Assessment of the influence of 660 and 808-nm PBM treatments on mitochondrial oxygen consumption of MG-63 osteoblast: a 3D cell culture study

Abstract

This study explores the dose-dependent effects of 660-nm and 808-nm photobiomodulation (PBM) on mitochondrial oxygen respiration rate activity in MG-63 osteoblast cells using an innovative 3D in vitro spheroid model. MG-63 osteoblast cells were grown to 80% confluence and seeded in fish gelatin hydrogel (LunaGel™) to form 3D spheroids within 3-7 days. Spheroids were seeded on Seahorse microplates and incubated in a LunacrossLinker™ (visible light crosslinking system) for 2 min to give hydrogel a mid-stiffness of 3.5 kPa. Cells were exposed to PBM either 660-nm or 808-nm at panel setting of 5 J/cm² and 15 J/cm² and then assessed immediate (15 min before analysing) and 24 h time points. Mitochondrial activity was determined using an XFe96 Seahorse analyzer. Data distribution was assessed, and parametric or non-parametric tests and compared the mitochondrial respiratory capacity across different experimental conditions. The study indicated that 660-nm and 808-nm PBM could modulate mitochondrial functions in osteoblasts. The maximal respiratory rate for the fluency assessed at 808-nm wavelength was increased when cells were assessed immediate post. Interestingly, the 660-nm PBM-treated cells showed a decrease in oxygen consumption rate (OCR) at the basal and maximal bioenergetic state at all time points (immediate and 24 h.) and fluency compared to the untreated control. The effects of 660-nm and 808-nm wavelengths on osteoblast mitochondrial function suggest that PBM demonstrates differential modulation of osteoblast metabolism and bioenergetics depending on the wavelength. These findings have practical implications in both research and clinical settings, offering insights into selecting specific wavelengths for therapeutic applications.

Keywords: Bio photonics; Cell signalling; Lasers; Molecular biology; Osteoblasts; Photo initiators.

Fig. 1

(A) Schematic flowchart for the isolated MG-63 and ATP assay using the Seahorse XFe96 Analyser. The real-time analyzer measures metabolic responses of MG-63 to modulators of Mito Stress Assay (MST). Basal OCR and extracellular acidification rates / proton production rates (PPR) were calculated using 2-minute mix and 4-minute measure cycles. Cycles of Oligomycin A with cycles of 4 as penetration within the spheroid to allow an observed effect. (B) Agilent Seahorse XFe96 Spheroid Microplate design showing space for cut steel straw to block wells

Fig. 2

Shows the results of viability testing showing viability of cell in hydrogel and with laser

Fig. 3

(A) Basal Respiration and (B) Maximal Respiration showing oxygen consumption rate (OCR) kinetics increased post-photobiomodulation, indicating trend for improved metabolism after 808-nm wavelength immediate and 24-hour treated cells at 5 J/cm² and 15 J/cm². The 660-nm PBM group showed a decrease in the immediate and 24-hour treated cells at 5 J/cm² and 15 J/cm². (sample size n = 9–10 and statistical significance for each result)