M. Hulsmann, M. Heckendorff & F. Schafers
Department of Operative Dentistry, Preventive Dentistry and Periodontology, University of Gottingen, Gottingen, Germany.

Introduction.
It has been shown in numerous investigations that mechanical instrumentation of the root canal does not result in perfectly cleaned canal walls as it leaves behind uninstrumented areas as well as debris and a smear layer of organic and inorganic material (McComb & Smith 1975, Se n et al.1995). In order to enhance the degree of cleanliness copious irrigation of the canal with a wide variety of irrigants has been proposed, including chelating agents (Harrison1984). Chelating agents act on calcified tissue by substituting sodium ions which combine with dentine to give soluble salts for the calcium ions that are bound in a less soluble combination (Weine 1982). The most popular of the chelating agents is EDTA, introduced by Nygaard-Ostby (Nygaard-Ostby 1957). EDTA is a liquid solution of the sodium salt of ethylenediamine tetraacetic acid with a pH of 7.3. Ina normal concentration it removes 10.6 g of calcium from 100 g of calcium (Nicholson et al.1968).
Although introduced into endodontics with the aim to facilitate preparation of calcified and narrow root canals by softening root-canal dentine, it also has been suggested as a useful irrigant owing to its capacity to remove the smear layer. Vonder Fehr&Nygaard-Ostby (1963) presented a combination of Cetavlon and EDTA, and this modification has been known and commercially distributed as EDTAC. The action of this solution has been described to be self-limiting and not exceeding a depth of 50 mm after 48-h exposure (Von der Fehr & Nygaard-Ostby1963).
Stewart et al. (1969) added urea peroxide to EDTA. In contact to sodium hypochlorite, this combination produces foam which lifts debris out of the root canal. A commercial product using this mixture has been known as RC-Prep (Premier-Dental, Norristown, PA, USA). Although EDTA has a relatively low surface tension (Tasman et al.2000) surfactants and disinfectants have been added to the ETDA solution (Brannstrom 1984) to enhance the removal of the superficial smear layer so that the antiseptic components can penetrate the dentinal tubules. This product has been marketed as Tubulicid (Dental Therapeutics, Nacka, Sweden).
Studies on the softening effect of EDTA gave controversial results. Von der Fehr & Nygaard-Ostby (1963) described a depth of demineralization of 20-30 mmafter 5-minworking time of EDTAC. Patterson (1963) reported that a 10% EDTA solution was able to lower the Knoophardness of dentine significantly from 42 to 7. Ameasurable softening effect of EDTA on dentine was confirmed by the studies of Fromme et al. (1970) and Pawlicka (1982).
Controversially, Fraser (1974) came to the conclusion that a softening effect of three different chelators, amongst these RC-Prep, could be found only in the coronal and middle third of the root canal but not in the apical part. These findings were confirmed by a further study using Largal Ultra (Septodont, St. Maur-des-Fossee- Cedex, France), a liquid chelating agent containing EDTA and cetrimide (Fromme et al. 1970). The latter suggest that the demineralizing effect of EDTA depends on the contact of large volumes of the solution with dentine which may occur in the coronal part of wide root canals but not in the narrow apical third. Wandelt (1965) also questioneda softening effect of EDTA as he could not find any signs of demineralization of root dentine under the microscope. The action of EDTA is self-limiting, the demineralization stops when an equilibrium between the calcium ions in dentine and in the chelating agent has been reached (Nygaard-Ostby1957, Seidberg & Schilder 1974).
Goldberg & Spielberg (1982) demonstrated that the extent of demineralization is related to the time of exposure to EDTA, reaching its maximum after 15 min. Although the first effects of the chelator were observed after 5 min an extended working period has been suggested to increase the cleansing effect (McComb & Smith 1975, Goldberg & Spielberg1982).
EDTA in several investigations has been reported to be effective in cleansing the root-canal walls (McComb & Smith 1975, Goldberg & Abramovich 1977, Ram 1980, Goldman et al.1981, Berg et al. 1986, Ciucchi et al. 1989, Garberoglio & Becce 1994, Stewart 1998, Hottel et al. 1999, O’Connell et al. 2000). Nevertheless, the cleaning ability seems to be better in the middle and coronal parts of the root canal (Goldman et al. 1981, O’Connell et al. 2000). The effect of liquid chelators has been shown to be confined to prepared root-canal surfaces whereas no effect on organic tissues in uninstrumented root canals could be detected (Koskinen et al.1980).The combination of EDTA and sodiumhypochlorite (0.5-5.25%) has been shown to be even more effective in smear layer removal than the use of these two solutions alone (Baumgartner &Mader1987) as well as in antibacterial efficacy (Bystroem & Sundqvist 1985). The combined use of EDTA and ultrasound did not enhance the dissolving capability of EDTA (Ciucchi et al.1989).
Heling et al. (1999) and Buck et al. (1999) described a limited antibacterial activity of RC-Prep dependent on the type of bacteria. Patterson (1963) also reported on a limited antibacterial activity and only slight inflammatory reactions in contact with vital tissue. By addition of Cetavlonthe antibacterial as well as the inflammatory potential of EDTA is increased (Weine 1982), probably owing to a lower surface tension and thus deeper penetration of the liquid into dentine (Von der Fehr & Nygaard-Ostby1963). The antibacterial effect and intratubular disinfection of EDTA have been questioned (Orstavik & Haapasalo1990). Recently, the use of EDTA has been shown in vitro to inhibit the substrate-adherence capacity of macrophages when leaking into the periapical tissues thus reducing both periapical inflammatory reactions and periapical healing (Segura et al. 1997).
The ability of EDTA to remove the smear layer has been demonstrated to result in a higher permeability of dentine (Cohen et al. 1970, Brannstrom 1984), a reduced leakage of the definite root-canal filling (Cergneux et al. 1987, Petschelt et al. 1987,Wennberg & Orstavik 1990, Behrend et al.1996), and a larger number of filled lateral canals (Goldberg et al. 1986). In SEM studies it has been shownthat the use of EDTAresulted inwideningof dentinal tubules (Goldberg & Abramovich 1977, Hottel et al. 1999).
As the majority of experimental studies on chelating agents has focused on liquid chelators (EDTA, EDTAC) or one single paste chelator (RC-Prep), it was the aim of the present investigation to compare three chelating pastes with different compositions. The parameters investigated were changes in dentine microhardness, weight loss and cleaning ability.

