INVESTIGATION OF ROOT CANAL DEBRIDEMENT …€¦ · Final irrigation following the last instrument was ... Group 2 (MTAD): 1 mL BioPure MTAD (Dentsply, Tulsa Dental, tulsa, oK) was

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Article Http://dx.doi.org/10.5504/bbeq.2012.0125 Mb

Biotechnol. & Biotechnol. eq. 27/2013/1

Biotechnol. & Biotechnol. eq. 2013, 27(1), 3547-3552Keywords: electron microscopy, in vitro, low-temperature atmospheric pressure plasma, smear layer, surface analysis

IntroductionDuring chemo-mechanical debridement, the removal of all pulp tissue, dentin debris, and the smear layer is challenging due to the complex nature of root canal anatomy. Drawbacks in conventional irrigation materials and techniques have resulted in a demand for new and improved methods. Both the antibacterial properties and the debridement effectiveness of root canal irrigants are important. in conventional irrigation protocols, sodium hypochlorite (naocl) is not able to dissolve inorganic components, so it has been used in association with chelating agents such as ethylenediaminetetraacetic acid (eDtA) (24). however, the most important disadvantage of this combined regimen is the inadvertent erosion of intraradicular dentin (26). Minimal erosion of intraradicular dentin has been reported with use of naocl and a mixture of tetracycline, acid, and detergent (MtAD), which is also effective for smear layer removal (26) and eradication of resistant microbes (21).

In recent years, the flushing activation of irrigants has gained importance since it provides cleaner root canals (4) and presence of fewer bacteria (15). For example, passive ultrasonic irrigation (PUi) was reported to improve the activity of irrigation solutions in inaccessible areas by acoustic streaming (18) and to result in better canal debridement than that provided by conventional needle irrigation (28).

Preliminary reports of a new method called low-temperature atmospheric pressure plasma (ltAPP) indicated effective

results in disinfection and sterilization, biofilm removal and intracoronal bleaching (14, 30). Furthermore, surface treatment using ltAPP was encouraging in the surface and tissue engineering, cleaning/etching, adhesion enhancement, and biomaterial development (5, 17). nevertheless, there remains a lack of information about its effectiveness on root canal dentin and removal of debris and smear layer.

The purpose of this study was to compare the efficacy of ltAPP with the current materials (MtAD, naocl, eDtA) used with or without PUi activation on smear and debris removal in root canals using scanning electronic microscopy (SeM).

Materials and MethodsSample selectionFifty-five extracted human mandibular premolars with uniform root canal width, single straight root canals, mature apexes, and similar anatomic forms were used for this study.

Root canal preparationthe teeth were decoronated to obtain a standardized root canal length of 15 ± 0.5 mm and randomly divided into 5 test groups (n = 10 per group) and 1 control group (n = 5). the teeth were shaped using Profile NiTi rotary instruments (Dentsply, Maillefer, Ballaigues, Switzerland) to an apical size of #40/.06 according to the manufacturer’s instructions. except for the negative control group, root canals in all groups were irrigated with 2 ml of naocl between each instrument change. its concentration was 1.3 % in the groups with MtAD, whereas it was 2.5 % in the groups without MtAD. Solutions were

INVESTIGATION OF ROOT CANAL DEBRIDEMENT EFFICACY OF LOW TEMPERATURE ATMOSPHERIC PRESSURE PLASMA COMPARED TO THE CURRENT TECHNIQUES

Bulem Üreyen Kaya, Ayşe Diljin Keçeci, Hilmi Egemen Güldaş Süleyman Demirel University, Faculty of Dentistry, Isparta, Turkeycorrespondence to: Bulem Üreyen Kayae-mail: [email protected]

ABSTRACTThis study aimed to compare the efficacy of low-temperature atmospheric pressure plasma (LTAPP) with the current materials (MTAD, NaOCl, EDTA) used with or without passive ultrasonic irrigation (PUI) on smear and debris removal. Mandibular premolars (n = 55) were prepared to an apical size of 40/.06 and randomly divided into 5 different test groups (n = 10 per group) and 1 control group (n = 5) with the following final irrigation protocols: NaOCl + EDTA; NaOCl with PUI; MTAD; MTAD with PUI; LTAPP; and saline (control). Presence of smear layer and debris was evaluated at ×2000 (for each third) using a four-level scoring system. The data were analyzed statistically by Kruskal–Wallis and Bonferroni–Dunn tests (P = 0.05). The results showed that NaOCl + EDTA and MTAD with PUI irrigation were the most effective methods for smear layer removal, whereas MTAD irrigation with or without PUI, for debris removal (P < 0.05). Smear and dentin scores showed significant differences among the groups in all thirds (P < 0.05). It could be concluded that LTAPP did not appear effective on smear layer and debris removal as expected. PUI improved only the efficacy of MTAD.

