- Email: [email protected]
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin
Basic Research—Technology
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin Monika Marending, Dr med dent,* Frank Paqué, Dr med dent,* Jens Fischer, Dr med dent, PD,† and Matthias Zehnder, Dr med dent, PhD, PD* Abstract To get the root canal system free of organic debris and the smear layer, it has been recommended to irrigate with a NaOCl solution during instrumentation, followed by a rinse with a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and a final irrigation with NaOCl. However, both hypochlorite and EDTA weaken dentin through dissolution of its organic and inorganic components, respectively. EDTA exposes the organic dentin matrix, which could then be attacked more easily by hypochlorite. It was the aim of this study to assess the impact of different irrigation sequences of NaOCl (2.5% w/v; total exposure time, 24 minutes) and EDTA (17%; 3 minutes) on the elastic modulus and flexure strength of standardized human root dentin bars (n ⫽ 11 per group). Exposures to solely EDTA (3 minutes), NaOCl (24 minutes), and water were used as control treatments. Specimens were subjected to 3-point bending tests; modulus of elasticity and flexure strength values were compared between groups with one-way analysis of variance followed by Fisher probable least-squares difference test. The alpha-type error was set at .05. The 24-minute exposure to the hypochlorite solution caused a significant drop in flexure strength compared with water- or EDTA-treated controls (P ⬍ .05), whereas the elastic modulus remained unaffected. In contrast, the short exposure to EDTA as is clinically recommended did not affect the mechanical dentin parameters under investigation, regardless of the irrigant sequence that was used. (J Endod 2007;33:1325–1328)
Key Words Dentin, EDTA, flexure strength, modulus of elasticity, NaOCl
From the *Division of Endodontology, Department of Preventive Dentistry, Periodontology, and Cariology and †Clinic for Fixed and Removable Prosthodontics and Material Science, University of Zürich Center for Dental Medicine, Zürich, Switzerland. Address requests for reprints to Dr Matthias Zehnder, Division of Endodontology, Department of Preventive Dentistry, Periodontology, and Cariology, University of Zürich Center for Dental Medicine, Plattenstrasse 11, CH-8032 Zürich, Switzerland. E-mail address: [email protected] 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2007.08.005
JOE — Volume 33, Number 11, November 2007
T
he primary goal of an endodontic treatment is to obtain a clean root canal system free of microbiota and debris, which can then be sealed with a microbial-tight root canal filling. The chemomechanical preparation concept relates to the use of chemically active irrigating solutions in combination with mechanical cleansing. To this end, sodium hypochlorite (NaOCl) solutions remain the most widely recommended irrigants in endodontics on the basis of their unique capacity to dissolve necrotic tissue remnants and their excellent antimicrobial potency (1). In addition to hypochlorite, the use of a chelating agent has been advocated to rid the root canal system of the so-called smear layer consisting of dentin particles embedded in an amorphous mass of organic material that forms on the canal walls during the instrumentation procedure (2). It is believed that removing this layer could (a) dissolve attached microbiota and their toxins from root canal walls, (b) improve the seal of root canal fillings, and (c) reduce the potential for bacterial survival and reproduction (1, 2). Calcium-chelating agents such as ethylenediaminetetraacetic acid (EDTA) or citric acid are used to dissolve the smear layer (2). However, these agents interfere with sodium hypochlorite and thus have to be used separately (3). There is much dispute regarding the ideal sequence, volume, and concentration of irrigating solutions during a root canal treatment. Most clinicians use NaOCl solutions in the w/v range of 1%–5% and EDTA at 17%. To reduce stress on endodontic instruments, it is advocated to keep the root canal system flooded with an aqueous solution during mechanical preparation (4). For that purpose, sodium hypochlorite is recommended to maximize working time on necrotic tissue remnants and microbiota. A chelating solution could then be used as the final rinse to remove the smear layer. However, an alternating irrigating regime with hypochlorite and EDTA has also been recommended, with a final NaOCl flush after EDTA (5, 6). Dentin is composed of approximately 22% organic material by weight. Most of this consists of type I collagen, which contributes considerably to the mechanical properties of dentin (7). Sodium hypochlorite is a nonspecific oxidizing agent (8). It is known to fragment long peptide chains and to chlorinate protein terminal groups; the resulting N-chloramines are broken down into other species (9, 10). Therefore, along with its desired therapeutic effects, the use of sodium hypochlorite can have undesired consequences for the dentin substrate. A 2-hour exposure of dentin to NaOCl solutions of more than 3% (w/v) significantly decreases the elastic modulus and flexure strength of human dentin compared with a control exposure to physiologic saline, thereby possibly contributing to the weakening of root canal–treated teeth (11, 12). The mineral phase in dentin protects the collagen matrix. It is therefore conceivable that demineralizing agents used for the removal of the smear layer can expedite the destructive hypochlorite effect (13). In other words, using a chelating agent before sodium hypochlorite exposes the organic dentin matrix, which can then be attacked by the subsequent hypochlorite rinse (14). Overzealous use of irrigants might thus make root canal–treated teeth more prone to fracture. In this context, it appears important to notice that vertical root fractures are among the most common causes for the extraction of endodontically treated teeth (15, 16). It was the aim of the current study to assess the impact of different irrigation regimes on root dentin mechanical properties. The hypothesis tested here was that an EDTA exposure before a final NaOCl treatment would affect the mechanical integrity of root dentin more than a corresponding EDTA exposure without subsequent application of NaOCl. Standardized human root dentin bars were used for this study. The outcome
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin
1325
Basic Research—Technology variables assessed were modulus of elasticity and flexure strength after exposure to the irrigants. Modulus of elasticity is a measure of the linear deformation of a material during mechanical stress, whereas flexure strength is related to the maximum force that a material can bear before it breaks.
