Effects of Autoclave Sterilization on Cyclic Fatigue Resistance in 5 Types of Rotary Endodontic Instruments: An In Vitro Study

Background

Root canal preparation is one of the most important active phases in the treatment of endodontic infections. Its purpose is to remove pulp remnants, microorganisms, and microbial toxins mainly through mechanical preparation of the root canal system and chemical disinfection [1]. Rotary nickel-titanium (NiTi) endodontic instruments are increasingly used in root canal treatment and have replaced stainless steel manual files. NiTi rotary files are essential for root canal preparation and provide faster and more predictable instrumentation due to high flexibility and cutting efficiency, which ensure the preservation of the initial form of the root canal [2,3]. During the preparation of the root canal, the endodontic file is exposed to cyclic and torsional stress [1]. Cyclic fatigue is the cyclic loading of stress to produce deformation or fracture of the instrument. The sudden fracture of instruments inside the root canal is a major concern in clinical practice and this complication can compromise the entire endodontic treatment [4]. Considering that cyclic fatigue is the most common cause of instrument fracture, it has become a frequent topic of study among endodontic practitioners [5,6]. It can be said with certainty that almost no metal or alloy has introduced as many revolutionary new therapeutic possibilities in dental medicine and endodontics as the NiTi alloy [7]. The NiTi alloy has 3 different, temperature-dependent, microstructure phases: austenite, martensite, and the R-phase [8]. The conventional NiTi alloy is primarily at room temperature in the austenite phase. Austenitic NiTi instruments have limited flexibility and low resistance to fatigue; therefore, their use is restricted in severely curved canals [7,8]. In attempts to make soft and ductile instruments that can be easily deformed, manufacturers apply a specific thermomechanical process which results in obtaining a NiTi alloy that primarily contains a stable martensite phase, an R-phase, or a mixed form in clinical conditions [8–10]. These changes result in improved physical characteristics of the rotating NiTi files, such as greater dentin cutting efficiency inside the root canal, as well as resistance to fracture [11,12]. Furthermore, electropolishing surface treatments have beneficial effects on prolonging the fatigue life of rotary NiTi instruments by reducing surface irregularities that serve as points of stress concentration and crack initiation [13,14]. Although the number of uses of NiTi rotary instruments in clinical practice is limited, to reuse these instruments both for reasons of economy and to avoid cross-contamination, they have to be exposed to repeated cycles of sterilization in an autoclave [15,16]. There are conflicting opinions about the effect of autoclaving on the efficiency of various NiTi rotary systems. It was believed that repeated sterilization in an autoclave leads to a decrease in cutting efficiency, initiation of irregularities, and deepening of cracks on the surfaces of the working parts of the instruments [17]. However, thanks to the finishing of new-generation instruments, it is possible that the heat of sterilization improves the flexibility of a deformed instrument and can return it to its original shape [18,19]. A positive effect of sterilization can only be expected for instruments that have been thermally treated in the finished production procedure [18,20].

Therefore, this study aimed to evaluate the effect of autoclave sterilization on the cyclic fatigue resistance of 5 types of NiTi rotary files, to compare the resistance of conventional rotary instruments with treated instruments, and to examine the effect of the root canal curvature radius on the fatigue resistance of sterilized instruments.

Material and Methods

INSTRUMENTS:

A total of 480 instruments were tested in this study. Five types of NiTi rotary instruments were included: conventional ProTaper Universal (Dentsply Sirona, Ballaigues, Switzerland) (#25, 0.04 taper), electropolished BioRace (FKG, Dentaire, Switzerland) (#25, 0.04 taper), M-wire ProTaper Next (Dentsply Sirona, Ballaigues, Switzerland) (#25, 0.06 taper), twisted and thermally treated Twisted File (SybronEndo, Orange, CA, USA) (#25, 0.04 taper), and thermally treated and control memory HyFlex CM (Coltene, Whaledent, Altstätten, Switzerland) (#25, 0.04 taper). All instruments were 25 mm in length. Each type included 96 instruments divided into 4 groups: group I – 24 unsterilized instruments made up this group; group II – 24 instruments were subjected to 1 sterilization cycle; group III – 24 instruments were subjected to 3 sterilization cycles; group IV – 24 instruments were subjected to 5 sterilization cycles.

STERILIZATION:

The instruments were sterilized in an autoclave (Cliniclave 45M, MELAG, Berlin, Germany). Each cycle was performed at a temperature of 134°C for a duration of 43 minutes (including 23 minutes of sterilization and 20 minutes of drying).

