The Application of a Fluoride-and-Vitamin D Solution to Deciduous Teeth Promotes Formation of Persistent Mineral Crystals: A Morphological Ex-Vivo Study.

Background: The use of effective, low-cost, and easy-to-use products for early caries management will avoid loss of dental vitality and impairment in oral function. The ability of fluoride to re-mineralize dental surfaces has been widely reported as well as vitamin D demonstrated to have significant potential in improving the remineralization of early lesions on enamel surfaces. The aim of the present ex vivo study was to evaluate the effect of a fluoride and vitamin D solution in terms of formation of mineral crystals on the enamel of primary teeth, and their permanence over time on dental surfaces. Methods: Sixteen extracted deciduous teeth were cut to obtain 64 specimens that were divided into two groups. The first consisted of immersion of specimens for 4 days in a fluoride solution (T1); in the second group, the specimens were immersed for 4 days (T1) in fluoride and Vitamin D solution, and for a further 2 (T2) and 4 days (T3) in saline solution. Then, samples were morphologically analyzed by using Variable Pressure Scanning Electron Microscope (VPSEM) and underwent 3D surface reconstruction. Results: After a 4-day immersion in both solutions, octahedral-shaped crystals were formed on the enamel surface of primary teeth, demonstrating any statistically significant differences in terms of number, size, and shape. Moreover, the binding of the same crystals seemed to be strong enough to be maintained until 4 days in saline solution. However, a partial dissolution was observed in a time-dependent manner. Conclusions: A topical application of fluoride and Vitamin D promoted the formation of persistent mineral crystals on enamel surfaces of deciduous teeth and should be further studied to be potentially used as an alternative strategy in preventive dentistry.

Effect of Ciprofloxacin-Loaded Niosomes on Escherichia coli and Staphylococcus aureus Biofilm Formation.

Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli, Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation.

Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

A Different Exosome Secretion Pattern Characterizes Patient-Derived Colorectal Cancer Multicellular Spheroids and Their Mouse Xenografts.

Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes' role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them "in situ" in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs' exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.

Mechanical and Metallurgical Evaluation of 3 Different Nickel-Titanium Rotary Instruments: An In Vitro and In Laboratory Study.

An in-depth evaluation of the mechanical and metallurgical properties of NiTi instruments is fundamental to assess their performance and to compare recently introduced instrument with widespread ones. According to this, since there are no data on this topic, the aim of the study was to mechanically and metallurgically evaluate an instrument recently introduced into the market (ZenFlex (ZF)), by comparing it with two well-known instruments with similar characteristics: Vortex Blue (VB) and EdgeSequel Sapphire (EES). According to this, 195 instruments were selected: 65 ZF, 65 VB and 65 EES. Each group was divided in subgroups according to the mechanical tests (i.e., cyclic fatigue resistance, torsional resistance and bending ability; (n = 20)) and the metallurgical test (differential scanning calorimetry (n = 5)). A scanning electron microscopy was performed to verify the causes of fracture after mechanical tests (cyclic fatigue and torsional tests). According to results, VB showed the highest flexibility and cyclic fatigue resistance in comparison to the other instruments, with a statistically significant difference (p < 0.05). Regarding torsional resistance, EES showed the lowest value of torque at fracture, with a statistically significant difference, whilst the comparison between ZF and VB showed no statistically significant difference (p > 0.05). DSC analysis pointed out that VB had the highest austenite start and finish temperatures, followed by ESS and then ZF. ESS sample showed the highest martensite start and finish temperatures followed by VB and ZF. Considering the results, it can be concluded that VB showed the best mechanical performance during static tests in comparison to ESS and ZF. This is fundamentally due to the interaction of parameters such as instrument design and heat-treatments that are able to enhance its mechanical performance.

New Strategies and Instruments for Root Canal Shaping Procedure: An Overview.

