Ultrastructural Analysis of the Surface of Endodontic Instruments after Immersion in Irrigating Solutions

Summary Introduction Separation (fracture) of endodontic instruments in the root canal during chemomechanical instrumentation is a complication that can compromise the final outcome of endodontic treatment. One of the most common factors that cause fatigue of endodontic instruments and consequent fracture is surface corrosion. The aim of this study was to investigate the ultrastructure of surface corrosion of endodontic instruments made of stainless steel and nickel-titanium after immersion in the most commonly used root canal irrigants. Material and Methods The study included 48 nickel-titanium and stainless steel endodontic hand files. All instruments were immersed in 5.25% sodium hypochlorite, 0.2% CHX and 17% EDTA. Surface corrosion was analyzed using a scanning electron microscope (SEM). Results Nickel-titanium instruments showed significantly higher susceptibility to corrosion after immersion in 5.25% sodium hypochlorite compared to stainless steel instruments (p<0,001). After immersion in 0.2% CHX corrosion damage was observed on both nickel-titanium and stainless steel instruments but the difference was not statistically significant (p=0.096). No corrosion was observed in both types of instruments after immersion in 17% EDTA. Conclusion The use of 5.25% NaOCl and 0.2% CHX as root canal irrigating solutions can cause serious corrosion changes on the surface of nickel-titanium and stainless steel endodontic instruments.


INTRODUCTION
Endodontic instrument separation (fracture) in the root canal during chemomechanical preparation is a complication that can compromise the final outcome of endodontic therapy [1]. Weakening of instruments' structure is one of important factors that affect safety of their use. Numerous studies that have examined clinical use of endodontic instruments have concluded that metal fatigue is the first anomaly which occurs during clinical use and that, combined with sudden loads during root canal instrumentation, may lead to fracture [1,2]. In addition, the literature data indicate surface corrosion of endodontic instruments as one of the first factors that can cause fatigue of the material [3]. Corrosion can start during chemomechanical instrumentation or chemical disinfection and sterilization of instruments [1]. Corrosion is caused by the contact of metal with different solutions when various electrochemical reactions occur and affect surface integrity making instruments more prone to fracture [4]. Corrosion pits and surface porosity can also reduce the cutting efficiency of endodontic instruments [5].
The aim of this study was to evaluate the ultrastructure of surface corrosion of stainless steel and nickel-titanium endodontic files after immersion in the most commonly used root canal irrigants.

MATERIAL AND METHODS
The study included nickel-titanium (Ni-Ti) ("I-FLEX", "IMD", USA) and stainless steel (NTI-Kahla GmbH, Germany) hand endodontic files. New instruments were taken out of the packages, and in order to remove all debris received from manufacturers, the files were cleaned in the ultrasonic bath (JUS-S01, JEOL) with distilled water for 15 minutes at the frequency of 28kHz. Then after, the corrosion behaviour of endodontic instruments was assessed using potentiodynamic metod in the three most commonly used irrigating solutions: 5.25% sodium hypochlorite (NaOCl) (prepared in the laboratory); 0.2% chlorhexidine gluconate (CHX) (R4, Septodont, France, diluted to 0.2%); 17% ethylenediamine tetraacetic acid (EDTA) (prepared in the laboratory).
All solutions used in this study were freshly prepared and stored in adequate conditions. Forty-eight instruments (24 Ni-Ti and 24 stainless steel instruments) were tested. Instruments were divided into the 6 groups according to the material and used irrigating solution ( Table 1).
The experiments were carried out in an ordinary, threecompartment cylindrical glass cell. The counter electrode was a Pt foil and the reference electrode was a saturated calomel electrode (SCE). All potentials were referred to SCE. The working electrode-endodontic instrument was placed into the cell in such way that only working part of the instrument was immersed in the solution, whereas the handle was above the solution. The instruments were immersed 15 seconds before the potential rise as set by the software. Anodic E-I polarization curves were recorded using the software Par Stat by means of the linear sweep technique (sweep rate 0.2mV/s). The potential value that showed sharp rise of the current was assigned as pitting potential. The sharp increase of the current was a result of local dissolution of metal and forming pits. Electrochemical testing was performed at the Department of Production Engineering, Faculty of Mechanical Engineering, University of Niš, and Department of Physical Chemistry and Electrochemistry, Faculty of Technology and Metallurgy, University of Belgrade.
After electrochemical testing (published results) [6], the instruments were prepared for scanning electron microscopic examination (SEM; JEOL-JSM 5300). In order to obtain adequate visualization of the working parts of instruments, the handles of the instruments were cut off, and their working parts were fixed to the aluminum stubs with a fixing agent (Dotite paint xc 12 Carbon JEOL, Tokyo, Japan) and sputter coated with gold/palladium (in the unit JFC 110 Ion Sputter JEOL). SEM examination was completed at the Institute for Biomedical Research of the Faculty of Medicine in Nis.
Ultrastructure of the surface corrosion changes was analysed using modified score presented by Linsuwanont et al. [7]: score 3 -continuous corrosion of the entire surface of the instrument; score 2 -clearly limited corrosion fields; score 1 -individual corrosion pits; score 0 -no visible corrosion changes. Surface of the working parts of the instruments was observed at three levels: apical, middle and coronal. At each level an appropriate score was estimated, and final score represented the mean value of all three scores for each instrument individually and also within the groups.
Statistical analysis was performed using χ 2 and Fisher Exact test, a p value of p<0.05 was considered statistically significant.

