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Clinical and Ultrastructural Effects of Different Liners/Restorative Materials

on Deep Carious Dentin: A Randomized Clinical Trial

Article  in  Caries Research · January 2013

DOI: 10.1159/000345648 · Source: PubMed

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 Original Paper
Caries Res 2013;47:243–250
DOI: 10.1159/000345648
Clinical and Ultrastructural Effects of Different
Liners/Restorative Materials on Deep Carious
Dentin: A Randomized Clinical Trial
D.J. Corralo a M. Maltz b
a
Faculty of Dentistry, University of Passo Fundo, Passo Fundo, and b Department of Social and Preventive Dentistry,
Faculty of Dentistry, Federal University of Rio Grande do Sul, Bom Fim, Brazil
Key Words
Calcium hydroxide ⴢ Carious dentin ⴢ Deep carious lesions ⴢ
Glass ionomer cement ⴢ Stepwise excavation
Abstract
We evaluated the effect of calcium hydroxide cement (CHC)
and glass ionomer cement (GIC) on carious dentin and bac-
terial infections after partial caries removal and sealing. Sixty
permanent teeth with deep lesions underwent partial caries
removal, the application of CHC, GIC or wax, i.e. negative
control (NC), and were then sealed for 3–4 months. After the
partial caries removal and the sealing period, the dentin was
clinically assessed (colour and consistency) and analysed by
scanning electron microscopy to assess dentin organization
and bacterial infections. The effect of the treatment in each
group was assessed by the Wilcoxon and ␹2 tests, differences
among groups by the Kruskal-Wallis test and the correla-
tions between variables by Spearman correlation. No clin-
ical symptoms or radiographic signals of pulpits or pulp ne-
crosis were observed during the study. Dentin darkening
was observed after the sealing period in the CHC and NC
groups (p ! 0.05). However, there was no difference in the
colour after treatment among the 3 groups (p 1 0.05). Dentin
hardening occurred in all groups after treatment (p ! 0.05),
also with no differences (p 1 0.05). Dentin samples showed
better organization after the sealing period than after partial
caries removal, with total or partial obliteration of dentinal
tubules (CHC p ! 0.03, GIC p ! 0.05, NC p ! 0.01) and a reduc-
© 2013 S. Karger AG, Basel
0008–6568/13/0473–0243$38.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
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Received: August 10, 2012
Accepted after revision: November 7, 2012
Published online: January 19, 2013
tion of bacterial infections (CHC p ! 0.03, GIC p ! 0.05, NC
p ! 0.03). No differences were observed. Correlations be-
tween the different criteria, except for colour and bacterial
infection, were detected in all cases. Partial caries removal
and sealing resulted in dentin hardening, decreased bacte-
rial numbers and dentin reorganization, irrespective of the
dentin protection used. Copyright © 2013 S. Karger AG, Basel
Stepwise excavation has been widely used in the treat-
ment of deep carious lesions to prevent pulp exposure
[Magnusson and Sundell, 1977; Leksell et al., 1996;
Bjørndal et al., 2010]. This approach consists of preserv-
ing a layer of carious dentin over the pulp. A protective
liner is then applied, and the tooth is sealed for a certain
period of time. The purpose of this procedure is to pro-
mote the formation of tertiary dentin before the complete
excavation, thus making pulp exposure less likely [Kidd,
2004].
The covering of the innermost layer of carious dentin
with a protective liner has been extensively discussed.
Calcium hydroxide cement (CHC) and glass ionomer ce-
ment (GIC) have been commonly applied on carious den-
tin to induce remineralisation. CHC is often the preferred
cement for pulp protection because it is alkaline and bio-
compatible, induces pulp-dentin remineralisation and
reduces the risk of bacterial infection [Eidelman et al.,
1965; Aponte et al., 1966; Leung et al., 1980; Bjørndal et
Fundacao Coordenaco de Aperfeicoamento
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Marisa Maltz
Faculdade de Odontologia – UFRGS
Departamento de Odontologia Preventiva e Social
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Ramiro Barcelos, 2492 , Bom Fim 90035-003 (Brazil)
E-Mail marisa.maltz @ gmail.com
al., 1997; Maltz et al., 2002; Pinto et al., 2006]. A signifi-
cant increase in phosphorus content was observed in car-
ious dentin treated with CHC [Eidelman et al., 1965]. Re-
garding the incidence of bacterial infection, controlled
studies show controversial results [Leung et al., 1980; Pin-
to et al., 2006]. GIC has been used for the restoration and
protection of dentin in Atraumatic Restorative Treatment
(ART). ART has been described as a one-section ap-
proach to treat dentin decay, in which caries excavation
is done with manual instruments and without anaesthe-
sia, and cavity sealing with GIC is considered to be the
final restoration [Frencken et al., 1994, 1996]. GIC is the
material of choice because it is compatible and adhesive
to dental tissues and it releases fluoride, which increases
dentin remineralisation [Frencken et al., 1996]. Carious
dentin treated with GIC showed a decrease in the amount
of bacteria, an increase in calcium concentration and a
more compact arrangement of collagen fibres which op-
timized the mineralisation process [Massara et al., 2002;
