Low-level laser therapy enhances osteogenic differentiation of gingiva-derived stem cells in 2D and 3D cultures

Modulation of cellular activity by low-level laser therapy (LLLT) has been widely studied, particularly in regenerative medicine. This study examined the effects of LLLT on the viability, osteogenic differentiation, and mineralization of gingiva-derived mesenchymal stem cells (GMSCs) in two-(2D) and three-dimensional (3D) cultures. GMSCs were treated with LLLT at 980 nm and 808 nm using different energy densities and irradiation frequencies. Cell viability was assessed using a colorimetric assay and live/dead staining. Osteogenic differentiation was assessed through alkaline phosphatase activity and real-time polymerase chain reaction (RT-PCR) analysis of RUNX2 and COL1A1 mRNA expression. Mineralization was analyzed using Alizarin Red S staining. LLLT enhanced cell viability without inducing significant morphological changes, with more pronounced effects in 3D spheroids than in 2D monolayers. Osteogenic differentiation and mineralization were significantly increased in LLLT-treated groups. Gene expression analyses confirmed the upregulation of key osteogenic markers, reinforcing LLLT's role in promoting osteogenesis. These findings indicate that LLLT is a non-invasive and effective approach to promoting osteogenic differentiation and mineralization. Notably, its effects were more evident in 3D culture systems, which better mimic in vivo conditions. This study highlights the therapeutic potential of LLLT in tissue engineering and regenerative medicine, emphasizing its applications in bone regeneration.