RESULTS
Ultrastructural analysis of instruments surfaces showed the most intensive corrosion changes on the Ni-Ti instruments after immersion in 5.25% sodium hypochlorite. Erosive metal surfaces were observed along the entire working part of all tested instruments in this group (score 3) ( Table 2, Figures 1 and 2). Sensitivity to 5.25% NaOCl was also seen in stainless steel instruments. Continuous surface corrosion and limited fields of corrosion were observed on the working surfaces of these instruments, so the total score of this group was 2.33 (Table 2, Figure 3). Fisher Exact test showed significantly higher sensitivity of Ni-Ti instruments compared to stainless steel after immersion in 5.25% sodium hypochlorite (p<0.001).
SEM analysis of the Ni-Ti instruments after immersion in 0.2% CHX showed limited corrosion fields and individual corrosion pits. The total score in this group of instruments was 1.5 ( Table 2, Figures 4 and 5). Stainless steel instruments showed higher vulnerability to 0.2% CHX. These instruments had higher number of limited corrosion fields compared to individual corrosion pits, so the total score in this group was 1.83 (Table 2, Figure  6). However, Fisher Exact test did not show statistically significant difference in sensitivity to 0.2% CHX between both types of instruments (p=0.096).
Ultrastructural analysis of Ni-Ti instruments after immersion in 17% EDTA did not show corrosion defects on the surface of working parts of the instruments so this group was assigned score 0 (Table 2, Figure 7). Corrosion damages were not observed on the surfaces of the stainless steel instruments after immersion in 17% EDTA, so the average score for this group was also 0 (Table 2, Figure 8).

