Scanning Electron Microscopic Evaluation of Surface Defects of Remover Retreatment File After Single and Multiple Uses

Summary

Here, we present a protocol for evaluating the surface characteristics of endodontic retreatment files after repeated use in retreatment procedures, utilizing scanning electron microscopy to identify and analyze potential surface defects.

Abstract

This study aimed to evaluate surface defects of Remover rotary Nickel-Titanium (NiTi) files after single and multiple uses in conventional endodontic retreatment procedures using scanning electron microscopy (SEM). Eighty acrylic blocks, simulating root canals with a 1.5 mm internal diameter, a 5 mm radius of curvature, and a 55° curvature, were utilized. After chemomechanical preparation and obturation, 24 new Remover files (N30, 7%, L23) were randomly assigned to three groups: single use, triple use, and six uses. The files were operated at 600 rpm with a torque of 2.5 Ncm, cleaned, and sterilized after each use.

SEM analysis at magnifications of 100x, 250x, and 500x revealed surface defects, including tip deformation, microcracks, fracture, unwinding, surface pitting, and blade disruption. Deformation was observed in 75% of the files after a single use and in 100% of the files after three and six uses. Microcracks were absent after single use but appeared in 25% and 87.5% of files after three and six uses, respectively, showing a statistically significant increase (p < 0.001). Surface pitting also significantly increased among groups (p = 0.004).

No fractures were observed in any group. The most common defects were tip deformation (91.7%) and surface pitting (70.8%). The findings suggest that repeated use of NiTi files significantly increases surface defects, elevating the risk of fatigue fractures. Thus, the results recommend limiting the reuse of Remover files to a maximum of 3x. Further research is needed to correlate defect types with anatomical factors and to assess file effectiveness in retreatment scenarios.

Introduction

Endodontic retreatment is a procedure performed when a previously treated tooth fails to heal or develops new pathologies, such as persistent infection, reinfection, or missed anatomy. The procedure involves the removal of the existing root canal filling material, thorough cleaning and disinfection of the canal system and subsequent refilling^1,2.

Nickel-titanium (NiTi) instruments are of great importance in improving and facilitating endodontic procedures due to their flexibility and high cutting efficiency^3,4. The superelasticity of NiTi instruments permits them to better adapt to canal curvature, exhibit less wear, and have a higher resistance to fracture^5,6. However, one of the major concerns with NiTi files is that they can fracture without visible deformation³.

The most common cause of fracture in NiTi rotary instruments is cyclic fatigue⁷. Cyclic fatigue occurs due to alternating tensile and compressive stresses on opposing surfaces of the instrument as it rotates continuously in a curved root canal without binding^8,9. Fracture due to cyclic fatigue results from metal exhaustion¹⁰. Several factors influence the occurrence of fracture due to cyclic fatigue, including the physical properties of the instrument^11,12, the root canal morphology¹³, repeated clinical use, and the sterilization process^14,15. Therefore, to improve the fatigue resistance of NiTi rotary files, various modifications in the manufacturing method and core diameter, as well as changes in the cutting-edge and cross-sectional designs, have been attempted¹⁶. The Remover file is a new generation file produced by thermal treatment and a special electropolishing process called C-wire. Its design features are claimed to increase fatigue resistance. The file has a 30/100 mm non-cutting (inactive) tip and a minimally invasive core diameter. It is manufactured with a variable triple helix cross section that is symmetrical for the first 3 mm and then becomes asymmetrical towards the shaft. In addition, it is designed to preserve periradicular dentin by having a 7% taper in the first 10 mm, followed by a 0% taper towards the shaft¹⁷.

Cyclic fatigue fractures in NiTi rotary files typically occur without any visible plastic deformation^18,19,20. As a result, these fractures cannot be evaluated clinically, and structural changes must be examined under high magnification using tools such as a stereomicroscope or scanning electron microscope (SEM)²¹. Due to the impracticality of performing such examinations on a routine basis, manufacturers recommend that files be used only once^22,23. However, due to the high cost of NiTi files, many clinicians choose to reuse them²⁴. Therefore, it is important to investigate the effects of clinical reuse on these files. One clinical study showed that rotary instruments can be safely reused up to 4x²⁵. However, other studies have evaluated much higher reuse rates and there is no consensus on how many times a file can be safely reused^24,26.