Wambier et al., 2007]. However, these studies did not in-
clude control groups, and it is therefore unclear whether
these effects could be attributed to GIC.
Caries arrest has been observed after incomplete exca-
vation and cavity sealing without the use of CHC or GIC
as liners [Besic, 1943; Jeronimus et al., 1975; Handelman
et al., 1976; Mertz-Fairhurtz et al., 1986, 1998; Casa-
grande et al., 2009]. The lack of access to the oral environ-
ment significantly affects the surviving microbiota [Pad-
dick et al., 2005]. Based on these observations, the hy-
pothesis of this study was that sealing of carious dentin
arrests the lesion progression irrespective of the dentin
protection used. The aim of this randomized clinical
study was to assess the clinical and ultrastructural effects
of different liners on deep carious dentin after incomplete
excavation and sealing.
Materials and Methods
This was a prospective, double-blind, placebo-controlled,
three-arm parallel study. A convenience sample was selected from
patients who were treated at the Dental Clinic of the Federal Uni-
versity of Rio Grande do Sul between April 2002 and April 2003.
Eligible patients were those presenting with at least 1 tooth with
active carious lesions at risk of pulp exposure during direct com-
plete excavation (primary caries lesion involving 62/3 of the en-
tire dentin thickness determined radiographically). Signs and
symptoms indicative of pulp vitality, i.e. a positive response to
thermal stimulation during a cold test (CS68쏐; Chemical Special-
ties Industrial and Commerce Ltd, São Paulo, Brazil) and an ab-
sence of apical pathosis were additional requirements for inclu-
sion. Teeth with spontaneous pain or sensitivity to percussion
were excluded. The study sample included 60 permanent poste-
244 Caries Res 2013;47:243–250
rior teeth with deep carious lesions (38 occlusal and 22 occluso-
approximal, 47 molars and 13 premolars) from 44 patients aged
11–35 years (mean 17.62 8 5.57 and median 18) (fig. 1). The num-
ber of teeth per patient varied from 1–3. The size of the sample
was calculated on the basis of a pilot study involving 10 teeth. The
difference in the dentin consistency before and after cavity lining
and sealing was 55%, and a number of 11 teeth was calculated for
each group. A level of significance of 5% for a two-tailed test, and
a power of the study of 80% were adopted. Considering the design
effect for clustering of 1.5, the sample size increased to 17 teeth.
Assuming possible losses of 15%, the number of teeth per group
was adjusted to 20.
This study was approved by the Ethics Committee of Federal
University of Rio Grande do Sul, Brazil, and free informed con-
sent was obtained from all patients or their legal guardians. All
patients received dental treatment at the Dental Clinic of the Fed-
eral University of Rio Grande do Sul.
Intervention
Patients were treated under local anaesthesia. A rubber dam
was used (disinfected with iodated alcohol 0.05%), and access to
lesions was gained with a No. 245 carbide bur whenever neces-
sary. Necrotic fragments were removed with a sterile sharp exca-
vator. Decayed tissue was completely removed from the sur-
rounding cavity walls in accordance with the hardness criteria.
Soft dentin was left on the pulpal wall to avoid pulp exposure.
The cavity was washed with saline and dried with sterile swabs.
Clinical analysis and sample collection were then performed (de-
scribed below) and the teeth were randomly assigned to 1 of 3
experimental groups. The treatment group was kept in a sealed
dark envelope and a person other than the operator selected an
envelope before the placement of the line/restorative materi-
als. There were 3 experimental groups: (1) CHC group: A calci-
um hydroxide-containing liner was applied (Dycal쏐, Dentsply,
Petrópolis, Rio de Janeiro, Brazil) and the cavity was restored
with modified zinc oxide-eugenol cement (IRM쏐, Caulk/Dent-
sply, Petrópolis, Rio de Janeiro, Brazil). (2) GIC group: The cav-
ity was filled with a conventional GIC (Vitromolar쏐, DFL, Rio de
Janeiro, Brazil). (3) Negative control (NC) group: A sterile wax
layer was placed and the cavity was restored with modified zinc
oxide-eugenol cement (IRM).
After 3–4 months, pulp sensitivity was reassessed clinically
and radiographically (by cold test and periapical radiograph). Af-
ter anaesthesia, prophylaxis, placement of a rubber dam and asep-
sis, the temporary restorations were removed, the colour and con-
sistency of dentin evaluated and another dentin sample collected.
The remaining carious dentin was removed and teeth were then
restored with a light-cured composite resin (Charisma쏐, Kulzer,
São Paulo, Brazil).
Clinical Analysis
Examinations were performed by 2 calibrated examiners. The
colour and consistency of dentin were recorded after incomplete
excavation and immediately after cavity reopening. Dentin col-
our was classified according to the following criteria: (1) yellow,
(2) light brown or (3) dark brown. The consistency of dentin was
classified as: (1) soft, if it could be easily removed by the excavator,
(2) leathery, if it could be removed when the excavator was firmly
used, or (3) hard, when hardness was similar to sound dentin
[Maltz et al., 2002].
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 Clinical and radiographic
exams
Selection and invitation to join
the study (44 patients)

