Adverse effects of human pulps after direct pulp capping with the ...

Dental Materials (2005) 21, 599–607
Adverse effects of human pulps after direct pulp
capping with the different components from
a total-etch, three-step adhesive system
Maria de Lourdes Rodrigues Accorinte a , Alessandro D. Loguercio b, *,
Alessandra Reis b , Antonio Muench a , Vera Cavalcanti de Araújo c
a Department of Dental Materials, School of Dentistry, University of São Paulo, SP, Brazil
b Department of Dental Materials and Operative Dentistry, School of Dentistry,
University of Oeste de Santa Catarina, Joaçaba, SC, Brazil
c Department of Oral Pathology, School of Dentistry, University of São Paulo, SP, Brazil
Received 3 March 2004; received in revised form 10 June 2004; accepted 3 August 2004
KEYWORDS
Biocompatibility;
Pulp therapy;
Adhesive system;
Calcium hydroxide;
Resin monomer;
Primer;
Cytotoxicity
* Corresponding author.
E-mail address: aloguercio@hotmail.com (A.D. Loguercio).
www.intl.elsevierhealth.com/journals/dema
Summary Objectives. The objective was to evaluate the response of human pulps
capped with different components from a total-etch three-step adhesive system.
Methods. Direct pulp capping was performed in 25 caries-free human premolars
scheduled for extraction due to orthodontic treatment. The teeth were randomly
divided in five groups, and capped with the following materials: Group 1—acidC
primerCadhesive were used as recommended; Group 2—only primer was applied;
Group 3—only bonding resin (light-cured for 10 s); Group 4—only composite resin
(light-cured for 40 s); Group 5—calcium hydroxide. After capping, all teeth were
restored with ScotchBond Multi Purpose Plus and Z-100 was placed incrementally.
After 60 days, the teeth were extracted and processed for light microscopic
examination (H/E) according to a histological score system. These were subjected to
non-parametric tests (a!0.05).
Results. Overall, the histological features showed that groups 1–4 were quite
similar and inferior to group 5. In groups 1–4 the pulp response varied from acute
inflammatory cell infiltrate with varying degrees to necrosis. The groups 3 and 4
showed a trend towards better pulp response, since a normal connective tissue could
be observed in more than half of the sample. All teeth from group 5 showed normal
connective tissue below an amorphous dentin bridge.
Significance. Adhesive components (primer or adhesive) as well as a composite
should be avoided for pulp capping. Ca(OH)2 should be the first choice for pulp
capping.
Q 2004 Academy Dental Materials. Published by Elsevier Ltd. All rights reserved.
0109-5641/$ - see front matter Q 2004 Academy Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2004.08.008

600
Introduction
The advent of adhesive dentistry has broadened the
range of possibilities of using resin-based materials
for a wide variety of clinical applications in
dentistry. Amongst them, the possibility of direct
pulp capping with bonding agents has increased.
Measuring the biocompatibility of a material is not
simple, and the methods of measurement are
evolving rapidly as more is known about the
interaction between dental materials and oral
tissues and as technologies for testing improve
[1]. The American Dental Association and American
National Standards Institute (ADA/ANSI) have recommended
a sequence of biocompatibility tests of
dental materials. This sequence consists of ‘initial’,
‘secondary’, and ‘usage’ tests [2] in that order.
However, usage tests in humans, as the present
investigation, are the most important ones since
they provide direct information about the biocompatibility
of dental materials.
It is likely that resin components produce more
detrimental effects to human pulps than they do in
monkeys and other animals. When bonding agents
are applied over human pulps, no sign of any
mineralized tissue formation and odontoblast-like
cell differentiation is observed [3]. The observation
of chronic inflammation with the presence of
macrophages and giant cells adjacent to the pulp
exposure site is common, and also necrosis of
connective tissue. Foreign body reaction is also
evident around particles of resin found dispersed
within the pulp [4–6]. This chronic inflammatory
response seems to preclude dentin bridge formation
[4–8].
