Basic Research—Technology

Dentin Demineralization When Subjected to BioPure

Abstract
In the present study, the demineralizing ability of BioPure
MTAD (Dentsply/Tulsa, Tulsa, OK), 17% EDTA, and 5%
citric acid on radicular dentin was quantitatively and lon-
T he two targets of root canal instrumentation are, first, to clean and shape the root
canal system and, second, to allow the placement of a hermetic filling (1). To date,
the cleaning of the root canal system is linked to bacterial control and the dissolution of
gitudinally analyzed. 3 mm thick disks were obtained at organic pulpal tissue. Moreover, a smear-free dentin seems desirable (1). Combina-
the root cervical third from 9 maxillary human molars, and tions of decalcifying agents (generally organic acids) and sodium hypochlorite (NaOCl)
a standardized smear layer was produced. Cosite images have been recommended because no single irrigator is capable of providing the con-
of the dentin surface were obtained after several cumu- ditions mentioned earlier (3, 4).
lative demineralization times. Sixteen images were ob- With the aim of improving the root dentin cleanness and disinfection, Torabinejad
tained in each dentin sample for each experimental time, et al (5) developed a new solution, the so-called BioPure MTAD (Dentsply/Tulsa, Tulsa,
at 1000⫻ magnification. An image processing and anal- OK), which contains a mixture of a tetracycline isomer, citric acid, and a detergent.
ysis sequence measured sets of images, providing data of BioPure MTAD represents an innovative approach for the simultaneous removal of
area fraction for thousands of tubules over time. Thus, it smear layer and disinfection of the root canal system. BioPure MTAD is commercially
was possible to follow the demineralization phenomenon available as a two-part set that is mixed on demand. In recent times, many reports have
and quantitatively analyze the effect of the various sub- been focused on BioPure MTAD properties. To date, an English PubMed search for
stances. The nonparametric Kruskal-Wallis H-test was “BioPure MTAD” produced more than 20 papers published since 2003.
used to analyze the data. Based on the present results, it BioPure MTAD is a biocompatible material (6) and has similar solubilizing effects
can be concluded that the demineralization kinetics pro- on pulp and dentin to those of EDTA (5, 7, 8). A major difference between BioPure
moted by both 5% CA and BioPure MTAD was signifi- MTAD and EDTA is a high binding affinity to dentin of the doxycycline present in BioPure
cantly faster than by 17% EDTA. (J Endod 2007;33: MTAD that allows for an extended antibacterial effect. Torabinejad et al (9) reported
1364–1368) that BioPure MTAD possessed superior bactericidal activity compared with NaOCl or
REDTA when tested against Enterococcus faecalis. However, Baumgartner et al (10)
Key Words reported that the combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the
Chelators, cosite microscopy, demineralization, dentin,
canals contaminated with E. faecalis.
digital image analysis
In addition to these desirable properties, a few experiments have shown that
BioPure MTAD is an effective solution for the removal of the smear layer (5, 7).
According to a recent study (11), a longer period of time is required for bacteria to
From the *Veiga de Almeida University and †Rio de Janeiro penetrate when either EDTA or MTAD is used for smear layer removal.
State University, ‡Department of Materials Science and Met- In addition, the microstructural dentin changes promoted by BioPure MTAD do
allurgy, Catholic University of Rio de Janeiro, Rio de Janeiro, not seem to cause significant damage to the dentin matrix when used as a final rinse in
Brazil. conjunction with low concentrations of NaOCl (6). Unlike EDTA and citric acid (12, 13,
Address requests for reprints to Dr Gustavo André De-
Deus, R. Desembargador Renato Tavares, 11 ap. 102-Ipanema,
14), minimal erosion of intraradicular dentin has been reported when NaOCl and
22411-060 Rio de Janeiro, RJ, Brazil. E-mail address: Biopure MTAD were used in an equivalent sequence (7). For optimal removal of
endogus@[Link]. endodontic smear layers and to avoid inadvertent erosion of the intraradicular dentin,
0099-2399/$0 - see front matter a revised clinical protocol has been proposed that involves the use of an initial rinse with
Copyright © 2007 by the American Association of
Endodontists.
