Evaluation of surface characteristics of new rotary nickel-titanium instruments – SEM-EDS analysis

SUMMARY Introduction Modern endodontic procedure implies the use of rotary Ni-Ti instruments during chemomechanical treatment of root The aim of this study is to analyze the surfaces of new (unused) rotary endodontic instruments using the SEM-EDS method and determine how frequently manufacture defects or impurities appear on their working surfaces. Material and method Five new different sets of rotary endodontic Ni-Ti instruments were included in this study: K3, Mtwo, ProTaper Universal, HyFlex and BioRaCe. The working part of endodontic instrument was analyzed using SEM-EDS method (magnifications ×150 to ×2000), which determined the morphological characteristics of the instrument surface and chemical composition of the found impurities. Statistical analysis was performed using the Fisher’s test (p < 0.05). Results The results of SEM-EDS analysis showed that there is no new instrument without defects on its surface. The most common defects were observed in K3 (27.43%) and ProTaper Universal group (27.21%) and the least were in BioRaCe instruments (7.67%). The most common type of defect in tested instruments was fretting. In addition, the presence of debris and metal strips was found on all instruments, while corrosion of the working part was observed only in K3, ProTaper Universal and Mtwo systems in a small percentage. Conclusion Based on the results of this research, it can be concluded that manufacturing defects were noticed in all examined instruments. The most common defect is pitting. Impurities such as debris and metal strips have also been registered. No organic debris was observed on electropolished surface of BioRaCe instruments, but a small percentage of other types of defects were registered.


INTRODUCTION
The application of rotary nickel-titanium (Ni-Ti) instruments with shape memory properties, biocompatibility and corrosion resistance, has introduced a new era into the endodontic procedure. Rotary Ni-Ti instruments have enabled faster and more efficient preparation i.e. reduced the possibility of procedural errors during cleaning and shaping of root canals of different morphology [1]. Numerous innovations in instrument design, in recent years related to the surface and heat treatment of Ni-Ti alloys, have affected the efficiency and required safety during endodontic treatment [2][3][4].
In the production of endodontic instruments, Ni-Ti alloys are used in the ratio 56: 44 = Ti: Ni, which achieves their equiatomic relationship. Although only one manufacturer (Dentsply, Maillefer Instruments SA, Ballaigues, Switzerland) published the absolute composition and detailed technological manufacturing process, it is assumed that this is the best ratio that gives the alloy super-elastic properties [5].
The production of Ni-Ti rotary endodontic instruments is much more complicated compared to the process of making steel instruments by cold twisting of pre-profiled wire cones [2]. Ni-Ti instruments are created by specific grinding, i.e. by carving a certain profile into the central stem of the Ni-Ti wire [5]. Newer production techniques include a combination of heat treatment of alloy and simultaneous twisting, for greater flexibility and better resistance to torsion and cyclic fatigue [3]. Although modern computer technology is used in the process of making a complicated design of Ni-Ti instruments, surface defects often occur in the form of fretting, pitting, cracks and impurities that can increase vulnerability to fracture [6]. It has been observed that surface defects act as points of stress concentration, leading to the initiation and spread of cracks i.e. frequent fractures during instrument activation [7].
Various metal residues and impurities of organic and inorganic origin can be found on the surface of new endodontic instruments. During instrumentation, these metal shavings can be incorporated into dentinal wall or pushed into periapical tissue and cause an allergic reaction [8]. The use of instruments with organic impurities also carries the risk of potential cross-infection [9].
The aim of this study is to analyze the surfaces of new rotary endodontic instruments using Scanning Electron Microscopy with Energy-Dispersive Spectrometry (SEM-EDS) to determine how frequently manufacture defects or impurities appear on their working surfaces.
Scanning Electron Microscopy with Energy-Dispersive Spectrometry (SEM-EDS) was performed in the Laboratory for SEM, Faculty of Mining and Geology, University of Belgrade, using the type of SEM -JEOL JSM-6610LV, Japan. The instruments were analyzed without any preparation (directly from the factory packaging). Images were made using Secondary Electron Detector (SE images -second electron) at magnifications ranging from 150x to 2000x. Chemical analysis was performed on unpolished samples using the EDS detector (type X-Max Large Area Analytical Silicon Drifted spectrometer, Oxford Instruments) using internal standards. Obtained chemical composition is presented as the content of chemical elements in weight percent (wt%), normalized to 100%. Detection limit for most elements was about 0.1 wt%. This type of chemical analysis is considered semi-quantitative, because it was performed on unpolished surfaces.