A chronic inflammatory response may be directly
related to the cytotoxic effects of the bonding
agent’s components. Acid products, from cements
or even etchants, were blamed for moderate to
severe pulp inflammation observed in human pulps
when applied on dentin [9–12]. These studies were
reviewed [13] and the role of bacteria in detrimental
pulpal responses started being emphasized.
Also, acid etching is a short procedure that is
completely rinsed off by water and buffered by
surrounding dentin and/or dentinal fluids [14,15].
Therefore, the rationale behind the use of dentin
bonding materials instead of the commonly used
calcium hydroxide compounds has been that prevention
of leakage by micromechanical interlocking
of adhesive systems to cavity walls is more critical
for the healing processes than the material used for
capping [16,17].
Besides the acid etchants, the total-etch, threestep
adhesive systems contain other components,
which are shared between the primer and adhesive
bottles. After acid etching, a significant increase in
dentin permeability due to smear layer removal and
opening of dentinal tubules can facilitate the
ingress of resin monomers towards pulp, mainly in
deep dentin [17–20].
Monomers like Bis-GMA, HEMA, UDMA, TEGDMA,
initiators and solvents were already associated to
cytotoxic effects on fibroblast and odontoblastslike
cells [21–27]. All these components individually
or in combination ultimately compromise the pulp
healing as reported by several studies [4–8]. To
date, no study has addressed the individual role of
each bottle (primer and adhesive) on the pulp
response of human teeth. It is likely that if one of
these components were more of a problem, pulp
capping with adhesive systems could be successfully
performed by simply excluding that component
from the capping procedure. Therefore
the aim of this study was to address the individual
role of each adhesive component (bottles) on the
pulp response of human teeth.
Material and methods
M.L.R. Accorinte et al.
Twenty-five human premolars scheduled to be
extracted for orthodontic reasons were selected
from patients ranging from 15 to 25 years old. All
teeth were clinically and radiographically examined
to assure absence of proximal caries and periapical
lesions. The patients and their parents signed
consent forms after receiving a thorough explanation
about the experimental rationale, clinical
procedures and possible risks. The parents and
adult volunteers were asked to read and sign a
consent form allowing the clinical procedure. Both,
the consent form and the research protocol were
performed according to the Human Subject Review
Committee from the University of São Paulo, Brazil.
For the thermal testing, ENDO-ICE frozen gas
(Coltène/Whaledent Inc., Mahwah, NJ) was applied
for 5 s on the buccal surface of the teeth scheduled
for the pulp therapy, and adjacent teeth. After
local anesthesia (Citanest 3%; Merrel Lepetit, São
Paulo, Brazil) rubber dam isolation was installed
and each tooth was pumiced with rubber cup at low
speed. Mesio-occlusal cavities, with 0.5 mm beyond
the cementum–enamel junction, were prepared by
means of sterile diamond burs (# 1095, KG
Sorensen, Barueri, São Paulo, Brazil) at high speed
under water/spray coolant. The dimensions of
the cavity were approximately: occlusal depth,
3.0G0.2 mm; axial depth, 4.0G0.5 mm; proximal
faciolingual width, 3.0G0.2 mm. Pulp exposure

Human pulps direct capped with adhesive components 601
was performed in the center of the pulpal floor by
means of a round diamond bur under water cooling
(#1014, f 1.2, KG Sorensen, Barueri, São Paulo,
Brazil). One bur was used for each cavity. The teeth
were then divided into five experimental groups
(nZ5) as shown in Fig. 1. All materials employed
are described in Table 1.
The pulp hemorrhage was controlled by abundant
irrigation with saline solution followed by the
application of a damp cotton pellet embedded in
saline solution and held in place for 1 min.
In group 1, a metal matrix band was installed and
enamel, dentin and pulp were conditioned with
phosphoric acid for 20 s. The acidic agent was
rinsed off, slightly dried in such way that the dentin
stayed visibly moist with a shiny surface. Two coats
of the primer were applied and air-dried for 20 s.
