Fig. 6

Sorafenib attenuates the progression of the BMP-induced In vivo Ossification (BIO) model. a, b Representative pictures of capillary-like structures formed on Matrigel (left) of ligament cells overexpressing empty vector plasmids (OE-Ctrl) and LOXL2 plasmids (OE-LOXL2) with the treatment of sorafenib (90 nmol/L and 900 nmol/L) or DMSO (Vehicle); alongside quantification (right). Scale bars = 100 μm. Values are means ± SD (n = 3). ***P < 0.001, *P < 0.005 by ANOVA. c, d Representative reconstructed 3D micro-CT transverse position images depict the ossification site following a 2-week treatment with 50 mg/kg Sorafenib, an LOXL2 enzyme inhibitor [(2-Chloropyridin-4-yl)methanamine hydrochloride] and a methylcellulose carrier alone (Vehicle). Colored bars within the images signify the density of ossified tissue, with higher density indicated at the top and lower density at the bottom. Quantitative analysis of structural parameters related to heterotopic bone formation, including Bone Volume (BV) and Trabecular Thickness (Tb.th), is presented. Scale bar = 2 mm. Data is reported as mean values ± SD (n = 6). Statistical analysis was performed using ANOVA, with triple asterisks indicating high significance (***P < 0.01), and “ns” denoting non-significance. e, f Safranin O-Fast Green (SOFG) staining of sections from the ossification site following a 2-week treatment period, accompanied by the quantification of the cartilage area ratio within the ossified tissue. The scale bar represents 100 μm. Data is presented as means ± SD (n = 6). Statistical analysis was conducted using ANOVA, with triple asterisks indicating high significance (***P < 0.001) and “ns” signifying non-significance. g–j The left panel displays representative images depicting the presence of SP7 and RUNX2 within the ossification site, while the right panel presents quantitative analysis. The scale bar corresponds to 50 μm. Error bars represent mean values ± SD (n = 6). Statistical significance was determined by ANOVA, with ***P < 0.001 and “ns” indicating non-significance. k, l Representative images demonstrate immunofluorescence (IF) staining for EMCN (green) and CD31 (red) within the ossification site, followed by the corresponding quantitative analysis (right). The scale bar represents 50 μm. Data is reported as means ± SD (n = 6). Statistical analysis was performed using ANOVA, with double asterisks indicating significance (**P < 0.01) and “ns” indicating non-significance. All experiments were conducted with three independent biological replicates