A total of 540 recordings of apical and middle thirds of instruments were made from two different directions. Three SE images were taken for each surface of the instrument. Two researchers reviewed the images and their results were reconciled by Cohen Kappa analysis.
A qualitative analysis of various irregularities and errors present on the working surface of Ni-Ti instruments was applied in accordance with recommendations of Kristina Egert et al. [10]. The instruments registered the presence of: pitting, fretting, microfractures, complete fractures, metal flash, metal strips, blunt cutting edge, disruption of cutting edge, corrosion and presence of debris.
Statistical analysis of obtained results was performed using the Fisher's test (p <0.05).

RESULTS
The results of SEM-EDS analysis of the new Ni-Ti sets are presented in Tables 2-4 and Figures 1-9. The analysis of SE images showed the existence of surface contamination on the working part of tested instruments, and the subsequent EDS analysis determined its chemical composition. This way, a division was made into instruments contaminated with debris and instruments contaminated with metal strips. Examples of SEM-EDS analysis with the appearance of performed spectrum are presented in Figures 1 and 2.
SEM-EDS analysis in point 1 (spectrum 1, Figure 1, Table  2) shows that dominant element in the examined sample was carbon (88.1 wt%) with low oxygen content (1.5 wt%), and impurity on this ProTaper Universal instrument is characterized as a debris of organic origin. Nickel and titanium contents (spectrum 1) reflect the composition of instrument.
Based on SEM-EDS nalysis, the impurities on K3 instrument are characterized as a combination of debris of organic origin and contamination with metal strips. The quantity of nickel and titanium in analysis 1 (spectrum 1, Figure 2) and slightly more quantity of these two elements in analysis 2 (spectrum 2, Figure 2) represents the distribution of these elements in the structure of instruments, just like in the previous case.
The results of SEM analysis indicate the most frequent occurrence of defects and impurities in systems   The most common defects on the working surface of new ProTaper Universal instruments were changes in the form of fretting (apical and middle third, 100%) and pitting (apical third 83.3% and middle third 88.8%) (Table 4, Figure 6). Metal strips were detected on the apical (50%) and middle third (38.8%). A defect on the cutting edge (disruption of its continuity) was observed on one, the most conical instrument (Sx) (apical and middle third), and corrosion on the apical (11.1%) and middle part (11.1%) ( Figure  7). Contamination in the form of debris was noticed in apical third (100%) and in middle third of slightly more than a half (55.5%) of ProTaper Universal sets. The most common defects on new HyFlex instruments were the appearance of fretting in the form of debris on apical and middle segments of all instruments (100%) (Figure 8). A defect in the form of a microfracture was observed on the apical part of the instrument (25-0.08) as well as the appearance of metal strips (25-0.04).
The results of SEM analysis of BioRaCe sets show the most frequent occurrence of metal strips (apical 50% and middle third     38.8%) and fretting (apical 33.3% and middle third 27.7%) (Table 4, Figure 9). Debris contamination detected by EDS analysis was observed on the apical (11.1%) and middle surface (22.2%) of the instruments. A statistically significant difference in the presence of fretting was observed between K3, MTwo, ProTaper and Hyflex instruments between the apical (by p < 0.05) and middle third (by p < 0.05).
Significant differences related to the presence of metal strips were observed between K3 and HyFlex instruments (by p < 0.05), between ProTaper Universal and HyFlex (by p < 0.05), MTwo and HyFlex (by p < 0.05) and BioRaCe and HyFlex instruments (for p < 0.05)). The difference was also significant in the occurrence of metal strips in apical third between K3 and BioRaCe group of instruments (by p < 0.05) between K3 and MTwo (by p <0.05) and K3 and ProTaper group, respectively (by p < 0.05).
In the middle third, a statistically significant difference in the occurrence of metal strips was observed between the HyFlex group and K3, BioRaCe, ProTaperUniversal and MTwo instruments (by p < 0.05). The difference was also significant between the apical and middle third of K3 instruments (by p < 0.05).