the greater bearing surface centered on the support (ie, with the tubules parallel to the cross-head). The cross-head speed of the testing machine was set to 0.5 mm/min, and the bars were tested until failure. The elastic modulus was calculated from the slope (m) of the load-displacement curves within the linear elastic region by using the formula
Materials and Methods Preparation of Dentin Specimens Human root dentin bars were prepared from extracted upper third molars as described earlier (17). The teeth that were used had been stored in a 0.2% thymol solution at 5°C for a maximum of 1 year. In brief, dentin bars were prepared by using a saw microtome (SP1600; Leica Microsystems, Glattbrugg, Switzerland) to a profile of 0.8 mm ⫻ 1.2 mm; their length was adjusted to 10 mm. The orientation of the dentinal tubules was perpendicular to the greater bearing surface of the bars and was equal for all specimens. The dentin bars were stored in sterile saline solution until further usage. The treatment plan of any of the involved patients, who had given informed consent that their extracted teeth can be used for study purposes, was not altered by this study. Because the teeth were taken from the pooled third molars of the department’s collection of extracted teeth, the age and gender of the donors were not known. Test and Control Treatments The 55 specimens were randomly divided into 5 groups of 11 specimens each. Specimens were immersed individually in polypropylene containers containing 5 mL of the respective solutions at 37°C in a water bath. Care was exercised that the hypothesis under investigation was addressed with the appropriate controls (Fig. 1). Exposure times and volumes for both chemical agents (NaOCl and EDTA) were kept constant between the 2 experimental sequences (groups I and II) and the control treatments with solely NaOCl (group III) or EDTA (group IV). Immersion in tap water for 30 minutes (group V) was used as the positive control. Three-Point Bending Tests Mechanical tests were performed with a universal testing machine (Zwick, Ulm, Germany) directly after treatment with test and control solutions. The dentin bars were kept moist with physiologic saline solution during all manipulations. Before they were transferred to the testing apparatus, their width and depth were measured with a sliding caliper. A specimen holder with 2 cylindrical supports with a radius of 1 mm and a span of 7 mm was used. Specimens were placed with
Figure 1. Flow chart depicting the treatments in the experimental (I and II) and control groups (III to V).
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Marending et al.
E⫽
l3m 4bh3
with the support span (l), the width (b), and the height (h) of the specimen. The flexure strength () was calculated according to the formula ⫽
3Fl 2bh2
with F representing the load at fracture. Registration of the load at fracture and calculation of modulus of elasticity as well as flexure strength were performed by means of a software program (testXpert; Zwick).
Data Presentation and Analysis Data were distributed evenly and thus are presented as means and standard deviations. Parametric statistical methods were applied to compare mean values between groups: one-way analysis of variance (ANOVA) followed by Fisher probable least-squares difference (PLSD) test.
Results Mean elastic modulus values were statistically similar between all groups under investigation (Table 1), indicating the absence of an EDTA and/or NaOCl effect on this outcome variable under the current conditions. In contrast, sodium hypochlorite exposure significantly reduced flexure strength (Table 1). This effect, however, was not influenced by the use of EDTA either before or after the last hypochlorite exposure (P ⬎ .05). A 3-minute exposure to EDTA without hypochlorite caused no significant effect on flexure strength compared with a water treatment (P ⬎ .05).