CYCLIC FATIGUE TESTING:

After the sterilization procedure, the instruments were subjected to a cyclic fatigue test. For the purposes of the experiment, a special metal block made of stainless steel was constructed in accordance with the research of Plotino et al [19]. This block contained 2 machine-cut artificial canals for instrument testing, 19 mm in length and 1.4 mm in internal diameter, with a 45-degree angle of the curvature, one with a curvature radius of 5 mm and the other with a curvature radius of 2 mm. In both canals, the center of the curve was 5 mm away from the tip of the instrument. The instruments of each group were divided into 2 subgroups of 12 instruments. One subgroup was tested for cyclic fatigue in a curvature with a 5 mm radius, and the other subgroup was tested in a curvature with a 2 mm radius. The metal block with artificial canals was placed on a base with feet and could move in 2 directions for easier placement of instruments in the canal. The canals were covered with tempered glass to allow observation of the rotating instrument and prevent the broken fragment from falling out. The handpiece was fixed in a metal ring holder perpendicular to the canal. The instruments were inserted into the canal to the tip and rotated using an electric endomotor (X-smart plus, Dentsply Sirona, Ballaigues, Switzerland). Glycerin was used to reduce the friction of instruments with stainless steel walls. All instruments were continuously rotated to the right, with constant resistance and speed, as recommended by the manufacturer (ProTaper Universal – 250 rpm, 2.5 Ncm torque; BioRace – 600 rpm, 1.0 Ncm torque; ProTaper Next - 300 rpm, 2.0 Ncm torque; Twisted File – 500 rpm, 2.0 Ncm torque; HyFlex CM – 500 rpm, 2.5 Ncm torque). The rotation of the instruments was analyzed visually, and the fracture was registered visually and by sound. Rotation time until the instrument broke was measured in seconds with a digital stopwatch. The number of cycles to fracture (NCF) was calculated according to the formula:

A higher value of NCF meant that the file is more resistant to cyclic fatigue.

The length of the fractured fragment (FL) was measured using a Vernier caliper with an accuracy of 0.02 mm.

Means and standard deviation were calculated for each subgroup. Within each type of instrument, the influence of the number of sterilization cycles on the change in NCF and FL values was analyzed. A comparison of the values of NCF and FL was then made after the cyclic fatigue test in canals with 2 different curvature radii: 2 mm and 5 mm. First, an analysis of the changes in NCF and FL values was performed with an increase in the number of sterilization cycles for each type of instrument. After that, the results of the treated instruments (BioRace, ProTaper Next, Twisted File, and HyFlex CM) were compared to the results of the conventional ProTaper Universal, and then all the types of instruments were compared to one another.

SEM EVALUATION:

After the cyclic fatigue test, the fractured fragments were subjected to ultrasonic cleaning (Sonic 4G, 40KHz, Sonic, Niš, Serbia) in 70% alcohol to prepare them for ultrastructural evaluation. The samples were mounted to aluminum cylindrical stubs with a fixing agent (Dotite paint xc 12 Carbon JEOL, Tokyo, Japan) and sputter-coated with gold/palladium (JFC 1100E Ion Sputter JEOL). Fractured surfaces were examined by scanning electron microscopy (SEM) (JEOL-JSM-5300, Tokyo, Japan). The ultrastructure of the fracture surface as well as the appearance of initial cracks on the perimeter of the section were analyzed.

STATISTICAL ANALYSIS:

The results of the statistical analysis are shown in tabular form, while the statistical calculations were performed using SPSS, version 20. The results are shown in the form of arithmetic mean (X) and standard deviation (SD). The normality of the distribution was tested with the Kolmogorov-Smirnov test. A comparison of the arithmetic means of 2 independent samples was performed with the t test, while in cases of irregular data distribution, the non-parametric Mann-Whitney U test was used. A comparison of 3 or more groups was performed by ANOVA, after which the Tukey post hoc test was used to determine the significance between individual groups. The statistical hypothesis was tested at the level of significance for risk of α=0.05, ie, the difference between the samples was considered significant if p<0.05.