1.3% NaOCl for a period of 20 minutes followed by the use of BioPure MTAD as the final
doi:10.1016/[Link].2007.07.009 rinse for a cumulative period of 5 minutes (5, 7).
So far, two SEM investigations conducted by Torabinejad et al (5, 7) have repre-
sented the most important source of information concerning the demineralizing ability
of BioPure MTAD. In those studies, prepared root sections were examined under
scanning electron microscopy in a long-established approach in which the smear-
removal ability was quantified by a scoring model. However, Gulabivala et al (15),
Hülsmann et al (16), and De-Deus et al (14, 17) have pointed out that the main factor
leading to the lack of conclusions regarding the smear layer removal is the qualitative
and nonreproducible character of most studies. In addition, the magnifications used in
the SEM differ widely; in some studies, such data are not presented at all or different
magnifications were used during the investigation. A certain observer bias may occur in
the SEM when working with higher magnifications because only a small area of the root
canal wall can be observed. This area may be adjusted on the screen by chance or be

1364 De-Deus et al. JOE — Volume 33, Number 11, November 2007
 Basic Research—Technology
selected by the SEM operator. It is a common finding that most SEM At this point, the samples were randomly divided into 3 groups
operators tend to select clean canal areas with open dentinal tubules according to the chelating agent used as follows (n ⫽ 3 per group): (1)
rather than areas with large bulk of debris or smear layer (15, 16). G1: BioPure MTAD, (2) G2: 17% EDTA (pH 7.7), and (3) G3: 5% citric
Moreover, SEM investigations do not allow longitudinal observa- acid (pH 2.0). EDTA and citric acid were freshly prepared by the man-
tions of the dentinal morphology. In this kind of study, the status of the ufacturer (Formula & Ação Ltda., Sao Paulo, Brazil).
dentinal surface before the application of the chelator is unknown be-
cause the evaluation is not performed in the same sample. In an impor- Experimental Procedure (Cosite Microscopy)
tant article, Peters et al (18), using a high-quality microcomputed to- The experiments were developed in an Axioplan 2 Imaging motor-
mography method, showed that the amount of uninstrumented canal ized microscope (Carl Zeiss Vision Gmbh, Hallbergmoos, Germany). An
areas after preparation is 35% or more of their total. So, it is possible to Epiplan 100⫻ HD objective lens was used coupled to a 1300 ⫻ 1030
conclude that uninstrumented dentinal walls are smear free, and this pixels Axiocam HR digital camera (Carl Zeiss), leading to a total mag-
fact highlights the importance of a longitudinal character in smear- nification of approximately 1000X, and a resolution of 0.1 ␮m/pixel.
removal investigations, in which the status of the dentinal surface is well In the cosite microscopy experiment, a special holder allowed
known before the initial application of the chelator. application of the chelating solutions without removing the dentin sam-
It is worth mentioning that atomic force microscopy (AFM) has ple from the microscope. A motorized specimen stage was used to
been used for longitudinal evaluation of the dentin demineralization automatically acquire 16 image fields at specific x-y positions of a given
process (14, 19). This approach showed relevant advantages such as sample for several cumulative demineralization times (0, 15, 30, 60,
the observation of the process in near real time as the samples were 180, and 300 seconds). Thus, it was possible to follow the same fields
immersed in the chelating substance during observation. However, lim- with high reproducibility of the x-y positions and autofocus, allowing the
itations caused by specific characteristics of AFM precluded obtaining observation of the effect of demineralization in the very same regions. A
accurate quantitative results. Watari (20) used AFM to obtain quantita- pilot test was performed for the first methodologic article (17) in which
tive results regarding relief measurements such as roughness. a number of 16 image fields per tooth was defined. This decision was
Cosite optical microscopy (CSOM) was recently introduced (17), based on the following criteria: (1) the available dentin area and (2) the
and it represents an efficient method for direct comparison of the demi- amount of useful data obtained per image. The details of the procedure
neralizing power of the solutions used in Endodontics. The accuracy were described by De-Deus et al (17). The complete image acquisition
and reproducibility of CSOM have been verified previously (17), and it sequence was controlled by a special routine implemented under the
proved to be fast and robust. Moreover, the method provides quantita- AxioVision 4.5 software (Carl Zeiss Vision).