Enrollment
Excluded
There was no exclusion after
clinical and radiographic
selection

Randomized
(n = 60 teeth)

Allocation

Allocated to CHC Allocated to GIC Allocated to NC

(n = 20) (n = 20) (n = 20)
Follow-up

Lost to follow-up after Lost to follow-up after Lost to follow-up after

3–4 months (n = 1) 3–4 months (n = 1) 3–4 months (n = 1)
Analysis

Fig. 1. Flow chart showing the number of Analysed CHC Analysed GIC Analysed NC
patients that were enrolled, allocated to (n = 19) (n = 19) (n = 19)
groups and lost to follow-up, and the final
number of cases analysed in the 3 trials.

Sample Collection Statistical Analysis

Before sample collection, the cavities were divided into 2
halves: for occlusal lesions in a mesiodistal direction and for oc-
cluso-approximal lesions in a buccopalatal/buccolingual direc-
tion. After incomplete excavation, a sample was collected from
the mesial or buccal half. Another sample was taken from the dis-
tal or palatal half after the sealing period of 3–4 months. This was
done to ensure that the initial and final samples were collected
from the same depth [Eidelman et al., 1965; Fairbourn et al., 1980;
Massara et al., 2002]. Samples were obtained with a sterile dentin
excavator and stored in a 2% glutaraldehyde solution (pH 7.0,
phosphate buffer, 0.2 M) for a minimum of 24 h.
Cohen’s kappa was used to assess intra- and inter-examiner
reproducibility. The Kruskal-Wallis test was applied to investigate
inter-group differences in relation to the variables colour, tissue
organization and bacterial infection at baseline and after 3–4
months. The ␹2 test was used for dentin consistency, and the Wil-
coxon test was used for intra-group comparisons at the 2 experi-
mental periods. Spearman correlation was used to test correla-
tions between clinical (colour and consistency) and ultrastruc-
tural (tissue organization and bacterial infection) variables. A
p value of !0.05 was considered statistically significant.