In previous studies that have evaluated the reuse of NiTi files, the primary focus has been on the effects of root canal widening and shaping on the fracture resistance of the files. A review of the literature, therefore, reveals that there is only one study that specifically evaluates the repeated use of retreatment file systems²⁷. The aim of this study is to evaluate the impact of repeated use on the surface characteristics of the Remover file using scanning electron microscopy (SEM). It is hypothesized that increased clinical use will result in an increase in surface defects, thereby elevating the risk of fatigue fractures. The specific objective is to analyze the changes in surface defects of the Remover file after single and multiple uses, and to discuss the implications of these changes for clinical practice.

Protocol

1. Sample procurement

1. Procure 80 acrylic blocks with an internal diameter of 1.5 mm, a radius of curvature of 5 mm, a 55° curvature, and a working length of 16 mm.

2. Cleaning and shaping procedure

1. Set the endomotor to a torque of 2.0 Ncm and a speed of 300 rpm.
   1. Attach a 10/.04 taper file to the motor and use it in a back-and-forth motion until the working length (16 mm) is reached, ensuring that it does not bind.
   2. Irrigate the canals with 5.25% NaOCl.
   3. Attach a 15/.04 tapered file to the motor and use it in a back-and-forth motion until the working length (16 mm) is reached, ensuring that it does not bind.
   4. Repeat steps 2.1.2 and 2.1.3 with 20/.04, 25/.04, 30/.04, and 35/.04 taper files, used sequentially at the working length (16 mm).
   5. Dry the canals with paper points.

3. Obturation

1. Check the fit of a guttapercha cone to the canal.
2. Inject the bioceramic canal sealer into the canal and fill it with bioceramic sealer.
3. Insert the appropriate gutta-percha cone into the sealer-filled canal. Cut the gutta-percha 2 mm below the canal orifice using a heat tool.
4. Take a periapical radiograph to verify the canal fillings (see Figure 1).
5. Store the specimens in an incubator at 37 °C and 100% humidity for 2 weeks.

4. Retreatment procedure

NOTE: A total of 24 new Remover files (23 mm) were used in the present study. The files were randomized into three groups of eight samples each. In determining the number of samples and files used in this research, the quota sampling method was used, considering the budget and the sample sizes of other reports in the literature²⁷.

1. Operate the files at 600 rpm and 2.5 Ncm torque according to the manufacturer's instructions. Use the files with a back-and-forth motion without applying apical pressure until they are 3 mm short of the working length.
2. Remove the file from the canal when resistance is felt and irrigate with 5.25% NaOCl solution.
3. Repeat steps 4.1 and 4.2 until the desired length is reached.
4. Clean and sterilize the instruments in an autoclave for 18 min at 134 °C before shaping the specimen.
   NOTE: The files in the first group were used for retreatment in eight curved canals. The files in the second group were used for retreatment 3x each, and the files in the third group were used for retreatment 6x each. The procedures were repeated in group 2 and group 3 according to the number of uses.

