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Appl. Sci. 2020, 10, 4290 7 of 9 showed that aPDT with Photoactive+ and 635 nm diode laser showed the greatest reduction in the bacterial count, but when activated by LED (420–480 nm) did not significantly reduce the bacterial count. In addition, our results showed that R1–Blue was better than the Blue–R1 treatment. This can be related to the phenomenon that this complex was more activated by red wavelength rather than blue one. It seemed that that the chlorophyllin part of this mixture had no contribution as an antimicrobial photosensitizer when activated by LED. If a blue laser with a more accurate wavelength were used, it may have had an effect on bacterial reduction. In agreement with our results, Afrasiabi et al. in assessing the efficacy of chlorophyllin–phycocyanin mixture activated by 635 nm diode laser against Streptococcus mutans cultured on enamel slabs concluded that this mixture can reduce the number of living bacteria within the biofilms of S. mutans [22]. Luksiene et al. evaluated the effect of chlorophyllin-Sodium–Copper activated by blue light (400 nm) with power density of 20 mW/cm2 on Bacillus cereus (Gram-positive bacteria) which led to a significant reduction in bacteria [23]. In addition, Caires et al. used chlorophyllin-Sodium–Copper using a homemade LED device at wavelength of 625 nm and power density of 8.3 mW/cm2 during 1 h for inactivation of Staphylococcus aureus and Escherichia coli. The results of their studies showed efficacy for inactivation of Gram-positive bacteria. The results of our study were in line with above-mentioned studies, as it focused on E. faecalis which is a Gram-positive bacterium [24]. Our result showed that chlorophyllin–phycocyanin mixture at concentration of 1000 μg/mL has a very negligible cytotoxicity (p > 0.05) and can be used safely as a natural compound for aPDT. Fimple et al. in assessing the PDT for eradication of polymicrobial infection in endodontic concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized. On the other hand, due to minimally invasive manner of this technique it can be beneficial to be used as an adjunct treatment [25,26]. This study carried out using a standard strain of E. faecalis in in vitro condition which can be considered as a limitation of the study. Anatomic complexity of root structure and morphology, oxygen presence, heterogeneity of clinical isolates of E. faecalis with different behavior and resistance properties can be the reasons which may limit direct extrapolation of this study to embrace clinical conditions. Nonetheless, the irradiation timing values used would find clinical acceptance and it may be possible to extend laser irradiation to assess the degree antimicrobial efficacy. However, the use of aPDT, based on Photoactive+ as adjunctive to conventional chemo-mechanical debridement of infected root canal system provides additional benefits. Further high-quality randomized clinical trials focused on the standardized aPDT parameters and high methodological quality are needed. 5. Conclusions The results of this study demonstrate that the efficacy of aPDT by Photoactive+ is dependent on the light source wavelength with the superiority of red laser compared to the blue light source. It is recommended to use the Photoactive+ as a PS accompanied by red diode lasers or combination of red diode laser first following the blue wavelength. The chlorophyllin–phycocyanin mixture can be applied safely due to very negligible cytotoxicity. Author Contributions: Conceptualization, N.C.; methodology, N.C.; software, A.B.; validation, S.B.; formal analysis, M.P.; investigation, N.C.; resources, A.B.; data curation, A.B.; writing—original draft preparation, N.C.; writing—review and editing, S.P. and S.B; visualization, T.S.; supervision, I.R.B.; project administration, A.B.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported, in part, by the Tehran University of Medical Sciences & Health Services grant No. 97-03-97-40368. Acknowledgments: This research was supported, in part, by Tehran University of Medical Sciences & Health Services grant No. 97-03-97-40368. The publication was supported by funds from the National Research Development Projects to finance excellence (PFE)-37/2018-2020 granted by the Romanian Ministry of Research and Innovation. Conflicts of Interest: The authors declare no conflicts of interest.PDF Image | Antimicrobial Photodynamic Therapy Chlorophyllin–Phycocyanin
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