Ultrastructural Analysis
Analyses were carried out blindly, by a single calibrated exam-
iner, following routine scanning electron microscopy (SEM) pro-
cedures [Massara et al., 2002]. The samples were scanned using a
scanning electron microscope (JSM 5.800, Jeol, Peabody, EUA) at
magnifications of !3,000–!6,000. Dentin organization and bac-
terial infection of dentin were classified according to the criteria
described in table 1. To assess reproducibility, repeated analyses
were performed in 25% of the samples.
Results
The intra-examiner kappa scores for clinical criteria
were 0.80 and 0.77, and the inter-examiner score was 0.77.
The intra-examiner kappa score for SEM criteria was
0.89.
Three patients were lost to follow-up (1 from each
group); thus, data from 57 teeth were available for analy-
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Effect of Different Liners/Restorative Caries Res 2013;47:243–250 245

Materials on Deep Carious Dentin
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Table 1. Criteria used for assessing the ultrastructural aspects Table 2. p values of the clinical (colour and consistency) and mi-
and bacterial infection of dentin by SEM analysis croscopic (dentin structure and bacterial infection) changes ob-
served in the carious dentin after partial caries removal and after
Criteria the sealing period in the experimental groups

Ultrastructural aspects of dentin samples Treatments

1 Organized dentin (mineralised inter- and intra-tubular CHC GIC NC
dentin), total or partial obliteration of dentinal tubules
(fig. 2b, d, f) PCR SP PCR SP PCR SP
2 Predominant organized dentin and fewer disorganized Colour
dentin areas Yellow 8 3 5 4 8 3
3 Disorganized dentin and fewer organized areas Light brown 9 5 8 7 7 4
Brown 2 11 6 8 4 12
4 Predominant disorganized dentin (inter-tubular dentin p 0.002* 0.257* 0.006*
with a clearly exposed collagen matrix, presenting well-
defined loosely organized collagen fibres, and dentin in- Consistency
ter- and intra-tubular lower or not mineralised), en- Soft 8 1 16 2 9 1
larged dentinal tubules (fig. 2a, c, d) Leathery 11 10 3 8 10 11
Hard 0 8 0 9 0 7
Microbial infection of dentin samples p 0.001** 0.001** 0.001**
0 No microorganisms on dentin (fig. 2b) Ultrastructural aspect
1 Few microorganisms scattered on dentin (most of the 1 3 6 2 10 1 6
dentin areas with no microorganisms) or little area of 2 4 5 12 7 3 10
dentin with high numbers of microorganisms 3 7 7 3 2 10 1
4 5 1 2 0 5 2
2 Few microorganisms uniformly scattered on dentin p 0.026* 0.005* 0.002*
3 High numbers of microorganisms (fig. 2a) Bacterial infection
0 0 0 0 0 0 0
1 8 14 9 16 6 14
2 6 4 8 2 7 3
3 5 1 2 1 6 2
p 0.028* 0.050* 0.033*
sis (fig.  1). Three teeth from the GIC group presented
pulpal exposure during cavity reopening, and were treat- PCR = Partial caries removal; SP = sealing period.
ed with direct pulp capping. Absence of apical pathosis * Kruskal-Wallis test, ** ␹2 test used for dentin consistency.
(detected by radiographic examination) and pulp sensi-
tivity to cold were observed in the 57 monitored cases.

Clinical Observations
At baseline, the 3 groups were similar with regard to
dentin colour (p = 0.411). Concerning dentin consistency,
the hardness was lower in the GIC group than in the oth-
er groups at baseline (p ! 0.05). After the sealing period,
dentin darkening was observed in the CHC and NG
groups, but it was not evident in the GIC group. Dentin
hardening was observed in all groups after treatment, ir-
respective of the liner used (table 2). No differences were
observed among the 3 groups regarding dentin colour
(p = 0.322) and consistency (p = 0.918) after 3–4 months
of sealing (table 2).
Ultrastructural Observations
At baseline, the dentin samples from the CHC and
NC groups were similar in relation to tissue organization
(p 1 0.05), while the samples from the GIC group showed
a more organized structure (p ! 0.05). After the sealing
period, however, there were no differences among the 3
groups (p = 0.135). In all groups, dentin structure was
more organized after 3–4 months than at baseline, with
total or partial obliteration of dentinal tubules. All
groups showed a reduction in the level of bacterial infec-
tion after 3–4 months. The 3 experimental groups
showed similar bacterial numbers at baseline (p = 0.373)
as well as after the sealing period (p = 0.684) (table  2;
fig. 2).
Significant correlations among all studied criteria
were observed, except for colour versus bacteria (ta-
ble 3).
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246 Caries Res 2013;47:243–250 Corralo /Maltz

