Figure 5

Effects of anti-PTX3 antibody on human osteoblast primary cultures. (a) Graph displays the percentage of proliferating osteoblasts in treated and control primary cultures (Ig 0.1 μg/ml versus PTX3 0.1 μg/ml P<0.0001; Ig 1 μg/ml versus PTX3 1 μg/ml P<0.001). (b) Table showed the main characteristics of primary osteoblast cultures after 72 h of treatment with PTX3 antibody (cellular cluster IgG 0.1 μg/ml versus PTX3 0.1 μg/ml *P=0.0048; IgG 1 μg/ml versus PTX3 1 μg/ml P=0.0501; HA microcrystals IgG 0.1 μg/ml versus PTX3 0.1 μg/ml **P=0.0017; IgG 1 μg/ml versus PTX3 1 μg/ml **P=0.0035; RANKL IgG 0.1 μg/ml versus PTX3 0.1 μg/ml *P=0.0354; IgG 1 μg/ml versus PTX3 1 μg/ml *P=0.0317; RUNX2 IgG 0.1 μg/ml versus PTX3 0.1 μg/ml *P=0.0330; IgG 1 μg/ml versus PTX3 1 μg/ml *P=0.0418). Well-differentiated osteoblasts forming numerous cellular cluster (arrows) in control cultures (1 μg/ml rabbit IgG; 72 h). Scale bar represents 200 μm for panel (c) image and 100 μm for panel (d) image. (e) SEM-EDX analysis showed the presence of well-differentiated osteoblast and micro-HA calcifications (arrows) in cell cultures treated with 1 μg/ml rabbit IgG (72 h). Scale bar represents 30 μm. (f and g) After 72 h, the treatment with 1 μg/ml of anti-PTX3 antibody induced a strong reduction of osteoblasts size and the appearance of cells with fibroblast–mesenchymal characteristics. Scale bar represents 200 μm for panel (f) image and 100 μm for panel (g) image. (h) Electron micrograph shows ultrastructural characteristics of fibroblast–mesenchymal-like cells. Scale bar represents 30 μm. (i) rhPTX3 induce a significant increase of cell proliferation in primary osteoblast cultures derived from OP patients (P=0.0023) (j) rhPTX3 induce a significant increase of HA crystal formation in primary osteoblast cultures derived from OP patients (P<0.001)