5. SEM analysis

1. Sample preparation and loading
   NOTE: Take the necessary precautions to avoid contamination when handling the sample (e.g., wear gloves). Do not place the sample in a gold sputtering system as the surface is nickel-titanium.
   1. Mount the sample on an SEM stub using conductive double-sided carbon tape.
   2. Attach the stub to the stage and tighten the side screw (see Figure 2).
2. SEM operation
   1. Open the SEM sample chamber and remove the stage.
   2. Place the sample stub on the stage and secure it in place.
   3. Insert the sample stage into the sample chamber and close the chamber.
   4. Switch on the pumps and wait for the system notification of the vacuum.
   5. Open the SEM software and select the required operating voltage between 1 kV and 30 kV.
3. Image analysis
   1. Have a trained investigator take images of the 4 mm distal end, which is the active part (area of interest), at standard magnifications of 100x, 250x, and 500x. Use an unused Remover file as a reference to evaluate the surface characteristics of the samples (see Figure 3).
   2. To commence the Auto Focus function, select the key icon within the SEM software. The resulting focused image of the sample is the desired endpoint.
   3. Set the magnification to the minimum zoom level of 50x.
   4. Enable the fast scan mode for efficient image acquisition.
   5. Adjust the focus using the coarse focus mode until a preliminary focus is achieved.
   6. Gradually increase the magnification to observe the desired feature. Use the coarse focus knob to achieve a rough focus, followed by the fine focus knob for precise focusing. Repeat this step for each magnification increase.
   7. Increase the magnification until the desired feature is observed. Adjust the coarse focus knob to roughly focus the image at this magnification. Then, use the fine focusing knob to improve the focus to obtain a focused image at the desired magnification. Repeat this step each time the magnification level is increased.
   8. Once the desired magnification is reached, refine the focus using the fine focus knob for optimal clarity.
   9. For enhanced image clarity, further increase the magnification to a near-maximum level and adjust the focus using the fine focus knob. If clarity is still not sufficient, adjust the stigmation in both the x and y axes. Continue fine-tuning the focus and stigmation until the clearest image is obtained at the higher magnification.
   10. After achieving a high-quality image of the sample, return to the desired magnification level. Capture the image by pressing the photo button. Choose either slow photo mode for higher quality and resolution, or fast photo mode for quicker capture.
   11. Repeat these steps for each sample.
   12. Download the images to the computer.
   13. Have two calibrated examiners analyze all SEM images by reviewing the images on a computer screen and recording the presence and type of deformations that occur in the files. The deformations include tip deformation, microcracks, fracture, unwinding, surface pitting, and blade disruption (Figure 4, Figure 5, Figure 6, Figure 7, and Figure 8).
   14. Have the same examiners analyze the collected data twice at 1 week intervals.
       NOTE: Differences of opinion in the interpretation of SEM images of the samples between the observers are to be discussed until a consensus is reached.

6. Statistical analysis

1. Present descriptive statistics as counts and percentages.
2. Perform analyses using statistical analysis software. Evaluate the differences between groups using the Fisher-Freeman-Halton Exact test. Set a type 1 error rate of 0.05 (two-tailed), and consider p < 0.005 statistically significant.

Results

Deformation was observed in 75% of files after single use and in 100% of files after three and six uses, but the differences between the groups were not statistically significant (Table 1). The evaluation of deformation types among groups is shown in Table 2. When the types of deformation were evaluated separately, no microcracks were observed after a single use, while microcracks were observed in 25% of the files after three uses and in 87.5% of the files after six uses; this difference was statistically significant (p < 0.001). Surface pitting was observed in 25% of the files after single use, in 87.5% after three uses, and in 100% of files after six uses; the difference between the groups was statistically significant (p = 0.004). Although unwinding, tip deformation, and blade disruption were less common or not observed after a single use, the differences between the groups were not significant. No fractures occurred in any group.

Figure 1: Radiographic evaluation after obturation: The periapical radiographic image was employed for the purpose of evaluating the quality and homogeneity of the root canal obturation procedure conducted on acrylic blocks. Please click here to view a larger version of this figure.

Figure 2: Scanning electron microscope sample holder: The scanning electron microscope sample holder is a specialized platform designed to securely hold and position samples within the microscope for imaging. Its function is to ensure that the sample remains stable under the electron beam, thereby facilitating precise and high-resolution surface analysis. Please click here to view a larger version of this figure.

Figure 3: Scanning electron microscopic image of unused Remover file: The scanning electron microscope image of an unused Remover file was used as a reference for evaluating the surface characteristics of the files after single, triple, and six uses. Please click here to view a larger version of this figure.

Figure 4: Scanning electron microscopic image of tip deformation: File tip deformation is defined as the alteration or bending of the tip of an endodontic file, which can occur as a result of mechanical stress during root canal procedures. Such deformation can impair the file's cutting efficiency and increase the risk of procedural errors. Deformation of the tip is frequently indicative of metal fatigue and may suggest that the file is approaching the end of its functional lifespan.(A,B) The distortions in the tip of the files that were used on three and six occasions, respectively, are indicated by red arrows. Please click here to view a larger version of this figure.