3548 Biotechnol. & Biotechnol. eq. 27/2013/1

delivered using a syringe and a 27-G needle that was inserted apically 1 mm short of the working length and moved slowly over a distance of 4 mm up and down in the apical half of the root canal. Final irrigation following the last instrument was performed as follows:

Group 1 (Positive control – NaOCl + EDTA): 3 mL 2.5 % naocl followed by 3 ml 17 % eDtA for 1 min and 3 ml distilled water for 1 min were applied.

Group 2 (MTAD): 1 mL BioPure MTAD (Dentsply, Tulsa Dental, tulsa, oK) was placed in the root canal for 5 min. the canal was then rinsed with 4 ml MtAD according to the manufacturer’s instructions.

Group 3 (NaOCl with PUI): EMS Mini Master Piezon (eMS, nyon, Switzerland) ultrasonic unit was used with the power setting at 5. Canals were filled with 2.5 % NaOCl and activated 3 times for 20 seconds using an eSi noncutting ultrasonic tip (endosoft eSi; eMS, nyon, Switzerland) with intermittent replenishment of the naocl solution.

Group 4 (MTAD with PUI): EMS Mini Master Piezon ultrasonic unit was used with the power setting at 5. canals were filled with BioPure MTAD and activated 3 times for 20 seconds using an eSi noncutting ultrasonic tip with intermittent replenishment of the MtAD solution.

Group 5 (LTAPP): A dental syringe was used for guiding the gas flow from the experimental LTAPP device. The needle served as the electrode, which was connected to a high-voltage sub-microsecond pulsed direct-current power supply (amplitudes of up to 10 kV, repetition rate of up to 10 khz). the gas used was he/o2 (ratio 99/1) with a flow rate of 10 L/min. ltAPP was applied for 5 min through the root canal. the gas flow was observed during this application.

Group 6 (negative control): Root canals were irrigated with 2 mL of saline between each file and 5 mL of saline was used for final irrigation.

Scanning electron microscopyA longitudinal groove was created along the root surface with a diamond disc, with care being taken to avoid touching the root canal. the roots were then split into 2 halves with a chisel. the specimens were serially dehydrated in ethanol solutions, mounted on metallic stubs, and then evaluated under a scanning electron microscope (Vega ii lSU; tescan, england). the amount of smear layer and debris remaining were scored at 3 different levels: the apical (0 mm – 5 mm), middle (5 mm – 10 mm), and coronal (11 mm – 14 mm) portions of the root halves by two investigators blinded to the treatment groups and level of the images. Presence of smear layer was evaluated at ×2000 on 495 micrographs (3 for each level) using a four-level scoring system based on the order of severity of smear layer presence. Score 0 = no smear layer on the surface of the root canal and all the dentinal tubules clear and open; score 1 = little or no smear layer on the surface of the root canal and less than 25 % of the dentinal tubules containing a smear layer; score 2 = 25 % to 50 % of the dentin tubules containing a

smear layer; score 3 = heavy smear layer, and more than 50 % of the dentinal tubules covered with a smear layer.

The presence of debris was defined as dentin chips, pulp remnants, and particles attached to the root canal wall (1) and was evaluated at ×2000 magnification using a four-level scoring system based on the order of severity of debris. Score 0 = clean root canal wall, no debris particles; score 1 = few small agglomerations of debris; score 2 = many agglomerations of debris covering less than 50 % of the root canal wall; score 3 = more than 50 % of the root canal wall covered by debris.

For the statistical evaluation, the data was obtained from the mean scores of two investigators for both parameters on each micrograph.

Statistical analysesObserver reproducibility. Fifteen images were evaluated three times in random order by two observers at weekly intervals without knowledge of the previous results. the weighted Kappa coefficient (Kw) was used to measure inter- and intra-observer reproducibility. Smear layer and debris removal. nonparametric data were analyzed statistically by Kruskal–Wallis and Bonferroni–Dunn tests to compare the overall efficacy of each method and their efficacy on each third (P < 0.05 was considered statistically significant).