Discussion The current study failed to demonstrate a significant effect of irrigant sequence on mechanical dentin properties. Consequently, the hypothesis that the short-term use of EDTA before sodium hypochlorite should increase the destructive effect of NaOCl on dentin was rejected. Storage of the teeth in 0.1% thymol for up to 1 year did not influence the outcome variables under investigation. In a pilot experiment, we compared modulus of elasticity and flexure strength between 7 dentin bars dissected from freshly extracted third molars with the corresponding values obtained with 7 randomly selected dentin bars dissected from molars of the extracted teeth collection (as used for the main study). There was no difference between fresh and thymol-stored teeth in either parameter (data not shown). The current data were obtained in a controlled laboratory environment, and direct clinical conclusions can therefore not be drawn. The dentin specimens used here were exposed to the irrigants under investigation from 4 sides; this does not correlate with the situation in the root canal. On the other hand, exposure times to hypochlorite were relatively short, irrigant volumes were low, and still there was a significant effect on flexure strength. This finding correlates well with published results (11, 12). However, the former studies used exposure times of 2 hours. It was
JOE — Volume 33, Number 11, November 2007
Basic Research—Technology TABLE 1. Modulus of Elasticity and Flexure Strength Values (Means ⫾ Standard Deviations) of Human Root Dentin Bars after Test and Control Treatments with 2.5% NaOCl and 17% EDTA Group I II III IV V
Modulus of Elasticity (GPa)
Flexure Strength (MPa)
Irrigant Sequence (21 min–3 min–3 min–3 min)
11.3 ⫾ 0.9A 11.8 ⫾ 0.8A 11.9 ⫾ 1.3A 11.8 ⫾ 2.0A 12.1 ⫾ 0.7A
147.0 ⫾ 19.7a 160.0 ⫾ 29.3ab 145.6 ⫾ 30.1a 181.1 ⫾ 35.9bc 195.4 ⫾ 44.9c
NaOCl–EDTA–NaOCl–H2O NaOCl–H2O–NaOCl–EDTA NaOCl–H2O–NaOCl–H2O H2O–EDTA–H2O–H2O H2O–H2O–H2O–H2O
EDTA, ethylenediaminetetraacetic acid. Identical superscript letters indicate that there was no significant difference.
found that 3% and 5% NaOCl significantly affect both flexure strength and modulus of elasticity (12). A 1% NaOCl solution has no such effect for up to 1 hour under conditions identical to those in the current study (18), which underlines the time- and concentration-dependency of hypochlorite actions on organic structures. In general, flexure strength is mainly affected by flaws or alterations on a specimen’s surface. By exposing the dentin surface to NaOCl or EDTA, the surface structure is degraded, which might explain the decrease in flexure strength. In contrast, the modulus of elasticity is determined by a specimen’s bulk properties. It appears that under the conditions of the current study, hypochlorite-mediated dentin degradation was limited to the surface of the specimens (13). Hence, the modulus of elasticity did not change in the present investigation. Values drop only after longer exposure times or at higher hypochlorite concentrations (18). The current finding that short-term exposure to EDTA does not weaken dentin should not lead to the erroneous conclusion that mechanical dentin properties are not affected by the dissolution of inorganic components. An exposure of human root dentin to 17% EDTA for 2 hours completely rids the exposed surface from calcium to a depth of approximately 150 m (19). The flexure strength of specimens such as those used in the current study is reduced by one third and the elastic modulus by half after a 2-hour exposure to 17% EDTA (17). However, in the present study, we chose short exposure times. An exposure time of 3 minutes to 17% EDTA should suffice to dissolve the inorganic parts of the smear layer (20). Although removal of smear layer might be desirable, we have to consider which irrigant to use last. If the last irrigant is EDTA, it is reasonable to believe that the organic part of the smear layer, ie, the collagen, remains on the surface of the root canal lumen (21, 22). Collagen can be important for the binding of bacteria including Enterococcus faecalis (23, 24). It might therefore not be prudent to use EDTA as the final irrigant. When used by themselves, EDTA and NaOCl can each affect the physical and mechanical properties of dentin adversely (11, 25). Not surprisingly, the combined removal of the inorganic as well the organic phase gives rise to damaging effects on the peritubular and intertubular dentin (22, 26). However, under the current conditions, this combined effect did not result in lowered modulus of elasticity and flexure strength values. This might have to do with 2 things; first, the exposure times of 3 minutes during the sequential use of EDTA and NaOCl were probably too short to disclose an impact on the mechanical dentin properties, and second, EDTA remnants on the dentin might have reduced hypochlorite and thus blocked its proteolytic effects (27). The second issue could have been corrected by applying a water rinse between EDTA and NaOCl. However, this is not usually done clinically and was thus not performed in the current study either. Future studies should aim to delineate guidelines for the time and concentration of endodontic irrigants to obtain a clean root canal system without producing any untoward effects to the tooth.
