Irrigating Solution Effects on the Ultrastructural Human Dentin

the Dentin. Abstract Introduction: The aim of this study was to evaluate the action of different endodontic irrigating solutions on the ultraestructural human dentin. Materials and Methods:

ance between toughness and stiffness [1]. Different solutions were tested alone and alternatively [2] to clean the root canal during the endodontic treatment.There is no single irrigating solution with all beneficial properties. In the clinical treatment, different solutions are applied according to their properties [3] and certain concentrations are considered biocompatible and effective according to clinical researches [4,5]. Sodium hypochlorite (0.5%, 1%, 2.5% and 5.25% NaOCl); ethylendiaminetetraacetic acid (15% and 17%EDTA), chlorhexidine (0.12%, 0.2% and 2% CHx) solutions and calcium hydroxide (0.1%, 0.5%, 1% and 2.5% Ca(OH) 2 suspensions in distilled water) are among the most used irrigating solutions during the mechanical preparation of the root canal.
Different effects on dentin of irrigating solutions during the endodontic treatment have been showed through scanning electron microscopy (SEM) studies [5,6].A study by scanning electron microscopy revealed increased efficiency of 17% EDTA respect to1% NaClO irrigants [6]. Too 1% Ca (OH) 2 respect to 0.2% CHx [7]. Some authors showed that 2.5% NaOCl completely dissolved organic remains and that 17% EDTA removed the smear layer by chelating mineral detached mainly in the middle and cervical thirds of dentin [8][9][10][11], resulting in a cleaner surface and tubular lumen.A study by SEM with 17% EDTA, 42% citric acid and 5.25% NaOCl showed the erosive effect of these solutions in apical intraradicular dentin [12] chequear. The aim of this study was to evaluate the action of different irrigating solutions on the ultrastructure of the root dentin to facilitate the selection of the most suitable solution. 16

Processing Substrates
Uniradicular upper incisors teeth without instrumentation were used. The crowns were removed and the root portions were sectioned with a high speed turbine cylindrical diamond stone with cooling along their long axis in the vestibule lingual direction under water spray coolant. The pulp was extracted and the two half roots were used for comparison. Half root of each tooth layed in 1 ml of each irrigation solution during thirty minutes; the other half was immersed in the same volume of DW (control) for the same time.
We worked in quadruplicate.The half roots were carefully washed with DW. Then they were processed according to the SEM protocol (1°-glutaraldehyde fixation 2°-100% ethanol and acetone de hydration; 3°-freeze dehydration up to the critical point of freezedrying; 4°-placing the roots on stubs; 5°-golden metallization). The specimens were mounted on aluminum cylinders 3 cm in diameter and gold-coated. They were observed using SEM (JSM-35CF; Scanning microscope, JEOL, Tokyo, Japan) at 20X magnification to select the area of observation in the middle third of the root dentin; a line was drawn on the major root axis in the middle of the canal, and a perpendicular line was drawn at 2 mm from the apex. Photomicrographs was standardized at 1500X y 4000X of the dentin wall of the canal and observed by two evaluators.

Quantitative Analysis
In the middle third of human dentin four parameters according to the measurement micrometric scale were quantified with an image analyzer as measuring scale Image Tool, sensibility 1/100 µm.T/A: number of dentin tubules per area in mm 2 (3.5 mm = 1µm; micrographic area = 50 x 33 µm) (13) TD: the biggest tubule hole open lumen [14]. ITS: intertubular space equivalent to the space between tubules in the area [15]. RD: relative density related to the relation between the tubular area and the area of the micrograph/ 100X [14][15][16] to 1500x. 20 measurements per parameter were made for each root (the treated and the controlones).

Statistical Analysis
The variables were analyzed with Anova factorial test. Levenne test was used to determine the homogeneity of the variances of T/A, TD, ITS and RD variables. The Tukey test was applied to compare results of different solutions with an alpha error α = 1%.
The parametric correlation of Pearson was used to compare TD, ITS and RD variables. The analysis between the treated and control roots was performed with the T test for independent samples previously analyzing the normality of the variables (Kolgomorov-Smirnov Test) with an alpha error α = 1%. Data were analyzed with the SPPSS statistical software 11.0 (Inc, Il USA).

Qualitative Analysis
The control dentin showed many cleared dentin tubules ( Figure 1A), and others with their holes partially filled with surface residues which were also observed in the intertubular spaces. At    In Figure 3A were observed dentin tubules cleared from residues, with reduced intertubular spaces, signs of erosion (X) and few but clearly visible collagen fibers after the use of 1% Ca(OH) 2 ; bigger tubular holes partially cleared and with few residues with respect to the control group ( Figure 3A). With DW (Figure 4-right) and 0.2% CHx ( Figure 4A) the root dentin showed surface and tubules of similar aspect and distribution (4000X); collagen fibers were evident in both (F) as well as cleared tubule holes (T) with similar diameters and some of them with their holes partially filled with surface residues (R).

Quantitative Analysis
All quantized values in the middle third of human root dentin Control showed inactivity presenting distilled water (Table 1).