The difference was also significant in the values of debris in the apical segment between K3 and ProTaper Universal group (for p < 0.05), K3 and MTwo group (for p < 0.05), and between K3 and HyFlex group of instruments (for p < 0.05). A statistically significant difference was also observed between K3 and BioRaCe group (for p < 0.05), ProTaperUniversal and BioRaCe group (for p < 0.05), MTwo and BioRaCe group (for p < 0.05), respectively, HyFlex and BioRaCe groups (for p < 0.05). In the middle third, the difference was significant between K3 and MTwo (for p < 0.05) and K3 and HyFlex instruments (for p < 0.05). In the ProTaper Universal group, a statistically significant difference in the occurrence of debris was observed between the apical and middle third (by p < 0.05).

DISCUSSION
SEM analysis of various surface irregularities, manufacturing defects and contamination of new Ni-Ti rotating instruments showed that there is not a single tested instrument without defects or impurities on the working surface. In this study, the presence of various defects and impurities was found in all new Ni-Ti instruments (five different commercial sets), with a slightly higher prevalence observed in their apical third. Although there is confirmation of their sterility on the factory packaging, the presence of defects and dirt on the active part of new Ni-Ti instruments is a proven reality, which is documented by the results of various studies [8,[10][11][12][13][14][15][16][17][18][19]. The complicated process of machining the initial Ni-Ti wire often causes the occurrence of surface deformations and cracks due to the traces of milling and machining, but also the appearance of polished surfaces on the cutting edges of instruments [5,10].
Changes on the surface can compromise blade efficiency of instruments and become sites for potential corrosion. Also, these points represent the sites of initiation of defects, contributing to degradation of mechanical properties and occurrence of micro or complete fractures during their clinical use [5,7,13]. Arens et al. presented an interesting study on the incidence of fractures after the first use of new Ni-Ti instruments (0.9%), while Shen cites inadequate manipulation and existence of manufacturing defects as the cause of this complication [12,13].
Due to the higher forces and speed that are necessary for the processing of Ni-Ti alloys, it is possible to cause burning sawdust and formation of hardened places. These are the hardened parts that are more difficult to process, and they represent the zones with higher probability of deformation and fractures [16].
The manner in which defects are formed during the formation of Anusavica and Phillips alloys has been attributed to the specific phase transformation and recrystallization of Ni-Ti alloy [20]. Recrystallization represents the change in the type of lattice depending on the temperature (e.g. titanium at 882 ° C changes from a hexagonal to a monoclinic structure), where the rate of crystallization affects the regularity of crystal structure [20].
The most common type of surface irregularities on working surface of new instruments in this study was the appearance of fretting. Clinical significance of fretting is potentially increased possibility of its screwing (due to the friction that is caused by uneven surface) and increased incidence of fracture [13].
Presence of metal strips as a consequence of the production process was observed on the work surface of all tested instruments. The correlation between the high prevalence of metal strips and the higher conicity of K3 instruments in this study (conicity greater than .06) is in accordance with the results of Marending et al. who indicated that metal strips are formed as a result of the production process of Ni-Ti instruments [11]. Using SEM analysis, Van Eldik et al. noticed the presence of a large amount of metal strips on the surface of new Ni-Ti instruments, immediately after opening the original packaging [14]. This type of contamination leads to a decrease in cutting efficiency, and metal strips can be retained in the dentinal walls of the canal or in the periapical tissue during instrumentation. Van Eldik proved that possible contamination of periapical tissue with these metal strips could reduce the course of tissue repair and compromise the success of endodontic therapy [14]. According to the results of Stefanescu et al. metal particles can be transported during instrumentation and active irrigation through the apical foramen and cause an allergic reaction of the periapical tissue [8]. It has been shown that metal ions as potential hapten allergens can cause type 1 reaction, with a possible immediate or delayed dermal or mucosal reaction. Allergic reactions in endodontics are extremely rare, but the consequences of allergic reactions such as symptoms of delayed apical healing, persistent discomfort after canal obstruction, can increase their number significantly [8].
The presence of debris was also observed on the working surfaces of all types of tested Ni-Ti instruments. Titanium alloys are difficult to machine due to their elasticity and require higher cutting forces compared to steel. Ni-Ti alloys are intensively glued to the tool with which they are processed, so the protection of materials is achieved by oxidizing the surface or metal coating, which are removed chemically after processing, but may still remain on their surface [16]. Electropolishing the surface of BioRaCe instruments increases cutting efficiency, while reducing defects in the production process and possible debris contamination [21]. The significant frequency of debris in the apical third of K3 and ProTaper Universal instruments in relation to their middle third confirms higher contamination of the apical segment due to the more complex production of thinner apical part. This finding is consistent with studies by Eggert and Alapati, which indicated a higher incidence of debris in the apical segment of new Ni-Ti instruments [10,22].