Chelator compositions.
The compositions of the three chelator pastes used in the present study according to the manufacturers’ information are as follows:

• Calcinase Slide (lege artis, Dettenhausen, Germany): 15% sodiumedetate,58-64% water and a water-soluble gel base, pH 8-9.
• RC-Prep (Premier-Dental, Norristown PA, USA): 15% EDTA and 10% urea peroxide in a water-soluble gel base, polyethylenglycol, cetylalcohol, propyleneglycol.
• GlydeFile (DeTrey/Dentsply, Konstanz, Germany): 15% EDTA,10% urea peroxide in an aqueous solution.

The investigations were performed on freshly extracted maxillary incisors and single rooted premolars with intact roots. Teeth showing signs of endodontic or restorative treatment of the root dentine were excluded from the study as were teeth showing root caries. Teeth were stored in distilled water and always kept wet.

Changes of microhardness.
The roots of the extracted teeth were cut horizontally into slices of 2-mm height. For this part of the study, 100 slices were prepared and randomly distributed into 10 groups. For each chelator paste,10 dentine slices were covered with 0.1mL paste for 30, 60, or 120 s followed by an irrigation with 5 mL H2O2 (5%) and 5 mL NaOCl (3%).This procedure was repeated five times, simulating root-canal enlargement of five instrument sizes resulting in a total working time of the chelator pastes of 2.5, 5 and 10 min. Microhardness of root dentine was measured at four points of the dentine near the root canal, before and after treatment (Fig.1). Ten slices served as controls and were only irrigated with hydrogen peroxide and sodiumhypochlorite as described. For evaluation of Vickers hardness, a Durimeter (Leitz,Wetzlar, Germany) was used with 490.3 mN (50 p) and an impression time of 10 s. Preoperative markings on the slices enabled pre- and postoperative measurements to be taken in direct neighbourhood close to the root canal (Figs 1 and 2).