tive data linked to the longitudinal observation of the dentinal substrate
changes. Image Analysis
Hence, the present work aimed to assess, both longitudinally and A previously developed image analysis routine (17) was used to
quantitatively, the demineralizing ability of BioPure MTAD through enhance image contrast, discriminate (17, 19, 24), and measure open
CSOM and digital image analysis. Both 17% EDTA and 5% citric acid dentin tubules in each acquired image. Then, the ratio between the total
were used as reference solutions to compare the results. The null hy- area of open tubules and the area of the full image field, the so-called
pothesis tested was that there is no difference in demineralization of area fraction (AF), was measured. The change of AF over demineraliza-
dentin irrigated with either BioPure MTAD or EDTA or 5% citric acid. tion time was used to quantify the process. The routine was applied
without operator influence to the vast majority of the images acquired
for different samples at different times. All steps were implemented as a
Materials and Methods macroroutine under the KS400 3.0 software (Carl Zeiss Vision). The
Specimen Selection and Preparation initial images (0 seconds) with the standardized smear layer were not
This study was revised and approved by the Ethics Committee, analyzed because of the low contrast of the tubules covered by the smear
Nucleus of Collective Health Studies, Rio de Janeiro State University, Rio layer, which prevented their discrimination. As an overtime evaluation,
de Janeiro, Brazil. Nine maxillary human molars were selected from the each specimen served as its own control.
tooth bank of Rio de Janeiro State University. Each specimen was
mounted in an epoxy resin cylinder (Arazyn 1.0; Ara Química, Sao Data Presentation and Analysis
Paulo, Brazil) to facilitate manipulation and improve the metallographic Data are presented as tubule AF in percentage of the whole dentin
preparation. Following a protocol described previously (21), dentin area (17). The preliminary analysis of the pooled data from the exper-
discs approximately 3 ⫾ 0.3 mm thick were cut at the middle third level imental groups (SPSS for Windows, Version 8.0; SPSS Inc, Chicago, IL)
of the crowns but above the root canal using a low-speed saw (Isomet; did not show normal distribution (Kolmogorov-Smirnov test). Further
Buhler, Ltd, Lake Bluff, NY) with a diamond disc (A 125 mm ⫻ 0.35 statistical analysis was performed with nonparametric test methods us-
mm ⫻ 12.7 mm, 33°C), with continuous water irrigation in order to ing the Kruskal-Wallis H-test with Bonferroni correction to compare the
prevent overheating. The dentin surfaces were carefully inspected to AF values between the groups at the respective experimental times. The AF
ensure that they were free of coronal enamel or pulpal exposures. and the time were used as factors and the level of significance was set at
The decision to perform the present evaluation on crown dentin p ⬍ 0.05. Origin 6.0 (Microcal Software, Inc, Northampton, MA) was
instead of on intraradicular dentin was based on a pilot study in which also used as analytic tool.
the difficulty to obtain useful disks from intraradicular dentin was de-
termined. In addition, crown dentin can provide more homogeneous Results
than intraradicular dentin (22), and this represents a desirable feature The columns of the image montage in Figure 1 show the evolution
in comparative assessments. of demineralization over time for 17% EDTA, Biopure MTAD, and 5%
A standard metallographic procedure (grinding with SiC paper CA. In each column, an image field at a specific x-y position of a sample
[200, 300, 400, and 600] grits and 3 ␮m diamond paste) was used on is shown for 5 cumulative demineralization times.
the pulpal surface of the tooth to prepare them for the experimental Figure 2 shows the increase of AF of open tubules against time for
process and to produce a standardized smear layer (14, 17, 19, 23). each group. Based on the present data the following statements can be

JOE — Volume 33, Number 11, November 2007 Dentin Demineralization and BioPure MTAD 1365
Basic Research—Technology

Figure 1. Time evolution of a given dentin region during demineralization with each chelator. The columns show the evolution of demineralization over time for 17%
EDTA, 5% CA, and BioPure MTAD (from left).