DISCUSSION
Chemomechanical instrumentation of the root canal is essential during endodontic treatment and involves procedures of cleaning and shaping of the root canal space and use of irrigating solutions. Most commonly used irrigation solutions are: sodium hypochlorite (NaOCl), hydrogen peroxide (H 2 O 2 ), citric acid, ethylenediamine tetraacetic acid (EDTA), chlorhexidine gluconate (CHX), saline solution, etc [5]. Although the use of irrigants during root canal preparation is essential, chemical and electrochemical aggressiveness of these solutions can damage surface of the instruments [8].
Electrochemical techniques based on determination of pitting potential and current density can accurately define sensitivity of metals to different solutions [9]. The surface ultrastructure also plays an important role in determining corrosion behaviour of the tested endodontic instruments in certain solutions [10]. Corrosion on the microscopic level is directly related to the weakening of the structure of instruments that reduces cutting efficiency and make instruments more susceptible to fracture [9].
Corrosion behaviour of nickel-titanium and stainless steel instruments can be affected by numerous factors. Stokes et al. [11] investigated corrosive effect on Ni-Ti instruments of five different manufacturers. They reported that both corroding and non-corroding files were present in the same packages and these results confirmed that severity of corrosion could depend on manufacturing process and quality control. The negative impact of sterilization on corrosion has also been demonstrated [10]. The presence of residual protein substances on the surface of endodontic instruments can also increase the severity of surface attack and dissolution of metal surface [9].
Sodium hypochlorite (NaOCl) is the most commonly used solution for root canal irrigation in endodontic practice. It is used in concentration range from 0.5% to 6% [12]. It has a wide spectrum of antimicrobial activity, and due to its ability to dissolve organic part of dentin it is used for removing smear layer as well as pre-soaking solution in the cleaning procedures after clinical use [13,14]. However, sodium hypochlorite contains active and aggressive Clions that promote the occurrence of corrosion pits and weakening of the instrument structure [15]. It has been shown that NaOCl is corrosive for many metals and selectively removes nickel from Ni-Ti alloys [16]. Studies have shown measurable release of titanium after immersion of Ni-Ti instruments in NaOCl for 30 to 60 minutes [17]. Sensitivity of nickel-titanium and stainless steel endodontic instruments to NaOCl has been reported in numerous studies [1,5,13]. In the study of Stokes et al. [14] corrosion of endodontic instruments was visually confirmed after immersion in 5.25% NaOCl. A significant difference between different manufacturers was observed, but there were no significant differences between nickeltitanium and stainless steel instruments. Berutti et al. [4] found that instruments immersed in NaOCl had significantly reduced resistance to fracture due to early cycle fatigue and occurrence of unexpected fractures in these instruments was significantly higher than in the control group of instruments. SEM analysis of the fractured surface revealed limited corrosion fields, pits and cracks. The effect of Cland Fions on the corrosion of Ni-Ti and stainless steel was studied by Amaral et al. [18] and Aboud et al. [19] for the purpose of electrochemical dissolution and removal of fractured endodontic instruments from root canals. The current study revealed intensive continuous corrosion damages of instruments after immersion in 5.25% sodium hypochlorite and this was in accordance with pitting potential values obtained in electrochemical analysis [6]. Chlorhexidine gluconate (CHX) represent frequently used root canal irrigant due to its prolonged antimicrobial effect that may last up to 12 weeks [20]. It is used in a concentration range from 0.1% to 2%. However, literature data indicate the potential for surface corrosion of instruments after immersion in CHX [5,6]. Corrosion potential of CHX depends on its acidic pH (5.72) as acidic environment increases the corrosion rate [21]. In the current study, a visibly damage of the surface of Ni-Ti and stainless steel instruments was observed after immersion in 0.2% CHX. Such visible damage in the form of limited fields and fissures can act as weak points where further loads on instruments can lead to undesirable cracks that propagate [8].
Ethylenediamine tetraacetic acid (EDTA) is a chelating agent that is used in endodontic practice at concentrations from 15% to 17%. Due to its ability to dissolve inorganic part of dentin, it is used as a lubricant in the preparation of narrow and curved root canals and for removal of inorganic part of the smear layer [22]. The results of SEM analysis from the current study revealed no negative effects of EDTA on the surface structure of Ni-Ti and stainless steel instruments, and that was in accordance with the results of electrochemical testing from previous study [6]. In a study published by Fayyad and Mahran [23] there was no visible change in surface roughness of endodontic instruments after immersion in 17% EDTA. According to Reinhard et al. [24] EDTA has the ability to protect and passivize instruments because it forms complexes with metal ions at pH values less than 4 thus creating an inhibiting barrier for oxidation and corrosion.

CONCLUSION
The use of 5.25% NaOCl and 0.2% CHX as root canal irrigants may cause serious corrosion damage on the surface of Ni-Ti and stainless steel endodontic instruments. The application of 17% EDTA did not cause corrosion changes in both types of instruments. To minimize the risk of damage it is recommended that irrigants should be rinsed out from the files immediately after their use and files should be replaced frequently.

NOTE
The paper was given as poster presentation at the Rosov Pin 2014, The Second Regional Roundtable: Refractory, Process Industry and Nanotechnology, held on October 23-24, 2014 in Fruška gora.