Fig. 7.

Investigation of the potential molecular mechanisms of the regulatory effects of GO/Ga nanocomposites on osteoblastogenesis and osteoclastogenesis in MC3T3-E1 and RAW 264.7 cells. (a) Expression features of BMP/SMAD signaling molecules during the osteoblast differentiation of MC3T3-E1 cells after coculture with different nanomaterials for 7 d. (b) Expression features of RANKL-stimulated MAPK signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. (c) Expression features of RANKL-stimulated NF-κB signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. The expression levels of NFATc1 and c-Jun were also confirmed after coculture for 3 d with the stimulation of RANKL (50 ng/mL). Overall, the relative expression levels of p-Smad 1/5, p-JNK, p-P38, p-ERK, p-IkBα, p-P65, NFATc1 and c-Jun were calculated and normalized by β-actin in terms of the gray band intensities as confirmed by ImageJ software. (d) Luciferase reporter gene assessment of BMP-2 and NFATc1 in MC3T3-E1 and RAW264.7 cells, respectively. Stably transfected cells were treated with different nanomaterials for 6 h with specific stimulation, and the luciferase activities were confirmed by a Pierce™ Gaussia Luminescence Assay kit. (e) Molecular docking of Ga³⁺ with JNK/P38 kinases as demonstrated in binding mode figures using PyMOL visualization software. (f) Schematic diagram of the regulatory mechanisms of GO/Ga nanocomposites involved in osteoblastogenesis and osteoclastogenesis (Created with BioRender.com). *p < 0.01 compared with CTRL and GaNPs, **p < 0.05 and ***p < 0.01 compared with CTRL, ^#p < 0.01 compared with GO, ^##p < 0.05 compared with GO.