Comparison Among Four Commonly Used Demineralizing Agents For Root Conditioning. A Scanning Electron Micros

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Comparison among four commonly used

demineralizing agents for root conditioning. A
scanning electron microscopy
Nathalia Godoy do AMARAL1, Maria Lúcia Rubo de REZENDE2, Fabiana HIRATA1, Marcus Gustavo Silva RODRIGUES3,
Adriana Campos Passanezi SANT’ANA2, Sebastião Luiz Aguiar GREGHI2, Euloir PASSANEZI4
1- DDS, Graduate student, Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, Bauru, SP, Brazil.
2- DDS, MSc, PhD, Associate Professor, Department of Prosthodontics, Discipline of Periodontology, Bauru School of Dentistry, University of São Paulo,
Bauru, SP, Brazil.
3- DDS, MSc, Graduate student, Department of Prosthodontics, Discipline of Periodontology, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil.
4- DDS, MSc, PhD, Professor, Department of Prosthodontics, Chairman of the Discipline of Periodontology, Bauru School of Dentistry, University of São
Paulo, Bauru, SP, Brazil.
Corresponding address: Prof. Dr. Maria Lúcia Rubo de Rezende - Faculdade de Odontologia de Bauru - Departamento de Prótese - Al. Otavio Pinheiro
Brizola, 9-75 - Bauru - SP - Brazil - 17012-901 - Fone/Fax: +55-3223-4679 - e-mail: malurezende@usp.br

!
ABSTRACT
D ental roots that have been exposed to the oral cavity and periodontal pocket environment

Demineralizing agents have been used as an adjunct to the periodontal treatment aiming
at restoring the biocompatibility of roots. Objective: This study compared four commonly
used demineralizing agents for their capacity of removing smear layer and opening dentin
tubules. Methods: Fifty fragments of human dental roots previously exposed to periodontal

!"#$%& %&
!"!'($ '($!")* !+,
phosphoric acid for 3 min; 5) Control: rubbing of saline solution for 3 min. Scanning electron
/
0 1 0 2 4 66,
of the specimens from the groups PA and control; in 80% from EDTA group; in 33.3% from
TC-HCl group and 0% from CA group. The mean numbers of exposed dentin tubules in a
$%&=)!>?#@#"=!B!?!+"*=#?G!"'($=))?+@
=#!?@+$
0
pairs of groups: TC-HCl and Control; TC-HCl and EDTA; CA and Control; and CA and EDTA.
$ 01 0 =6#?6+,"
$%&=66>?66G,"*=66!?66@,"'($=66?66, =6?6,$

1$%& $
0 0
I$%&I*I'($$ 0 1
one of them for root conditioning.
Key words: Demineralization. Root scaling. Smear layer. Scanning electron microscopy.
J Appl Oral Sci. 469 2011;19(5):469-75

Comparison among four commonly used demineralizing agents for root conditioning. A scanning electron microscopy
INTRODUCTION diameter of dentin tubules exposed after root

conditioning in order to relate these parameters
One of the goals of periodontal therapy is the 0
predictable regeneration of the periodontium collagen exposure13,15. Labahn, et al.17 (1992) have
in areas previously affected by periodontal found a time-dependent increase in the mean dentin
disease5,23,27. Histological and ultrastructural studies 0

have demonstrated that dental roots that have been & 2
exposed to the oral cavity or to the periodontal al.25 (2007) stressed that the exposure of the dentin
/
0 1, 1
changes in their mineral density 27 and root
0
contamination by bacteria and its products1. Scaling
and root planing alone are not able to fully eliminate events 25 $
the etiological contaminants and produce a compact 0
smear layer covering the instrumented surface2,5 favoring migration and attachment of gingival
0 5.
00 4,6,7,18,20.
These alterations have become the rationale for There still is a remarkable controversy concerning
the use of demineralizing agents as adjunct to to the type of chemical conditioner, time of its
periodontal therapy due to their potential for
removing smear layer and exposing the underlying justifies the search for parameters that can

0 0 support the option for this procedure in periodontal
and modifying dentin permeability, restoring the $0 /
biocompatibility of the roots9,26. no standardized study comparing several chemical
V 0 root conditioners for their ability of smear layer
the root surface can exert neutralizing effects on 0 $
endotoxins from periodontal pathogens in vitro, 0
0
00 reliable data to analyze and compare diseased
and attachment11. W dental root surfaces treated by manual scaling
% 0
conditioned dental roots are more effective in demineralizing agents.

