Over time, manufacturers have progressively perfected methods for the production of endodontic instruments for scouting, pre-flaring [1], preparing the glide path and shaping the endodontic canal [2]. Every endodontic file with a cutting capacity can also be employed for shaping. Here, the term “shaping step” refers to the shaping of the coronal, medium and apical parts following the scouting, pre-flaring and glide path steps [3].

The main steps of endodontic treatment are scouting, pre-flaring, glide path production, canal shaping and canal finishing, and each step requires an instrument that has different features that correspond to different physical and mechanical characteristics [4].

The sterilization process includes several phases: pre-sterilization, drying, packaging, heat sterilization and storage of the sterile material [5].

Pre-sterilization is the disinfection, decontamination and cleaning of dental instruments. This phase involves immersing the instruments in decontaminating and disinfecting liquids or cleansing them with washer-disinfectors. Decontamination reduces the microbial load on endodontic instruments, while cleansing removes organic and inorganic residues (to avoid the removal of debris by hand brushing, the use of ultrasonic trays is recommended) [6].

The subsequent phases involve the rinsing, drying and packaging of instruments, followed by heat sterilization (autoclave at 134 degrees at 2 atmospheres) to eliminate spores. The last phase is the storage of the instruments.

Other sterilization procedures after the pre-sterilization phase include glass bead sterilization or treatment with 2% glutaraldehyde (glutaraldehyde was used for high-level disinfection instead of sterilization but is not recommended because of its toxicity) [7].

An important limit to the reuse of endodontic instruments after sterilization lies in the inability to perfectly clean the blades from the debris created following the canal shaping phase [8]; the removal of debris by manual cleaning or with the help of ultrasonic trays does not allow to remove all debris of an organic and inorganic nature. And among these the prions which represent one of the contaminants that can resist decontamination procedures and are only partially inactivated and denatured at the temperatures reached by autoclaves used in dentistry; remember that prions are responsible for spongiform encephalopathy disease in humans [9, 10, 11, 12].

Walker et al. [13] in 2007 they described the theoretical possibility of transmission of the pathology through prions through dental and endodontic procedures, indicating however low the risk of transmission [13, 14, 15]. Contamination by prions would potentially be triggered by the contact of the blades of the endodontic instruments with the pulp nerve tissue [16]; In addition, in patients with spongiform encephalopathy prions can be found at the trigeminal level, as demonstrated by Guiroy et al. [17, 18].

The WHO guidelines report the following methods as methods for the inactivation of the prion and its denaturation, as reported by Walker et al. [13]:

$\mathrm{✓}$ Immersion in sodium hypochlorite (20,000 ppm of available chlorine) for one hour;

$\mathrm{✓}$ Boiling in 1M sodium hydroxide for one hour;

$\mathrm{✓}$ Autoclaving under vacuum at 121 ${}^{\circ }$C for 30–90 minutes in the presence of 2M sodium hydroxide [19].

These methods alter the surface of the instruments and are not applicable to dental endodontic instruments as reported by Sonntag et al. [20].

The fracture of endodontic instruments inside the canal is a problem that is not always easy to solve [21, 22, 23]. The potential reuse of many endodontic instruments after sterilization procedures raises the question of how much these processes influence their physical and mechanical properties. There are three possible answers to this question: the instrument properties deteriorate, improve or are unaffected [24].

Some studies suggest that sterilization procedures have no influence on these instruments, but the results in the literature do not always agree. For example, D. Zhao et al. [25] reported an increase in the cyclic fatigue resistance of HyFlex CM, Twisted File and K3XF instruments (autoclave sterilization was performed at 134 ${}^{\circ }$C with a pressure of 30 psi for 5 min), and Viana et al. [26] reported that the average number of cycles (916–950 cycles to failure) before failure was higher for instruments that were heat sterilized. In contrast, a recent study on NiTi instruments conducted by Masoud Khabiri et al. [27] indicated that sterilization had no influence on cyclic fatigue, while Silvaggio and Hicks [28] demonstrated that 10 autoclave cycles did not increase the risk of fracture of the files. Resistance to cyclic fatigue is not the only physical and mechanical property affected by sterilization procedures; other effects include resistance to torsional fatigue, on which recent studies disagree: Casper et al. [29] reported the improvement of M-Wire alloys on torsional fatigue strength, whereas King et al. [30] reported the deterioration of Gt Series X instruments. Furthermore, sterilization procedures can negatively affect the cutting efficiency and have corrosive effects on the surface of instruments, as demonstrated in a 1999 study by Rapisarda et al. [31], who reported a reduction cutting efficiency for ProFile instruments of 20%–50% after 7–14 cycles autoclave.

Thus, the literature reports conflicting findings about changes in endodontic instruments after sterilization procedures. These disagreements may be due to the multitude of instruments and methods used in endodontics and the different methods of sterilization and disinfection [32].