JOE — Volume 33, Number 11, November 2007
Acknowledgements The authors thank Beatrice Sener and Bogna Stawarczyk for their help with the laboratory procedures.
References 1. Zehnder M. Root canal irrigants. J Endod 2006;32:389 –98. 2. Torabinejad M, Handysides R, Khademi A, Bakland L. Clinical implications of the smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:658 – 66. 3. Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy reconsidered. J Endod 2005;31:817–20. 4. Peters OA, Boessler C, Zehnder M. Effect of liquid and paste-type lubricants on torque values during simulated rotary root canal instrumentation. Int Endod J 2005;38: 223–9. 5. Yamada RS, Armas A, Goldman M, Lin P. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: part 3. J Endod 1983;9:137– 42. 6. Baumgartner J, Mader C. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 7. Currey JD, Brear K, Zioupos P. Dependence of mechanical properties on fibre angle in narwhal tusk, a highly oriented biological composite. J Biomech 1994;27:885–97. 8. Dychdala G. Chlorine and chlorine compounds. In: Block SS, ed. Chlorine and chlorine compounds. Philadelphia: Lea & Febiger, 1991:131–51. 9. Stoward PJ. A histochemical study of the apparent deamination of proteins by sodium hypochlorite. Histochemistry 1975;45:213–26. 10. Davies JM, Horwitz DA, Davies K. Potential roles of hypochlorous acid and n-chloroamines in collagen breakdown by phagocytic cells in synovitis. Free Radic Biol Med 1993;15:637– 43. 11. Sim T, Knowles J, Ng Y, Shelton J, Gulabivala K. Effect of sodium hypochlorite on mechanical properties of dentine and tooth surface strain. Int Endod J 2001; 34:120 –32. 12. Grigoratos D, Knowles J, Ng Y, Gulabivala K. Effect of exposing dentine to sodium hypochlorite and calcium hydroxide on its flexural strength and elastic modulus. Int Endod J 2001;34:113–9. 13. Oyarzun A, Cordero A, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002; 28:152– 6. 14. Di Renzo M, Ellis T, Sacher E, Stangel I. A photoacoustic FTIRS study of the chemical modifications of human dentin surfaces: II— deproteination. Biomaterials 2001; 22:793–7. 15. Vire D. Failure of endodontically treated teeth: classification and evaluation. J Endod 1991;17:338 – 42. 16. Caplan D, Weintraub J. Factors related to loss of root canal filled teeth. J Public Health Dent 1997;57:31–9. 17. Vollenweider M, Brunner TJ, Knecht S, et al. Remineralization of human dentin using ultrafine bioactive glass particles. Acta Biomater 2007 (in press) doi:10/1016/j.actbio.2007.04.003. 18. Marending M, Luder H, Brunner TJ, Knecht S, Stark W, Zehnder M. Effect of sodium hypochlorite on human root dentine: mechanical, chemical and structural evaluation. Int Endod J 2007 (in press) doi:10/1111/j.1365-2591.2007.01287.X. 19. Kawasaki K, Ruben J, Tsuda H, Huysmans M, Takagi O. Relationship between mineral distributions in dentine lesions and subsequent remineralization in vitro. Caries Res 2000;34:395– 403. 20. De-Deus G, Reis C, Fidel R, Fidel S, Paciornik S. Co-site digital optical microscopy and image analysis: an approach to evaluate the process of dentine demineralization. Int Endod J 2007;40:441–52. 21. Goldman M, Goldman LB, Cavaleri R, Bogis J, Lin P. The efficacy of several endodontic irrigating solutions: a scanning electron microscopic study—part 2. J Endod 1982;8:487–92.
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Basic Research—Technology 22. Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 2002;35:934 –9. 23. Hubble TS, Hatton JF, Nallapareddy SR, Murray BE, Gillespie M. Influence of Enterococcus faecalis proteases and the collagen-binding protein, ACE, on adhesion to dentin. Oral Microbiol Immunol 2003;18:121– 6. 24. Love R. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int Endod J 2001;34:399 – 405.
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25. Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutions on the microhardness and the roughness of root canal dentin. J Endod 2004;30:792–5. 26. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28:17–9. 27. Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003; 36:411–7.
