Dentine microhardness changes following conventional and alternate irrigation regimens: An in vitro study.

AIM: To compare the changes in microhardness of root dentin caused by two novel irrigation regimens with conventional irrigation.
MATERIALS AND METHODS: Forty extracted human permanent incisor teeth were selected. Decoronated roots were separated longitudinally to get 80 specimens that were embedded in autopolymerizing acrylic resin and grounded flat with silicon carbide abrasive papers. Of these, 60 root segments without any cracks or defects were selected and divided into four groups according to the irrigation regimen used (n = 15). Group I: 5% sodium hypochlorite (NaOCl) + 17% ethylenediaminetetraacetic acid (EDTA) + 0.2% chlorhexidine digluconate (CHX) (conventional). Group II: 6% Morinda Citrifolia Juice + 17% EDTA (MCJ). Group III: 5% NaOCl + Q Mix 2 in 1 (QMix). Group IV: Distilled water (control). Irrigation regimens were performed for 5 minutes. Dentin microhardness was measured with a Vickers indenter under a 200-g load and a 20-s dwell time at the midroot level of root dentin. The data were analyzed using Kruskal Wallis test and Dunn's multiple comparison tests.
RESULTS: A significant difference was seen in the median values of the four groups. The control group showed the least reduction in microhardness when comparison with the other groups. Except for Group III (Q Mix), the other groups that were tested (MCJ and conventional regimens) showed statistically significant difference from the control group.
CONCLUSION: Within the limitation of this study, it was concluded that NaOCl + Q Mix were least detrimental to root dentin microhardness when compared with MCJ and conventional irrigation regimens.

INTRODUCTION

An irrigant with the ability to remove the smear layer without causing erosion of the radicular dentin and also eradicate microbial biofilms would be considered close to ideal.[1] The irrigants that are used in routine endodontic practice are saline, sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), and chlorhexidine (CHX).[1] It is reported that endodontic irrigants are capable of altering the chemical composition of dentin by removing the calcium ions present in the hydroxyapatite crystals.[234] This, in turn, would lead to changes in the microhardness, permeability, and solubility characteristics of dentin, owing to the alteration of the original proportion of organic and inorganic components.[5] In addition, changes in the calcium ion content adversely affect the adhesion of resin-based root canal sealers to root dentin.[5] Hence, microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues.[6]

Murray et al.[7] proved that 6% Morinda Citrifolia Juice (MCJ), a phytotherapeutic agent, with high antibacterial efficacy can be used as a root canal irrigant; 6% MCJ is recommended as the initial irrigant to be followed with 17% EDTA, to produce a similar smear layer removing efficiency as that of 6% NaOCl + EDTA.[78] Another new irrigant that has been introduced, contains a combination of EDTA, CHX, and Cetrimide (Q Mix 2 in 1) (Q Mix™ 2 in; DENTSPLY Tulsa Dental Specialties, Tulsa, OK, USA). It has been assessed and compared with other irrigants for smear layer removal,[9101112] antibacterial potential,[9] cytotoxicity,[13] wetting with root canal sealer,[14] and bonding with fiber post. (11) However, no study has been reported for a comparative evaluation of changes in the root dentin microhardness caused by these novel irrigation regimens (Q Mix™ and MCJ) with the conventional irrigation regimen as suggested by Zender.[15] Hence, the aim of this study is to compare the changes in microhardness of dentin caused by these new irrigation regimens, using Vicker's microhardness test. The research hypothesis states that there is a significant difference between the tested groups compared with the control.