Results

CYCLIC FATIGUE RESISTANCE OF UNSTERILIZED INSTRUMENTS:

Within the group of unsterilized instruments tested at a 45-degree angle with a radius of 2 mm, ProTaper Universal showed a statistically significant lower resistance to cyclic fatigue compared to all the other types of instruments (353.25±9.57) (p<0.001). A significantly higher NCF was observed in the comparison between Twisted File (1446±33.11) and BioRace (910±37.25) (p<0.001), ProTaper Next (1256±101.25) and BioRace (910±37.25) (p<0.001), HyFlex CM (7486.66±1062.08) and Twisted File (1446±33.11) (p<0.001), and HyFlex CM (7486.66±1062.08) and ProTaper Next (1256±101.25) (p<0.001). A statistically significant difference was noted for unsterilized instruments tested at a 45-degree angle with a radius of 5 mm between all tested types of instruments (p<0.001), except between BioRace (1299.91±100.73) and ProTaper Next (1280±51.43) (Table 1).

CYCLIC FATIGUE RESISTANCE OF THE INSTRUMENTS AFTER ONE AUTOCLAVE CYCLE:

After 1 cycle of sterilization and testing of instruments in a curvature radius of 2 mm, a statistically significant higher NCF mean was observed for BioRace (894.75±13.24), ProTaper Next (1258.91±99.97), Twisted File (1439.50±27.31), and HyFlex CM (7552±1115.98) compared to ProTaper Universal (354±14.54) (p<0.001), as well as HyFlex CM compared to BioRace, ProTaper Next, and Twisted File (p<0.001). Testing in a 5 mm curvature radius showed a statistically significant difference between all the tested types of instruments (p<0.001), except between BioRace (1283±107.85) and ProTaper Next (1283.16±51.85) (Table 1).

CYCLIC FATIGUE RESISTANCE OF THE INSTRUMENTS AFTER 3 AUTOCLAVE CYCLES:

After 3 cycles of sterilization, testing of the instruments in a curvature radius of 2 mm showed that BioRace (1660±103.76), ProTaper Next (1230±33.69), Twisted File (1250±66.60), and HyFlex CM (8583.50±408.34) had a significantly higher mean value of NCF compared to ProTaper Universal (353.58±11.91) (p<0.001). A significantly higher NCF was observed for BioRace compared to Twisted File and ProTaper Next (p<0.001), and HyFlex CM compared to BioRace, ProTaper Next, and Twisted File (p<0.001). Testing in a 5 mm curvature radius showed a statistically significant difference between all the tested types of instruments (p<0.001), except between BioRace (2022±177.89) and Twisted File (1916±62.03) (Table 1).

CYCLIC FATIGUE RESISTANCE OF THE INSTRUMENTS AFTER 5 AUTOCLAVE CYCLES:

After 5 cycles of sterilization and testing of instruments in a curvature radius of 2 mm, a statistically significant higher mean of NCF was observed for BioRace (980±127.42), ProTaper Next (1210±208.39), Twisted File (1208±133.04), and HyFlex CM (9683.66±967.72) compared to ProTaper Universal (344.75±16.48) (p<0.001). A statistically significant higher NCF was also observed in ProTaper Next compared to BioRace (p<0.001), and HyFlex CM compared to BioRace, ProTaper Next, and Twisted File (p<0.001). Testing in a 5 mm curvature radius showed a statistically significant difference between all the tested types of instruments (p<0.001), except between BioRace (1800±80.92) and Twisted File (1458±100), as well as ProTaper Next (1750±373.01) and Twisted File (1458±100) (Table 1).

CYCLIC FATIGUE RESISTANCE OF THE INSTRUMENTS TESTED IN 2 DIFFERENT RADII:

The analysis showed that a different curvature radius had an influence on the occurrence of cyclic fatigue. The NCF value was higher in instruments tested in a curvature with a radius of 5 mm compared to a radius of 2 mm, both in unsterilized and sterilized instruments. This difference was statistically significant in BioRace, Twisted File, and HyFlex CM before sterilization, and also after 1, 3, and 5 autoclave cycles, as well as for ProTaper Next after 5 cycles of sterilization (p<0.001) (Table 1).

FRAGMENT LENGTH ANALYSIS:

The average lengths of the fractured fragments (FL) are shown in Table 1. The mean of FL changed compared to the mean of the NCF – with an increase in the value of the NCF, the average FL decreased, and vice versa. This relationship between the NCF and FL was observed in all types of instruments, except the Twisted File, where with a decrease in the value of the NCF, there was also a decrease in the FL. The FL mean was higher for the instruments tested in a curvature radius of 5 mm compared to those tested in a curvature radius of 2 mm. A statistically significant difference was observed in all ProTaper Next instruments, BioRace instruments sterilized over 3 cycles, as well as in unsterilized instruments, and Twisted File instruments sterilized over 3 and 5 cycles.