Figure 1. Localization of the pre- and postoperative measuring points for evaluation of changes in microhardness.



Figure 2. SEM of the pre- and postoperative measuring points in direct neighbourhood and near to the root canal (magnification: 700x).

Loss of weight.
For the determination of weight loss, 200 dentine slices were prepared as described before, dried in a desiccator (Novus-Desiccator, Wertheim, Germany) for 5 days, weighed (R 160 P, Sartorius, Gottingen, Germany), and randomly distributed into10 groups.
For each of the three chelator pastes, 60 slices were covered with 0.1mL of the chelator, applied with a syringe, for 3,6, or 9 min. Then the slices were irrigated with 5 mL H2O2 (5%) and 5 mL NaOCl (3%) to remove the chelator. Twenty slices served as controls and were only irrigated. Finally, the slices were dried in the desiccator for 5 days and weighed again. The optimal time for desiccation had been evaluated in preliminary tests, showing that no further changes in weight of the slices occurred after 5 days.

Canal cleanliness.
For the evaluation of root-canal cleanliness after use of a chelator paste, 35 maxillary incisors with intact roots, closed apices, and no signs of carious decay or endodontic treatment of root-canal dentine were selected. An access cavity was prepared and length was determined by inserting an endodontic file through the apical foramen. Working length was determined 1mm coronal to the root tip and the size of the first instrument binding at working length was determined. All root canals were enlarged five ISO-sizes using stainless steel Hedstroem-files in a filing motion with 0.1mL of the chelator paste for each instrument size resulting in a total of 0.5 mL paste for each root canal. Most of the preparations were finished with ISO-sizes 50-60.Working time for each file was 1min, resulting in an identical total working time of 5 min for instruments and chelators in all groups. After each instrument size, the root canals were irrigated with 5 mL H2O2 (5%) and 5 mL NaOCl (3%). Five teeth served as controls and were prepared as described but without use of a chelator.
Following enlargement, the teeth were split longitudinally and evaluated under the SEM (DSM 960, Zeiss, Oberkochen, Germany).The roots were coded and mixed so that during the SEM evaluation the type of chelating agent could not be identified by the evaluator (M.H.) nor the SEM operator (F.S.). The root half was placed in the SEMand the SEM-beam adjusted to the coronal, middle or apical third of the root under a 20x magnification by the SEM operator.
A transparent grid was placed on the SEM screen and cleanliness of the root-canal walls was evaluated for10 pre-selected squares of the grid separately for the coronal, middle and apical third of the canal wall under a 1000x magnification using a four-score system with reference photographs. The four scores were defined as follows:

• Score I : dentinal tubules completely opened;
• Score II : more than 50% of dentinal tubules opened;
• Score III: less than 50% of dentinal tubules opened;
• Score IV: all of the dentinal tubules covered with smear layer.

Statistical analysis.
The results for weight loss and changes in microhardness were statistically analysed for differences between the different working times for each chelator using the Kruskal-Wallis test. In cases of significance pair tests were undertaken using the Wilcoxon-Mann-Whitney test. Comparisons between the results for the apical, middle and coronal parts of the roots were performed using the Friedmanntestand the Wilcoxon test. The level of significance for all of the statistical tests was P < 0.05.

Changes of microhardness Regardless of working time (2.5,5,10 min) no significant differences between the three chelator pastes were found. Mean loss of hardness was 4.4% for Calcinase- Slide after 2.5 min, 10.0% after 5 min and 21.2% after 10 min working time. The results for RC-Prep were 5.6% (2.5 min), 11.5% (5 min) and 18.6% (10 min) and for Glyde-File 5.5% (2.5 min), 11.1% (5 min) and 15.1% (10 min). For each of the pastes microhardness decreased significantly with increasing working time (Kruskal- Wallis test, P-values for all chelator pastes and working times <0.0013). Compared to the controls (mean loss of microhardness: 1%) the results for the chelating agents differed significantly regardless of working time (all P-values <0.0022) (Fig. 3).

