Porphyrins and phototherapy of oral bacteria

Abstract: In the mid-1990s researchers became interesting in blue light-based therapies using light- emitting diodes (LED) light. Blue light therapy has been successfully used in treatment of acne, as well as shown wound healing effects. It has also shown significant antibacterial effects, especially on black-pigmented bacteria. Recent studies have shown that a toothbrush equipped with blue light LED emitting in the 412 nm region can reduce plaque and gingival inflammation up to 50 % after 4 weeks of intervention. According to the World Health Organization gingivitis is common in children and adolescents worldwide, and 5–20% of the adult population have periodontal destructive disease. Persistent gingival inflammation has been reported to be a risk factor for developing periodontitis. Thus, it is of great interest to prevent and treat gingival inflammation. Studies have shown that 85% of adults in Sweden brush their teeth at least twice a day, but it has also been reported that the majority don’t use a proper technique or have a low motivation for keeping a sufficiently good oral health. In this context there is a need to emphasize the importance of repeated oral hygiene instructions and to strengthen the motivation for maintaining oral health. This thesis is based on four papers. In Paper I it is demonstrated that the periopathogen Aggregatibacter actinomycetemcomitans on its own is able to produce red fluorescence. According to the literature, bacteria that are able to emit red fluorescence can be killed by phototherapy due to their endogenous porphyrins which raised an interest to investigate if A. actinomycetemcomitans could be killed by blue light. In an in vitro pilot study it was demonstrated that 410 nm blue light could inhibit the growth of A. actinomycetemcomitans. In Paper II a new sensitive method using high performance liquid chromatography - tandem mass spectrometry (LC/MS/MS) was developed for chemical analysis of porphyrins in microorganisms and it was used to identify and quantify porphyrins in the oral pathogens A. actinomycetemcomitans and Porphyromonas gingivalis, as well as in the fungi Saccharomyces cerevisiae. In Paper III chemical porphyrin profiles of A. actinomycetemcomitans and P. gingivalis are determined and their changes during bacterial growth investigated. It was shown that the porphyrin content changes drastically during cultivation and thus points out the need for standardized culturing protocols when performing phototherapy experiments in-vitro. In Paper IV a randomized controlled trial was conducted with the aim to investigate the phototherapeutic effect on dental plaque and gingival inflammation of toothbrushes with incorporated 450 nm LEDs. In both the intervention and the control groups there were significant reductions in plaque and gingival inflammation (p<0.001). There was a larger decrease in all three clinical indices for the blue light intervention group when compared to the control where the amount of plaque was reduced by 62% and 51% respectively. This difference was established at a level of p=0.058, and there was no significant difference observed in the GI and BOP indices (p>0.29) between the blue light group and the control group. For the blue light intervention groups there were significant decrease in all four inflammatory markers used in the data evaluation, i.e. in IL-1β and IL-8 in GCF, as well as in MMP-8 and TIMP-1 in saliva. This is in contrast to the control group which only showed a statistical significant decrease in the concentration of MMP-8. These differences in clinical indices as well as in inflammatory markers suggest that there could be a weak effect from the 450 nm LED illumination. However, the conclusion must be that at a level of p=0.05 there was no significant adjunctive effect to tooth brushing found for the 450 nm LED. Combined with what is stated in the literature a suggestion is to use toothbrushes equipped with 405 nm LED for further studies.