made: (1) generally, all images were smear free after ⬇ 15/30 seconds whereas 5% citric acid was more effective than BioPure MTAD at both
of etching and one can observe the enlargement of the dentinal tubules, 180 seconds and 300 seconds; (4) there is a clear saturation on the
as expected in the typical evolution of demineralization; (2) BioPure demineralizing ability of BioPure MTAD after 30 seconds; (5) there is a
MTAD and 5% citric acid were more effective than 17% EDTA for all trend of saturation on the demineralizing ability of 5% citric acid after
experimental times (p ⬍ 0.05); (3) BioPure MTAD was more effective 30 seconds; and (6) the demineralization kinetics promoted by both
than 5% citric acid at both 15 seconds and 30 seconds (p ⬍ 0.05), 5% citric acid and BioPure MTAD was clearly faster than for 17% EDTA.

1366 De-Deus et al. JOE — Volume 33, Number 11, November 2007
 Basic Research—Technology
or a relatively weaker chelator with the opposite behavior? The answer
requires a better understanding of the mechanism of dentin matrix
destruction and its effects on the adaptation of the filling and achieved
sealing as well as a possible influence on root strength.
The erosive effects of EDTA and citric acid have been reported in
several studies (12, 13, 14, 26, 27). On the other hand, the manufac-
turer (28) and Torabinejad et al (7) reported that, unlike the use of
EDTA as the final rinse, minimal erosion of intraradicular dentin has
been reported when NaOCl and BioPure MTAD were used in a similar
sequence. The citric acid and doxycycline present in BioPure MTAD are
responsible for its chelating ability. However, De-Deus et al (14) re-
ported that CA led to strong erosive effects.
Possibly, some kind of chemical interaction between the compo-
nents of BioPure MTAD is responsible for the self-limiting effect. Tor-
abinejad et al (7) reported that apparently BioPure MTAD reacts with
the dentin surface differently when compared with CA or EDTA, and
these findings are in agreement with the current results.
The use of BioPure MTAD with doxycycline affinity to dentin may
Figure 2. Time evolution of the open tubule AF. Data points are the average of 48
measurements from 3 different samples. The frame highlights the experimental lead to a different effect on the dentin structure when compared with the
times for which all the solutions were statistically different. 17% EDTA and 5% CA (29). Krause et al (30) point out that in the
BioPure MTAD preparation, CA may serve to remove the smear layer,
thus allowing doxycycline to penetrate the dentinal tubules and exert an
antimicrobial effect. However, some studies have shown that both doxy-
Discussion cycline and CA have acid etching ability (31, 32).
Based on the present results, the null hypothesis tested was plainly Analyzing the present results, it is apparent that 5% CA and BioPure
rejected. It can be straightforwardly observed in the graph of Figure 2 MTAD have comparable effects. So, despite its self-limiting effect, it is
that under the same experimental conditions, both BioPure MTAD and not clear that BioPure MTAD caused minimal dentin erosion. More
5% CA were more effective than 17% EDTA at all experimental times. investigation is necessary to clarify whether the clinical protocol for the
Regarding the data obtained, two interesting points can be drawn: use of BioPure MTAD really prevents dentin erosion. Transmission elec-
(1) the fast and sharp increase of the tubular area fraction promoted by tron microscopy can be considered the ideal method to assess the
BioPure MTAD and 5% CA and (2) the full saturation of the deminer- effects of the irrigation protocols on the dentin matrix (33).
alizing ability of BioPure MTAD after 30 seconds. This project is a part of a larger study comparing the demineral-
The first point, the faster demineralization kinetics of BioPure ization power of the demineralizing agents available in current end-
MTAD and 5% CA, can be viewed as positive from a clinical point of view odontic practice. In the present study, BioPure MTAD was evaluated
because smear removal is fast and efficient. The faster effect of CA has through CSOM and digital image analysis. The method provides quan-
been reported in a few earlier studies. Recently, De-Deus et al (14) titative data linked to the longitudinal observation of the dentinal sub-
using a longitudinal observation method (AFM) found that CA had the strate changes.
