
dentine occasionally. In the 2 cases that were clinically diagnosed as
deep caries, bacteria were abundantly seen in longitudinal or trans­
versal pockets within the partially-degraded secondary dentine. These
observations do not lend credit to the long-held belief that bacterial
infection is only present in superficial dentine and that discoloured,
leathery or tertiary dentine is free from bacterial invasion [6,11,37], an
idea that had been doctrinally-accepted by many up to recent years
[29,33].
Observations from the present work, on the contrary, confirmed the
results of earlier studies that bacteria were universally identified from
deep, firm, secondary dentine and tertiary dentine. The previous results
were based on classical histobacteriological staining, electron micro­
scopy and bacterial culture [5,13,14]. In those studies, bacterial growth
had also been observed occasionally in specimens harvested with
aseptic methodology from clinically-sound dentine after complete ex­
cavation of the partially-demineralised tissues [13,14,16]. Regrettably,
only the works of Massler [6,9] were cited in the recent literature as the
rationale for the selective excavation approach; none of the afore­
mentioned classical studies that demonstrated bacteria in the leathery
8
Journal of Dentistry 100 (2020) 103430
and hard dentin was referenced.
Although one may consider the presence of a caries-infected zone
and a caries-affected zone, these zones do not correspond with softened
dentine and uninfected dentine, respectively. The present investigation
confirms that bacteria are present throughout the “leathery” or “firm”
dentine left by selective excavation. This is in line with classical studies
describing the occurrence of bacteria in meticulously-cleaned dentine
in approximately one-third of the cases [13]. The statement that firm or
leathery carious dentine is only presumably infiltrated by micro-or­
ganisms, and should be referred to as “contaminated” tissue instead of
“infected tissue” [38,39] is therefore a misconception. Although con­
taminant bacteria are not pathogens by definition [40], bacteria that
heavily infiltrated demineralised dentine are without doubt infectious
micro-organisms. Although one may challenge that the bacteria found
in deep dentine could have originated from saliva leakage that occurred
over time, factors such as the clinical intact status of the restorations,
absence of bacteria from the lateral walls, as well as formation of bio­
films on the pulpal wall of the cavity preclude the possibility of new
infection.
Pulpal changes such as formation of tertiary dentine and reduction
of the odontoblast layer were present in all cases. These features are
attributed primarily to the carious process [4,41,42]. The presence of
inflammatory cells, congested capillaries in the subodontoblastic layer
and polymorphonuclear leukocytes in the afferent vessels, are in­
dicative of unceasing irritation caused by the presence of noxious sti­
muli derived from bacteria that resided within the secondary/tertiary
dentine. It should be mentioned that no severe inflammatory pulpal
changes were observed in any of the specimens and the reactions
ranged from the presence of scattered chronic inflammatory cells to
exiguous, localised accumulation of inflammatory cells. This is not
surprising, given that ten of the cases had medium caries (i.e. caries
extending to one-half of the remaining dentin thickness) and only two
teeth had deep caries (i.e. caries extending to two-thirds of the re­
maining dentin thickness). Untreated medium/deep carious lesions
exhibit changes that are similar to those observed in restored teeth in
the present study [4,41]. Severe inflammatory responses are only ob­
served when the infection front reaches the innermost part of the ter­
tiary dentine, where bacteria are on the verge of penetrating the dental
pulp [4,43]. Migration of inflammatory cells into the dental pulp is the
result of pulpal response to slow, chronic diffusion of bacterial by-
products from the overlying dentine. Manifestation of chronic in­
flammation was further supported by the identification of eosinophilic,
large, homogenous immunoglobulin-containing Russell bodies in the
plasma cells. These intracellular inclusions are indicative of active
synthesis of immunoglobulins in hyper-functioning plasma cells.
The presence of congested capillaries in the subodontoblastic layer
is another sign of irritation. Capillaries contain little or no blood in
uninflamed dental pulps. They are usually flattened and obscured in H&
E-stained sections [3]. However, capillaries are readily identified from
injured dental pulps and they are a sign of hyperaemia. Likewise, the
presence of a large number of polymorphonuclear leukocytes in the
arterioles and in the smaller vessels afferent to the pulp directly beneath
the cavity is indicative of chemotaxis caused by active irritation [44].
It is important to stress that all the teeth under investigation did not
develop post-operative pain. These teeth were asymptomatic over the
entire observation period, and the subjects all responded normally to
pulp vitality tests prior to extraction. The complete absence of clinical
symptoms in teeth even when dental pulps show varying degrees of
pathologic changes is not unreasonable. It is well known that symptoms
appear during the late stages of the carious process; in some instances,
the pulp may undergo necrosis without symptoms [4,41].