   
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 D

a b

c d

F T

e f

Fig. 2. SEM photomicrographs (!3,000) of dentin samples (a, c, e) after partial caries removal showing in-
creased bacterial infection (B) and disorganized dentin structure with exposure of inter-tubular dentin collagen
fibre (F). Dentin samples after a 3- to 4-month sealing period with CHC (b), GIC (d) and wax (f) showing more
organized dentin (D) with total or partial obliteration of dentinal tubules (T) and a reduction in bacterial infec-
tion.
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Effect of Different Liners/Restorative Caries Res 2013;47:243–250 247

Materials on Deep Carious Dentin
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Table 3. Correlations between the clinical (dentin colour and con-
sistency) and ultrastructural (dentin structure and microbial in-
fection) variables in the CHC, GIC and NC groups
Variables Spearman coefficient
correlation
Colour ! consistency 0.351*
Colour ! structure –0.250*
Colour ! bacterial infection –0.096
Structure ! bacterial infection 0.497*
Structure ! consistency –0.374*
Consistency ! bacterial infection –0.332*
* Significance correlation 5%.
Discussion
Our study assessed the clinical and ultrastructural
outcomes of the use of CHC and GIC for the lining/res-
toration of deep carious lesions. Incomplete carious den-
tin excavation and sealing for 3–4 months resulted in
dentin hardening, reduction of bacterial contamination
and dentin reorganization, irrespective of the liner/res-
toration used. This was the first controlled trial compar-
ing these cements that are widely used in clinical prac-
tice.
Clinically, active dentin lesions are light brown or yel-
low and soft [Miller and Massler, 1962; Nyvad and Fejer-
skov, 1986]. Spontaneous pain or painful reactions to
sweets, acids and provocative stimuli (probing, heat or
cold) are generally associated with this type of lesion,
while inactive lesions are not normally associated with
painful reactions. The dentin is often hard and pigment-
ed as a consequence of specific biochemical processes
[Dreizen et al., 1964; Kidd et al., 1993; Lynch and Beigh-
ton, 1994; Bjørndal et al., 1997; Kleter, 1998; Bjørndal and
Larsen, 2000; Maltz et al., 2002; Massara et al., 2002; Pin-
to et al., 2006; Orhan et al., 2008]. Miller and Massler
[1962] observed the presence of mixed clinical character-
istics in certain lesions, which may indicate an ongoing
inactivation process; leathery lesions, which they consid-
ered to be inactive, do not represent entirely controlled
lesions [Nyvad and Fejerskov, 1986]. In our study, active
dentin lesions had a soft or leathery consistency and were
yellow or light brown in colour. In some cases, a dark
colour accompanied the soft or leathery consistency,
along with a history of painful reaction to sweets and heat
or cold (table  1). After the sealing period, all lesions
showed dentin hardening irrespective of the experimen-
248 Caries Res 2013;47:243–250
tal group, which indicated that the liner/restorative mate-
rial itself did not play a role in the arrestment process.
With regard to the colour, lining with CHC and wax, an
inert material, resulted in dentin darkening, while lining/
restoration with GIC did not. Microbiologic studies by
Orhan et al. [2008] and Lula et al. [2011] demonstrated
that dentin colour is not correlated to the degree of infec-
tion before and after dentin sealing. Dentin colour ap-
pears not to be an adequate criterion for defining lesion
activity while dentin consistency appears to be a more
reliable parameter [Kidd et al., 1993; Lynch and Beighton,
1994; Orhan et al., 2008].
In several studies, clinical signs of lesion arrestment
have been observed as a result of the use of a variety of
liners. Besic [1943] showed dentin darkening and harden-
ing after sealing carious lesions without the use of a lining
material, by applying cotton and gutta-percha under the
temporary filling. Lesion arrestment, as classified by
clinical criteria, was also observed in dentin caries lined
with zinc oxide-eugenol [King et al., 1965], CHC [Law
and Lewis, 1961; Eidelman et al., 1965; Fisher, 1977; Fair-
bourn et al., 1980; Leung et al., 1980; Bjørndal et al., 1997;
Bjørndal and Thylstrup, 1998; Maltz et al., 2002; Pinto et
al., 2006; Orhan et al., 2008], GIC [Kreulen et al., 1997;