strongest time-effect relationship when compared with EDTA and Because the goal of the present work was restricted to a direct
EDTAC. However, to the best of the authors’ knowledge, this faster effect longitudinal and quantitative assessment of the chelating ability of Bio-
has not been reported for BioPure MTAD so far. However, because of Pure MTAD, the application of these results to the clinical situation is
the current concern regarding erosive effects stemming from the asso- not straightforward. The limitations of the present result are directly
ciation of chelators with NaOCl, one cannot state that the fastest or most linked to the requirements of the light optical microscopy tech-
powerful solution is the best indicated for smear layer removal. niques. The chelator solution was applied to a flat horizontal dentin
The second point, the self-limiting effect of BioPure MTAD, sug- surface, different from the clinical situation, in which the contact
gests a novelty in relation to its well-known properties. The self-limiting between the chelating substance and the dentin surface is affected by
effect of EDTA has already been shown (13, 14). Once all the chelating the vertical position of the teeth and the intrinsic anatomical vari-
ions have reacted with the calcium ions of the dentin, equilibrium is ability of the root canal system. Moreover, in the present method, a
established and demineralization stops. However, the few reports about rinsing procedure was not performed and this is also not in line with
the smear-removal ability of BioPure MTAD do not point out a self- the clinical situation. However, for a direct comparison of the che-
limiting effect. Considering the concerns mentioned earlier regarding lating ability of the solutions the present methodological model
erosive effects, a solution that presents a self-limiting effect associated to proved to be fast, robust, reliable and statistically sound. Moreover,
efficiency is very attractive. the method provides longitudinal information about the dentin mor-
The consequences of dentin matrix destruction remains undefined phology and does not depend on the traditional descriptive scores of
although Park et al (25) speculated that an increased coronal leakage in SEM studies.
samples treated with EDTA compared with those treated with BioPure Under the conditions of the present ex vivo evaluation, the follow-
MTAD might be caused by the erosive property of EDTA and the length ing conclusions can be drawn: (1) the demineralization kinetics pro-
of dentin exposure to this solution. moted by both 5% citric acid and BioPure MTAD was clearly faster than
Future research should try to answer the following question: what for 17% EDTA, and (2) CSOM represents a powerful approach to com-
is the best clinical indication? Either a very strong chelator that removes pare directly longitudinally and quantitatively the ability of the deminer-
completely the smear layer but that might also destroy the dentin matrix alizing solutions.

JOE — Volume 33, Number 11, November 2007 Dentin Demineralization and BioPure MTAD 1367
Basic Research—Technology
Acknowledgments 15. Gulabivala K, Patela B, Evans G, Yuan Ling N. Effects of the mechanical and chemical
procedures on root canal surfaces. Endod Topics 2005;10:103–22.
The authors wish to thank Dr. Carlos Cleber Garcia for his 16. Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment:
essential assistance. mode of action and indications for their use. Int Endod J 2005;36:810 –30.
17. De-Deus G, Reis CM, Fidel RAS, Fidel SR, Paciornik S. Co-site digital optical micros-
copy and image analysis: an approach to evaluate the process of dentin demineral-
ization. Int Endod J 2007;40:1–12.
References 18. Peters OA, Schonenberger K, Laib A. Effects of four Ni-Ti preparation techniques on
1. Schilder H. Filling root canals in three dimensions. Dent Clin North Am root canal geometry assessed by micro computed tomography. Int Endod J
1967;11:723– 44. 2001;34:221–30.
2. Shahravan A, Haghdoost A, Adl A, Rahimi H, Shadifar F. Effect of smear layer on 19. Paciornik S, De-Deus G, Reis CM, Pinho Mauricio MH, Prioli R. In situ atomic force
sealing ability of canal obturation: a systematic review and meta-analysis. J Endod microscopy and image analysis of dentin submitted to acid etching. J Microsc
2007;33:96 –105. 2007;3:235– 42.