JOE — Volume 33, Number 11, November 2007
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin Monika Marending, Dr med dent,* Frank Paqué, Dr med dent,* Jens Fischer, Dr med dent, PD,† and Matthias Zehnder, Dr med dent, PhD, PD* Abstract To get the root canal system free of organic debris and the smear layer, it has been recommended to irrigate with a NaOCl solution during instrumentation, followed by a rinse with a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and a final irrigation with NaOCl. However, both hypochlorite and EDTA weaken dentin through dissolution of its organic and inorganic components, respectively. EDTA exposes the organic dentin matrix, which could then be attacked more easily by hypochlorite. It was the aim of this study to assess the impact of different irrigation sequences of NaOCl (2.5% w/v; total exposure time, 24 minutes) and EDTA (17%; 3 minutes) on the elastic modulus and flexure strength of standardized human root dentin bars (n ⫽ 11 per group). Exposures to solely EDTA (3 minutes), NaOCl (24 minutes), and water were used as control treatments. Specimens were subjected to 3-point bending tests; modulus of elasticity and flexure strength values were compared between groups with one-way analysis of variance followed by Fisher probable least-squares difference test. The alpha-type error was set at .05. The 24-minute exposure to the hypochlorite solution caused a significant drop in flexure strength compared with water- or EDTA-treated controls (P ⬍ .05), whereas the elastic modulus remained unaffected. In contrast, the short exposure to EDTA as is clinically recommended did not affect the mechanical dentin parameters under investigation, regardless of the irrigant sequence that was used. (J Endod 2007;33:1325–1328)
Key Words Dentin, EDTA, flexure strength, modulus of elasticity, NaOCl
From the *Division of Endodontology, Department of Preventive Dentistry, Periodontology, and Cariology and †Clinic for Fixed and Removable Prosthodontics and Material Science, University of Zürich Center for Dental Medicine, Zürich, Switzerland. Address requests for reprints to Dr Matthias Zehnder, Division of Endodontology, Department of Preventive Dentistry, Periodontology, and Cariology, University of Zürich Center for Dental Medicine, Plattenstrasse 11, CH-8032 Zürich, Switzerland. E-mail address: [email protected] 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2007.08.005
JOE — Volume 33, Number 11, November 2007
T
he primary goal of an endodontic treatment is to obtain a clean root canal system free of microbiota and debris, which can then be sealed with a microbial-tight root canal filling. The chemomechanical preparation concept relates to the use of chemically active irrigating solutions in combination with mechanical cleansing. To this end, sodium hypochlorite (NaOCl) solutions remain the most widely recommended irrigants in endodontics on the basis of their unique capacity to dissolve necrotic tissue remnants and their excellent antimicrobial potency (1). In addition to hypochlorite, the use of a chelating agent has been advocated to rid the root canal system of the so-called smear layer consisting of dentin particles embedded in an amorphous mass of organic material that forms on the canal walls during the instrumentation procedure (2). It is believed that removing this layer could (a) dissolve attached microbiota and their toxins from root canal walls, (b) improve the seal of root canal fillings, and (c) reduce the potential for bacterial survival and reproduction (1, 2). Calcium-chelating agents such as ethylenediaminetetraacetic acid (EDTA) or citric acid are used to dissolve the smear layer (2). However, these agents interfere with sodium hypochlorite and thus have to be used separately (3). There is much dispute regarding the ideal sequence, volume, and concentration of irrigating solutions during a root canal treatment. Most clinicians use NaOCl solutions in the w/v range of 1%–5% and EDTA at 17%. To reduce stress on endodontic instruments, it is advocated to keep the root canal system flooded with an aqueous solution during mechanical preparation (4). For that purpose, sodium hypochlorite is recommended to maximize working time on necrotic tissue remnants and microbiota. A chelating solution could then be used as the final rinse to remove the smear layer. However, an alternating irrigating regime with hypochlorite and EDTA has also been recommended, with a final NaOCl flush after EDTA (5, 6). Dentin is composed of approximately 22% organic material by weight. Most of this consists of type I collagen, which contributes considerably to the mechanical properties of dentin (7). Sodium hypochlorite is a nonspecific oxidizing agent (8). It is known to fragment long peptide chains and to chlorinate protein terminal groups; the resulting N-chloramines are broken down into other species (9, 10). Therefore, along with its desired therapeutic effects, the use of sodium hypochlorite can have undesired consequences for the dentin substrate. A 2-hour exposure of dentin to NaOCl solutions of more than 3% (w/v) significantly decreases the elastic modulus and flexure strength of human dentin compared with a control exposure to physiologic saline, thereby possibly contributing to the weakening of root canal–treated teeth (11, 12). The mineral phase in dentin protects the collagen matrix. It is therefore conceivable that demineralizing agents used for the removal of the smear layer can expedite the destructive hypochlorite effect (13). In other words, using a chelating agent before sodium hypochlorite exposes the organic dentin matrix, which can then be attacked by the subsequent hypochlorite rinse (14). Overzealous use of irrigants might thus make root canal–treated teeth more prone to fracture. In this context, it appears important to notice that vertical root fractures are among the most common causes for the extraction of endodontically treated teeth (15, 16). It was the aim of the current study to assess the impact of different irrigation regimes on root dentin mechanical properties. The hypothesis tested here was that an EDTA exposure before a final NaOCl treatment would affect the mechanical integrity of root dentin more than a corresponding EDTA exposure without subsequent application of NaOCl. Standardized human root dentin bars were used for this study. The outcome
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin
1325
Basic Research—Technology variables assessed were modulus of elasticity and flexure strength after exposure to the irrigants. Modulus of elasticity is a measure of the linear deformation of a material during mechanical stress, whereas flexure strength is related to the maximum force that a material can bear before it breaks.
