FIGURE 1.

Illustration of the three-dimensional culture system. A, the BM cells on the ST2 cell layer formed on the collagen gel (Gel) containing the MLO-Y4 cells were co-cultured. A combination of Dex (10 nm) and 1,25D₃ (10 nm) was added to the culture for the induction of RANKL-dependent osteoclastogenesis. B, effect of the MLO-Y4 cells in the gel on osteoclastogenesis. The indicated number of MLO-Y4 cells were seeded into the three-dimensional culture system. At the end of the culture, osteoclastogenesis was evaluated based on the relative TRAP activity in the culture medium. The data are expressed as the mean ± S.E. (n = 6). *, p < 0.01 versus MLO-Y4 (−) 1,25D₃ (+) group. Microphotographs of TRAP⁺ cells on gels containing the indicated number of MLO-Y4 cells are shown. Bar, 100 μm. C, effects of UV light exposure on MLO-Y4 cell viability and RANKL mRNA expression. MLO-Y4 cells in the collagen gel solution were exposed to UV light for the indicated time and culture for 2 days. Cell viability (left panel) and RANKL mRNA expression (right panel) were evaluated by Cell Counting kit-8 and real-time RT-PCR, respectively. The data are expressed as the mean ± S.E. (n = 6). *, p < 0.01 versus MLO-Y4 (+) UV light exposure (−) group (left panel) and as the mean ± S.E. (n = 3). *, p < 0.01 versus 125D₃ (+) UV light exposure (−) group (right panel). D, effect of MLO-Y4 cell death induced by UV light exposure on osteoclastogenesis. MLO-Y4 cells exposed to UV light were used in the three-dimensional culture system. Osteoclastogenesis was evaluated based on the TRAP activity. The data are expressed as the mean ± S.E. (n = 6). *, p < 0.05 versus MLO-Y4 (+) UV light exposure (−) group. Microphotographs of TRAP⁺ cells on gels containing MLO-Y4 cells exposed to UV light for the indicated times are shown. Bar, 100 μm. E, effect of the MLO-Y4 cells in the gel on osteoclastogenesis in the absence of the ST2 cell layer. The indicated number of MLO-Y4 cells was seeded into the gel, and BM cells were seeded directly onto the gel. The osteoclastogenesis was induced by Dex and 1,25D₃. Osteoclastogenesis was evaluated based on the relative TRAP activity in the culture medium. The data are expressed as the mean ± S.E. (n = 6).