Statistical Analysis
High dispersion of T/A was observed in control (p <0.01) ( Figure 5); in RD low dispersion was observed for both groups (p <0.001). When comparing among solutions, TD showed high dispersion ( Figure 6) specially with Ca (OH) 2 ; TD increased (p <0.001) and showed a negative difference. Low dispersion was observed in RD with all the solutions with respect to CHx; RD increased with Ca (OH) 2 (p <0.001) and lowered with NaOCl (p <0.01). ITS showed high dispersion (Figure 7) especially in root dentin with Ca (OH) 2 (p <0.001). T/A and TD decreased (p <0.001) where as ITS and RD increased with 1% NaOCl respect to control (Figure 8). RD decreased with 17% EDTA (p <0.001) (Figure 9). TD, RD and ITS increased with 1% Ca (OH) 2 (p <0.001) ( Figure 10). No significant differences were observed respect to control with 0.2% CHx (p ~ 0.01).

Discussion
This study shows the changes that endodontic solutions produced both on the structure and the ultrastructure of the root dentin: 1% NaOCl and 1% Ca (OH) 2 removed surface residues and cleared the dentinal tubule holes. The solvent and proteolytic action of NaOCl affected the intertubular collagen according to Agee et al. [17] and Miori Pascon et al. [18]. Ca (OH) 2 partially dissolved surface residues and showed signs of alteration in the ultraestructure. The intertubular spaces were damaged and peritubular dentin absence was observed.17% EDTA did not affect surface remnants although the intertubular dentin fibers became more evident because of calcium extraction. The organic matrix of the dentin would act as a limiting factor for the demineralizing effect of the solution.
Little erosion could be observed in different areas. O´Conell et al. [19] showed that chelating action increased on previously treated dentin with 2.5% NaOCl which produced protein denaturalization that would facilitate dentin demineralization.The lonely application of 17% EDTA according to Mota Moreira et al [20] produced demineralization on bovine dentin whereas 5.25% NaOCl dissolved matrix collagen. When alternated, the solutions produced dissolution and loss of tissues. Long-lasting treatments with the chelating agent also produced dentin weakening because of micro-hardness decrease and organic component loss [21].
The alternated use of different solutions was frequent because none of them isolated met all the requirements of the treatment for the right cleansing of the canal and the bonding of sealants to the dentin. Nevertheless it is important to consider the erosion that it could produce on the dentin tissue [22] and its influence on micro-hardness and posterior resistance [21,23]. Agee et al. [17] mentioned that when 17% EDTA removes the mineral component, it exposes the collagen fibers and tissue becomes more vulnerable to proteolytic degradation. Calcium and phosphorous give more strength and hardness to the matrix and collagen fibers give resistance and flexibility to tissue.The dentin organic componentcollagen-, gives resistance to fracture, whereas enamel, with a high mineral content, is more rigid and fragile.
The solution, its concentration, the alternate use of different solutions and their interactions, the contact time between dentin and the solutions and the areas of the root to be treated should be considered. To standardize the study, the most representative middle third of dentin was chosen. % 0.2 CHx does not modify neither the surface nor the dentin structure [24]. In re-treatment of roots with high amount of gram positive bacteria CHx was successfully applied as a final rinse, after drying the NaOCl remnantin the root canal.The statistical study of human substrates controls were widely dispersed; a heterogeneous distribution T/A dentin was evident. TD, RD and ITS had lower dispersion and the like, indicating a selection of homogeneous substrates young age.
After contacting the solutions 1% NaOCl significantly diminished T/A and TD and increased ITS and RD. 17% EDTA significantly diminished RD and slightly increased the rest of the parameters.
1% Ca (OH) 2 significantly increased TD, ITS and RD.0.2% CHx did not produce changes in the studied parameters during 30 minutes contact. Soares et al. [25] showed through SEM and a polarized light microscope that 0.12% CHx in contact with human dentin does not affect its morphological and mechanical properties because it does not produce collagen degradation. Other studies showed that its alternate use with 2.5% and 5% NaOCl affects the organic component and mechanical resistance of human dentin [11,24,26] which rises doubt about its biocompatibility. Ergucu et al. [27] stated that Na-OCl at low concentrations was biocompatible and adequate for the human root canal and dentinal tubules cleaning.
The statistical study of the parameters revealed low dispersion which a homogeneous response to the solutions action except to Ca (OH) 2 1% high dispersion in TD and ITS. The negative half show significant increase in value after contact with the solution.1% Ca (OH) 2 raised ITS, RD and specially TD. 1% NaOCl diminished T/A and TD. 17% EDTA diminished RD, increased ITS and TD. 0.2% CHx did not significantly modify the measured parameters with respect to the controls.The results of this study demonstrated the biocompatibility with dentin of 0.2% CHx, although it was not efficient in the dentin surface and canals cleaning under the experimental conditions. The other solutions modified the ultrastructure of dentin, specially 1%Ca (OH) 2 . For an optimal cleaning of the canals inhuman root dentin, 1% NaClO and 17% EDTA would be biocompatible and efficient agents used under low concentrations and at less contact time.

Conclusion
1% NaOCl and 1% Ca (OH) 2 endodontic solutions dissolved surface remnants, affected fibers of dentin and opened the lumen of tubular holes after 30 minutes contact with dentin. 1% NaOCl showed effective solvent action on surface remnants and 1% Ca (HO) 2 showed a greater action on the ultrastructure of dentin. It also produced significant changes on the intertubular and peritubular dentin surface.17% EDTA did not eliminate remnants of the dentin surface, extracted calcium, and exposed organic component and fibers. Quantified parameters showed significant changes with 1% Ca (OH) 2 in TD, ITS and RD, and with 17% EDTA in ITS and RD. 0.2% CHx was inefficient in its cleaning under the tested experimental conditions.