Cone-beam computed tomography-based three-dimensional analysis of root canal trajectories and shapes have clearly shown that root canals are more complex in 3D, compared to traditional 2D visualization,1-4 and consequently, these findings affect properties of the nickel-titanium (NiTi) files requiring the following factors.

Anti-Virulence Activity of the Cell-Free Supernatant of the Antarctic Bacterium Psychrobacter sp. TAE2020 against Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is an opportunistic pathogen often involved in airway infections of cystic fibrosis (CF) patients. Its pathogenicity is related to several virulence factors, such as biofilm formation, motility and production of toxins and proteases. The expression of these virulence factors is controlled by quorum sensing (QS). Thus, QS inhibition is considered a novel strategy for the development of antipathogenic compounds acting on specific bacterial virulence programs without affecting bacterial vitality. In this context, cold-adapted marine bacteria living in polar regions represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire. In this paper, we investigated the biological activity of a supernatant derived from a novel Antarctic bacterium (SN_TAE2020) against specific virulence factors produced by P. aeruginosa strains isolated from FC patients. Our results clearly show a reduction in pyocyanin and protease production in the presence of SN_TAE2020. Finally, SN_TAE2020 was also able to strongly affect swarming and swimming motility for almost all tested strains. Furthermore, the effect of SN_TAE2020 was investigated on biofilm growth and texture, captured by SEM analysis. In consideration of the novel results obtained on clinical strains, polar bacteria might represent potential candidates for the discovery of new compounds limiting P. aeruginosa virulence in CF patients.

Nickel-Titanium Rotary Instruments: An In Vitro Comparison (Torsional Resistance of Two Heat-treated Reciprocating Files).

AIM AND OBJECTIVE: The present study aims to evaluate the difference in torsional resistance of two reciprocating nickel-titanium (Ni-Ti) rotary files: WaveOne Gold and EdgeOne Fire. MATERIALS AND METHODS: A total of 40 nickel-titanium rotary instruments (n = 40): 20 WaveOne Gold Small (WOGS) and 20 EdgeOne Fire Small (EOFS) were divided into two groups. Each instrument was tested using a torsional resistance device already validated in previous studies to evaluate and compare torsional resistance. The static torsional test was implemented by blocking each instrument at 3 mm from the tip and rotating it until fracture with a reciprocating motion. Torque to fracture (TtF) and fragment length (FL) were measured and statistically analyzed. RESULTS: Statistical analysis of TtF found significant differences between the two groups (p<0.05). The EOFS showed higher TtF if compared to WOGS, with a mean value and a standard deviation of 3.05 ± 0.07 (N cm) against 2.97 ± 0.08 (N cm). Data for FL showed no significant differences (p>0.05) between the two groups. CONCLUSION: According to the results of this study, it is reasonable to assert that EOFS instruments showed a higher torsional resistance if compared to the WOGS. CLINICAL SIGNIFICANCE: As evidenced by this study, EOFS should be considered as a safer solution, in terms of torsional resistance, if compared to WOGS, reducing the risk of intracanal separation due to excessive torsional load.

Angle of Insertion and Torsional Resistance of Nickel-Titanium Rotary Instruments.

