Fig. 6.

Effect of rLOX-PP treatment of BMSCs on osteoclast number and fusion (a-d), and assessment of apoptosis (e-g): Bone marrow cells were induced with M-CSF (50 ng/mL) RANKL, (30 ng/mL) in the presence of either 550 nM (10 μg) rLOX- or 550 nM (8 μg) lysozyme. Culture media were changed every other day and rM-CSF and rRANKL were added to the growth medium throughout the experiment. The cells were grown for either 3, 5 or 7 days after initiation of rLOX-PP treatment. This was followed by TRAP staining of cell layers. Digital images of 20–36 randomly selected non-overlapping areas of each well were taken for analyses of TRAP staining with inverted microscope at 100X magnification. The total number of osteoclasts per well in each treatment group was calculated. The numbers of nuclei in each osteoclast were counted and the data were divided into 6 groups. The percentage of cells falling into each category was calculated and compared among different treatment conditions (a-d). White bars, cells treated with M-CSF and RANKL only; black bars, cells treated with M-CSF, RANKL and rLOX-PP; gray bars, cells treated with M-CSF, RANKL, and lysozyme. Data represent mean ± S.D., n = 3, ^*p˂0.05. (e-g) Bone marrow cells were grown under conditions to promote osteoclast development in the presence or absence of rLOX-PP or lysozyme as in (A-D). Caspase-3 activity was measured in cell lysates. Data are means ±S.D., n = 3, ^*p˂0.05 compared to no treatment controls