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Comparison among four commonly used

demineralizing agents for root conditioning. A
scanning electron microscopy
Nathalia Godoy do AMARAL1, Maria Lcia Rubo de REZENDE2, Fabiana HIRATA1, Marcus Gustavo Silva RODRIGUES3,
Adriana Campos Passanezi SANTANA2, Sebastio Luiz Aguiar GREGHI2, Euloir PASSANEZI4

1- DDS, Graduate student, Hospital for Rehabilitation of Craniofacial Anomalies, University of So Paulo, Bauru, SP, Brazil.
2- DDS, MSc, PhD, Associate Professor, Department of Prosthodontics, Discipline of Periodontology, Bauru School of Dentistry, University of So Paulo,
Bauru, SP, Brazil.
3- DDS, MSc, Graduate student, Department of Prosthodontics, Discipline of Periodontology, Bauru School of Dentistry, University of So Paulo, Bauru, SP, Brazil.
4- DDS, MSc, PhD, Professor, Department of Prosthodontics, Chairman of the Discipline of Periodontology, Bauru School of Dentistry, University of So
Paulo, Bauru, SP, Brazil.
Corresponding address: Prof. Dr. Maria Lcia Rubo de Rezende - Faculdade de Odontologia de Bauru - Departamento de Prtese - Al. Otavio Pinheiro
Brizola, 9-75 - Bauru - SP - Brazil - 17012-901 - Fone/Fax: +55-3223-4679 - e-mail: malurezende@usp.br

           ! 

ABSTRACT

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

diameter of dentin tubules exposed after root

conditioning in order to relate these parameters
    0  
collagen exposure13,15. Labahn, et al.17 (1992) have
found a time-dependent increase in the mean dentin
 0 
     
  & 2 
al.25 (2007) stressed that the exposure of the dentin
 1           

0   
           
events 25 $        
0    
favoring migration and attachment of gingival

00 4,6,7,18,20.
There still is a remarkable controversy concerning
to the type of chemical conditioner, time of its
         
justifies the search for parameters that can
support the option for this procedure in periodontal
 $0 /
no standardized study comparing several chemical
root conditioners for their ability of smear layer
    0   $
         0  
reliable data to analyze and compare diseased
dental root surfaces treated by manual scaling
0  
demineralizing agents.

INTRODUCTION
One of the goals of periodontal therapy is the
predictable regeneration of the periodontium
in areas previously affected by periodontal
disease5,23,27. Histological and ultrastructural studies
have demonstrated that dental roots that have been
exposed to the oral cavity or to the periodontal
/     
0 1,
changes in their mineral density 27 and root
contamination by bacteria and its products1. Scaling
and root planing alone are not able to fully eliminate
the etiological contaminants and produce a compact
smear layer covering the instrumented surface2,5
 0       5.
These alterations have become the rationale for
the use of demineralizing agents as adjunct to
periodontal therapy due to their potential for
removing smear layer and exposing the underlying
    
0  0 
and modifying dentin permeability, restoring the
biocompatibility of the roots9,26.
V   0       
the root surface can exert neutralizing effects on
endotoxins from periodontal pathogens in vitro,
0
00  
and attachment11. W   
    %  
conditioned dental roots are more effective in
  
0 1 
0
and associated proteoglycans25.
In vivo animal 22 and human histological
studies12,13  0 
    
root surface. The most used demineralizing agents
for these purposes are citric acid9,14,17,28, phosphoric
acid23, ethylenediaminetetraacetic acid (EDTA)18 and
tetracycline hydrochloride17. Nevertheless, the great
variability of protocols employed by clinicians and
researchers has prevented consistent comparisons
among them. Clinical trials have also provided
insufficient evidence that acid conditioning of
     
      %
 $        
 0[    0 0 \ 19 ]#66G 
concluded that the use of citric acid, tetracycline
or EDTA to modify the root surface provides no
0
    
     
     W  
  
    
lack of controls, non-calibrated examiners, masked
reference standards and small sample sizes, among
others, reduced the observational quality of relevant
studies. As a consequence, Mariotti19 (2006) stated
    0
carefully considered.
Some authors have measured the number and

J Appl Oral Sci.