Figures 1–5 present the SEM of the fracture surfaces of the analyzed instruments. Signs of initial fatigue were observed in all 5 tested types of instruments. Magnification at ×200 showed the initial cracks on the periphery of the sections (white arrows) (Figures 1A, 2A, 3A, 4A, and 5A), while higher magnification (×2000) revealed a pliant pattern of the fractured surface with gaps, pits, and cracks (black arrows) (Figures 1B, 2B, 3B, 4B, and 5B).

Discussion

In clinical practice, due to the risk of cross-contamination, instruments are subjected to repeated autoclave sterilization [22]. Sterilization must also be provided for all new instruments that have not been pre-sterilized. The simultaneous use of chemical agents for rinsing, or contact with cleaning and disinfecting agents, as well as the sterilization process in an autoclave, affect the occurrence of instrument fatigue, and changes to surface roughness [2,23]. Previously published studies indicate the influence of autoclave sterilization on the cyclic fatigue and strength of NiTi instruments, but the results are controversial [24]. The results published in existing studies seem incomplete and the results differ for different types of instruments [25].

According to data from the literature, there are 2 ways of testing endodontic instruments for cyclic fatigue: static and dynamic tests [26]. The static test implies that the instrument is inserted into a simulated canal on the model to a certain depth, and then rotated until a fracture occurs. In this type of testing, the point of maximum stress is usually located in the center of the curve. With this kind of test, better information is obtained about the impact of different instrument blade designs or the finishing of the NiTi alloy itself on cyclic fatigue, which is important for the optimization and development of new instruments. In the dynamic test, the instrument is moved in a simulated canal with a certain amplitude of vertical movements to simulate the clinical situation. Due to the movement of the instrument, the localization of the point of maximum stress varies along the file resulting in a higher resistance to cyclic fatigue [27]. Li et al found that vertical movement of instruments by 1 to 3 mm resulted in a 15% increase in time to fracture [28]. According to Keleş et al, fatigue resistance was significantly increased in the dynamic model for all instrument systems tested in their study; even so, temperature did not influence the cyclic fatigue resistance results [26]. Shen and Cheung also gave priority to systems for laboratory studies that closely imitate in vivo conditions [1]. However, the dynamic test is more sensitive to procedural errors because it is difficult to accurately determine the amplitude of axial movements, as well as the exact angle and path at which the instrument enters the artificial canal on the model [27]. According to research by Hülsmann et al, most studies in the existing literature were conducted on a static model (88%), while only 12% of studies were conducted on a dynamic model [27].

This study was performed on a static model device. The files were subjected to multiple autoclave sterilization and the results of the cyclic fatigue resistance were analyzed in relation to a different NiTi alloy treatment.

In this study, no statistically significant difference was observed in the resistance of conventional ProTaper Universal before and after its exposure to sterilization. It only showed a slight decrease in the NCF value after 5 sterilizations cycles. The results obtained agree with the results of authors who examined other conventional instruments such as Mtwo, Hero642, and K3 [29,30]. Another study, conducted by Elbatal et al, showed that there was no statistically significant difference between the mean of the NCF of ProTaper Universal exposed to the first, second, and third sterilization cycle [31]. However, 1 study reported an increase in the cyclic fatigue resistance of conventional ProFile instruments after 5 sterilization cycles [19]. Because ProTaper Universal is obtained by cutting technology that is standard for conventional instruments, and is not exposed to heating processes during production, the change in mechanical properties of this type of instrument after exposure to sterilization processes in an autoclave could not be expected [7,8].

In this study, electropolished BioRace showed significantly greater resistance to cyclic fatigue compared to conventional ProTaper Universal before and after exposure to sterilization cycles. The results also showed that autoclave sterilization did not significantly affect the cyclic fatigue resistance of electropolished BioRace. The NCF value increased until the third sterilization, and then showed a decrease in value after the fifth, at approximately initial values. It seems that the temperatures to which the rotating instruments are exposed during an autoclave sterilization cycle are not high enough to cause any serious changes in the microstructure of the electropolished NiTi alloy, which would negatively affect fatigue resistance [2]. Almohareb et al and Zhao et al confirmed that they found no detrimental effect of sterilization on the cyclic fatigue resistance of electropolished BioRace [2,32].

