Pulse frequency dependency of photobiomodulation on osteogenic differentiation of human dental pulp stem cells

Abstract

Photobiomodulation (PBM) therapy contributes to pain relief, wound healing, and tissue regeneration. The pulsed wave (PW) mode has been reported to be more effective than the continuous wave (CW) mode when applying PBM to many biological systems. However, the reason for the higher effectiveness of PW-PBM is poorly understood. Herein, it suggest using delayed luminescence (DL) as a reporter of mitochondrial activity after PBM treatment. DL originates mainly from mitochondrial electron transport chain systems, which produce reactive oxygen species (ROS) and adenosine triphosphate (ATP). The decay time of DL depends on the pulse frequencies of applied light, which correlate with the biological responses of human dental pulp stem cells (hDPSCs). Using a low-power light whose wavelength is 810 nm and energy density is 38 mJ/cm2, it find that a 300-Hz pulse frequency prolonged the DL pattern and enhanced alkaline phosphatase activity. In addition, it analyze mitochondrial morphological changes and their volume density and find evidence supporting mitochondrial physiological changes from PBM treatment. Our data suggest a new methodology for determining the effectiveness of PBM and the specific pulse frequency dependency of PBM in the differentiation of hDPSCs. In addition, duty cycle and pulse frequency of PW were empirically verified based-on-expanded biology experiment. To do such this, it applied 810 nm LPL of 128 W/cm2 energy density in vitro. Upon this value, CW irradiation did not induce any significant changes for differentiation of hDPSCs. However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the PW with 30 % of duty cycle and 300-3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency. Meanwhile, together with NIR, blue light has been known to induce differentiation of stem cells. In order to enhance the effectiveness of PBM the blue light is incorporated into NIR-PBM. PBM has almost been used in the region of red or near infrared (NIR) whereas the blue light does not frequently due to inducing disrupt cellular processes. Photoacceptor for NIR is cytochrome c oxidase and is Flavin for blue light in the mitochondrial electron transport chain system. The delayed luminescence (DL), in which radiate light after expose light to cells, originate from cytochrome c oxidase in the region of NIR and from flavins in the region of blue light. Correlation between PBM and NIR and blue light-induced DL for hDPSCs was employed to investigate an optimal excitation to differentiate the cells. The DL showed that the NIR with blue-light-pre-irradiation exerted better excitation than the others (only NIR or blue light) in pulsed wave mode that their energy density was 89 mJ/cm2. In blocking complex I with rotenone, the DL was all decreased in excitation for NIR, blue light and blue-then-NIR light, while Antimycin A-bloocked-complex III DL was preserved for only NIR. It suggests that photoacceptor of blue light may be in complex I and III simultaneously whereas in complex III for NIR. As for differentiation of hDPSCs, it showed that alkaline phosphatase (ALP) activity was highly expressed for blue-then-NIR light. ROS and RT-PCR data support the result of ALP activity. Based on the results in vitro, PBM was applied to Beagles teeth in order to verify formation of dentin in vivo. A dentin formation was produced to be up to 20 % increment compared to the control that was not exposed to NIR and also induced dense periodontal ligament (PDL) after 8 weeks of exposure of NIR to teeth. It was observed that odontoblast subjected to NIR light exposure was active, generating new tubular dentin along with older dentin. Notably, such active odontoblast was not observed in controls, even though odontoblast-like layer whereby dentin structure was there in SEM analysis. It suggests that such a strategy might facilitate treatments relating to the diseases of dentin and PDL in mature permanent teeth. In summary, PW-PBM was more effective than CW-PWM in differentiating hDPSCs into osteogenesis. Duty cycle 30% and pulse frequency 300 Hz was effective among PW-frequencies. Blue-light-incoporated NIR induced much more differentiation than when only NIR applied. Beagle test verified dentin formation by means of PW-PBM.