Figure 5: Scanning electron microscopic image of microcrack: A microcrack in an endodontic file is defined as a minor fracture or fissure that develops on the surface of these instruments, typically made from a nickel-titanium (NiTi) alloy, as a consequence of mechanical stress during use. Such microcracks have the potential to compromise the structural integrity of the file, thereby increasing the risk of file separation or fracture during endodontic procedures. The presence of microcracks is frequently identified through the utilization of advanced imaging techniques, such as scanning electron microscopy, which plays a pivotal role in the assessment of the suitability for reuse of endodontic files.(A,B) These images present the formation of microcracks at 500x and 250x magnification, respectively. The presence of cracks is indicated by red arrows. (C) A microcrack at 20,000x magnification. Please click here to view a larger version of this figure.

Figure 6: Scanning electron microscopic image of unwinding: File unwinding can be defined as the distortion or deformation of the helical structure of a rotary endodontic file, whereby the twisted metal begins to untwist or lose its original shape. This phenomenon typically occurs as a result of excessive torsional stress or fatigue during root canal procedures. The unwinding of the file can have a detrimental impact on its cutting efficiency, thereby increasing the risk of instrument failure, such as fracture. Consequently, it is of critical importance to monitor this phenomenon during endodontic treatments.(A,B) The tip unwinding of the files used 3x and 6x are indicated by red arrows. Please click here to view a larger version of this figure.

Figure 7: Scanning electron microscopic image of blade disruption: File blade disruption is defined as damage or irregularities that occur on the cutting edges or blades of an endodontic file. This disruption can manifest in a number of ways, including chipping, bending, or fragmentation of the blades. Such damage is typically the result of mechanical stress, repeated use, or improper handling during root canal procedures. (A,B) The blade disruption of the files are indicated by red arrows. Please click here to view a larger version of this figure.

Figure 8: Scanning electron microscopic image of surface pitting: Surface pitting is defined as the formation of small, localized depressions or cavities on the surface of a material, often observed under high magnification, such as with scanning electron microscopy. In the context of endodontic files, surface pitting may result from repeated use, mechanical stress, or chemical reactions that occur during clinical procedures. (A,B) The surface pitting of the files is indicated by red arrows. Please click here to view a larger version of this figure.

Deformation	n (%)
Deformation	22 (91.7)
Type of Deformation
Unwinding	5 (20.8)
Microcracks	9 (37.5)
Tip deformity	22 (91.7)
Blade disruption	3 (12.5)
Surface pitting	17 (70.8)
Fracture	-

Table 1: Presence of deformation: The total amount of deformation observed in the samples, both in numerical form and as a percentage, as well as the frequency of occurrence of the different types of deformation.

	Single Use	Triple Uses	Six Times of Uses	p
	n (%)a	n (%)a	n (%)a	valueb
Deformation	6 (75.0)	8 (100.0)	8 (100.0)	0.304
Type of Deformation
Unwinding	-	1 (12.5)	4 (50.0)	0.083
Microcracks	-	2 (25.0)	7 (87.5)	<0.001
Tip deformity	6 (75.0)	8 (100.0)	8 (100.0)	0.304
Blade disruption	-	1 (12.5)	2 (25.0)	0.747
Surface pitting	2 (25.0)	7 (87.5)	8 (100.0)	0.004
Fracture	-	-	-	-

Table 2: Deformation types by groups: This table compares the occurrence of deformation types based on the frequency of use.