Results and DiscussionRoot canal level, taper and size are the most significant factors for the outcome of irrigation. in order to form ultrasonic acoustic streaming, the apical preparation must be at least size 40 (2). the preparation dimensions used in this study were intended to allow adequate penetration of solutions to the apical third and to prevent contact between the ultrasonic needle and the root canal wall. however, there is always a risk of touching the canal walls, especially with a rigid irrigant needle, which would automatically trigger the formation of a new and undesirable smear layer (4). in the present study, a noncutting nickel-titanium ultrasonic tip was used to minimize these negative effects.

in this study the average Kw values for intra-observer reproducibility were 0.90 and 0.88 for the smear and debris scores, respectively. the average Kw value for inter-observer reproducibility was 0.89 for smear evaluation and 0.88 for debris evaluation.

the frequencies of the smear layer scores in the irrigation groups and their significance are shown in Table 1 (H = 101.326, P < 0.001). The Bonferroni–Dunn test indicated that the negative control group had the highest smear scores, whereas groups 1 and 4 had the lowest ones (P < 0.05). Ultrasonic activation of naocl (group 3) did not show a superior performance to non-activated naocl (group 1) (P < 0.05). However, smear layer removal was superior in ultrasonically activated MtAD (group 4) to the non-activated MTAD (group 2) (P < 0.05). Group 5 had a score of 0 only

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TABLE 1Distribution of the smear and debris scores according t o the irrigation groups*

GroupsSmear layer scores (0–3)

0 n (%)

1n (%)

2n (%)

3n (%)

Gr. 1. (NaOCl + EDTA)4 19 (21.1) 30 (33.3) 24 (26.7) 17 (18.9)Gr. 2. (MTAD)3 6 (6.7) 31 (34.4) 24 (26.7) 29 (32.2)Gr. 3. (NaOCl + PUI)2 1 (1.1) 15 (16.7) 37 (41.1) 37 (41.1)Gr. 4. (MTAD + PUI)4 13 (14.4) 25 (27.8) 41 (45.6) 11 (12.2)Gr. 5. (LTAPP)2 3 (3.3) 4 (4.4) 42 (46.7) 41 (45.6)Gr. 6. (Negative control)1 0 3 (6.7) 4 (8.9) 38 (84.4)

GroupsDebris scores (0–3)

0 n (%)

1n (%)

2n (%)

3 n (%)

Gr. 1. (NaOCl + EDTA)b 8 (8.9) 62 (68.9) 11(12.2) 9 (10.0)Gr. 2. (MTAD)c 38 (42.2) 33 (36.7) 8 (8.9) 11(12.2)Gr. 3. (NaOCl + PUI)b 16(17.8) 47 (52.2) 13 (14.4) 14 (15.6)Gr. 4. (MTAD + PUI)c 33 (36.7) 40 (44.4) 9 (10.0) 8 (8.9)Gr. 5.(LTAPP)b,c 8 (8.9) 73 (81.1) 8 (8.9) 1 (1.1)Gr. 6. (Negative control)a 0 11(24.4) 22 (48.9) 12 (26.7)

*Superscript same letters and numbers indicate non-significant difference (P > 0.05)

in 3 of the 90 (3.3 %) samples (Fig. 1, Fig. 2) and it was not significantly different from that in group 3 (P > 0.05).

Fig. 1. Smear and debris scores in representative SeM micrographs from groups 1–3. NaOCl + EDTA group showing: (a) smear: 0, debris: 0 at the coronal level; (b) smear: 0, debris: 0 at the middle level; (c) smear: 2, debris: 1 at the apical level; MtAD group showing: (d) smear: 0, debris: 1 at the coronal level; (e) smear: 2, debris: 1 at the middle level; (f) smear: 3, debris: 3 at the apical level; NaOCl + PUI group showing: (g) smear: 1, debris: 1 at the coronal level; (h) smear: 2, debris: 1 at the middle level; (i) smear: 3, debris: 3 at the apical level.