Previously published studies have investigated the influence of instrument access on cyclic fatigue resistance. However, no studies have evaluated the relationship between angulated access and torsional resistance. The aim of this study was to investigate the influence of the angle of access on the torsional resistance of endodontic instruments. One hundred and eighty instruments were selected: 90 F-One Blue 25/04 and 90 HeroShaper 25/04 instruments. Three subgroups (n = 30) for each instrument type (A and B) were established according to the angle of insertion of the instruments inside the artificial canal (0°, 10° and 20°). The tests were performed using a custom-made device consisting of the following: a motor that can record torque values of 0.1 s; interchangeable stainless-steel canals with different curvature (0°, 10° and 20°) that allow the instrument's angulated insertion and keep it flexed during testing procedures; and a vise used to secure the instrument at 3 mm from the tip. Torque limit was set to 5.5 Ncm, and each instrument was rotated at 500 rpm until fracture occurred. Torque to fracture (TtF) was registered by the endodontic motor, and the fragment length (FL) was measured with a digital caliper. Fractographic analysis was performed using a scanning electron microscopy (SEM) evaluation to confirm the cause of failure. TtF values and fragment length (FL) values were statistically analyzed using one-way analysis of variance (ANOVA) test and the Bonferroni correction for multiple comparisons across the groups with significance set to a 95% confidence level. Regarding the F-One Blue instruments, the results showed a higher TtF for group A3 (20°) than for group A1 (0°) and group A2 (10°), with a statistically significant difference between group A3 and the other two groups (p < 0.05), whereas no statistically significant difference was found between group A1 and group A2 (p > 0.05). Regarding the HeroShaper instrument, the results showed the highest TtF for group B3, with a statistically significant difference between the three subgroups B1, B2 and B3 (p < 0.05). The results showed that the torsional resistance increases as the angle of instrument access increases with a varying intensity, according to the crystallographic phase of the instrument selected.

Biocompatibility and Antibiofilm Properties of Calcium Silicate-Based Cements: An In Vitro Evaluation and Report of Two Clinical Cases.

Calcium silicate-based cements have reached excellent levels of performance in endodontics, providing predictable and successful results. To better assess the properties of these bioactive materials, the present study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured on ProRoot MTA and Biodentine samples or in the presence of both cement extracts. Cell viability assay, measurement of reactive oxygen species (ROS), immunofluorescence analysis, as well as morphological evaluations were conducted. Moreover, Streptococcus mutans was used to assess the biofilm forming ability on ProRoot MTA and Biodentine disks. Finally, both cements were applied in vivo to treat immature permanent teeth affected by reversible pulpitis. Results: Cell viability assay demonstrated that Saos-2 cells had a dose- and time-dependent cytotoxicity to both analyzed cements, although cells exposed to ProRoot MTA showed a better cell vitality than those exposed to Biodentine (p < 0.001). Both cements demonstrated ROS production while this was greater in the case of Biodentine than ProRoot MTA (p < 0.001). Immunofluorescence images of the cytoskeleton and focal adhesions showed no differences in Saos-2 cells grown in the presence of ProRoot MTA eluate; whereas in the Biodentine groups, cells showed a morphology and focal adhesions more similar to that of the control sample, as the eluate concentration decreased. Morphological analysis revealed that Saos-2 cells were more flattened and exhibited better spreading when attached to ProRoot MTA disks than to Biodentine ones. The antibiofilm properties showed a time-dependent powerful inhibition of S. mutans superficial colonization and an antibiofilm effect of both cements. Clinically, complete root formation of the treated elements was achieved using the two studied cements, showing stable results over time. ProRoot MTA and Biodentine was demonstrated to be biocompatible and to possess antibiofilm properties. Their clinical application in vital pulp therapy provided successful outcomes after 2 years of follow-up.

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons.

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

In Vitro Evaluation of a Recently Developed Rotary File: AF Rotary / Avaliação in vitro de um arquivo rotativo desenvolvido recentemente: AF Rotary

Objective: Aim of the present study is to evaluate mechanical properties of a newly developed rotary file, AF Rotary (Fanta Dental, Shanghai, China), and to compare these features with a worldwide spread rotary file, Protaper Gold F2 (PTG F2; Dentsply Maillefer, Ballaigues, Switzerland). Material and Methods: 60 AF Rotary 25.06 and 60 PTG F2 were divided in three groups of 20 files each. A group underwent to cyclic fatigue resistance tests, second group underwent to torsional resistance tests, third group underwent to bending tests. The statistical analysis was performed using t student test with significance level set at 95% (p < 0.05). Results: AF Rotary showed significantly higher torsional resistance and cyclic fatigue resistance (p < 0.05) when compared to the PTG F2. AF Rotary also showed significantly lower bending resistance (p < 0.05) when compared to the PTG F2. Conclusion: Therefore, within the limitations of this study, the results show better performances for AF Rotary compared to PTG F2 regarding the cyclic fatigue test, the torsional test and the bending test. Since the PTG F2 is a commonly used and widely investigated instrument it could be possible to state that the AF Rotary exhibits remarkable in vitro performances. (AU)