0 1
0
and associated proteoglycans25. MATERIAL AND METHODS
In vivo animal 22 and human histological
studies12,13 0 Specimen preparation
$%
%
root surface. The most used demineralizing agents for extraction due to advanced periodontal disease
for these purposes are citric acid9,14,17,28, phosphoric at the Bauru School of Dentistry, University of São
acid23, ethylenediaminetetraacetic acid (EDTA)18 and * w
tetracycline hydrochloride17. Nevertheless, the great signing an informed consent form. The selected
variability of protocols employed by clinicians and
researchers has prevented consistent comparisons no history of scaling and root planing in the previous
among them. Clinical trials have also provided 6 months; 2) proximal attachment loss of 5 mm or
insufficient evidence that acid conditioning of more; 3) absence of decay lesions or restorations
near the cementoenamel junction (CEJ). The freshly
% 1 0
$ 0 0
0[ 0 0 \ 19 ]#66G gently removed using manual scalers (Figure 1A).
concluded that the use of citric acid, tetracycline $ 6,
or EDTA to modify the root surface provides no + $0
0

'|
W high speed bur (Figure 1B). The diseased parts of

lack of controls, non-calibrated examiners, masked ]} ~)
"
reference standards and small sample sizes, among } 4* 4*w
others, reduced the observational quality of relevant absence of remnants of the periodontal ligament
studies. As a consequence, Mariotti19 (2006) stated (Figure 1A). Then, each root received a second
0 #

carefully considered. apical direction, resulting in radicular dishes
Some authors have measured the number and (Figure 1C). On the mesial and distal surfaces of
J Appl Oral Sci. 470 2011;19(5):469-75

AMARAL NG, REZENDE MLR, HIRATA F, RODRIGUES MGS, SANT’ANA ACP, GREGHI SLA, PASSANEZI E
the dishes, 2 grooves separated by a distance of

) $ 0
bur, determining an area measuring 2 mm x 4 mm B6,
] ( ' 1 $ 0
0 #6/24 of $ 0 %
Gracey curettes (Hu-Friedy; Hu-Friedy do Brasil, W / % ( %
Rio de Janeiro, RJ, Brazil) (Figure 1F) and then,

@6
0
] $

5 groups of 10 fragments each according to the

@6, &]* 4
" A B
Farmácias e Drogarias, Bauru, SP, Brazil) for 3
" # $%&
solution of tetracycline hydrochloride at 50 mg/mL
(Laboratório Teuto, Anápolis, GO, Brazil) for 3 min;
3) EDTA: demineralization by a gel of EDTA at 24%
]*"wVW2w\ 4!")
* !+,
]* 4
"
( !"@ C D
!
0 0
sterile cotton pellet changed every 30 s (Figure
&"

by conventional scanning electron microscopy
(SEM).
SEM analysis E F
$ 4'\
analysis as described by Braidotti, et al.8 (2000)
and observed at a JSM-5600 LV scanning electron
microscope (JOEL, Tokyo, Japan). Digital images
/ 666 #666

and at zero tilt angle.
Quantitative and Qualitative Measurements

$ 4'\ G H
0 V |
Figure 1- Specimen preparation. A: extracted teeth in
(available from http://rbs.info.nih.gov/ij/). The

core of the groove-delimited area on each specimen
$ absence of periodontal ligament remnants (*); B: tooth
examined for general morphologic characteristics crown being transversely cut at the cementoenamel with

a water-cooled high-speed bur; C: radicular dish obtained
666 $

666 / apical direction; D: pencil marks made 4 mm apart from
01 0 each other on the mesial and distal surfaces of the dishes
$