Working surface defects in the form of pitting were observed only in two groups of new instruments, but in a high percentage (K3 and ProTaper Universal). The appearance of pitting occurs during the production process, as during melting of elemental nickel and titanium, the rates of their mutual diffusion during heating differ, which leads to the formation of void spaces [23]. Nickel atoms diffuse faster into titanium than titanium atoms in the opposite direction. Thus, the mass transport is not balanced which can lead to the formation of void spaces in the nickel after alloying. These cavities are known as the Kirkendal porosity or Kirkendal effect [23].
Nagumo presented the evidence on the significance that these defects have on mechanical characteristics of Ni-Ti instruments, as well as the exact mechanism of their influence [24]. He observed that alloy could absorb hydrogen from saliva and form hydride bonds with Ni-Ti lattice atoms that are stable at room temperature. This change in the molecular structure leads to the change in physical properties of the alloy, causing hydrogen porosity. Asaoka also pointed out that diffusion of hydrogen through a Ni-Ti alloy forms hydride phases on the surface of a material that has a more brittle structure [25]. This newly formed hydride layer on the active surface of Ni-Ti instrument is of different thickness thus causing microcracks during clinical work. By providing an absolutely dry working field, this mechanism is not important, but it can have an impact during the process of cleaning and sterilization of instruments, when the instruments are exposed to a longer action of ionizing liquids [25].
Corrosion of the working part of Ni-Ti instruments was not observed on HyFlex and BioRaCe instruments, and in other groups it was observed in a small percentage. The low degree of corrosion on Ni-Ti instruments confirms the resistance of this alloy to corrosion, but also the non-exposure of new instruments to corrosive factors [26].
Findings of defects on the surface of new Ni-Ti instruments in the form of blunting of the cutting edge, disruption of the blade edge and microfracture, only confirm the problems of their production. Microfractures on new instruments are, according to research by Marending and Barbakow, the result of manufacturing process of larger and more conical but less flexible instruments [11]. According to the most researchers, cracks or microfractures are the most dangerous defects that a file can have [24,25]. If instruments with this defect are activated in the canal, during rotation and screwing, they break immediately. Microfracture affects high sensitivity of the instrument to the accumulation of cyclic fatigue and inevitable fractures [11,24,25].
Subsequent heat treatment of finished Ni-Ti instruments (HyFlex) potentially offers the most promising method of manufacturing rotating instruments [27]. These instruments do not have the shape memory that traditional Ni-Ti instruments have, and a special thermomechanical procedure significantly increases their flexibility [28]. The research results in this study show the lowest contamination of Hyflex system with metal strips. The low prevalence of this contamination can be explained by their specific heat treatment that reduces irregularities on their surface. Heat treatment, apart from the change in microstructure (increased flexibility), also leads to the appearance of a cleaner and more regular surface of these instruments [28,29].
Following the results of our study, a significantly lower prevalence of defects in BioRaCe group is observed. This finding is in accordance with the results of a research on a significant reduction of surface irregularities of electropolished instruments [3]. Electropolishing creates a homogeneous oxide layer during the production process, which reduces the appearance of surface defects and increases resistance to corrosion and fracture [27]. Electropolished surface of instruments is visibly brighter than the untreated surface [26]. By introducing current through the solution, a thin passive layer is formed and the surface dissolves into the electrolyte, which also leads to the selective removal of surface defects [4].
In order to improve microstructure of the working surface of Ni-Ti instruments and improve mechanical properties, flexibility, fatigue resistance, i.e. cutting efficiency, manufacturers have used various techniques in recent years (ionic application, plasma immersion, titanium oxide coating, thermal nitriding, thermal treatments and cryogenic treatments, electropolishing) [3,26,28,30].

CONCLUSION
Based on the results of this study, it can be concluded that production defects or impurities (one or more) were observed on all new tested instruments. The most common type of irregularity was the existence of fretting, debris and metal strips on the working part of instruments. No organic debris was observed on electropolished surface of BioRaCe instruments. The results of our study indicate that cleaning and sterilization of instruments before the first use is mandatory. However, further research is needed in order to start manufacturing instruments without defects and impurities.