Discussion

This study evaluated the presence and types of microscopic defects on the external surfaces of Remover files after single, triple, and six-times use in acrylic blocks simulating curved canals. Ideally, human teeth are recommended for use in studies evaluating the fracture resistance of files to better simulate clinical use²⁸. In their study, Peters and Barbakow²⁹ found an increase in fracture initiation and propagation rates in instruments used in blocks compared to extracted canals, emphasizing the need for careful evaluation. However, to ensure standardization and reproducibility, in vitro studies often use stainless steel, ceramic, or acrylic blocks^30,31,32. In addition, studies investigating the fracture resistance of NiTi rotary files have reported an increased risk of surface deterioration and fracture in curved canals compared to straight canals^33,34. Therefore, acrylic blocks with an internal diameter of 1.5 mm, a radius of curvature of 5 mm, and a 55° curvature were used in this study. After shaping the root canals, obturation was completed using a bioceramic-based root canal sealer and the single-cone technique. The preference for the bioceramic sealer is based on previous studies that have shown that canals sealed with bioceramic sealers are more difficult to retreat compared to other types of sealers³⁵. This allows the most clinically risky conditions for file breakage to be assessed. The literature indicates that surface defects on NiTi rotary files may not be visible to the naked eye, requiring evaluation at higher magnifications^36,37. In the present study, routine SEM magnifications of 100x, 250x, and 500x were used to examine the surface of the files.

Previous studies have shown that repeated use reduces the fracture resistance of files. However, there is no consensus on the number of times files can be reused without fracture. Wolcott et al.²⁵ concluded that ProTaper files can be safely used up to four times. Troian et al.³⁸ found that K3 files remained relatively unchanged after the fifth use. In addition, Shen et al.²² reported that new files could deform on first use, especially in narrow and curved canals, and that repeated use increased deformation. They demonstrated that a set of ProTaper files could treat an average of 16.88 canals, but this number dropped to 2.83 when only molars were considered. These results highlight the significant difference between the use of files in curved versus straight canals and the shorter life of files in curved canals. Similarly, Ankrum et al.³⁴ used ProTaper rotary files in the treatment of 15 severely curved molars and found that the failure rate increased to 6.0%. Some researchers evaluate fracture incidence based on the number of teeth, while others evaluate fracture incidence based on the number of canals rather than the number of teeth^25,34,39. Typically, molars have three or four canals. In a four-canal molar, if two instruments break, the separation incidence based on the number of teeth would be 200% (2/1), whereas based on the number of canals it would be 50% (2/4). The first incidence is certainly not convincing. Therefore, the separation incidence derived from the number of canals is considered more accurate than that derived from the number of teeth due to the variable number of canals in different teeth³³. Consequently, this study evaluated the effect of using the file for retreatment in 1, 3, and 6 canals on surface defect morphology.

In vitro studies evaluating canal preparation have investigated file fracture and the formation of surface defects; however, no other studies have assessed the effect of the retreatment procedure on file surfaces^40,41. Similarly, studies evaluating the effects of clinical reuse have employed canal preparation procedures but have not examined the effects of file reuse in retreatment^33,42,43. The only study evaluating the impact of repeated use on the surface characteristics of retreatment file systems was conducted by Saglam et al.²⁷ in 2015. The researchers assessed the properties of three different systems after 1, 3, and 5 uses and concluded that repeated use led to increased deformation in all three systems. This finding is consistent with the results of the present study. These results also align with previous studies that evaluated the surface characteristics of Reciproc files after single use⁴¹. Similarly, Yared et al. found no significant difference between new and used ProFile files when assessing the effect of repeated use on file fracture resistance⁴⁴. On the other hand, the results of our study do not correlate with those of You et al., who evaluated the lifespan of NiTi rotary files in curved canals²⁴. They concluded that reciprocating files could be safely used up to 6x. However, in our study, the percentage of deformation that increased the risk of fracture was significantly higher for files used 6x. This inconsistency in results is likely due to differences in the methodologies of the studies.

In this study, when deformation types were examined separately, the most common surface defects were tip deformation and surface pitting (91.7% and 70.8%, respectively). These results are consistent with findings from previous studies^24,33,45. When comparing the frequency of deformation types between groups, unwinding, tip deformation, and blade disruption were less common or not observed after a single use, and the differences between groups were not statistically significant. While no microcracks were observed in files used once, microcracks were observed in 25% of files used 3x and 87.5% of files used 6x. This difference was statistically significant (p < 0.001). There was also a significant difference in the percentage of surface pitting between the groups (p = 0.004; 25%, 87.5%, and 100%, respectively). Studies have shown that these surface defects significantly increase the risk of file breakage^46,47. Therefore, the null hypothesis that surface defects increase with repeated clinical use and the risk of fracture increases should be partially accepted. Although deformation was observed in all file groups, deformation that significantly increased the risk of fracture was more common with repeated use.

