Photobiomodulation therapy at red and near-infrared wavelengths for osteogenic differentiation in the scaffold-free microtissues

One of the novel strategies for bone tissue regeneration is photobiomodulation (PBM) which depends on the red and near-infrared light absorption by mitochondria and may trigger bone tissue regeneration via the production of intracellular ROS and ATP, NO release, etc. It is also important to identify the changes in those signal molecule levels in an in vivo mimicking platform such as 3-Dimensional (3D) Scaffold Free Microtissues (SFMs) that may serve more natural osteogenic differentiation responses to PBM. Herein, we aimed to increase the osteogenic differentiation capability of the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVECs) on 3D SFMs by triple light treatment at 655 and 808-nm of wavelengths with the energy densities of 1, 3, and 5 J/cm2. We performed the analysis of cell viability, diameter measurements of SFMs, intracellular ROS production, NO release, ATP activity, temperature measurements, DNA content, ALPase activity, calcium content, and relative gene expressions of ALP, Collagen, and Osteopontin by qRT-PCR. It was found that both wavelengths were effective in terms of the viability of SFMs. 1 and 5 J/cm2 energy densities of both wavelengths increased the SFM diameter with significant changes in intracellular ROS, ATP, and NO levels compared to the control group. We concluded that PBM therapy was successful to induce osteogenesis. 1 J/cm2 at 655 nm of wavelength and 5 J/cm2 at 808 nm of wavelength were the most effective energy densities for osteogenic differentiation on SFMs with triple light treatment.