Fig. 2. Smear and debris scores in representative SeM micrographs from groups 4–6. MTAD + PUI group showing: (a) smear: 2, debris: 3 at the coronal level; (b) smear: 2, debris: 2 at the middle level; (c) smear: 2, debris: 3 at the apical level; ltAPP group showing: (d) smear: 3, debris: 2 at the coronal level; (e) smear: 3, debris: 1 at the middle level; (f) smear: 3, debris: 1 at the apical level; negative control group showing: (g) smear: 3, debris: 3 at the coronal level; (h) smear: 3, debris: 3 at the middle level; (i) smear: 3, debris: 3 at the apical level.

the smear scores related to the canal level for the 6 groups are shown in Table 2. The Kruskal–Wallis tests indicated a


significant difference among the groups according to the canal level (h = 57.67, h = 38.964 and h = 33.898 for coronal, middle and apical thirds respectively; P < 0.001 for all thirds). in the coronal third, the negative control group had the highest smear scores (P < 0.05). The samples of group 1 demonstrated the cleanest root canal wall, while the results for them were not significantly different from those in groups 2 and 4 (P > 0.05). Groups 3 and 5 showed a similar performance in the coronal third (P > 0.05). In the middle third, the results for group 5 were not significantly different from those for the negative control group, which had the highest smear scores (P > 0.05). The samples of group 1 demonstrated the cleanest root canal wall, but this was not significantly different from group 4 (P > 0.05). There were no significant differences neither between groups 4 and 2, nor between groups 4 and 1 (P > 0.05). Group 3 showed similar effectiveness to groups 2 and 5 (P > 0.05). In the apical third, the most successful protocol in terms of smear layer removal was the use of MTAD with PUI (group 4) (P < 0.05). the results for group 5 did not show any effect on the smear layer and it was not significantly different from group 2 (P> 0.05).

in terms of debris removal, there were statistically significant differences among the groups (H = 70.240, P < 0.001) (Table 1). Most effective debris removal was obtained in group 2 and group 4 (P < 0.05). None of the specimens in the negative control group were free of debris. Ultrasonic activation did not improve the effectiveness of NaOCl and MTAD (P > 0.05) (Fig. 1, Fig. 2).

the debris scores for the 6 groups related to canal level are shown in Table 2. The Kruskal–Wallis tests showed significant differences among the groups at the coronal, middle, and apical levels (h = 48.484, h = 18.784, and h = 33. 898; P < 0.001, respectively). PUI did not improve the activity of naocl (group 3) or MtAD (group 4) in the coronal and middle thirds (P > 0.05). Group 5 showed no significant differences from groups 1, 3 and 4 in the coronal and middle thirds (P > 0.05). In the middle third, group 2 had the least debris scores, but these were not significantly different from the ones observed in group 4 (P > 0.05). In the apical third, while the negative control group had the highest debris scores, they were not significantly different from the ones in group 5 (P > 0.05). Ultrasonic activation of MTAD (group 4) was the

TABLE 2Distribution of smear and debris scores on three canal levels

Groups ScoreSMEAR LAYER DEBRIS

Coronal Middle Apical Total Coronal Middle Apical Total

Positive control NaOCl + EDTA

(Gr. 1)

Score 0 8 7 4 19 3 3 2 8Score 1 13 14 3 30 25 23 14 62Score 2 9 6 9 24 2 4 5 11Score 3 0 3 14 17 0 0 9 9

MtAD(Gr. 2)


NaOCl + PUI(Gr. 3)


MTAD + PUI(Gr. 4)


ltAPP(Gr. 5)


negative control (Gr. 6)


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most effective protocol in terms of debris removal in the apical third (P < 0.05). Group 1 was not significantly different from group 2 or group 3 (P > 0.05). None of the samples in group 5 and the negative control group were free of debris in the apical third.

the open system design with unsealed root apex used in the present study allows a comparison among the root canal irrigation protocols. however, tay et al. (22) suggested that in vivo conditions can be better simulated with a closed system, in which gas entrapment (vapor lock) and a significantly higher debris score occurs compared to an open system. thus, further studies can be designed considering this issue. Another point in our study design was the difference in concentrations and volumes of the irrigants. Although they could be standardized, the instructions of the manufacturers and clinical use of conventional protocols were preferred. thus, MtAD was used with lower concentration of naocl and ultrasonic activation was applied with a nonspecific amount of the solutions.

In the present study, without a final flush with 17 % EDTA solution, the smear layer was seen to cover the root canal surface in the coronal, middle, and apical thirds, even when the PUi was used. the necessity of chelating or acid solutions to remove the smear layer was demonstrated by the results of the NaOCl + PUI group in the present study, as well as in a previous study (12). Mello et al. (13) showed that a continuous rinse with 5 mL of 17 % EDTA for 3 min can efficiently remove the smear layer from all areas of the root canal walls. in our study, eDtA was applied for 1 min based on the lower application time limits of previous studies; this may be the reason for the partial removal of the smear layer as reported in a recent study (16).