the greater bearing surface centered on the support (ie, with the tubules parallel to the cross-head). The cross-head speed of the testing machine was set to 0.5 mm/min, and the bars were tested until failure. The elastic modulus was calculated from the slope (m) of the load-displacement curves within the linear elastic region by using the formula
Materials and Methods Preparation of Dentin Specimens Human root dentin bars were prepared from extracted upper third molars as described earlier (17). The teeth that were used had been stored in a 0.2% thymol solution at 5°C for a maximum of 1 year. In brief, dentin bars were prepared by using a saw microtome (SP1600; Leica Microsystems, Glattbrugg, Switzerland) to a profile of 0.8 mm ⫻ 1.2 mm; their length was adjusted to 10 mm. The orientation of the dentinal tubules was perpendicular to the greater bearing surface of the bars and was equal for all specimens. The dentin bars were stored in sterile saline solution until further usage. The treatment plan of any of the involved patients, who had given informed consent that their extracted teeth can be used for study purposes, was not altered by this study. Because the teeth were taken from the pooled third molars of the department’s collection of extracted teeth, the age and gender of the donors were not known. Test and Control Treatments The 55 specimens were randomly divided into 5 groups of 11 specimens each. Specimens were immersed individually in polypropylene containers containing 5 mL of the respective solutions at 37°C in a water bath. Care was exercised that the hypothesis under investigation was addressed with the appropriate controls (Fig. 1). Exposure times and volumes for both chemical agents (NaOCl and EDTA) were kept constant between the 2 experimental sequences (groups I and II) and the control treatments with solely NaOCl (group III) or EDTA (group IV). Immersion in tap water for 30 minutes (group V) was used as the positive control. Three-Point Bending Tests Mechanical tests were performed with a universal testing machine (Zwick, Ulm, Germany) directly after treatment with test and control solutions. The dentin bars were kept moist with physiologic saline solution during all manipulations. Before they were transferred to the testing apparatus, their width and depth were measured with a sliding caliper. A specimen holder with 2 cylindrical supports with a radius of 1 mm and a span of 7 mm was used. Specimens were placed with
Figure 1. Flow chart depicting the treatments in the experimental (I and II) and control groups (III to V).
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Marending et al.
E⫽
l3m 4bh3
with the support span (l), the width (b), and the height (h) of the specimen. The flexure strength () was calculated according to the formula ⫽
3Fl 2bh2
with F representing the load at fracture. Registration of the load at fracture and calculation of modulus of elasticity as well as flexure strength were performed by means of a software program (testXpert; Zwick).
Data Presentation and Analysis Data were distributed evenly and thus are presented as means and standard deviations. Parametric statistical methods were applied to compare mean values between groups: one-way analysis of variance (ANOVA) followed by Fisher probable least-squares difference (PLSD) test.
Results Mean elastic modulus values were statistically similar between all groups under investigation (Table 1), indicating the absence of an EDTA and/or NaOCl effect on this outcome variable under the current conditions. In contrast, sodium hypochlorite exposure significantly reduced flexure strength (Table 1). This effect, however, was not influenced by the use of EDTA either before or after the last hypochlorite exposure (P ⬎ .05). A 3-minute exposure to EDTA without hypochlorite caused no significant effect on flexure strength compared with a water treatment (P ⬎ .05).
