Synergistic effects of integrin binding peptide (RGD) and photobiomodulation therapies on bone-like microtissues to enhance osteogenic differentiation.

Bone tissue engineering aims to diversify and enhance the strategies for bone regeneration to overcome bone-related health problems. Bone mimetic peptides such as Gly-Arg-Gly-Asp-Ser (RGD) are useful tools for osteogenic differentiation. Similarly, photobiomodulation (PBM) at 600-800 nm of wavelength range improves bone tissue healing via the production of intracellular reactive oxygen species (ROS), ATP synthesis, and nitric oxide (NO) release. Besides, traditional monolayer cell culture models have limited conditions to exhibit the details of a mechanism such as a peptide or PBM therapy. However, scaffold-free microtissues (SFMs) can mimic a tissue more properly and be an efficient way to understand the mechanism of therapy via cell-cell interaction. Thus, the synergistic effects of RGD peptide (1 mM) and PBM applications (1 J/cm2 energy density at 655 nm of wavelength and 5 J/cm2 energy density at 808 nm of wavelength) were evaluated on SFMs formed with the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVEC) for osteogenic differentiation. Cell viability assays, mechanistic analysis, and the evaluation of osteogenic differentiation markers were performed. Combined therapies of RGD and PBM were more successful to induce osteogenic differentiation than single therapies. Especially, RGD + PBM at 655 nm group exhibited a higher capability of osteogenic differentiation via ROS production, ATP synthesis, and NO release. It can be concluded that the concomitant use of RGD and PBM may enhance bone regeneration and become a promising therapeutic tool to heal bone-related problems in clinics.

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.