Objetivo: O objetivo do presente estudo é avaliar as propriedades mecânicas de uma lima rotativa recém-desenvolvida, AF Rotary (Fanta Dental, Shanghai, China), e comparar essas características com uma lima rotativa mundialmente difundida, Protaper Gold F2 (PTG F2; Dentsply Maillefer, Ballaigues, Suíça). Material e Métodos: 60 AF Rotary 25.06 e 60 PTG F2 foram divididos em três grupos de 20 arquivos cada. Um grupo foi submetido a testes de resistência à fadiga cíclica, o segundo grupo foi submetido a testes de resistência à torção, o terceiro grupo foi submetido a testes de flexão. A análise estatística foi realizada por meio do teste t student com nível de significância de 95% (p <0,05). Resultados: AF Rotary apresentou resistência torcional e resistência à fadiga cíclica significativamente maiores (p <0,05) quando comparado ao PTG F2. AF Rotary também apresentou resistência à flexão significativamente menor (p <0,05) quando comparado ao PTG F2. Conclusão: Portanto, dentro das limitações deste estudo, os resultados mostram melhores desempenhos do AF Rotary em relação ao PTG F2 no que diz respeito ao teste de fadiga cíclica, ao teste de torção e ao teste de flexão. Uma vez que o PTG F2 é um instrumento comumente usado e amplamente investigado, pode-se afirmar que o AF Rotary exibe desempenhos in vitro notáveis. (AU)

Essential Oils Biofilm Modulation Activity, Chemical and Machine Learning Analysis. Application on Staphylococcus aureus Isolates from Cystic Fibrosis Patients.

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus (S. aureus) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and "green alternatives" to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients' isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed different biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies.

The effect of low and ultra-low oxygen tensions on mammalian embryo culture and development in experimental and clinical IVF.

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O2) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O2 (20%) affects in vitro embryo development and intracellular redox balance, the use of low (5% O2, physiologic) and ultra-low (close or less to 5% O2) O2 applied to in vitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O2, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O2 in embryo culture, and examines the impact of multiple factors on IVF outcomes. ABBREVIATIONS: O2: oxygen tension; IVF: in vitro fertilization; IVC: in vitro culture; ET: embryo transfer; ROS: reactive oxygen species; ARTs: assisted reproductive technologies.

SEM BSE 3D Image Analysis of Human Incus Bone Affected by Cholesteatoma Ascribes to Osteoclasts the Bone Erosion and VpSEM dEDX Analysis Reveals New Bone Formation.

Bone erosion is considered a typical characteristic of advanced or complicated cholesteatoma (CHO), although it is still a matter of debate if bone erosion is due to osteoclast action, being the specific literature controversial. The purpose of this study was to apply a novel scanning characterization approach, the BSE 3D image analysis, to study the pathological erosion on the surface of human incus bone involved by CHO, in order to definitely assess the eventual osteoclastic resorptive action. To do this, a comparison of BSE 3D image of resorption lacunae (resorption pits) from osteoporotic human femur neck (indubitably of osteoclastic origin) with that of the incus was performed. Surface parameters (area, mean depth, and volume) were calculated by the software Hitachi MountainsMap© from BSE 3D-reconstructed images; results were then statistically analyzed by SPSS statistical software. Our findings showed that no significant differences exist between the two groups. This quantitative approach implements the morphological characterization, allowing us to state that surface erosion of the incus is due to osteoclast action. Moreover, our observation and processing image workflow are the first in the literature showing the presence not only of bone erosion but also of matrix vesicles releasing their content on collagen bundles and self-immuring osteocytes, all markers of new bone formation on incus bone surface. On the basis of recent literature, it has been hypothesized that inflammatory environment induced by CHO may trigger the osteoclast activity, eliciting bone erosion. The observed new bone formation probably takes place at a slower rate in respect to the normal bone turnover, and the process is uncoupled (as recently demonstrated for several inflammatory diseases that promote bone loss) thus resulting in an overall bone loss. Novel scanning characterization approaches used in this study allowed for the first time the 3D imaging of incus bone erosion and its quantitative measurement, opening a new era of quantitative SEM morphology.