The adhesive was subsequently applied and lightcured
for 20 s at 450 mW/cm 2 (Ultralux electronic,
Dabi Atlante, Ribeirão Preto, SP, Brazil). Increments
of Z-100 (3M ESPE, St. Paul, MN, USA) were
used to restore the cavities. Each increment was
light-cured for 40 s. A radiometer (Model 100P—
Demetron Research Corp., Kerr, Danbury, CT, USA)
Figure 1 Experimental design.
was used to check the light intensity immediately
before each clinical section. When necessary,
the material excesses were removed using an
ultra-fine diamond bur at high speed and water
cooling (KG Sorensen, Barueri, SP, Brazil).
In group 2, one coat of the primer solution from
ScotchBond Multi Purpose Plus system was applied
and air-dried for 10 s. Immediately after, a 2 mm
increment of Z100 was placed over the primer and
light-activated for 40 s. In group 3, only the
adhesive from the ScotchBond Multi Purpose Plus
system was applied, followed by light curing for
10 s. Over this layer, a 2 mm thick composite resin
increment was placed and light-cured for 40 s. In
group 4, over the exposure site, one increment of
Z100 was applied (liner), followed by light-curing
for 40 s. Care was taken to avoid composite resin
insertion in the pulp chamber in all groups. Then,
the cavities from groups 2–4 were restored as
already described for group 1 (Fig. 1).
In group 5, calcium hydroxide powder was
applied on pulp exposure by means of an amalgam
carrier after homestasis (group 1). Calcium hydroxide
cement (Dycal, Dentsply, Petropólis, RJ, Brazil)
Table 1 Products, commercial name and composition.
Product/commercial name Composition
ScotchBond Multi Purpose Plus (3M ESPE, St. Paul, MN, USA) 1. Etchant: 35% phosphoric acid
2. Primer: water (40%), HEMA (47%) and
polialkenoic acid copolymer (13%)
3. Adhesive: Bis-GMA (65%), HEMA (34%) and
initiators/accelerators (1%)
Z-100 (3M ESPE, St. Paul, MN, USA) Bis-GMA, TEGDMA and silica/zirconium
filler
Calcium hydroxide powder Labrynth Prod., Diadema, SP, Brazil Ca(OH)2
Saline solution Labrynth Prod., Diadema, SP, Brazil 2% NaCl

602
Table 2 Scores used during the histological exams.
Scores Inflammatory cell response
1 None or a few scattered inflammatory cells present in the pulp beneath the exposure
site
2 Polymorphonuclear leukocytes (acute) or mononuclear lymphocytes (chronic) in an
inflammatory lesion
3 Severe inflammatory lesion appearing as an abscess or dense infiltrate involving one
third or more of the coronal pulp
4 Completely necrotic pulp
Scores Soft tissue organization
1 Normal or almost normal tissue morphology below the exposure site and throughout
the pulp
2 Lack of normal tissue morphology below the exposure site, with deeper pulp tissue
appearing normal
3 Loss of general pulp morphology and cellular organization below the exposure site
4 Necrosis in at least the coronal third of the pulp
Scores Dentin bridge formation
1 New barrier tissue directly adjacent to some portion of the restorative material
2 New dentin bridge some distance from the material interface
3 No evidence of any dentin tissue formation in any of the tissue sections
was applied in the occlusal cavity floor. Then the
restorative procedure was conducted as described
in group 1 (Fig. 1).
The observation period was adapted to the
orthodontics treatment plans. During 60 days, the
patients were frequently asked about the presence
of sensitivity. After this period the extraction of
each tooth was performed under local anesthesia.
Although 60 days is not usual in biocompatibility
tests, several studies have demonstrated a great
percentage of dentin bridge formation in human
teeth capped before 60 days [6,8,28].
The teeth had their roots sectioned in about
2 mm in order to facilitate fixation in 10% buffered
formalin solution for 72 h. They were decalcified
in 20% formic acid for 6–8 weeks, prepared
according to normal histological techniques and
embedded in paraffin. Six-micron thick sections
were cut with a microtome parallel to the main
vertical axis of the tooth. The sections, mounted
on glass slides were stained with hematoxylin and
eosin (H/E). The sections were blindly evaluated
by an experienced pathologist according to the
criteria described in Table 2. The scores attributed
to each group were subjected to nonparametric
Kruskal–Wallis. This test is considered
very powerful for several independent samples
[29]. Means were considered significantly different
when a!0.05 [29]. The correlation between the
histological features (scores 1–4) and the frequencies
of pain for each group was performed by a
Spearman test (a!0.05). This test was performed
considering the histological features ranks (1–5)
for the five groups and the corresponding ranks of
the frequencies of pain.