Discussion The current study failed to demonstrate a significant effect of irrigant sequence on mechanical dentin properties. Consequently, the hypothesis that the short-term use of EDTA before sodium hypochlorite should increase the destructive effect of NaOCl on dentin was rejected. Storage of the teeth in 0.1% thymol for up to 1 year did not influence the outcome variables under investigation. In a pilot experiment, we compared modulus of elasticity and flexure strength between 7 dentin bars dissected from freshly extracted third molars with the corresponding values obtained with 7 randomly selected dentin bars dissected from molars of the extracted teeth collection (as used for the main study). There was no difference between fresh and thymol-stored teeth in either parameter (data not shown). The current data were obtained in a controlled laboratory environment, and direct clinical conclusions can therefore not be drawn. The dentin specimens used here were exposed to the irrigants under investigation from 4 sides; this does not correlate with the situation in the root canal. On the other hand, exposure times to hypochlorite were relatively short, irrigant volumes were low, and still there was a significant effect on flexure strength. This finding correlates well with published results (11, 12). However, the former studies used exposure times of 2 hours. It was
JOE — Volume 33, Number 11, November 2007
Basic Research—Technology TABLE 1. Modulus of Elasticity and Flexure Strength Values (Means ⫾ Standard Deviations) of Human Root Dentin Bars after Test and Control Treatments with 2.5% NaOCl and 17% EDTA Group I II III IV V
Modulus of Elasticity (GPa)
Flexure Strength (MPa)
Irrigant Sequence (21 min–3 min–3 min–3 min)
11.3 ⫾ 0.9A 11.8 ⫾ 0.8A 11.9 ⫾ 1.3A 11.8 ⫾ 2.0A 12.1 ⫾ 0.7A
147.0 ⫾ 19.7a 160.0 ⫾ 29.3ab 145.6 ⫾ 30.1a 181.1 ⫾ 35.9bc 195.4 ⫾ 44.9c
NaOCl–EDTA–NaOCl–H2O NaOCl–H2O–NaOCl–EDTA NaOCl–H2O–NaOCl–H2O H2O–EDTA–H2O–H2O H2O–H2O–H2O–H2O
EDTA, ethylenediaminetetraacetic acid. Identical superscript letters indicate that there was no significant difference.
found that 3% and 5% NaOCl significantly affect both flexure strength and modulus of elasticity (12). A 1% NaOCl solution has no such effect for up to 1 hour under conditions identical to those in the current study (18), which underlines the time- and concentration-dependency of hypochlorite actions on organic structures. In general, flexure strength is mainly affected by flaws or alterations on a specimen’s surface. By exposing the dentin surface to NaOCl or EDTA, the surface structure is degraded, which might explain the decrease in flexure strength. In contrast, the modulus of elasticity is determined by a specimen’s bulk properties. It appears that under the conditions of the current study, hypochlorite-mediated dentin degradation was limited to the surface of the specimens (13). Hence, the modulus of elasticity did not change in the present investigation. Values drop only after longer exposure times or at higher hypochlorite concentrations (18). The current finding that short-term exposure to EDTA does not weaken dentin should not lead to the erroneous conclusion that mechanical dentin properties are not affected by the dissolution of inorganic components. An exposure of human root dentin to 17% EDTA for 2 hours completely rids the exposed surface from calcium to a depth of approximately 150 m (19). The flexure strength of specimens such as those used in the current study is reduced by one third and the elastic modulus by half after a 2-hour exposure to 17% EDTA (17). However, in the present study, we chose short exposure times. An exposure time of 3 minutes to 17% EDTA should suffice to dissolve the inorganic parts of the smear layer (20). Although removal of smear layer might be desirable, we have to consider which irrigant to use last. If the last irrigant is EDTA, it is reasonable to believe that the organic part of the smear layer, ie, the collagen, remains on the surface of the root canal lumen (21, 22). Collagen can be important for the binding of bacteria including Enterococcus faecalis (23, 24). It might therefore not be prudent to use EDTA as the final irrigant. When used by themselves, EDTA and NaOCl can each affect the physical and mechanical properties of dentin adversely (11, 25). Not surprisingly, the combined removal of the inorganic as well the organic phase gives rise to damaging effects on the peritubular and intertubular dentin (22, 26). However, under the current conditions, this combined effect did not result in lowered modulus of elasticity and flexure strength values. This might have to do with 2 things; first, the exposure times of 3 minutes during the sequential use of EDTA and NaOCl were probably too short to disclose an impact on the mechanical dentin properties, and second, EDTA remnants on the dentin might have reduced hypochlorite and thus blocked its proteolytic effects (27). The second issue could have been corrected by applying a water rinse between EDTA and NaOCl. However, this is not usually done clinically and was thus not performed in the current study either. Future studies should aim to delineate guidelines for the time and concentration of endodontic irrigants to obtain a clean root canal system without producing any untoward effects to the tooth.