Differences in cyclic fatigue lifespan between two different heat treated NiTi endodontic rotary instruments: WaveOne Gold vs EdgeOne Fire.

BACKGROUND: Aim of this study is to investigate the cyclic fatigue resistance of the Gold treated WaveOne Gold and the Firewire treated EdgeOne Fire instruments. The null hypotesis was that there were no differences between the lifespan of Gold treated and FireWire treated instruments when subjected to cyclic fatigue tests. MATERIAL AND METHODS: 40 new NiTi instruments with a length of 25 mm were tested: 20 Wave One Gold Medium (WOG), tip size 35 and variable taper (Dentsply Maillefer, Ballaigues, Switzerland) and 20 Edge One Fire (EOF) (EdgeEndo, Albuquerque, New Mexico) tip size 35 and the same variable taper. A mobile support for the electric handpiece and a stainless-steel block containing the artificial canals were used. The same artificial root canal with a 90 degrees angle of curvature and 2 mm radius of curvature was used for all the tested instruments and the WOG counter-clock wise reciprocating motion with an engaging angle of 150° and a disengaging angle of 30° at 300 rpm, was selected for the test. All instruments were inserted at the same length (18mm) and then rotated in the same reciprocating motion until fracture occurred: the time was stopped as soon as the fracture was visible and video-recorded with a 1/100 sec chronometer. Differences among groups were statistically evaluated with an analysis of variance test ANOVA (significance level was set at p<0.05). RESULTS: Mean values of time to fracture (TtF) for EOF instruments were 28,00 seconds (SD +/- 2,64) and for WOG instruments were 14,67 seconds (SD +/- 2,41). Statistical analysis found significant differences between the TtF of the two instruments (p<0,05). CONCLUSIONS: Firewire instruments resulted to be about two times more resistant to cyclic fatigue when compared with identical instruments made with Gold treatment. Key words:Endodontics, NiTi, Waveone Gold, EdgeOne Fire, Cyclic Fatigue.

Role of the Flat-Designed Surface in Improving the Cyclic Fatigue Resistance of Endodontic NiTi Rotary Instruments.

The aim of this study was to investigate the role of the flat-designed surface in improving the resistance to cyclic fatigue by comparing heat-treated F-One (Fanta Dental, Shanghai, China) nickel-titanium (NiTi) rotary instruments and similar prototypes, differing only by the absence of the flat side. The null hypothesis was that there were no differences between the two tested instruments in terms of cyclic fatigue lifespan. A total of 40 new NiTi instruments (20 F-One and 20 prototypes) were tested in the present study. The instruments were rotated with the same speed (500 rpm) and torque (2 N) using an endodontic motor (Elements Motor, Kerr, Orange, CA, USA) in the same stainless steel, artificial canal (90° angle of curvature and 5 mm radius). A Wilcoxon-Mann-Whitney test was performed to assess the differences in terms of time to fracture and the length of the fractured segment between the flat- and non-flat-sided instruments. Significance was set at p = 0.05. The differences in terms of time to fracture between non-flat and flat were statistically significant (p < 0.001). In addition, the differences in terms of fractured segment length were statistically significant (p = 0.034). The results of this study highlight the importance of flat-sided design in increasing the cyclic fatigue lifespan of NiTi rotary instruments.