Figure 3. Differences in microhardness before and after use of a chelating paste (in percentage of preoperative value). For each paste there was a significant decrease in microhardness with increasing working time and when compared to the respective controls.



Figure 4. Differences in weight of dentine slices before and after treatment with a chelator paste (in percentage of preoperative weight). For each of the pastes there were no significant differences in weight loss with regard to different working times of the pastes. After 6 and 9 min Calcinase resulted in a significant higher weight loss than RC-Prep, after 6 min Glyde-File removed significantly more dentine than RC-Prep.



Figure 5. Results for root-canal cleanliness after use of Calcinase-Slide. The best results were achieved in the coronal and middle parts of the root canal. For the coronal part the results were significantly better than for Glyde-File, RC-Prep and the control group, in the middle part significantly better than for the control group.



Figure 6. Results for root-canal cleanliness after use of RC-Prep.



Figure 7. Results for root-canal cleanliness after use of Glyde-File.



Figure 8. Root-canal wall (middle third) after use of Calcinase-Slide (magnification1000x). Cleanliness was rated score I.



Figure 9. Root-canal wall (middle third) after use of RC-Prep (magnification 1000x). Cleanliness was rated score I.



Figure 10. Root-canal wall (middle third) after use of Glyde-File (magnification1000x). Cleanliness was rated score I.

Loss of weight.
The results for the loss of weight after different working times of the chelator pastes are summarized in Fig. 4. The highest amount of weight loss was observed for an application time of 3 min for Calcinase- Slide (0.3%) and RC-Prep (0.3%), for Glyde-File an application time of 6 min resulted in the greatest weight loss (0.25%).
For all three chelating agents the differences between the three working times were not statistically significant (Kruskal-Wallis test, Calcinase-Slide P ј 0.89, Glyde-File P ј 0.29, RC-Prep P ј 0.08).
After 3 min no significant differences were found between the groups, after 6- and 9-min specimens treated with Calcinase-Slide showed a significantly higher weight loss than specimens treated with RC-Prep (Wilcoxon-Mann-Whitney test, P ј 0.0089 and P ј 0.0271), after 6 min Glyde-File removed significantly more substance than RC-Prep (P ј 0.0108). The differences between all experimental groups and the control group were statistically significant for all working times.

Canal cleanliness.
The results of the SEM evaluation of root-canal cleanliness are presented in Figs 5-7. For all groups root-canal cleanliness decreased from the coronal to the apical third. Whereas in the coronal part a high number of scores I and II could be found (Figs 8-10), scores III and IV dominated in the apical third. The Kruskal-Wallis test showed significant differences for the coronal and middle parts of the root canals (P ј 0.0007 and P ј 0.0289, respectively). For these two sections, pair wise comparisons between the groups were performed using the Wilcoxon-Mann-Whitney test with P < 0.05. In the coronal third of the root canals the results for Calcinase- Slide showed statistically significant differences when compared to the other chelator pastes (Glyde-File: P ј 0.0024, RC-Prep: P ј 0.0002) and the control group (P ј 0.0051). In the middle part of the root canals, the results for Calcinase-Slide were significantly better than for the control group (P ј 0.0310) and for Glyde-File (P ј 0.0269). In the apical part no significant differences between the four groups existed.

Discussion.
Since the introduction of EDTA solutions into endodontics by Nygaard-Ostby (1957) their benefits have been debated. Initially, liquid chelators such as EDTA solutions in different concentrations or with different additional detergents and surfactants were used in order to soften dentine and thus facilitate root-canal enlargement. Recently, the use of paste-type chelating agents has been proposed with the primary aim to serve as gliding agents for rotary nickel-titanium files. Until now comparative studies on the properties of chelating pastes have not been published. Therefore, it was the aim of the present study to compare the changes in weight and microhardness and the degree of root-canal cleanliness after application of three chelator pastes in vitro. Three separate evaluations were undertaken using dentine disks or instrumented root canals. Great care was taken to achieve standardized working times and standardized amounts of the respective chelators. As the dimensions (diameter, thickness, preoperative weight) of the dentine slices could not be standardized the results for weight loss and changes in microhardness were presented as relative changes in percentage and analysed statistically. Cleaning ability was investigated using the SEM with a four-score index for the degree of remaining smear layer. Cleanliness was rated separately for the coronal, middle and apical parts of the root canals.