MATERIAL AND METHODS

Specimen preparation
$%
  % 
for extraction due to advanced periodontal disease
at the Bauru School of Dentistry, University of So
*  w        
signing an informed consent form. The selected
   
no history of scaling and root planing in the previous
6 months; 2) proximal attachment loss of 5 mm or
more; 3) absence of decay lesions or restorations
near the cementoenamel junction (CEJ). The freshly
1  0 
0       0      
gently removed using manual scalers (Figure 1A).
$        6,  
 + $0
   '|  
high speed bur (Figure 1B). The diseased parts of
 
  
  ]}  ~) 
 "
}  4* 4*w    
absence of remnants of the periodontal ligament
(Figure 1A). Then, each root received a second
   #     
  
apical direction, resulting in radicular dishes
(Figure 1C). On the mesial and distal surfaces of

470

2011;19(5):469-75

AMARAL NG, REZENDE MLR, HIRATA F, RODRIGUES MGS, SANTANA ACP, GREGHI SLA, PASSANEZI E

     

$   0 
     B6,
 0   
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 W / %  ( % 

the dishes, 2 grooves separated by a distance of

)    
bur, determining an area measuring 2 mm x 4 mm
] ( ' 1 $   
 0  #6/24 of
Gracey curettes (Hu-Friedy; Hu-Friedy do Brasil,
Rio de Janeiro, RJ, Brazil) (Figure 1F) and then,
           
 @6    
   0
  ] $
          
5 groups of 10 fragments each according to the
     
           
  @6, &]*  4
 "
Farmcias e Drogarias, Bauru, SP, Brazil) for 3
" # $%&       
solution of tetracycline hydrochloride at 50 mg/mL
(Laboratrio Teuto, Anpolis, GO, Brazil) for 3 min;
3) EDTA: demineralization by a gel of EDTA at 24%
]*"wVW2w\ 4!")
*     !+,
  ]*   4
 "  
(  !"@ 
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sterile cotton pellet changed every 30 s (Figure
&"    
         
    
by conventional scanning electron microscopy
(SEM).

SEM analysis

$         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'\      
      0 V  |  
(available from http://rbs.info.nih.gov/ij/). The
core of the groove-delimited area on each specimen
   $  
examined for general morphologic characteristics
    
 
 666 $
    
  
666   /   
 01 0  
 $
  
 #666
  /    ]#GG#)6
m2    0
  0     
  0 

J Appl Oral Sci.

Figure 1- Specimen preparation. A: extracted teeth in

 

   
  
absence of periodontal ligament remnants (*); B: tooth
crown being transversely cut at the cementoenamel with
a water-cooled high-speed bur; C: radicular dish obtained

  

apical direction; D: pencil marks made 4 mm apart from
each other on the mesial and distal surfaces of the dishes
where grooves were made (E), delimiting an area of 2 mm x
4 mm approximately; F: scaling of the area determined in E;
G: mesial and distal halves of the dishes separated before
receiving the burnishing of the demineralizing agents with
a sterile cotton pellet (H)

471

2011;19(5):469-75

Comparison among four commonly used demineralizing agents for root conditioning. A scanning electron microscopy


 66@

RESULTS
Group CA
        
    (0      
observed on the examined surfaces (Figure 2A).
( 0  1 666
field and the average percentage of the area
 0 0  6#,] >G+
m2 to 159 m2    
   #666
(Figure 2B).

