IADR Abstract Archives
Effects of Blue Light on Oxidative Stress Responses in Gingival Fibroblasts
Objectives: Photobiomodulation describes the application of light to influence cellular responses. UV-free blue light has been assessed as a potential periodontal disease therapy, due to its bactericidal effects on periopathogenic bacteria. However, few studies to date have elucidated the effects of UV-free blue light on cells of the periodontium. Therefore, this study investigated the effects of UV-free blue light irradiation on the viability and oxidative stress responses in gingival fibroblasts.
Methods: Primary human gingival fibroblasts were cultured in collagen type 1 gels (1mg/mL) and serum starved for 24h, prior to irradiation. Irradiation doses ranged from 3-90J/cm2 generated from combinations of radiant power (mW) and treatment duration. Collagen gels were digested with collagenase A (2mg/mL) to release gingival fibroblasts, prior to RNA extraction and cell counting. Cell cultures were assayed immediately (superoxide radical generation via fluorescent imaging) or 24h post-irradiation (lactate dehydrogenase release; caspase-3 activity; metabolic activity; proliferation; morphology). Enzymic antioxidant (SOD1, SOD2, CAT, NRF2, KEAP1) expression were quantified by RT-qPCR.
Results: Irradiation doses >9J/cm2 induced a significant dose-dependant reduction in metabolic activity and modulated superoxide radical generation. LDH release was significantly elevated, while caspase-3 activity was significantly reduced, at irradiation doses >60J/cm2. Changes in cell morphology and membrane disruption were also present at irradiation doses >36J/cm2. Cell counts revealed a dose-dependent reduction in fibroblast viability and total cell number, compared to untreated controls. Differential antioxidant gene expression was shown in treated samples (>3J/cm2).
Conclusions: There is clear evidence of metabolic alterations in human gingival fibroblasts irradiated with UV-free blue light, as a likely consequence of elevated oxidative stress. These observations are most apparent at irradiation doses >9J/cm2. As expected, high doses (>36J/cm2) induced potent cytotoxic responses and support a negative correlation between cell health and irradiation doses. As doses <30J/cm2 induced limited adverse effects, these doses should be investigated further as potential periodontal therapies.
Methods: Primary human gingival fibroblasts were cultured in collagen type 1 gels (1mg/mL) and serum starved for 24h, prior to irradiation. Irradiation doses ranged from 3-90J/cm2 generated from combinations of radiant power (mW) and treatment duration. Collagen gels were digested with collagenase A (2mg/mL) to release gingival fibroblasts, prior to RNA extraction and cell counting. Cell cultures were assayed immediately (superoxide radical generation via fluorescent imaging) or 24h post-irradiation (lactate dehydrogenase release; caspase-3 activity; metabolic activity; proliferation; morphology). Enzymic antioxidant (SOD1, SOD2, CAT, NRF2, KEAP1) expression were quantified by RT-qPCR.
Results: Irradiation doses >9J/cm2 induced a significant dose-dependant reduction in metabolic activity and modulated superoxide radical generation. LDH release was significantly elevated, while caspase-3 activity was significantly reduced, at irradiation doses >60J/cm2. Changes in cell morphology and membrane disruption were also present at irradiation doses >36J/cm2. Cell counts revealed a dose-dependent reduction in fibroblast viability and total cell number, compared to untreated controls. Differential antioxidant gene expression was shown in treated samples (>3J/cm2).
Conclusions: There is clear evidence of metabolic alterations in human gingival fibroblasts irradiated with UV-free blue light, as a likely consequence of elevated oxidative stress. These observations are most apparent at irradiation doses >9J/cm2. As expected, high doses (>36J/cm2) induced potent cytotoxic responses and support a negative correlation between cell health and irradiation doses. As doses <30J/cm2 induced limited adverse effects, these doses should be investigated further as potential periodontal therapies.