## 4 aPDT Performance Based on In Vitro Studies  
Laboratory outcomes are promising for aPDT against several oral microorganisms, such as _Streptococcus mutans_, _Fusobacterium nucleatum_, _Porphyromonas gingivalis_, and _Prevotella intermedia_[42; 43]. This approach opens the doors to investigate the effectiveness of aPDT against putative endodontic pathogens. Figure 4 shows the clinical protocol of aPDT applied to complement the disinfection of the root canal. The reported literature covers either in vitro studies where the therapy was applied against planktonic cultures of endodontic pathogens strains or monospecies biofilms, or studies where the aPDT effectiveness was assessed against endodontic bacterial strains grown inside root canals of extracted teeth. These in vitro studies explored the potential effect of aPDT against specific putative endodontic pathogens (mainly _E. faecalis_) and provide optimal parameters as a variable controlled in this type of designed study. The in vitro outcomes can pave the way for well-designed clinical studies and help support any discussion on the biological plausibility of aPDT, but they offer a low level of scientific evidence.  
Figure 4: Schematic sequence of the antimicrobial photodynamic therapy clinical protocol applies for root canal disinfection. (**A**) Pre-treatment tooth diagnosis with infected canals. (**B**) Access to root canal system to remove the infective pulpal tissues. (**C**) Chemical debridement and irrigation with 2.5% of sodium hypochlorite (NaOCI). (**D**) Mechanical instrumentation using rotary files to remove the infective radicular dentine. (**E**) A final rinse with 0.9% of saline. (**F**) A photosensitizer applied inside the canal for 1 min. (**G**,**H**) A red light-emitting diode (LED; 660 nm; 200 mW), used to activate the photosensitizer.  
Consequently, one of the pioneers in in vitro studies was conducted in 2002, where _Streptococcus intermedius_ biofilms were grown inside the root canal of extracted teeth for 48 h [44]. Toluidine Blue Ortho (TBO) at concentrations of 12.5, 25, 50, and 100 \(\upmu\)g/mL was used to disinfect the root canal, using different energy doses ranged between 2.1 and 21 J/cm\({}^{2}\). It was found that the concentration of the PS and light dosimetry are determinants concerning the antibacterial reduction. aPDT using TBO was found effective in reducing the _S. intermedius_ biofilm by around 5-log [44].  
Azulene-based paste (25 \(\upmu\)g/mL) as a PS and at an energy dose of 1.8 J/cm\({}^{2}\) was found to inhibit the _E. faecalis_ biofilm [45]. aPDT using chlorine and polyethyleneineineine along with the root canal treatment was more effective than each approach alone in inhibiting the growth of _Proteus mirabilis_ and _Pseudomonas aeruginosa_[46]. The importance of the PS's concentration can be observed in another study where the effectiveness of 6.25 \(\upmu\)g/mL of methylene blue ortho (MB), combined with an energy density of 30 J/cm\({}^{2}\), against _E. faecalis_ biofilm was limited to \(<\) 1-log reduction [47]. However, MB applied at a high concentration of 100 \(\upmu\)g/mL combined with an energy dose of 16.2 J/cm\({}^{2}\) promoted an around 2.5-log reduction [48]. It worth noting that the applied energy density is approximately half of the value in the previous study. This outcome highlights the relevance of PS concentration selection.  
Often, in vitro studies evaluate aPDT against early-grown endodontic biofilms. Early-grown biofilms may not represent the complexity and maturity of biofilms found inside the oral cavity. The lack of laboratory models that mimic the clinical scenarios may not result in reliable outcomes [49]. Therefore, the use of mature biofilms grown for an extended time (proximally 21 days) is recommended [50]. In an earlier investigation, 30-day _E. faecalis_ biofilms grown inside extracted teeth were treated with aPDT [50]. The outcome of the aPDT (MB; 25 \(\upmu\)g/mL) was similar to conventional irrigation [50].