Effect of different cleaning methods on surface contamination of unused endodontic files: a scanning electron microscopy analysis

Abstract

Background

The aim of this study is to analyze the residual organic film and debris on the surface of new endodontic files following different mechanical cleaning protocols.

Methods

Fifty new nickel-titanium (NiTi) rotary instruments were used. Initial scanning electron microscope (SEM) images were obtained and debris amounts were scored between 0 and 3. Files were then divided into five groups, each subjected to a different cleaning protocol: 1. group: Autoclave sterilization without mechanical cleaning, 2.group: Ultrasonic cleaning, 3.group: Brushing and ultrasonic cleaning, 4.group: Cleaning with 0.2% chlorhexidine-impregnated sponge and ultrasonic cleaning, 5. group: Cleaning with 70% alcohol-impregnated gauze and ultrasonic cleaning. Post-cleaning Scanning Electron Microscope images were taken, and surface debris was re-scored. Data normality was assessed using visual (histogram, Q–Q plot) and statistical (Shapiro–Wilk test) methods. As the distribution was non-normal, non-parametric tests were used: the Fisher-Freeman-Halton test for intergroup comparisons and the Wilcoxon Signed-Rank test for within-group comparisons.

Results

According to the Fisher-Freeman-Halton test, a statistically significant difference was found between groups in terms of post-cleaning scores (p < 0.001). All groups except Group 1 showed a significant decrease in scores after cleaning. The Wilcoxon Signed-Rank test revealed statistically significant differences between pre- and post-cleaning scores in Groups 2, 3, 4, and 5 (p = 0.008, p = 0.004, p = 0.006, and p = 0.004, respectively). Significant reductions in median contamination scores were observed in these groups. In Group 1, no statistically significant difference was found (p = 0.083). Numerically, the lowest median post-cleaning score (1.0) was seen in Groups 3, 4, and 5. The highest median percentage change was observed in Groups 4 and 5 (67%), followed by Group 3 (58%), and Group 2 (33%). No change was observed in Group 1.

Conclusion

Within the limitations of this study, it was shown that autoclave sterilization without mechanical cleaning is not sufficient for effective cleaning. Mechanical cleaning prior to sterilization is necessary for proper decontamination of endodontic files. Sponge and gauze-based methods were found to be the most effective, while brushing also provided a notable improvement in surface cleanliness.

Introduction

Proper cleaning and sterilization of endodontic files are essential for effective infection control and the prevention of contamination in endodontic treatments [1]. Due to the complexity of file surfaces, complete removal of residual debris is particularly challenging [2]. Cleaning of dental instruments refers to the meticulous pre-sterilization removal of contaminants such as dirt and organic matter to ensure the instruments are macroscopically clean [3, 4]. Several studies have reported the presence of organic and inorganic residues even on unused endodontic instruments [5, 6]. These contaminants may originate from the manufacturing process or packaging procedures [6]. Therefore, it is recommended that all endodontic instruments be thoroughly cleaned and sterilized prior to their initial use [3].

Most endodontic instruments are not supplied in a sterile condition by the manufacturer. While some manufacturers indicate on the packaging that the instruments should be sterilized before use, others provide no information regarding the sterility of the product. This lack of clarity may lead some clinicians to assume that the instruments are sterile upon delivery and use them directly without additional sterilization [7].

Studies have demonstrated the presence of metal debris, oily residues, and even epithelial cells on the surfaces of new endodontic files. Researchers have noted that these particles may be transferred into the root canal system during instrumentation, potentially posing a risk of contamination [8]. Moreover, it has been reported that milling marks, metallic residues from the manufacturing process, and carbon and sulfur deposits resulting from the oxidation of machine oils can serve as adhesion sites for dentin particles. This contamination may reduce the efficiency of the file and negatively impact the overall success of the endodontic treatment [3].

Various methods have been evaluated in previous studies for cleaning endodontic files [9]. These methods include using dry cotton rolls or gauze, either alone or soaked in antiseptic agents such as alcohol [10–12]; employing sponges as mechanical cleaners alone or in combination with detergents or antiseptic solutions [13]; and brushing the instruments with hand brushes [8].

In recent years, ultrasonic cleaners have gained popularity due to their ability to clean dental instruments rapidly and effectively. Advantages of these systems include a high cleaning capacity and reduced operation time compared to manual techniques. However, the effectiveness of ultrasonic cleaning is influenced by the initial preparation of the instruments and the proper adjustment of the cleaning duration [14, 15].