Results
M.L.R. Accorinte et al.
The percentage of scores observed for each group is
shown in Table 3. Overall, the histological features
from groups 1–4 were quite similar and inferior to
group 5 (Fig. 2(a)–(e)). No correlation between the
histological scores and the frequencies of pain was
observed (pO0.05).
(1) Inflammatory infiltrate: An intense and chronic
inflammatory response was observed in groups
1 (80%) and 2 (100%). In 60% of the cases from
groups 3 and 4 and 100% from group 5, none, or
a few scattered inflammatory cells were presented
in the pulp beneath the exposure site.
Completely necrotic pulp was observed in 40%
of the cases from group 3 and 20% from group 4.
(2) Soft tissue response: A connective tissue with
no signs of normality, below the exposure site,
was seen in most cases from group 1 (80%) and
group 2 (100%). The majority of the teeth from
groups 3 and 4 (60%) showed normal or quite
normal connective tissue morphology below the
exposure site and throughout the pulp. In
group 3, the formation of a ‘hybrid layer’ was
noticed between the adhesive and pulp tissue.
Pulp necrosis occurred in 40 and 20% of

Human pulps direct capped with adhesive components 603
Table 3 Percentage of scores attributed for each group in each criteria and multiple comparisons, as well as frequency of pain (%) and correlation (**).
Groups Inflammatory cell response (ICR) (*) Soft tissue organization (STO) (*) Dentin bridge formation (DBF) (*) Pain (%) (**)
1 2 3 4 1 2 3 4 1 2 3
1-Adhesive 20 80 0 0 b 20 80 0 0 e 0 0 100 h 40
system
2-Primer 0 100 0 0 b 0 100 0 0 e 0 0 100 h 0
3-Adhesive 60 0 0 40 a,b 60 0 0 40 d,e 0 0 100 h 40
4-Composite 60 20 0 20 a,b 60 20 0 20 d,e 0 0 100 h 20
5-Calcium 100 0 0 0 a 100 0 0 0 d 100 0 0 g 0
hydroxide
(*) Different letters for each criteria means statistical different (p!0.05) (letters a and b for ICR; letters e and d for STO and letters h and g for DBF); (**) Spearman’s correlation between
ranks of pain frequencies versus ranks of ICR, STO and DBF was not significant (pO0.05).
specimens from group 3 and 4, respectively. In
group 5, a loose connective tissue with several
blood vessels delineated by an odontoblastic
layer in the pre-dentin, was seen in all
specimens.
(3) Dentin bridge formation: There was no evidence
of any dentin tissue formation in any of
the tissue sections in specimens from groups 1–
4. In group 5, an amorphous dentin bridge
formation with irregular contour and varied
mineralization degree was observed in all
specimens. Along with the dentin bridge, a
similar odontoblastic layer was also observed.
(4) Pain: Pain occurred in 40% of the cases in group
1, 40% from group 3 and 20% of specimens from
group 4. In group 3, two teeth had to be avulsed
before the 30-day period due to severe pain.
Discussion
The present investigation did not aim to stain
bacteria because they cannot be stained very well
with the Brown and Brenn technique. Added to this,
many bacteria are lost during tissue processing due
to the histologic procedure: with fixation and
decalcification, the staining ability of bacteria
may be reduced [30] or they may adhere to the
restoration that is removed during the process [31].
A large number of studies have reported that as
long as bacteria are not present, bonding agents can
be successfully used for pulp capping [32–35]. Itis
interesting to note, however, that most of the
articles reporting acceptable biocompatibility of
adhesive agents were conducted either in monkeys
or rodents [36]. Although animal studies, in
general, are necessary and their results have
generated much valuable information, a certain
caution must be exercised if results from usage
tests in animals are extrapolated to humans [37].