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Acknowledgements The authors thank Beatrice Sener and Bogna Stawarczyk for their help with the laboratory procedures.
References 1. Zehnder M. Root canal irrigants. J Endod 2006;32:389 –98. 2. Torabinejad M, Handysides R, Khademi A, Bakland L. Clinical implications of the smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:658 – 66. 3. Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy reconsidered. J Endod 2005;31:817–20. 4. Peters OA, Boessler C, Zehnder M. Effect of liquid and paste-type lubricants on torque values during simulated rotary root canal instrumentation. Int Endod J 2005;38: 223–9. 5. Yamada RS, Armas A, Goldman M, Lin P. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: part 3. J Endod 1983;9:137– 42. 6. Baumgartner J, Mader C. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 7. Currey JD, Brear K, Zioupos P. Dependence of mechanical properties on fibre angle in narwhal tusk, a highly oriented biological composite. J Biomech 1994;27:885–97. 8. Dychdala G. Chlorine and chlorine compounds. In: Block SS, ed. Chlorine and chlorine compounds. Philadelphia: Lea & Febiger, 1991:131–51. 9. Stoward PJ. A histochemical study of the apparent deamination of proteins by sodium hypochlorite. Histochemistry 1975;45:213–26. 10. Davies JM, Horwitz DA, Davies K. Potential roles of hypochlorous acid and n-chloroamines in collagen breakdown by phagocytic cells in synovitis. Free Radic Biol Med 1993;15:637– 43. 11. Sim T, Knowles J, Ng Y, Shelton J, Gulabivala K. Effect of sodium hypochlorite on mechanical properties of dentine and tooth surface strain. Int Endod J 2001; 34:120 –32. 12. Grigoratos D, Knowles J, Ng Y, Gulabivala K. Effect of exposing dentine to sodium hypochlorite and calcium hydroxide on its flexural strength and elastic modulus. Int Endod J 2001;34:113–9. 13. Oyarzun A, Cordero A, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002; 28:152– 6. 14. Di Renzo M, Ellis T, Sacher E, Stangel I. A photoacoustic FTIRS study of the chemical modifications of human dentin surfaces: II— deproteination. Biomaterials 2001; 22:793–7. 15. Vire D. Failure of endodontically treated teeth: classification and evaluation. J Endod 1991;17:338 – 42. 16. Caplan D, Weintraub J. Factors related to loss of root canal filled teeth. J Public Health Dent 1997;57:31–9. 17. Vollenweider M, Brunner TJ, Knecht S, et al. Remineralization of human dentin using ultrafine bioactive glass particles. Acta Biomater 2007 (in press) doi:10/1016/j.actbio.2007.04.003. 18. Marending M, Luder H, Brunner TJ, Knecht S, Stark W, Zehnder M. Effect of sodium hypochlorite on human root dentine: mechanical, chemical and structural evaluation. Int Endod J 2007 (in press) doi:10/1111/j.1365-2591.2007.01287.X. 19. Kawasaki K, Ruben J, Tsuda H, Huysmans M, Takagi O. Relationship between mineral distributions in dentine lesions and subsequent remineralization in vitro. Caries Res 2000;34:395– 403. 20. De-Deus G, Reis C, Fidel R, Fidel S, Paciornik S. Co-site digital optical microscopy and image analysis: an approach to evaluate the process of dentine demineralization. Int Endod J 2007;40:441–52. 21. Goldman M, Goldman LB, Cavaleri R, Bogis J, Lin P. The efficacy of several endodontic irrigating solutions: a scanning electron microscopic study—part 2. J Endod 1982;8:487–92.
Impact of Irrigant Sequence on Mechanical Properties of Human Root Dentin
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Basic Research—Technology 22. Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 2002;35:934 –9. 23. Hubble TS, Hatton JF, Nallapareddy SR, Murray BE, Gillespie M. Influence of Enterococcus faecalis proteases and the collagen-binding protein, ACE, on adhesion to dentin. Oral Microbiol Immunol 2003;18:121– 6. 24. Love R. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int Endod J 2001;34:399 – 405.
1328
Marending et al.
25. Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutions on the microhardness and the roughness of root canal dentin. J Endod 2004;30:792–5. 26. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28:17–9. 27. Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003; 36:411–7.
JOE — Volume 33, Number 11, November 2007