Furthermore, there is no consensus in the literature regarding whether ultrasonic cleaning alone is sufficient or if it should be preceded by additional mechanical cleaning. The reported efficacy of ultrasonic cleaning varies depending on several factors, including the type of instrument, surface characteristics, duration of application, and chemical composition of the cleaning solution. Consequently, there is no standardized protocol, and the need for uniform cleaning procedures is frequently emphasized in literature.

A review of the current literature indicates that the majority of studies have focused on evaluating the cleaning efficacy of various methods applied to previously used endodontic files. However, there is a noticeable lack of research specifically addressing the surface cleanliness of new instruments [3, 6, 9, 14–21].

In light of this gap, the present study aims to assess the presence of residual organic and inorganic debris on the surfaces of new endodontic files subjected to different mechanical cleaning protocols.

The first null hypothesis (H₀₁) is that there is no statistically significant difference in surface cleanliness scores among endodontic files subjected to different cleaning protocols. The second null hypothesis (H₀₂) is that the cleaning protocols involving mechanical cleaning prior to autoclave sterilization do not result in significantly lower surface contamination scores compared to autoclave sterilization alone.

Materials and methods

This study aimed to evaluate the effectiveness of different mechanical cleaning protocols in removing surface contamination from new nickel-titanium (NiTi) rotary endodontic files using scanning electron microscopy (SEM) analysis. The sample size was determined based on similar in vitro studies in the literature [7, 22]. An a priori power analysis was initially performed using G*Power software (version 3.1; Heinrich Heine University, Düsseldorf, Germany). Assuming a medium effect size (Cohen’s f = 0.25), an alpha level of 0.05, and a statistical power of 0.80, the required total sample size for five groups exceeded 200, which was not feasible for this type of in vitro investigation. Therefore, in line with previously published protocols, the study was conducted with 10 specimens per group (total n = 50). In addition, a post hoc power analysis was performed using G*Power software (version 3.1; Heinrich Heine University, Düsseldorf, Germany) to evaluate the statistical adequacy of the selected sample size. Based on a one-way ANOVA with five independent groups, an alpha level of 0.05, and a large effect size assumption (Cohen’s f = 0.45), the achieved statistical power was calculated to be 0.67. A total of 50 new 25 mm long 10.04 NiTi rotary files (EndoArt Smart Gold, İnci Dental, Turkey) were used.

Prior to experimental procedures, baseline SEM images of each file were obtained. The blister packs were opened using sterile latex gloves, and each file was handled by the shank only, using sterile cotton pliers to avoid contact with the blades. Files were positioned in a custom holder with the “10.04” marking on the shank facing upwards to ensure consistent imaging of the same blade surface. Representative regions of each file were analyzed and photographed at 250x, 500x, and 1000x magnifications. SEM images were acquired using high vacuum mode, with a spot size of 5.0 and an accelerating voltage ranging between 15.00 kV and 20.00 kV. An Everhart-Thornley detector (ETD) was used in secondary electron (SE) mode. The working distance ranged from 8.7 mm to 11.9 mm. Horizontal field width (HFW) was 1.66 mm at 250x magnification, 829 μm at 500x, and 414 μm at 1000x. All images were obtained from the apical portion of the instruments. Specifically, the examined area extended from the tip of the file, encompassing approximately 4 mm at 250x, 3 mm at 500x, and 2 mm at 1000x magnification. For each sample, images were captured at all three magnification levels and archived.

The amount of surface debris was evaluated independently by two experienced endodontists using a subjective scoring system ranging from 0 to 3. In cases of disagreement, the evaluators re-examined the images together and reached a consensus. Inter-rater agreement was assessed using Cohen’s kappa coefficient. The kappa value was 0.827 for pre-cleaning scores and 0.738 for post-cleaning scores, indicating strong and substantial agreement, respectively. The debris scoring was based on the classification by Popovic [23].

• Score 0: Completely clean surface.

• Score 1: Presence of an organic film; mild contamination.

• Score 2: Presence of an organic film and small amount of debris; moderate contamination (defined as debris covering ≤ 50% of the observed surface).

• Score 3: Presence of an organic film and a large amount of debris; heavy contamination (defined as debris covering > 50% of the observed surface).

Following initial imaging, the files were randomly divided into five groups and subjected to different cleaning protocols. All procedures were performed by a single operator to minimize variability.

• Group 1: Files removed from their blister packs were sterilized in an autoclave (Trans Getinge, Sweden) at 134 °C for 45 min, without any mechanical cleaning.

• Group 2: Files removed from their blister packs were cleaned in an ultrasonic device (Hy Technology, Turkey) at 90 °C with a power output of 480 W and a frequency of 40 kHz filled with distilled water for 10 min.