3. Goldman LB, Goldman M, Kronman JH, Lin PS. The efficacy of several irrigating 20. Watari F. In situ quantitative analysis of etching process of human teeth by atomic
solutions for endodontics: a scanning electron microscopic study. Oral Surg Oral force microscopy. J Electron Microsc 2005;54:299 –308.
Med Oral Pathol Endod 1981;52:197–204. 21. Grégoire G, Joniot S, Guignes P, Millas A. Dentin permeability: self-etching and
4. Yamada RS, Armas A, Goldman M, Lin OS. A scanning electron microscopic com- one-bottle dentin bonding systems. J Prosthet Dent 2003;90:42–9.
parison of a high volume final flush with several irrigating solutions: part 3. J Endod 22. Mjör I, Nordhal I. The density and the branching of dentinal tubules in human teeth.
1983;9:137– 42. Arch Oral Biol 1996;41:401–12.
5. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the 23. De-Deus G, Paciornik S, Mauricio MHP. Evaluation of the effect of EDTA, EDTAC and
smear layer. J Endod 2003;29:170 –5. citric acid on the microhardness of root dentin. Int Endod J 2006;39:401–7.
6. Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT- 24. Paciornik S, Mauricio MH. Digital imaging. In: Vander Voort GF, ed. ASM Handbook:
tetrazolium method. J Endod 2003;29:654 –7. Metallography and Microstructures. Materials Park, OH: ASM International;
7. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various 2004:368 – 402.
concentrations of sodium hypochlorite on the ability of BioPure MTAD to remove the 25. Park DS, Torabinejad M, Shabahang S. The effect of MTAD on the coronal leakage of
smear layer. J Endod 2003;29:233–9. obturated root canals. J Endod 2004;30:890 –2.
8. Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing 26. Cergneux M, Ciucchi B, Dietschi JM, Holz J. The influence of the smear layer on the
action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod sealing ability of canal obturation. Int Endod J 1987;20:228 –32.
2003;29:334 –7. 27. Meryon SD, Tobias RS, Jakeman KJ. Smear removal agents: a quantitative study in vivo
9. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of and in vitro. J Prosthet Dent 1987;57:174 –9.
MTAD: An in vitro investigation. J Endod 2003;29:400 –3. 28. Dentsply Tulsa Dental. BioPure MTAD. Available at: [Link]
10. Baumgartner JC, Johal S, Marshall JG. Comparison of the antimicrobial efficacy of Accessed March 24, 2007.
1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. J 29. Baker PJ, Evans RT, Slots J, Genco RJ. Susceptibility of human oral anaerobic bacteria
Endod 2007;33:48 –51. to antibiotics suitable for topical use. J Clin Periodontol 1985;12:201– 8.
11. Ghoddusi J, Rohani A, Rashed T, Ghaziani P, Akbari M. An evaluation of microbial 30. Krause TA, Liewehr FR, Hahn CL. The antimicrobial effect of MTAD, sodium hypo-
leakage after using MTAD as a final irrigation. J Endod 2007;33:173– 6. chlorite, doxycycline, and citric acid on Enterococcus faecalis. J Endod 2007;
12. Takeda FH, Harashima T, Kimura Y, Matsumoto K. A comparative study of the removal 33:28 –30.
of smear layer by three endodontic irrigants and two types of laser. Int Endod J 31. Smith J, Wayman B. An evaluation of the antimicrobial effectiveness of citric acid as
1999;32:32–9. a root canal irrigant. J Endod 1986;12:54 – 8.
13. Çalt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 32. Demirel K, Baer PN, McNamara TF. Topical application of doxycycline on periodon-
2002;28:17–9. tally involved root surfaces in vitro: Comparative analysis of substantivity on cemen-
14. De-Deus G, Paciornik S, Pinho Mauricio M, Prioli R. Real-time atomic force micros- tum and dentin. J Periodontol 1991;62:312– 6.
copy of root dentin during demineralization when subjected to chelating agents. Int 33. Tay FR, Pashley DH, Loushine RJ, et al. Ultrastructure of smear layer-covered intrara-
Endod J 2006;39:683–92. dicular dentin after irrigation with BioPure MTAD. J Endod 2006;32:218 –21.

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