The heterogeneity of the results of the various studies could also depend on the high heterogeneity of the measurement apparatuses, in particular for the measurement of cyclic fatigue, Plotino [33] reports in a 2009 literature review as an ideal hypothetical model the curved canals of natural teeth, but the problem as set out by the author of the review and in the standardization of measurement.

All instruments for the measurement of cyclic fatigue that reproduce the artificial canal through a curved cylindrical tube are subject to wear and the endodontic instruments used within them have a degree of freedom following an unpredictable trajectory and without a precise measurement of radius and angle of curvature. In the realization of the measuring apparatus for cyclic fatigue, both the angle and the radius of curvature of the artificial canal are fundamental, which differ in the various studies on the resistance to cyclic fatigue [33].

NiTi endodontic instruments consist of a crystalline lattice with a body-centered cubic shape which takes the name of austenite which, following thermal variations or the application of forces, becomes a monoclinic structure called martensite [34].

For NiTi alloys the martensitic transformations are of the displastic type, obtained by cooling the austenitic phase with the release of heat [35]. The transformation from austenite to martensite consists of 2 moments: in the first we have all the atomic movements necessary to produce the new structure and in the second the adaptation of shape and volume with a change in the crystal lattice that can take place with the slip, which is permanent, or the twinning which is reversible (and is preferable if you want to get a return of shape) [36].

Following a lowering of the temperature which varies according to the type of alloy, the crystal lattice acquires an easily deformable martensitic type configuration, with a low yield point; while upon heating, the alloy forms an austenitic lattice [37]; Under the application of a force, the austenitic alloy behaves in a similar way, passing to a martensitic crystalline form through an intermediate transformation phase that can accommodate greater stress without increasing the tension. Therefore, an endodontic instrument while working inside the endodontic canals is subjected to deformation forces in which there is the transition from the austenitic to martensitic phase through a transformation phase. In the transformation phase, the instrument will show superplasticity characteristics before reaching the martensitic phase which could be followed by yielding and rupture [38, 39].

Previous studies on the subject by the same research group have focused on the torsional property and surface alterations of endodontic instruments subjected to sterilization, focusing neither on cyclic fatigue nor deformation [7, 40, 41]. To date, no other systematic reviews have addressed these topics (cyclic fatigue, deformation, torsional fatigue, corrosion or surface alterations, cutting efficiency) in relation to sterilization.

The purpose of this review is to provide the widest and most up-to-date overview of the topic through a systematic methodology involving the screening and selection of articles [42, 43]. Our hypothesis is that not all alterations induced by the autoclave have a negative effect on rotating endodontic instruments.

The following systematic review was performed in accordance with the PRISMA protocol (Preferred Reporting Items for Systematic reviews and Meta-Analyses): the outcomes, the data extraction process and the methods for their quantitative and qualitative synthesis were previously agreed upon by 3 reviewers.

The protocol and methodology adopted for this review have been used for previous systematic reviews by the same research group.

Articles that were eligible for inclusion in this review were in vitro or clinical studies that evaluated the influence of disinfection and sterilization procedures on rotary endodontic instruments and endodontic files. Reports that were not written in English, were published before 1980 or were published in journals of national interest only were excluded. We selected only articles published in the last 4 decades because the instruments and sterilization methods have profoundly changed. Four research outcomes were defined:

$\bullet$ Primary outcome: influence of sterilization on the resistance to cyclic fatigue;

$\bullet$ Secondary outcome: influence of sterilization on the resistance to torsional fatigue;

$\bullet$ Tertiary outcome: alterations induced by sterilization procedures on the surface of endodontic instruments;

$\bullet$ Quaternary outcome: Reduction in cutting efficiency induced by sterilization procedures.

The studies were identified through bibliographic searches in electronic databases and the manual review of the biobibliography of previous systematic reviews conducted on the subject.

The literature search was conducted in the “PubMed” and “Scopus” search engines. The original search was conducted between 12.12.2020 and 01.03.2021, and a final search was conducted on 10.04.2021 for a partial update of the records.

We searched for different terms using the following combinations: “Endodontic sterilization” “endodontic autoclave” “cyclic fatigue” AND “sterilization”; “torsional” AND “sterilization”; “cutting efficiency” AND “sterilization” “surface characteristics” AND “sterilization”; “Corrosion” AND “sterilization”. Bibliographic citations of previous systematic reviews on the subject were also analyzed. Table 1 describes the research methodology for keyword searches in databases and article selection.

The “records” obtained from the search were subsequently screened by two independent reviewers (M.D. and D.S.). Situations of uncertainty or disagreement between the 2 reviewers were resolved by a third supervisor. Agreement between the 2 screening reviewers was measured by the K statistic, and K = 0.68.

After applying the preliminary eligibility criteria (articles in English and published after 1979) through the application of filters in search engines, duplicates were removed with the aid of the EndNote X8 software (Thomson Reuters , Clarivate Analytics, Philadelphia, PA, USA). Then, the two reviewers independently removed articles and studies that were not related to the topics of the review (endodontics and sterilization) by analyzing the title and abstract. The inclusion and exclusion criteria applied to the studies are shown in the Table 1.