• Group 3: Files removed from their blister packs were held by the shank and rotated while manually brushed for 30 s with a soft nylon-bristle brush moistened with distilled water. They were then cleaned in an ultrasonic device (Hy Technology, Turkey) at 90 °C with a power output of 480 W and a frequency of 40 kHz filled with distilled water for 10 min.

• Group 4: Files removed from their blister packs were inserted 10 times into a sponge soaked with 0.2% chlorhexidine and then cleaned in an ultrasonic device (Hy Technology, Turkey) at 90 °C with a power output of 480 W and a frequency of 40 kHz filled with distilled water for 10 min. A new chlorhexidine-impregnated sponge was used for each individual instrument to eliminate the risk of cross-contamination and ensure methodological consistency.

• Group 5: Files removed from their blister packs were gently wiped with sterile gauze impregnated with 70% alcohol and then cleaned in an ultrasonic device (Hy Technology, Turkey) at 90 °C with a power output of 480 W and a frequency of 40 kHz filled with distilled water for 10 min.

After cleaning procedures, SEM imaging was repeated for each file, targeting the same regions previously photographed. Images were again captured at 250x, 500x, and 1000x magnifications (Figs. 1, 2, 3, 4 and 5). Post-cleaning contamination levels were assessed using the same procedure as the initial evaluation. All pre- and post-cleaning scores were recorded in an Excel spreadsheet.

Fig. 1.

Scanning electron microscope images at 1000x magnification showing the file from Group 1 The initial image has a baseline score of 3 (A). After autoclave sterilization without mechanical cleaning, the post-cleaning image has a score of 3 (B)

Fig. 2.

Scanning electron microscope images at 1000x magnification showing the file from Group 2. The initial image has a baseline score of 3 (A). After ultrasonic cleaning the post-cleaning image has a score of 2 (B)

Fig. 3.

Scanning electron microscope images at 1000x magnification showing the file from Group 3. The initial image has a baseline score of 3 (A). After brushing and ultrasonic cleaning the post-cleaning image has a score of 2. Brush residue is visible after cleaning (B)

Fig. 4.

Scanning electron microscope images at 1000x magnification showing the file from Group 4. The initial image has a baseline score of 2 (A). After cleaning with 0.2% chlorhexidine-impregnated sponge and ultrasonic cleaning, the post-cleaning image has a score of 0 (B)

Fig. 5.

Scanning electron microscope images at 1000x magnification showing the file from Group 5. The initial image has a baseline score of 3 (A). After cleaning with 70% alcohol-impregnated gauze and ultrasonic cleaning, the post-cleaning image has a score of 1 (B)

Statistical analyses were performed using SPSS version 29. The normality of the data was assessed using both visual methods (histogram and Q–Q plots) and statistical testing (Shapiro–Wilk test), considering the limited sample size. Descriptive statistics were presented as frequencies and percentages for categorical variables, and as medians and interquartile ranges for continuous variables. The Fisher-Freeman-Halton test was used to analyze the distribution of post-cleaning scores among the groups. The Wilcoxon Signed-Rank test was used to compare pre- and post-cleaning scores within each group. A Type I error level of 5% was considered statistically significant for all analyses.

Results

The distribution of cleanliness scores according to the different cleaning protocols is presented in Table 1, while Table 2 shows the median scores before and after cleaning, along with the percentage change.

Table 1.

Distribution of cleaning scores across different protocols

Cleaning Score	1 st Group Number (%)	2nd Group Number (%)	3rd Group Number (%)	4th Group Number (%)	5th Group Number (%)	p value*
0	-	-	1 (10.0)	2 (20.0)	-	< 0.001
1	-	1 (10.0)	8 (80.0)	5 (50.0)	6 (60.0)
2	3 (30.0)	7 (70.0)	1 (10.0)	2 (20.0)	4 (40.0)
3	7 (70.0)	2 (20.0)	-	1 (10.0)	-

* Fisher-Freeman-Halton Test

Table 2.