The absence of symptoms and normal response to pulp tests have
been the parameters usually adopted for evaluating clinical success of
selective excavation in clinical trials [32,45,46]. Although pulp vitality
was preserved in 88 % of the selective caries excavation cases after
36–45 months [32], it is prudent to point out that absence of symptoms
D. Ricucci, et al.
may coexist with bacterial infection. Pulpal inflammation can be pre­
sent for long periods (years) especially when caries is not particularly
deep, as supported by histological evidence [41].
It is important to reiterate that the ultimate biological goal in the
treatment of caries is to resolve pulp inflammation and to maintain
pulpal health. This goal can only be achieved when infected dentine is
completely removed by operative procedures (i.e. non-selective ex­
cavation). Complete caries removal should be followed by the place­
ment of a fluid-tight restoration to prevent bacterial recontamination.
Histologic analysis of successfully-restored carious teeth, extracted for
various reasons, reported pulps that are completely free of acute or
chronic inflammatory cells upon non-selective caries excavation.
Deposition of tertiary dentine represents a repair response of the pulp to
caries progression via the formation of calcified scar tissues [42]. Un­
inflamed dental pulps can only exist in the absence of stainable bacteria
on the cavity floor. When bacteria are present within a cavity, either
because of incomplete excavation or marginal leakage, varying
amounts of inflammatory cells are invariably observed in the pulp
connective tissue [41–43].
The questions as to whether the pulp will succumb to the persisting
dentinal infection by undergoing necrosis and how long the process
would take remain unanswered. The fate of the dental pulp is depen­
dent upon the number of remnant bacteria, their virulence, as well as
the host resistance. As long as the coronal restoration remains intact,
remnant bacteria may not have sufficient space to propagate to the
numbers that are required to aggravate inflammation. Conversely,
pulpal necrosis may occur if the host’s resistance abates or when the
bacteria-tight seal attributed to the restoration is lost.
With respect to the issue whether there is a risk of future reinfection
when viable bacteria exist in dentine that is in close proximity with the
dental pulp, the generally-accepted concept is that the remnant bacteria
will eventually die or undergo irreversible changes in their metabolic
activity that render them harmless to the dental pulp [27,33,47]. This
claim has not been substantiated by the results of the present study. On
the contrary, accumulation of pulpal inflammatory cells in all 12 cases,
together with the circulatory disturbances, are robust indicators that
residual bacteria in dentine are metabolically-active, releasing their by-
products into the dental pulp tissue. Although previous studies have
reported decrease in cariogenic bacterial counts under sealed restora­
tions [45,48,49], asaccharolytic anaerobic bacteria that dominate the
microbiota of deep caries adapt to the environmental changes by uti­
lising proteins and glycoproteins derived from demineralised collagen
and pulpal fluid in the dentinal tubules as nutrients [50–52]. These
alternative sources of nutrients enable the deep-seated bacteria to
propagate and initiate infection in the underlying dental pulp. Hence,
the presence of potentially-arrested caries does not necessarily mean
that bacterial infection is absent or under control.
5. Conclusion
The hypothesis that selective excavation arrests carious progression
and prevents infection of dental pulp without creating adverse pulpal
reactions cannot be validated by the results of the present study.
Knowledge on the pulpal response to active caries and the in­
flammatory responses associated with bacteria ingress into dentine is
paramount to assist clinicians in making an informed, rational choice
based on biologically-robust principles.
The fallacy of permanently leaving carious dentine under a filling is
well known to endodontists and generalists who routinely practise vital
pulp therapy. The present study demonstrated that “leathery” or “firm”
carious dentine is infected. From a clinical perspective, all deminer­
alised soft dentine has to be removed until hard sound dentine is en­
countered. If an exposure occurs, vital pulp therapeutic interventions
may be considered, with the guiding principle being localisation of the
advancing front of infection, both in the dentine and in the subjacent
pulp. No pulp healing can be expected to occur in the presence of
9
Journal of Dentistry 100 (2020) 103430
infection. The goal of caries removal is the elimination of infected tis­
sues, as well as protection of the uninfected pulp wound with bio­
compatible and potentially bioactive dental materials to prevent bac­
terial contamination.
CRediT authorship contribution statement
Domenico Ricucci: Conceptualization, Methodology,
Visualization, Writing - original draft. José F. Siqueira: Validation,
Formal analysis. Isabela N. Rôças: Data curation. Mariusz Lipski:
Investigation. Amal Shiban: Investigation. Franklin R. Tay:
Conceptualization, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ­
ence the work reported in this paper.
Acknowledgements
The authors deny any conflict of interest associated with the present
study. The study was not supported by any grant body or commercial
organization.
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