The literature indicates that the failure of NiTi files is influenced more by the way they are used than by the number of times they are used²². Therefore, all procedures in our study were performed by a single experienced endodontist. In addition, selection bias was minimized by ensuring that all materials in each group were of the same brand and quality. In similar studies, sample size calculations have typically involved working with approximately 10 to 12 teeth/instruments per group^47,48,49. Additionally, in the previous study evaluating the surface characteristics of retreatment files, assessments were conducted on three samples each²⁵. Based on these parameters and sample size calculations, our study used eight instruments per group. The small sample size could be considered a limitation of our study. However, it will serve as a reference for future research. One notable limitation of this study is the use of acrylic blocks as a substitute for human teeth. Although acrylic blocks provide a standardized and reproducible model for evaluating the surface characteristics of endodontic files, they do not fully replicate the complex anatomy and material properties of natural teeth. The utilization of acrylic blocks, with their uniform hardness and absence of dentinal tubules, may influence the behavior of NiTi files in a manner that differs from that observed in natural teeth, particularly in terms of file deformation and stress distribution. Therefore, the findings of this study may not be directly applicable to clinical practice, where the variability in canal morphology and dentin hardness can affect file performance. It would be beneficial for future studies to consider the use of extracted human teeth to more accurately simulate clinical conditions and enhance the generalizability of the results. A further limitation of this study is the use of unused remover files as a reference for SEM examination. As baseline images of surface defects were not captured for each file prior to use, there is a possibility that manufacturing defects may have been overlooked. This omission complicates the interpretation of surface changes observed after repeated use, as it remains unclear whether some defects were present before the file's initial application. Moreover, the present study concentrated exclusively on the surface characteristics of the Remover files following repeated use, without evaluating their clinical efficacy in retreatment procedures.

Consequently, while the study provides valuable insights into the mechanical degradation of these files, it does not offer direct evidence regarding their functional performance in the context of endodontic retreatment. It would be beneficial for future research to incorporate an initial baseline assessment and evaluate both the structural integrity and clinical efficacy of the files in a variety of clinical scenarios. Comprehensive and comparative studies are needed to further investigate this subject. In conclusion, the findings of this study indicate that Remover files display surface imperfections, including tip deformation and surface pitting, following repeated utilization in endodontic retreatment procedures. In particular, the frequency and severity of these defects increased significantly after three and six uses, with a notable rise in microcracks and surface pitting, which are associated with an elevated risk of fatigue fractures. The findings indicate that while Remover files demonstrate minimal deformation following a single use, their reuse beyond three instances markedly increases the risk of structural failure. From a clinical perspective, these findings highlight the necessity of limiting the reuse of these files to a maximum of 3x in order to maintain their efficacy and reduce the likelihood of fractures during retreatment procedures. Further research is required to elucidate the relationship between surface defects and various anatomical factors in clinical settings.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acrylic block	ArdaDent Medical,Ankara,Turkey		for obturation
DiaRoot Biosealer	DiaDent, South Korea	BS23101161	for obturation
DualMove Endomotor	MicroMega, Coltene, France	52002023	for preparation
EndoArt  Smart Gold	EndoArt, Inci Dental, Turkey	SGK10114	for initial preparation
Gutta Percha	EndoArt, Inci Dental, Turkey	GD23080701	for obturation
Quattro ESEM	Thermo Fisher Scientific, USA		SEM analysis
Paper Points	Dentsply Maillefer, Ballaigues, Switzerland	1I0305	for dry to root canal
Remover File	MicroMega, Besançon, France	891144/873757/	for retreatment procedure
Sodium Hypochlorite	Saba Chemical&Medical, Turkey	3010225	for irrigation
SPSS v29	IBM SPSS Corp, Armonk, New York, USA		Statistical analysis