Pre- and post-cleaning score changes across protocols

Protocol	Cleaning Score			p value**
	Pre-Cleaning Median (1st – 2nd Quartile)	Post-Cleaning Median (1st – 2nd Quartile)	Change in % Median (1st – 2nd Quartile)
1 st Group	3.0 (3.0–3.0)	3.0 (2.0–3.0)	0 (0–33)	0.083
2nd Group	3.0 (2.8–3.0)	2.0 (2.0–2.3)	33 (0–33)	0.008
3rd Group	2.5 (2.0–3.0)	1.0 (1.0–1.0)	58 (50–67)	0.004
4th Group	3.0 (2.0–3.0)	1.0 (0.8–2.0)	67 (33–75)	0.006
5th Group	3.0 (3.0–3.0)	1.0 (1.0–2.0)	67 (33–67)	0.004

**Wilcoxon Signed Rank Test

According to the Fisher-Freeman-Halton test, a statistically significant difference was observed between groups in terms of post-cleaning scores (p < 0.001). In Group 1, 70% of the samples scored 3 and 30% scored 2 after cleaning; a significant improvement in cleanliness was not achieved. In contrast, Groups 2, 3, 4, and 5 showed higher cleaning efficacy (Table 2). In group 2, 70% scored 2, 20% scored 3, and 10% scored 1; in group 3, 80% of samples scored 1 and 10% scored 0; in group 4, 50% scored 1, 20% scored 2, and 20% scored 0; in group 5, 60% scored 1 and 40% scored 2.

The Wilcoxon Signed-Rank test revealed statistically significant differences between pre- and post-cleaning scores in groups 2, 3, 4, and 5 (p = 0.008, p = 0.004, p = 0.006, and p = 0.004, respectively), indicating a significant reduction in median surface contamination scores. However, no significant difference was found in Group 1 (p = 0.083).

Numerically, the lowest median post-cleaning score (1.0) was achieved in groups 3, 4, and 5. The highest median percentage reduction was recorded in groups 4 and 5 (67%), followed by Group 3 (58%) and Group 2 (33%). Group 1 showed no change in median score before and after cleaning.

Discussion

In this study, the effectiveness of various mechanical cleaning methods in removing organic films and debris from the surfaces of new endodontic files was evaluated. The findings indicate that files supplied directly by the manufacturer may be contaminated during the production and packaging processes, highlighting the necessity of performing mechanical cleaning and sterilization prior to clinical use. Similar results have also been reported in previous studies that assessed the surface cleanliness of unused endodontic instruments [8, 15, 24].

Previous studies have reported that although newly purchased endodontic files are generally free from viable bacteria, manufacturers do not provide full sterility during the packaging process, and thus, files should be cleaned prior to use. Merdad et al. [5] evaluated the surface cleanliness of new files from five different brands using both microbial culture methods and SEM. They detected bacterial and fungal contamination in some files through microbial cultures, and SEM images revealed surface contamination in one out of every five files examined from each brand.

The Australian/New Zealand Standard AS/NZS 4187:2003 states that instruments must be macroscopically clean and free of protein residues, but does not specify how to assess such residues [3]. In fact, there is no universally accepted method for testing the cleanliness of an instrument [25]. In their study, Khullar et al. [21] stained residual debris on files with Van Gieson stain and examined them under a light microscope. Merdad et al. [5] employed microbiological culture techniques to assess sterility. In the present study, SEM was preferred due to its ability to provide high-resolution and high-magnification imaging [5, 14]. Furthermore, the positioning of the files was standardized in both imaging phases to ensure consistent visualization of the same surface region.

Based on the findings of the present study, files in group 1 exhibited a high level of surface contamination following the cleaning procedure. This indicates that autoclaving alone is ineffective in removing organic and inorganic residues. Files in group 2 showed a moderate level of contamination, suggesting that ultrasonic cleaning without prior mechanical treatment is also insufficient for complete removal of surface debris. In contrast, significantly better cleanliness was observed in the groups where mechanical cleaning was performed in combination with ultrasonic cleaning, and this difference was statistically significant. In light of these results, the first null hypothesis, which stated that no significant difference exists between autoclave sterilization and other cleaning methods, was rejected.

In the present study, the most effective cleaning results were statistically achieved in groups 3, 4, and 5. In terms of numerical values, the highest cleaning efficacy was observed in the groups treated with chlorhexidine-impregnated sponge and alcohol-soaked gauze, followed by the group that underwent mechanical cleaning with a nylon brush. Thus, the second null hypothesis, which stated that there is no significant difference among the various cleaning methods, was rejected.

There are numerous studies in the literature evaluating the mechanical cleaning of endodontic files after clinical use. While these studies emphasize the need for additional mechanical cleaning prior to sterilization, there is no consensus on which method should be preferred [6, 14, 15, 18, 20].

In the study by Murgel et al. [14], the cleaning effectiveness of three protocols was evaluated for both used and newly opened endodontic files: alcohol-soaked gauze, alcohol-soaked sponge, and ultrasonic cleaning with enzymatic solution. The study concluded that the least effective method was the alcohol-soaked sponge, while the combination of alcohol-soaked gauze and ultrasonic bath with enzymatic solution yielded the most successful results.