Subsequently, studies were included in the qualitative analysis based on the 4 outcomes decided in advance.

The extraction of data from the included studies involved the development of tables and was performed by the two reviewers.

The characteristics of the data were the following: first author, date of publication, bibliographic reference, type of endodontic instrument, method of sterilization and alterations in the instruments induced by sterilization.

A total of 1195 records were identified in the PubMed and Scopus databases. After screening the articles through the use of filters (restriction by year and language), 996 articles were obtained, and 578 were retained after the removal of duplicates. After the application of the eligibility criteria (all articles relating to the topic of sterilization in endodontics), we arrived at 161 articles, and 103 articles remained after the elimination of duplicates. After the application of exclusion and inclusion criteria, we obtained 51 articles, which were divided according to the topics of interest: outcome 1 (16 studies that investigate the influence of disinfection and sterilization procedures on resistance to cyclic fatigue); outcome 2 (7 articles investigating the influence of disinfection and sterilization procedures on resistance to torsional fatigue); outcome 3 (23 articles investigating the effects of sterilization and disinfection on the surface characteristics of endodontic instruments); outcome 4 (7 articles investigating changes in the cutting efficiency of instruments after sterilization and disinfection procedures). Some articles were included in multiple outcomes.

The studies included in the quantitative analysis were as follows. First outcome: Khabiri et al. 2017 [27], Arias et al. 2014 [44], Zhao et al. 2016 [25], Plotino, et al. 2012 [45], Mize et al. 1998 [46], Viana et al. 2006 [26], Sharroufna and Mashyakhy, 2020 [47], Das et al. 2019 [48], Alshwaimi 2019 [49], Arias et al. 2020 [50], Kim et al. 2020 [51], Li et al. 2015 [52], Yang et al. 2018 [53], Pedullà et al. 2011 [54], Bulem et al. 2013 [55], Özyürek et al. 2017 [56],Second outcome: Iverson et al. 1985 [57], Chenail et al. 1986 [58], Hilt et al. 2000 [59], Silvaggio et al. 1997 [28], Canalda-Sahli et al. 1998 [60], King et al. 2011 [30], Casper et al. 2011 [29]. Third outcome: Razavian et al. 2016 [61], Alexandrou et al. 2005 [62], Qaed et al. 2018 [63], Li et al. 2015 [52], Alexandrou et al. 2006 [64], Yang et al. 2018 [53], Yılmaz et al. 2017 [65], Nair et al. 2015 [66], Valois et al. 2008 [67], Spagnuolo et al. 2012 [68], Inan etal. 2007 [69], Can Saglam et al. 2015 [70], Das et al. 2019 [48], Cai et al. 2017 [71], Prasad et al. 2014 [72], Saglam et al. 2012 [73], Topuz et al. 2008 [74], Ametrano et al. 2011 [75], Fayyad and Mahran [76], Uslu et al. 2018 [77], Shahi et al. 2012 [78], Stokes et al. 1999 [79], Casella et al. 2011 [80]. Fourth outcome: Schafer et al. 2002 [81], Haikel et al. 1996 [82], Rapisarda et al. 1999 [31], Seago et al. 2015 [83], Haikel et al. 1998 [84], Morrison et al. 1989 [85], Neal et al. 1983 [86]. There are 4 articles included in the network meta-analysis for the first outcome: Sharroufna and Mashyakhy, 2020 [47], Kim et al. 2020 [51], Özyürek et al. 2017 [56], Plotino, et al. 2012 [45].

The entire selection and screening procedure, as described in Table 2 (Ref. [7, 40, 41, 87, 88, 89, 90]), is presented in the flowchart in Fig. 1.

The study of the literature shows that there are conflicting findings regarding the different effects of sterilization procedures. In sum, sterilization and disinfection procedures produce changes in physical and mechanical characteristics, but conclusions about whether the effects are favorable or unfavorable for clinical use are not always consistent between studies.

The alterations can be summarized as follows:

$\bullet$ There is a reduction in cutting capacity after autoclaving, and it becomes statistically significant after five autoclaving cycles;

$\bullet$ A corrosive effect is observed after the use of disinfectants based on sodium hypochlorite (12–48 hours) and after the application of heat sterilization;

$\bullet$ There is a potential shape recovery effect with regard to deformation and increased resistance to torsional and cyclic fatigue after sterilization, but not all studies agree.

Conceptualization, MD, AD, KZ, RA, GI; methodology, MD and DS; software, GI; validation, MD and AD; formal analysis, MD and AD; investigation, DS and AD and VCAC; resources, LLM; data curation, EL, BR and LL; writing—original draft preparation, MD and GT; writing—review and editing, MD, GT, CA and LLM.

Not applicable.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.

PRISMA, Preferred Reporting Items for Systematic reviews and Meta-Analyses; SEM, scanning electron microscopy; AFM, atomic force microscopy; AMR, arithmetic mean roughness; MH, maximum height; RMS, root mean square.