Therefore, the results of the present investigation
confirm that all components presented in the
adhesive solutions as well as the composite resin,
individually or in combination may result in degenerative
pulp alterations when placed directly over
pulp exposure sites. This is in agreement with
several studies where pulp capping was performed
with adhesive systems over human pulps [4–8].
As seen in Table 1, the main constituents of
primer and adhesive solution are HEMA and Bis-
GMA, respectively. The composite resin used has a
mixture of Bis-GMA and TEGDMA. In a ranking of
toxicity HEMA, which is the main component of
primer, is the least toxic substance in comparison
with other monomers such as Bis-GMA, UDMA and

604
Figure 2 (a) Adhesive systems capping after 60 days (group 1). Observe the chronic inflammatory response adjacent
the exposure site (black arrow). Below this site, the pulp morphology is normal (blue arrow) (HE, original magnification—
25.6!). (b) PrimerCcomposite resin after 60 days (group 2). There is a chronic inflammatory infiltrate subjacent to the
exposure site (HE, original magnification—100!). (c) AdhesiveCcomposite resin after 60 days (group 3). Normal pulp
tissue around the exposure site. No dentin bridge formation (HE, original magnification—25.6!). (d) Composite resin
after 60 days (group 4). Normal pulp tissue around the exposure site. No dentin bridge formation (HE, original
magnification—25.6!). (e) Calcium hydroxide after 60 days. Observe the formation of a thick dentin bridge (black
arrow) and the normal features of the tissue below it (HE, original magnification—25.6!).
TEGDMA after 24 and 72 h exposures [22]. Based on
such findings, one could expect worse results from
groups 3 and 4, where moieties with high molecular
weight are present. However, contrary to the
expected, groups 1 and 2 showed a trend towards
worse pulp response than groups 3 and 4. The high
percentage of HEMA in the primer, its lack of
polymer conversion due to the absence of photoinitiators,
its low molecular weight and hydrophilic
features, facilitate its diffusion through pulp tissue
and consequently avoid pulp healing [38].
When monomers are polymerized, as occurred
in groups 3 and 4, the amount of unreacted
product leached out is reduced and thus, their
cytotoxic action over cells is dramatically diminished
[24,27,28]. An evaluation of the cytotoxic
effect of three current total-etch, two-step
adhesive systems showed that both the acidic and
non-acidic components of un-polymerized adhesive
resins were responsible for the high cytotoxic
M.L.R. Accorinte et al.
effects on odontoblast-like cells [24]. When the
experimental materials were light-cured and rinsed
off in order to remove acidic agents as well as
residual unreacted monomers, the cytotoxic effects
of the adhesive resins decreased [24]. However,
when primers were applied to pulp wounds and the
residual monomers not rinsed out, as occurs
clinically, the cytotoxic effects of resinous monomers
promote pulp damage.
When the biocompatibility of the ScotchBond
Multi Purpose was compared to an aqueous solution
of HEMA (50%) on connecting tissue of a rat, the
HEMA solution showed the worst results and this
was attributed to the fact that the adhesive system
was light-cured, which gave rise to a decreased
number of resin particulates when compared with
HEMA [39].
Actually, the in vivo diffusion of spherical resin
globules into dentinal tubules of human teeth has
been previously observed [40]. These diffusing

Human pulps direct capped with adhesive components 605
resinous materials reached the pulp space and were
observed among odontoblast cells [40]. It seems
that the presence of resin particulates can trigger a
foreign body response characterized, by the presence
of mononuclear inflammatory infiltrate as
well as multinuclear giant cells [7].
It is reported that as low as 3.6 mmol/L of HEMA
can reduce cell metabolism to 50% after 24 h
exposure [22]. The concentration of HEMA in the
Scotchprep dentin primer, Scotchbond 2 dental
adhesive, and Gluma sealer is 4.09, 2.75, and
2.6 mmol/L, respectively [41]; figures much higher
than those reported to reduce cell metabolism to
50% [41]. Consequently, these high concentrations
of HEMA may impart remarkable cytotoxic effects
on cultured pulp cells [24].