The evaluation of the sterilization effect on ProTaper Next in this study did not reveal a statistically significant difference in resistance to cyclic fatigue. ProTaper Next instruments showed a slight increase in the NCF values after the first sterilization, followed by a discrete decline until the fifth sterilization. There are few studies that have dealt with the effect of heat during sterilization on instruments made of M-wire, and the results obtained are contradictory. The study of Özyürek et al found that exposure to autoclave sterilization increased cyclic fatigue resistance of ProTaper Next and ProTaper Gold [11]. Considering that the production of ProTaper Next instruments is based on M-wire technology, which involves a series of heat treatments to which the NiTi wire is exposed during the production process, this type of instrument is much more elastic and resistant to cyclic fatigue than conventional instruments [33]. A number of studies have reported results consistent with this claim [34,35]. However, the existing literature revealed that repeated autoclaving did not affect the fatigue resistance of unused rotating instruments made of M-wire [30,36,37]. These authors claim that sterilization did not increase the resistance to cyclic fatigue, but it did not show a negative effect that reduced the ‘lifetime’ of these rotating instruments.

In recent technologies, an attempt has been made to twist finished profiles of nickel-titanium wires, supported by the processes of heating, cooling, and stress of the alloy. This is followed by changes in the crystal structure of the alloy, when a stable R-phase in nitinol is obtained [38]. Although Twisted Files mainly consist of austenite in the oral environment, the bending load values were significantly lower compared to conventional NiTi rotary files [18]. In this study, Twisted File instruments showed a constant decrease in NCF values with an increase in the number of sterilizations cycles. The results of this study are in contrast to the results obtained in the study by Zhao et al and Hilfer et al, who showed that the autoclave sterilization process did not affect the mean NCF values in this type of instrument [32,37].

The results of this study showed that sterilization in an autoclave significantly increased the cyclic fatigue resistance of the HyFlex CM rotating instrument. A significant increase in the NCF values accompanied an increase in the number of sterilization cycles, a finding which is consistent with the research of other authors [18,32,36]. In a study by Zhao et al, the authors evaluated the effect of autoclave sterilization on the cyclic fatigue resistance of heat-treated (HyFlex CM, K3KCF, TF), electropolished (Race), and conventional (K3) NiTi instruments [32]. It was determined that the resistance to cyclic fatigue was significantly improved in HyFlex CM and K3KCF, while in the other instruments the heat of sterilization had no effect. These results could be explained by the fact that these instruments consist of a specially designed, controlled memory wire created by applying a special thermomechanical process that makes the instruments incredibly flexible, with a memory effect [18,39–41].

A small number of studies reported a relationship between the length of the fractured fragment and the cyclic fatigue of instruments, and the authors did not find a statistically significant relationship between the length of the fractured fragments and cyclic fatigue. This study showed that the mean value of FL changed in direct opposition to the mean value of the NCF in all the types of instruments. The exception was the Twisted File, where a decrease in the value of the NCF was accompanied by a decrease in the FL. Existing data also indicate that the fracture of instruments occurred at ±0.5 mm from the point of maximum curvature of the canal [42]. This study showed similar results in instruments tested in an artificial canal with a curvature radius of 5 mm. However, instruments tested in a 2 mm radius had shorter fragments. A decrease in the curvature radius seems to influence the decrease in fragment length.

A limitation of the current study is that it was performed on a static model which does not simulate a clinical situation. Bearing in mind that in clinical conditions instruments are exposed to cyclic and torsional stress at the same time, it is possible that different results could be obtained in clinical conditions. Although most studies were performed at room temperature, the increase in the temperature of the environment to the level of body temperature could cause an increase in the resistance of instruments to cyclic fatigue, which is why it would be important to introduce standardized conditions for experiments in laboratory studies of this type, where 1 parameter, such as cyclic fatigue, is analyzed [43].

Conclusions

The findings from this study showed that autoclave sterilization of newer rotary endodontic instruments could increase resistance to cyclic fatigue. Autoclave sterilization significantly increased the resistance to cyclic fatigue of HyFlex CM, while sterilization procedures slightly affected ProTaper Universal, ProTaper Next, and BioRace. The resistance of Twisted file decreased with repeated autoclaving. Cyclic fatigue resistance was higher in instruments tested in a higher curvature radius.