Effect of photodynamic therapy and non-thermal plasma on root canal filling: analysis of adhesion and sealer penetration

Abstract Objective The aim of this study was to evaluate the effect of photodynamic therapy (PDT) and non-thermal plasma (NTP) on adhesion and sealer penetration in root canals. Material and Methods Sixty single-rooted premolars were used. The teeth were prepared using a crown-down technique. NaOCl and EDTA were used for irrigation and smear layer removal, respectively. The root canals were divided into three groups: control, PDT, and NTP. After treatments, the roots were filled using gutta-percha and either AH Plus (AHP) or MTA Fillapex (MTAF) sealers. Samples were sectioned at 4, 8, and 12 mm from the apex (1-mm slices)and analyzed by the push-out bond strength test (adhesion) and confocal laser scanning microscopy (sealer penetration). Data were statistically evaluated using Kruskal-Wallis, Dunn’s, and Spearman’s tests. Results Regarding AHP, bond strength was similar in the NTP group and in the control group, but significantly lower in the PDT group. As to MTAF, both therapies showed lower values than the control group. In the confocal analysis of AHP, maximum and mean penetration, and penetrated area were statistically higher in the control group than in the PDT and NTP groups. Penetrated perimeter was similar among groups. Regarding MTAF, all parameters yielded better results in the NTP than in the control group. The PDT and control groups showed similar results except for penetrated area. Conclusion PDT and plasma therapy affected the adhesion and sealer penetration of root canals filled with AH Plus and MTA Fillapex and there is no positive correlation between adhesion and sealer penetration.


Introduction
The basic requirements for root canal treatment are effective chemomechanical preparation and threedimensional obturation of the root canal system 1 . The complexity of the root canal system, with isthmuses, ramifications, and dentinal tubules, makes it impossible to eliminate microorganisms from root canals during preparation 24 . In addition to the routinely used chemical substances and instruments, other technologies have been proposed to promote antimicrobial activity in the root canal system, such as photoactivated disinfection and non-thermal plasma 7,15,20,27,28 . Adhesion and penetration are two important aspects to be considered in sealer selection. Adhesion of an endodontic sealer is defined as its capacity to adhere to root canal walls and promote the union of guttapercha cones to each other and to the dentin 2,26 . Sealer penetration into dentinal tubules is also a required feature, as it can improve the connection between sealer and dentin 13   The gas pressure was kept at 6 bar and 1000 V was applied to generate plasma.
During treatment, the distance between the tip of the plasma jet and the sample was approximately 5 mm. The teeth were exposed to the plasma for 1 min.

Root canal filling
In control and experimental groups (after photodynamic or plasma therapy), all roots were immediately filled with gutta-percha cones (medium, The adhesion area was calculated by using the following formula: where π=3.14, R is the coronal side radius, r is the apical side radius, and h is the thickness of the slice. The thickness of each slice was measured using a digital caliper (Vonder, Curitiba, PR, Brazil) and the coronal and apical radii were measured using a stereoscope (Leika MZ75, Meyer Instruments, Houston, TX, USA) and IM50 software (Leika IM50 Image manager, Houston, TX, USA).

Confocal microscopy
After the push-out test, the remaining gutta-percha was removed and the sections were polished manually with wet 1200-, 2400-and 4000-grit silicon carbide  Figure 1A). Then, the image of the slice captured with the stereoscope was selected ( Figure 1B), creating the final image ( Figure 1C).
To calculate maximum penetration, measurements of the penetration areas were recorded on the slice ( Figure 1D) and maximum penetration was registered (green line) ( Figure 1E). For average penetration depth, four points were selected ( Figure 1F) and sealer penetration was registered (green line) ( Figure   1G). Total perimeter of the root canal ( Figure 1H) was measured and the penetrated perimeter registered (green line) ( Figure 1I). As to the penetrated area, first the total area of the slice ( Figure 1J) was assessed and then the sealer penetrated area was calculated ( Figure 1K) 5,8 .

Statistical analysis
The Kolmogorov-Smirnov test was used to assess the normality of data. Since data were not normally distributed, the Kruskal-Wallis test was used for general comparison and Dunn's test for pairwise comparison.
The Spearman's test was used to correlate data. The significance level was set at 5%.        In the present study, PDT was applied for 90 s.

Results
This period was chosen because it was the minimum period found in the literature that antimicrobial activity was verified by the same parameters employed in the present study 12,19,25 .
A mixture of helium and oxygen (98% He and 2% O 2 ) was applied for 60 s for its antimicrobial properties.
Also, this mixture has non-thermal characteristics acting at room temperature and causing no damage to periapical and periodontal tissues. Additionally, short periods are clinically favorable.

Conclusion
PDT and plasma therapy affected the adhesion and sealer penetration in root canals filled with AH Plus and