MATERIALS AND METHODS

Preparation of samples

Forty human maxillary central incisors were selected. All the teeth were stored in 0.1% Thymol (w/v) solution until sterilization. They were cleaned of surface debris and were decoronated at the cemento-enamel junction using low-speed diamond disc (Horico, Berlin, Germany) under water cooling, after which the pulp tissue was removed using a barbed broach (Mani Inc-Tochigi Ken, Utsunomiya-shi, Japan). Each root was then sectioned longitudinally with low-speed diamond disc (Horico, Berlin, Germany) starting from the cervical to the apical area with, separating each root into buccal and lingual segments and making total 80 segments. The root segments were then horizontally embedded in autopolymerizing acrylic resin, leaving their dentin exposed to facilitate manipulation and improve metallographic preparation. The dentin surfaces of the mounted specimens were then grounded flat and smooth on a circular grinding machine with a series of ascending grades of silicon carbide abrasive papers (500, 800, 1000, 1200, 1500, and 2000 grit) under distilled water to remove any surface scratches and finally polished with fine grades of composite polishing kit (Microdont, Brazil) and 0.1-mm alumina suspension (Ultra-Sol R; Eminess Tec Inc, Monroe, NC) on a rotary felt disk. Total 60 specimens without cracks or other surface defects were selected after examining them under a dental operating microscope (Seiler Revelation Microscope, St. Louis, MO).

Specimen treatment

The specimens were randomly divided into four groups (n = 15). Each specimen was treated with 5 ml of each irrigant, which was delivered using a micro pipette for 5 minutes as per the protocol assigned in each group.

Group I (Conventional regimen): 5 ml of 5% NaOCl for 5 minutes followed by 5 ml of 17% EDTA for 5 minutes (Pulpdent Corp, Watertown, MA) and finally with 5 ml of 2% CHX (Sigma Aldrich, Inc., Steinem, Germany) for 5 minutes.

Group II (MCJ regimen): 5 ml of 6% MCJ for 5 minutes followed by rinsing with 5 ml of 17% EDTA for 5 minutes.

Group III (Q MixTM regimen): 5 ml of 5% NaOCl for 5 minutes followed by 5 ml of Q Mix™

Group 1V (Control): 5 ml of distilled water for total 5 minutes.

MCJ was freshly prepared by taking 6 ml of MCJ (Tahitian Noni International, Provo, UT) and diluting it to 100 ml with normal saline using a pipette as recommended by Murray et al.[7] Distilled water rinse was applied between each irrigant to minimize the potential interaction between these irrigants and also immediately after the final irrigant to prevent any substantive effect of the solutions.

Determination of microhardness

The surface hardness of the root dentin was determined in each specimen with a Vickers hardness tester (Matsuzawa MMT7, Matsuzawa SEIKI Co., Ltd., Tokyo, Japan). The indentations were made with a Vicker's diamond indenter at 40× magnification and recorded as 3 separate indentations each using a 200 g load and a 20-second dwell time. The indentations were placed at 0.5-mm level to the root canal wall at a depth of 100 μm from the pulp-dentin interface, at the mid-root level of the root dentin, without any overlap between them. The length of the two diagonals was used to calculate the microhardness value (Vickers Hardness Number [VHN]). The representative hardness values were obtained as the average of the results for the 15 indentations (VHN).

Statistical analysis

The microhardness data were tabulated, and, as the data were not normally distributed, nonparametric test was used for statistical analysis. Data were statistically analyzed by Kruskal-Wallis non-parametric test and the inter-group comparison of means was conducted using a Dunn's multiple comparison test. Data were analyzed using the SPSS 20.0.1 software (V.20.0.1; SPSS, Chicago, USA). Significance was established at P < 0.05 level.

RESULTS

Statistically significant difference was detected among the irrigating solutions (P < 0.0001) [Table 1]. All irrigating solutions, except for distilled water (control), decreased the dentin microhardness. When compared with the control group, the maximum reduction in microhardness was noted in Group I (conventional regimen), and minimum reduction in microhardness were noted in Group III (Q Mix™ regimen). The reduction in hardness among the tested group were as follows; Group 1 (Conventional regimen) > Group II (MCJ regimen) > Group III (Q Mix™ regimen) > Group IV (Control). Inter-group comparison showed that Group III (Q Mix™ regimen) differed significantly (P < 0.01) from Group I (Conventional regimen), but did not show a significant difference (P > 0.05) from Group II (MCJ regimen) and Group IV (Control) [Table 2]. Except with Q Mix™ regimen, the research hypothesis was accepted.

Table 1

Comparison of microhardness values obtained in each groups using Kruskal-Wallis analysis

Table 2

Inter-group comparison of microhardness values done by Bonferroni Dunn's post hoc analysis

DISCUSSION

This study aimed to evaluate the impact of Conventional, MCJ, and Q Mix™ irrigation regimens on the dentin microhardness. Studies reported in the dental literature have tested the individual effect of irrigants on microhardness of root dentin.[416] This would appear to be the first report where the microhardness of dentin is being tested with a combination of irrigants used in succession, thus simulating clinical conditions.