In a more recent study by Cayo-Rojas et al. [19], it was reported that ultrasonic cleaning was more effective than manual brushing with a nylon brush for files used in root canals. However, when enzymatic detergent was added, both methods showed significantly improved outcomes.

Additionally, several studies have shown that both manual brushing and ultrasonic cleaning may be insufficient for removing organic debris from contaminated endodontic files prior to autoclave sterilization [12, 26]. These findings contradict the current study’s results.

Possible reasons for these discrepancies may include whether the files were previously used in root canals, the duration of ultrasonic cleaning, the temperature of the ultrasonic bath, and the type of solution used (distilled water vs. enzymatic detergent). For instance, in Murgel et al.’s study [14], the files were used in root canals and cleaned for 5 min in an ultrasonic device containing Dri-Clave solution (temperature not specified). In contrast, in the present study, files were unused and cleaned in an ultrasonic device containing distilled water at 90 °C for 10 min. This temperature was selected based on findings in the literature indicating that elevated temperatures reduce the viscosity of the liquid, thereby enhancing the efficiency of cavitation and strengthening the dynamics of microbubbles, which in turn positively influence the overall cleaning performance [27].

In a study by Guandalini et al. [18], it was concluded that manual cleaning with a nylon brush in combination with enzymatic detergent, as well as ultrasonic cleaning with water or detergent-based solutions, provided high cleaning efficacy. In contrast, cleaning with alcohol and gauze was found to be less effective. Similarly, in the present study, the differences in outcomes may be attributed to the use of previously used instruments and enzymatic solutions in their study, as opposed to the use of new files and distilled water in the current investigation.

Parirokh et al. [22], also detected residual debris on new instruments. Their findings showed that while ultrasonic cleaning reduced organic residues, it had limited effectiveness in removing metallic particles. Furthermore, autoclave sterilization did not significantly alter the amount of residual debris. These results are consistent with the present study, which also found that none of the instruments were completely clean following sterilization alone. Without mechanical cleaning, substantial debris remained visible on the file surfaces.

Based on the findings of the present study, cleaning methods such as alcohol-soaked gauze, chlorhexidine-impregnated sponges, or manual brushing, which have previously been considered insufficient for cleaning used endodontic files, may provide adequate cleaning efficacy for new instruments. Although these results have important implications for clinical practice, several limitations of the study should be acknowledged.

First, only four mechanical cleaning methods were evaluated, and the effects of chemical cleaning agents were not analyzed. Future studies could investigate protocols that combine mechanical and chemical cleaning techniques in a comparative manner.

In addition, only one brand of endodontic file was included in this study. Broader investigations involving instruments from different manufacturers are recommended to better assess variations in contamination levels and cleanability.

Although there is no universally accepted gold standard for evaluating instrument cleanliness, SEM analysis was used in this study due to its ability to provide high magnification and detailed surface evaluation. However, the method’s limitation lies in its ability to examine only one side of the instrument, and the evaluation itself was subjective, representing another constraint of the study.

Conclusion

Proper cleaning and sterilization of endodontic files prior to clinical use is critical in minimizing the risk of cross-contamination. Within the limitations of this study, it was demonstrated that autoclave sterilization alone, without prior mechanical cleaning, is insufficient for effective decontamination.

Mechanical cleaning prior to sterilization is necessary to ensure proper cleanliness of endodontic instruments. Among the evaluated methods, sponge and gauze-based cleaning protocols were found to be the most effective, while manual brushing also resulted in a noticeable improvement in surface cleanliness.

Abbreviations

ANOVA

Analysis of Variance

CHX

Chlorhexidine (0.2% chlorhexidine solution)

ETD

Everhart-Thornley Detector

HFW

Horizontal Field Width

NiTi

Nickel-Titanium

Q–Q plot

Quantile-Quantile plot

SD

Standard Deviation

SE

Secondary Electrons

SEM

Scanning Electron Microscope/Microscopy

SPSS

Statistical Package for the Social Sciences

°C

Degrees Celsius

µm

Micrometer

W

Watt

KHz

Kilohertz

Kv

Kilovolt

Authors’ contributions

EA: Material preparation, Data collection, Writing- Original Draft Preparation, and Visualization. İÖ: Conceptualization, Methodology, Material preparation, Investigation. Resources, Writing – Review & Editing. MG: Data Curation, Formal Analysis, Investigation. HSÖ: Supervision, and Writing – Review & Editing. All authors have read and approved the final version of the manuscript.

Funding

No funding.

Data availability

The datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study does not require ethical approval as it did not involve the use of human subjects or human-derived tissues. All experimental procedures were conducted using synthetic materials, thereby exempting the research from the necessity of ethical review.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.