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.

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.

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.

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.

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.