The conventional irrigation regimen of NaOCl + EDTA + CHX in Group I, as recommended by Zender,[15] could be considered as the clinical standard for irrigation. The Group I was tested against two new irrigants. The first of these used was 6% MCJ followed by a rinse of 17% EDTA (MCJ regimen). Studies reported that this new irrigation regimen has good disinfecting and smear layer removal properties when compared with the conventional regimen.[78] In addition, the toxicity of MCJ is very less when compared with NaOCl.[17] The second new irrigant considered was Q Mix™. This has been proven to have good properties such as biocompatibility, antibacterial action, smear layer removal, and less impact on dentin microstructure.[91011121318]

The primary factors that govern the action of an irrigant are contact time and concentration. Optimum contact time that an irrigant solution must be kept in root canals to remove the smear layer is yet unclear. Yamada's[19] suggested a duration of 1 minute with EDTA was sufficient. However, Goldberg & Spielberg[20] advised a longer period of 15 minutes for optimal results. It has also been proven that EDTA and NaOCl have deleterious effects on root dentine if applied for longer durations.[21] Ulusoy & Görgül[22] and Sayin et al.,[6] used the root canal irrigants for 5 minutes in their microhardness tests, stating that this duration is more realistic in terms of clinical practice. Accordingly, in this study, all irrigating solutions were used for a contact time of 5 minutes. Another determinant that has a profound effect on the post-treatment microhardness values of dentin is the concentration of the irrigation solution.[23] As the concentration of NaOCl increases, its bactericidal and smear layer removal efficacy also increases.[1524] Thus, in this study, 5% NaOCl was chosen as the irrigant for Group I and Group III.

Knoop indenter microhardness test[225] and the Vickers indenter method[41626] have been used to measure the hardness of dentin. However, Vickers microhardness test was preferred in this study because of the method's suitability. Studies reported that microhardness of dentin declined when tested from superficial to deep regions.[2] This can be attributed to the fact that more number of widely opened dentinal tubules are found near the pulp, which offer least resistance to the microhardness testing indenter.[2] Pashley et al,[25] proposed an inverse correlation between dentin microhardness and tubular density. In this study, mid-root dentin region was used for testing to minimize the effect of the structural variations of different teeth and provide a reasonable baseline for evaluation.

Results of this study show that with Conventional and MCJ irrigation regimens, dentin microhardness was significantly reduced. The possible reason for the increased reduction of microhardness in Group II can be attributed to the increased acidic content of the MCJ and the use of 17% EDTA. Maximum reduction in microhardness is seen in the conventional regimen wherein all the three irrigants (NaOCl, EDTA, and CHX) were used separately. It has been documented that individually 5% NaOCl[25] and 17% EDTA[2526] can decrease dentin microhardness significantly. Even though, there was no significant difference between MCJ and Q Mix™ regimens; the microhardness values of MCJ regimen was lesser when compared with Q Mix™ regimen [Table 1]. A significant reduction did not occur in Q Mix™ regimen, probably because this product comprises a combination of various substances, including a surface active agent [Table 3]. However, the exact mechanism of action of the two new irrigants, and the reasons behind its less demineralizing effect is yet unclear. Only one study was cited that compared the effect of MCJ and other irrigants on root dentin microhardness,[8] and their result was in accordance with this study.

Table 3

Composition and application procedure of conventional and alternate irrigation regimens

Q Mix™ is superior to 17% EDTA and BioPure MTAD in smear layer removal and exposure of dentinal tubules in the root canal system in single-rooted teeth.[9101112] In addition, Q Mix™ is shown to be less toxic to the rat subcutaneous tissue than 3% NaOCl, 2% CHX, and 17% EDTA individually.[13] Q Mix™ been found to be more effective than BioPure MTAD and CHX against Enterococcus faecalis and plaque bacteria in planktonic and biofilm culture.[9]

CONCLUSION

Within the limitation of this in vitro study, it could be concluded that the use of Q Mix™ regimen will not hamper the root dentin microhardness when compared with MCJ and conventional irrigantion regimens.