Group TC-HCl
'   0 1>
$%&  666

(Figure 2C), residual smear layer and some debris
   !!!,   $ 
    1  0  $  
percentage of area occupied by exposed tubules
 66>,] 62 to 48.18 m2) at
 
 #666] #(

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 m2 to 5.82 m2)
#666 
 ] #

Group PA
4    
            
(Figure 2G) probably due to the precipitation of an
insoluble calcium phosphate layer. The mean number
1 0  666 
  
12.1 (Figure 2G) and these tubules accounted for a
mean area of 0.03% (ranging from 0 m2 to 46.25
m2 #666 
 ] #&

Figure 2- Panel of scanning electron microscopy (SEM)

micrographs. A: representative specimen of the Ca group
in which no smear layer can be noted and dentin tubules
   
  ! "#   
the central part of A showing dentin tubules widened; C:
representative specimen of the TC-HCl group at 1,000
showing absence of smear layer and exposed dentin

! $#          %
showing widened dentin tubules; E: representative specimen
of EDTA group presenting smear layer and few exposed

!&#  
of E; G: representative specimen of PA group at 1,000 of
'  
   

 

! 8#          <
showing discrete widening of the tubules; I: representative
specimen of the control group at 1,000 showing smear layer
' 
 !=#
the central part of I in which the tubules are not enlarged
enough to measure their corresponding area

Control group
        
smear layer, open tubules could be seen in 2
    0    
number of 2.3 tubules for this group at the 1,000
 
 
] #V
           
their corresponding area and the mean value for
  6,    #666
 
 ] #|
The mean number of exposed dentin tubules by
the different treatments decreased according to the
  $%&II*I'($I
    
  
   $%&

J Appl Oral Sci.

472

2011;19(5):469-75

AMARAL NG, REZENDE MLR, HIRATA F, RODRIGUES MGS, SANTANA ACP, GREGHI SLA, PASSANEZI E

Table 1- Mean number of exposed dentin tubules and corresponding area after conditioning with the four different
demineralizing agents
Citric acid

Tetracycline-HCl

Phosphoric
acid

EDTA

Control group
(saline)

Mean number of exposed

tubules

39.337.0A

43.825.2A

12.116.3Aa

4.47.5Ba

2.35.7Ba

Mean area occupied by

enlarged tubules (%)

0.120.17A

0.080.06Aa

0.030.05Ba

0.010.01Ba

0.000.00B

J 
  
 




  KNQN
\        
tested ranging from 0.5% to 200% and from 0.5 to
10 min16,30. \  
0  0@6
mL and 125 mg/mL during 3 to 4 min of application
by burnishing technique. Isik, et al. 16 (2000)
      0 @6 
mL and 125 mg/mL might alter dentin surfaces by
removing the smear layer and also maximize tubule
openings in a short period if repeated applications
w   
the concentration of 50 mg/mL for TC-HCl. It has
0 0  
   
 
of smear layer and exposure of the underlying
tubules due to demineralization action of fresh
acid solution28$    
burnishing technique and for changing the cotton
pellet at every 30 s in this study.
$&    
   %&     
of the reasons for the reduced cellular insertion
and for the unpredictability of the results, once
it could denature the organic matrix of dentin14.
V           
interfere on periodontal healing by its necrotizing
effect on the surrounding progenitor cells13. Thus,
EDTA at 12%-24%, neutral pH for 30 s to 3 min
          
    0      
biological structures7   2 
et al.25 (2007)   0
 
  0 
  %
    ]'V4'\ 
that both citric acid and EDTA treatments are able to
 1 
0  
      1
W 
  '($ 
of use, failed to properly remove smear layer and
expose dentin tubules. Five specimens (50%) from
the EDTA group had none of their dentin tubules
1 #   
layer.
It must be emphasized that even though CA
 $%&    
    0 
 0      $%&  

,and C; TC-HCl and EDTA; CA and C and CA and

EDTA (Table 1).
When comparing the area occupied by dentin
tubule openings, CA produced the greatest tubule
0$%&*'($  
]$ 0  $       
 