Herrera et al., 2000; Massara et al., 2002; Wambier et al.,
2007], and adhesive systems [Jeronimus et al., 1975; Han-
delman et al., 1976; Mertz-Fairhurst et al., 1998; Casa-
grande et al., 2009]. The majority of these studies did not
include a control group, which should be considered
when interpreting the results.
SEM analysis revealed structural dentin modifica-
tions after 3–4 months of sealing, irrespective of the lin-
er/restorative material used. Both cements and the inert
material promoted dentin reorganization, with a total or
partial obliteration of dentinal tubules and a reduction in
the incidence of bacterial infection. In other qualitative
SEM analyses, similar results were obtained after sealing
deciduous teeth with GIC [Massara et al., 2002; Wambier
et al., 2007]. The reduction in bacterial contamination
after sealing carious dentin with different liners was as-
sessed by bacteriological exams in 2 controlled studies. In
the study by Leung et al. [1980], CHC promoted a signif-
icantly higher reduction in bacterial counts than did wax.
On the other hand, Pinto et al. [2006] reported a similar
reduction in infection when comparing samples treated
with CHC and gutta-percha. Unlike microbiological
techniques, which specifically detect the growth of viable
microorganisms, the methodology used in our experi-
ment was not adequate for the assessment of bacterial vi-
ability, although it could reveal a pattern of contamina-
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tion. Significant reduction in bacterial infection was ob-
served after the sealing period in all groups.
Dentin colour, consistency, microscopic structure and
bacterial infection have been used as indicators of dentin
caries activity. Few authors have studied the correlation
among these variables. Pinto et al. [2006] observed a pos-
itive correlation between colour, consistency and bacte-
rial contamination. On the other hand, Kidd et al. [1993]
and Lynch and Beighton [1994] found contrary results
during the analysis of coronary dentin caries and root
caries, respectively. No correlation between colour and
contamination was observed in these studies, and only a
correlation between consistency and contamination was
reported. Soft samples were reported to contain a higher
level of microorganisms than moderate and hard sam-
ples. Orhan et al. [2008] reported similar results. In our
study, the correlations between colour, consistency, den-
tin organization and bacterial infection were evaluated.
Positive correlations between all the assessed variables
were observed (p ! 0.05), with the exception of colour
versus contamination (p 1 0.05). These results are in ac-
cordance with those of Kidd et al. [1993] and Lynch and
Beighton [1994], who suggested that colour is not a useful
criterion for the diagnosis of dentin caries because colour
is not related to dentin infection level and pigmented
lesions show variable degrees of contamination. Further-
more, in a recent paper, Lula et al. [2011] found no cor-
relation between dentin colour and colonization of Strep-
tococcus mutans and Lactobacillus spp. Indeed, an ob-
servation of soft but dark carious tissue is common in
clinical studies [Bjørndal et al., 1997; Maltz et al., 2002].
In conclusion, our findings indicated that the removal
of superficial parts of the necrotic and demineralised
dentin and cavity sealing promotes dentin caries arrest-
ment, irrespective of the use of a liner. The results suggest
that the effect of the use of CHC or GIC on dentin caries
arrestment is not superior to the use of an inert material.
Acknowledgments
The authors thank Luana Severo Alves for revising the manu-
script, the Microscopy Electronics Centre of Federal University of
Rio Grande do Sul for assistance during microbiological analyses,
DFL (Rio de Janeiro, Brazil), Kulzer (São Paulo, Brazil) and Caren
Serra Bavaresco for helping in treating the patients. The study was
supported by the Brazilian Ministry of Education through its
agency Coordenação de Aperfeiçoamento de Pessoal de Nível Su-
perior (CAPES).
Disclosure Statement
The authors state that there is no personal or financial conflict
of interests for the study development that might introduce bias
or affect their judgment.

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