#666 where grooves were made (E), delimiting an area of 2 mm x
/ ]#GG#)6 4 mm approximately; F: scaling of the area determined in E;
μm2 0 G: mesial and distal halves of the dishes separated before
0
receiving the burnishing of the demineralizing agents with
0
a sterile cotton pellet (H)
J Appl Oral Sci. 471 2011;19(5):469-75


66@ A B
RESULTS
Group CA

(0
observed on the examined surfaces (Figure 2A).
C D
( 0 1 666
field and the average percentage of the area
0 0 6#,] >G+
μm2 to 159 μm2
#666
(Figure 2B).
Group TC-HCl
' 0 1> E F
$%& 666

(Figure 2C), residual smear layer and some debris
!!!, $
1 0 $
percentage of area occupied by exposed tubules
66>,] 62 to 48.18 μm2) at

#666] #(
G H
Group EDTA
The majority of the specimens of EDTA group
]>6,
no exposed tubules 5 of them. The mean number of
1 0 )) 666
] #'

0.01% of the total (ranging from 0 μm2 to 5.82 μm2) I J
#666
] #
Group PA
4

(Figure 2G) probably due to the precipitation of an
insoluble calcium phosphate layer. The mean number
1 0 666
Figure 2- Panel of scanning electron microscopy (SEM)
12.1 (Figure 2G) and these tubules accounted for a micrographs. A: representative specimen of the Ca group
mean area of 0.03% (ranging from 0 μm2 to 46.25 in which no smear layer can be noted and dentin tubules
μm2 #666
] #&
! "#
the central part of A showing dentin tubules widened; C:
Control group
representative specimen of the TC-HCl group at 1,000×

showing absence of smear layer and exposed dentin
smear layer, open tubules could be seen in 2

! $# %
0
number of 2.3 tubules for this group at the 1,000× showing widened dentin tubules; E: representative specimen

] #V of EDTA group presenting smear layer and few exposed

!&#
their corresponding area and the mean value for of E; G: representative specimen of PA group at 1,000× of
6, #666 '

] #|
! 8# <
The mean number of exposed dentin tubules by showing discrete widening of the tubules; I: representative
the different treatments decreased according to the specimen of the control group at 1,000× showing smear layer
$%&II*I'($I '
!=#

the central part of I in which the tubules are not enlarged
$%&
enough to measure their corresponding area
J Appl Oral Sci. 472 2011;19(5):469-75

Table 1- Mean number of exposed dentin tubules and corresponding area after conditioning with the four different
demineralizing agents
Citric acid Tetracycline-HCl Phosphoric EDTA Control group

acid (saline)
Mean number of exposed 39.3±37.0A 43.8±25.2A 12.1±16.3Aa 4.4±7.5Ba 2.3±5.7Ba
tubules
Mean area occupied by 0.12±0.17A 0.08±0.06Aa 0.03±0.05Ba 0.01±0.01Ba 0.00±0.00B
enlarged tubules (%)
J

KNQN
,and C; TC-HCl and EDTA; CA and C and CA and \
EDTA (Table 1). tested ranging from 0.5% to 200% and from 0.5 to
When comparing the area occupied by dentin 10 min16,30. \
tubule openings, CA produced the greatest tubule 0 0@6
0$%&*'($ mL and 125 mg/mL during 3 to 4 min of application
]$ 0 $
by burnishing technique. Isik, et al. 16 (2000)
0 $%& 0 @6
$%& mL and 125 mg/mL might alter dentin surfaces by
differed only from group C. The exposure of dentin removing the smear layer and also maximize tubule
0 openings in a short period if repeated applications
w
DISCUSSION the concentration of 50 mg/mL for TC-HCl. It has
0 0
This study compared the 4 most commonly used