However, an other issue, also important to
address, is the duration of exposure. This feature
has a strong effect on the toxicity of dentin bonding
agents. For instance, the same depression in cell
metabolism (about 50%) can occur in pulp cells with
lower amounts of HEMA (1.0 mmol/L) when this
substance is maintained in place for 72 h [22]. This
is particularly relevant if we consider that there is
clinical degradation of hydrophilic bonding agents
due to water sorption and exposure to oral fluids
that not only compromise the mechanical properties
of the adhesive layer [42,43], but also favor the
release of increasing amounts of monomers and byproducts
to pulp tissues. This is likely to be
responsible for chronic inflammatory response of
pulps and also to necrosis that occurs after longer
periods of evaluation [4,6,7,44]. The adhesive
resin might promote more intense pulp damage
over time [22,24,27,45] since these substances are
not degraded over time by macrophages and giant
cells. A persistent inflammatory pulp response has
also been reported after application of adhesive
resins to the human pulp wound [4,6]. In addition,
Costa et al. [6] demonstrated a delayed pulp
healing associated to the lack of dentin bridge
formation, even at 300 days long-term evaluation.
Adhesive components inhibit proliferation of immunocomponent
cells causing a chemical immunosupression
which favors the development of pulp
pathologies [46].
As discussed earlier, when the bonding resin or
the composite were applied over pulp exposures
(groups 3 and 4, respectively), a trend towards
better pulpal response was observed; however, no
dentin bridging was found in these groups. Although
these components were light-cured prior to restoration
of the cavity, it is known that the conversion
of monomers to polymers is never complete [47].
All the organic components of the adhesive
systems, photo-initiators and constituents
generated during the setting process, are released,
in particular shortly after setting [22,48,49].
It is known that oxygen acts as an inhibitor of
resin polymerization [50]. It has been previously
reported that an unfilled resin cured, in room
atmosphere had a significantly greater thickness
of polymerization-inhibited material than did resin
cured in argon atmosphere [50]. Other studies
have demonstrated that the wet environment (as
occurs to monomers when in contact with the
pulp tissue) may preclude the adequate polymerization
of resinous materials [49,51]. An
addition of approximately 20 ml of water per mL
of adhesive solution reduces the conversion
degree of a water-free bonding resin from 53%
to about 25% [52]. The residual water, present in
the pulp tissue, may dilute the priming components
and preclude the production of highquality
polymers, compromising the integrity of a
dentin interface over time [53].
In spite of the relatively high number of
inflammatory pulps or necrosis without bridge
formation in groups 1–4, no correlation between
histological features and postoperative sensitivity
was observed. This finding is coincident with the
published literature, which indicates that, postoperative
symptoms can be absent despite various
inflammatory reactions [5,6]. In fact, this is not a
new finding, since an old study has already demonstrated
this [54].
Calcium hydroxide and its compounds are the
materials against which new candidates for capping
are tested because of a long record of success from
clinical and histologic studies. The ability of
calcium hydroxide to stimulate reparative dentin
formation, shown in the present investigation,
has already been observed in many other clinical
studies [4–8]. In addition its bacterial effect may be
attributed to effects on induction and up-regulation
of odontoblast-like cell differentiation for new
matrix deposition and especially its effect on
growth factors from the dentin matrix [55].
It must be emphasized that this study was
performed on sound teeth in a clinical situation.
Pulp exposure frequently occurs by a carious
process in which the level of inflammation is much
higher and more difficult to predict than in the
clinical evaluation of pulp therapies. Had inflamed
pulps been capped in this study the use of adhesive
systems for capping could have caused disastrous
effects in vital pulp. This fact, however, merits
further evaluation.
Based on the experimental conditions, it was
concluded that none of the adhesive constituents
are capable of inducing pulp healing. Thus, under

606
clinical conditions, exposed pulps should be capped
with calcium hydroxide.
Acknowledgements
The authors are grateful to the Department of
Dental Pathology and Dental Materials of School of
Dentistry, USP. This study was partially supported
by CNPq Grants (551049/2002-2; 350085/2003-0;
302552/2003-0 and 474225/2003-8).
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