Figure 4

Evaluation of PTX3 in bone head biopsies and osteoblast cell cultures. Immunohistochemical results were evaluated by a semiquantitative approach assigning a score from 0 to 3 according to the number of positive osteoblasts (Table 2). (a) Graph shows immunohistochemical results of PTX3 (OP versus OA P=0.0140; OP versus CTRL P<0.0001; OA versus CTRL P<0.0001). (OP n=25, OA n=25, CTRL n=15) (b and c) Graphs displays the correlation between PTX3 score and BV/TV or Tb.Th values for each OP patient. OP patients are numbered in increasing order of BV/TV or Tb.Th values, respectively. (f and g) OA patients showed high expression of PTX3 both in osteoblasts and bone marrow cells. (h and i) CTRL patients showed expression of PTX3 in almost all osteoblasts and bone marrow cells (g, square). Scale bar represents 200 μm for DFH images and 40 μm for EGI images. (j) Graph shows the results of immunostaining of primary confluent osteoblast cultures (OP n=3, OA n=3, CTRL n=3) (k) Graph shows the presence of micro-HA crystals in primary confluent osteoblast cultures (OP n=3, OA n=3, CTRL n=3). (l and m) Dual-colour immunostaining for PTX3 (red) and RUNX2 (green) on confluent osteoblasts culture derived from CTRL patients. (l) Images show numerous RUNX2-positive osteoblast expressing PTX3. Scale bar represents 50 μm. (m) High magnification image of RUNX2-positive osteoblast expressing PTX3. Scale bar represents 10 μm. (n) Representative images of western blotting assay. Western blotting analysis displayed remarkable difference in PTX3 expression among the groups. Table reported the ratio between the densitometric values of PTX3 and β-catenin for each experimental group. (OP n=3, OA n=3, CTRL n=3). (o) mRNA expression level of PTX3 relative to GADPH expression in osteoblast primary cultures derived from OP (0.39±0.22), OA (0.72±0.19) and CTRL (1.00±0.11) patients. (OP n=3, OA n=3, CTRL n=3)