chemical agents for root conditioning as adjunctive of smear layer and exposure of the underlying
therapy for teeth affected by periodontitis. The tubules due to demineralization action of fresh
presented data suggest that citric acid and acid solution28$
%& burnishing technique and for changing the cotton
are more effective in removing smear layer and pellet at every 30 s in this study.
1 0 $&
phosphoric acid and EDTA. %&
Since Register and Burdick23 (1975) compared of the reasons for the reduced cellular insertion
]& #%! and for the unpredictability of the results, once
min) and other chemical substances and found it could denature the organic matrix of dentin14.
V
attachment, several investigators have devoted interfere on periodontal healing by its necrotizing
considerable time studying conditioning agents to effect on the surrounding progenitor cells13. Thus,
improve periodontal regeneration. Unfortunately, EDTA at 12%-24%, neutral pH for 30 s to 3 min
numerous and often uncontrolled histological
and clinical studies have created controversy and 0
confusion about the positive or negative effects of biological structures7 2
those agents19. The inconsistency of these studies et al.25 (2007) 0

may be due to differences in experimental systems 0
%
and techniques. Nevertheless, there is a common ]'V4'\
acceptance that it is not possible to decontaminate that both citric acid and EDTA treatments are able to
periodontitis-affected root surfaces by mechanical 1
0
V 1
or ultrasonic scaling of root surface produces W
'($
a nonbiocompatible smear layer that must be of use, failed to properly remove smear layer and
removed to expose the underlying collagen in order expose dentin tubules. Five specimens (50%) from

00 the EDTA group had none of their dentin tubules
orientation4,5,9,11,13,14,16-18,25,27. 1 #
$ 0 layer.
and studied demineralizing agents since in vitro It must be emphasized that even though CA
studies of Terranova, et al.29 (1986) suggested its $%&
0
potential usefulness in regenerative procedures. 0 $%&
J Appl Oral Sci. 473 2011;19(5):469-75

not be considered different from the other groups.

00
$ 0 '($%
TC-HCl in second place in our analysis. Another from smear layer and presented exposed round

* 1 0

similar numbers of dentin tubules, but CA produced is no study comparing the number of exposed
greater enlargement as reflected by the area dentin tubules and their corresponding area
measurements. EDTA and PA had the same behavior
as saline solution on both evaluations, suggesting

$
0
'($ data stressing the advantages of using conditioning

agents on diseased root surfaces and provide
*
foamy surface, probably due to a chemical acid/ agent for root conditioning. Before extrapolation
0 0 of data to the clinical conditions can be done,
hydroxyapatite, leading to calcium phosphate 0 0
deposition on the roots surfaces. This occurrence can 1
be attributed to the extended time of acid contact tissue attachment should be further investigated
]!V 0 in in vivo surveys.
0/
tubule openings and annulling its demineralizing CONCLUSION
effect10,21

The comparison among four of the most
& frequently used chemical root conditioners
suggestive that phosphoric acid is not appropriate

0
2
0 %&
reinforced that acid etching plays a decisive role phosphoric acid and EDTA. This information can be
0 of value as an extra parameter for choosing one of
them for root conditioning.
i.e., adsorption and adhesion of blood elements

0 3. In this aspect, ACKNOWLEDGMENTS
Baker, et al.3 (2005) have clearly demonstrated the
'($ / /*|20
applied for 5 min on planed dentin root surfaces. Pereira Lauris, from Bauru School of Dentistry,
0 0 0 University of São Paulo, for the statistical analysis,
CA-treated than EDTA-treated dentin surfaces and Mr. Adriano Luis Martins from Laboratory of Electron
forces produced by three 5-min rinses in PBS under Microscopy of the Faculty of Dentistry of Piracicaba
agitation on a rotary shaker table partially removed ]V\* 4'\

0'($% 0 4
$
from CA-treated surfaces. The authors stressed (]*

0/
to this research.
0 1
0 REFERENCES
$
0
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!%w /(}4 * 4/[\'0
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00
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J Appl Oral Sci. 475 2011;19(5):469-75

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