0 $%&  
   $%& 
differed only from group C. The exposure of dentin
 0   

DISCUSSION
This study compared the 4 most commonly used
chemical agents for root conditioning as adjunctive
therapy for teeth affected by periodontitis. The
presented data suggest that citric acid and
 %&          
are more effective in removing smear layer and
 1     0   
phosphoric acid and EDTA.
Since Register and Burdick23 (1975) compared
     ]&   #%!
min) and other chemical substances and found
   
attachment, several investigators have devoted
considerable time studying conditioning agents to
improve periodontal regeneration. Unfortunately,
numerous and often uncontrolled histological
and clinical studies have created controversy and
confusion about the positive or negative effects of
those agents19. The inconsistency of these studies
may be due to differences in experimental systems
and techniques. Nevertheless, there is a common
acceptance that it is not possible to decontaminate
periodontitis-affected root surfaces by mechanical
  V    
or ultrasonic scaling of root surface produces
a nonbiocompatible smear layer that must be
removed to expose the underlying collagen in order
  
00      
orientation4,5,9,11,13,14,16-18,25,27.
$ 0  
and studied demineralizing agents since in vitro
studies of Terranova, et al.29 (1986) suggested its
potential usefulness in regenerative procedures.
J Appl Oral Sci.

473

2011;19(5):469-75

Comparison among four commonly used demineralizing agents for root conditioning. A scanning electron microscopy

not be considered different from the other groups.

$      0   
TC-HCl in second place in our analysis. Another

       * 1
similar numbers of dentin tubules, but CA produced
greater enlargement as reflected by the area
measurements. EDTA and PA had the same behavior
as saline solution on both evaluations, suggesting
   

  '($  
   
  
    *    
foamy surface, probably due to a chemical acid/
0    0     
hydroxyapatite, leading to calcium phosphate
deposition on the roots surfaces. This occurrence can
be attributed to the extended time of acid contact
]!V    0
         0/
tubule openings and annulling its demineralizing
effect10,21 
 
          
       &  
suggestive that phosphoric acid is not appropriate
     
2 
  
reinforced that acid etching plays a decisive role
   0     
         
i.e., adsorption and adhesion of blood elements

0      3. In this aspect,
Baker, et al.3 (2005) have clearly demonstrated the
   '($
applied for 5 min on planed dentin root surfaces.
0     0   0 
CA-treated than EDTA-treated dentin surfaces and
forces produced by three 5-min rinses in PBS under
agitation on a rotary shaker table partially removed

0'($%   0 
from CA-treated surfaces. The authors stressed
  
0/ 
  
   0 1
0   
$
    0    
1  0  
   
0   0   
   0
Citric acid and tetracycline behaved very similarly
in this particular aspect, suggesting that both can
be equally effective as conditioning agents.
The number and diameter of exposed dentin
 0  %   
in SEM micrographs by Herrero, et al.15]#66B
related increased numbers and diameters to better
 0 
causes higher exposure of the underlying dentin
favoring connective tissue attachment. Gamal and
Mailhot12 (2003) also observed that periodontal

J Appl Oral Sci.

 
00      
'($%        
from smear layer and presented exposed round
  0 

is no study comparing the number of exposed
dentin tubules and their corresponding area
      
    
$
 0  
data stressing the advantages of using conditioning
agents on diseased root surfaces and provide
    
agent for root conditioning. Before extrapolation
of data to the clinical conditions can be done,
  0      0 
1     
tissue attachment should be further investigated
in in vivo surveys.

CONCLUSION
The comparison among four of the most
frequently used chemical root conditioners

   
 0   
 0 %&
phosphoric acid and EDTA. This information can be
of value as an extra parameter for choosing one of
them for root conditioning.

ACKNOWLEDGMENTS
 / /*|20
Pereira Lauris, from Bauru School of Dentistry,
University of So Paulo, for the statistical analysis,
Mr. Adriano Luis Martins from Laboratory of Electron
Microscopy of the Faculty of Dentistry of Piracicaba
]V\*     4'\   
   4
 $ 
(]*
   
to this research.

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