Cleanliness.
The existence of a smear layer after endodontic procedures in the root-canal system was described first by McComb & Smith (1975). Brannstrom & Nyborg (1973) could demonstrate that bacteria were able to survive and multiply in the smear layer. As it consists of organic and inorganic compounds it may be dissolved by irrigants suchas citric acid or EDTA, whereas the most commonly used root-canal irrigant sodium hypochlorite is not able to remove the smear layer unless it is used in combination with ultrasonics (Lester & Boyde 1977, Cameron1983).The removal of smear layer seems desirable as it increases dentine permeability (Pashley et al. 1981) thus allowing better disinfection of deeper layers of the infected root-canal dentine (Orstavik &Haapasalo 1990). Additionally, it has been shown in several invitro studies that adaptation and adhesion of root-canal filling materials to the root-canal wall may be increased and thus leakage of the root-canal filling be decreased by removal of the smear layer (Cergneux et al. 1987, Petschelt et al.1987,Wennberg & Orstavik1990, Behrend et al.1996).
Nevertheless, some authors seriously question whether the smear layer really should be dissolved and removed completely (Sen et al. 1995, Perez et al. 1996, Peters et al. 2000). They argue that bacteria might reinvade the cleaned root canal from the dentinal tubules, which might be prevented by leaving the smear layer at least inside the entrances of the dentinal tubules. With regard to removal of the smear layer and cleanliness of the root-canal walls the present study confirmed the results from several recent investigations (McComb & Smith 1975, Ram 1980, Goldman et al. 1981, Berg et al. 1986, Abbott et al.1991, Garberoglio & Becce1994, Stewart1998, Calt & Serper 2000, O’Connell et al.2000).None of the canals investigated under the SEM were completely cleaned, but no superficial debris could be found and the majority of the dentinal tubules were opened in the middle and coronal part of the root canals. Many of the specimens under the SEM showed widening of the dentinal tubules as described by several investigators (Goldberg & Abramovich 1977, Hottel et al. 1999) for liquid EDTA preparations. This effect was more pronounced in the coronal and middle thirds than in the apical parts of the root canals where no significant differences to the control group existed. This probably is owing to the fact that more of the chelating pastes gets in contact to the canal walls coronally than apically. Similar results have been reported by Goldman et al. (1981), Ciucchi et al. (1989) and O’Connell et al. (2000) for the liquid chelators REDTA and EDTA in different concentrations (15 and 25%, respectively). On the other hand, these authors could demonstrate that EDTA alone did not have any effect on uninstrumented canal walls. As it does not affect soft tissues, the pulp tissue remnants and predentine remained in these areas (Goldman et al. 1981, O’Connell et al. 2000). Cleaning efficacy of EDTA could be enhanced by alternate irrigation with sodium hypochlorite (Baumgartner & Mader 1987, O’Connell et al.2000). Goldberg&Spielberg (1982) reported on a significant influence of the working time of EDTA on the canal wall cleanliness with a maximum effect after 15 min. This has not been evaluated in the present study for cleaningabilityof the chelating pastes, but for weight loss and changes in microhardness. In a recent evaluation using Glyde-File during rotary instrumentation of root canals Ahn & Yu (2000) found no influence of this chelator on the smear layer and a significantly superior performance of liquid EDTA. This finding could not be confirmed by the present investigation showing good results regarding the cleaning ability of Glyde-File at least for the coronal and middle part of the root canal.
The results for root-canal cleanliness are partly contradicting the results of two previous studies on rootcanal preparation with different rotary Ni-Ti instruments (Hulsmann et al. 2001, Versumer et al. 2002). Despite the use of a chelator paste (RC-Prep) root-canal cleanliness showed no satisfying results with smear layer covering large parts of the root-canal walls. This may be due to the fact that less paste was used than in the present investigation and that instrumentation was performed in severely curved root canals. In straight root canals, distribution of the chelator paste over the rootcanal walls may be easier to control than in curved canals. On the other hand, it could be confirmed that debris was removed nearly completely and only smear layer remained on the canal walls and that the degree of cleanliness decreased from the coronal to the apical part of the root canals.