Studies have reported conflicting results on whether ultrasonic agitation enhances debridement and antibacterial efficacy (3, 10) or not (7). PUI can be used through continuous flushing of irrigant from the ultrasonic hand-piece or through intermittent flushing using syringe delivery. Both of these methods were proved to be equally effective for the removal of dentin debris in 3 min (28). The intermittent flush method was preferred in the present study to control the depth of syringe penetration and the volume of irrigant. in addition, van der Sluis et al. (29) found that using the intermittent flush method has a cumulative effect over 3 activation cycles.

Ultrasonic agitation increases the temperature and hydrostatic pressure, producing impact waves in the solution and facilitating debris removal. Although ultrasonic irrigation is considered more effective than syringe irrigation in removing debris (16), this could not be completely confirmed in the present study. none of the irrigation protocols in the present study could completely remove debris similar to previous reports (8, 19). The efficacy of PUI can be related also to the size of the tip and the intensity of the ultrasonic activation. A weak power setting paired with a small tip size may not allow high amplitude of oscillation and proper acoustic streaming (9). Since the efficacy obtained with PUI in the present study

was not as high as expected, a thicker tip and higher power settings may need to be tested in future studies.

Because of variations in current PUi protocols, there exists a demand for the creation of a standard protocol. A recent study, in which an extra irrigation (5 ml of 17 % eDtA and 5 ml of 2.5 % naocl) was carried out before testing different irrigation methods, reported that ultrasonic agitation resulted in better removal of smear layer in the apical third of curved roots than did conventional irrigation (2). in contrast, in the present study, ultrasonic agitation did not increase the efficacy of debris and smear layer removal of either naocl, or MtAD. this difference can be attributed to the intensive use of irrigants by Blank-Goncalves et al. (2). there is no consensus in the literature for irrigation in terms of optimal volume, time of application, or activation method (10, 13).

An initial rinse of prepared root canals with dilute naocl was recommended to enhance the efficacy of MTAD to dissolve the smear layer (25). tay et al. (23) reported that the effect of this combined use was equal to that of eDtA. in the present study, this combination had similar effectiveness to that of eDtA, especially in the apical third; which is not in agreement with the results of some other studies (12, 20). in a recent study, Adiguzel et al. (1) reported that self-adjusting file (SAF) instrumentation with continuously replaced irrigant enhanced the efficacies of 17 % EDTA and MTAD for debris and smear layer removal in all thirds of the root. in the present study, MTAD + PUI was not found to be more effective than MtAD alone in all thirds. only one report has shown that ultrasonic agitation of MtAD resulted in better smear and debris removal, although without any significant difference (6).

Removing the smear layer was more successful in the coronal third than in the apical third, as found in previous studies (18, 24). PUI was observed to be beneficial in the apical third, although it did not seem to improve smear layer removal significantly along the root canal.

in our preliminary study, ltAPP was designed to evaluate its elimination effect on endodontic microorganisms, in which promising results were shown (27). We thought that this technique could also be effective on debris and smear removal, since a study showed that plasma treatment can generally change surfaces by modification or etching of the treated surface. Its effect can be modified by changing the temperature, quantity of high-energy ions, the plasma intensity, exposure time, which results in etching of the surface versus etching away of the collagen fibrils on dentin surface (17). However, within the limitations of this study, ltAPP was ineffective on root canal debridement. Further studies can be planned by taking into consideration the above-mentioned modifications on plasma treatment. clinical use of this technique may require additional materials and techniques for root canal debridement.

A weak point in the methodology of the present and the other mentioned studies is that they have not taken the microanatomy of root dentin into consideration during smear


and debris scoring. however, sclerotic dentin can change the scoring values of smear in SeM evaluation (11). Further investigations are needed.

ConclusionsBased on the results of this study, ltAPP did not appear effective on smear layer and debris removal as expected. therefore, different gas parameters will be experimented in further studies to understand its mechanistic implications and role in endodontic disinfection/debridement. PUi improved the efficacy of MTAD for smear or debris removal in the apical third, whereas it did not enhance the efficacy of NaOCl. None of the tested protocols were sufficient to obtain a complete debridement of the root canal system.

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INVESTIGATION OF ROOT CANAL DEBRIDEMENT …€¦ · Final irrigation following the last instrument was ... Group 2 (MTAD): 1 mL BioPure MTAD (Dentsply, Tulsa Dental, tulsa, oK) was