Weight loss.
Regarding weight loss, significant differences between the three pastes were found. Specimens treated with Calcinase- Slide showed higher weight loss than RC-Prep after 6- and 9-min working time, Glyde-File was significantly superior to RC-Prep after 6 min. Seidberg & Schilder (1974) using a gravimetric method demonstrated for EDTA a chelation of a determinable quantity of dentine. They found this process to be self-limiting, the most rapid chelation occurred within the first hour. A measurable weight loss of root dentine after use of liquid EDTA was also reported by Heling et al. (1965).

Microhardness.
The demineralizing effect of EDTA solutions has been controversial. The results of the present study indicate a reduction of microhardness of dentine as wellas ameasurable weight loss after treatment with a paste chelating agent. Weight loss as well as changes in microhardness were significantly related to the contact period between the chelator paste and dentine. No differences between the three different chelators could be found, indicating that the addition of carbamide peroxide (Glyde-File and RC-Prep) does not have any significant effect on softening of the dentine. Recently, it has been shown that 10% carbamide peroxide used as a bleaching agent does not significantly change microhardness or the mineral content of enamel (Potocnik et al. 2000).
Numerous investigators have measured the hardness of dentine and reported wide variations along different regions of a tooth. As with previous investigators Pashley et al. (1985) demonstrated a statistically highly significant inverse correlation between dentine microhardness and tubular density. Tubular density increased and microhardness decreased near the pulp chamber, presumably owing to a decrease in the amount of intertubular dentine and an increase in individual tubular diameter. Measurements of changes in microhardness in the present study therefore were performed near the root canal. Pre- and postoperative measurements were undertaken in the same region and the results are presented as relative changes, i.e. weight loss (%) in relation to preoperative values. Significant changes in microhardness have been reported previously in several studies for liquid chelators as EDTA solutions with a maximum after approximately 5 min of working time and no significant further increase after 24 h (Pawlicka 1982).Patterson (1963) even described a maximum demineralization after 5 days, thus proposing the use of such agents as intracanal dressings. The measurements in the present study were undertaken after 2.5, 5 and 10 min which should mimic real working times during instrumentation of root canals. In contrast to Pawlicka (1982), the results show a further statistically significant decrease of microhardness after 5 min. These differences to previous studies presumably are owing to the fact that the penetration ability of paste agents into dentine will be clearly lower than that of liquid EDTA solutions. However, other studies have shown that in narrow canals little, if any demineralization occurred (Patterson 1963, Wandelt 1965, Fraser 1974). Verdelis et al. (1999) also reported the low decalcification efficacy of RC-Prep owing to its low pH.
No significant differences between the three pastes could be found, but decrease in microhardness was significantly related to working time indicating a time dependent demineralization, which is in contrast to the results of previous studies (Patterson 1963, Wandelt 1965). Fraser (1974) described a softening effect of RCP rep to a depth of 30-40 mm from the root-canal border in the coronal third with even smaller values in the middle and apical third, in his study liquid chelators performed better than pastes. In the light of recent observations reported by Mjor et al. (2001) the tubular density and structure of dentine in the apical part of the root canal differ from that in the coronal and middle parts of root canal. There fore, it needs further comparative investigations on the differences between liquid and paste chelating agents in different parts of the root canal.
It remains unclear whether weight loss as well as changes in dentine microhardness really are of any clinical significance. Under the conditions of the present study, a relatively large volume of the chelating agent had intimate contact to a large amount of root dentine which is not comparable to the situation of a narrow and calcified root canal. Whereas it seems questionable if such low changes in microhardness really might affect the ease of preparation of such root canals it should be remembered that chelator pastes act additionally as lubricants thus enhancing the gliding capability of endodontic files (Weine1982).
Further studies are necessary to evaluate whether liquid or paste chelators should be preferred as adjuncts during root-canal preparation.

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