| Summary: | 碩士 === 中原大學 === 醫學工程研究所 === 90 === Osteoporosis occurs when the activity of bone resorption is larger than the activity of bone formation. The osteoclast is one of the most direct and powerful factors, which is involved in bone resorption. Our lab have been proved that pulsed electromagnetic fields (PEMFs) with specific parameters influence the inhibition of osteoclast differentiation and formation, and this study investigates the effect of PEMF on osteoclast apoptosis. First, a novel coculture system is established to develop osteoclast-like cells (OCLs), which are exposed to PEMFs for different stimulation times, and OCL apoptosis rates are assayed at different times after PEMF exposure. We expect that PEMFs have effects on treatment or prevention of osteoporosis by inducing osteoclast apoptosis.
In this study, bone marrow cells (BMCs) obtained from 10~12 -month-old adult female Wistar rats were the major cells in this coculture system. Osteoblasts (OB) were obtained from new-born ICR mice and new-born Wistar rats, and were individually cocultured with BMCs. OB density was 1x106 cells/6-cm dish, and BMC densities were 2x107, 4x107, and 8x107 cells/6-cm dish, giving a total 6 of types of coculture models. 10 nM 1α, 25(OH)2D3 was added in culture medium in coculture periods to enhance OCL differentiation. On the eighth day, purified OCLs were acquired after isolation by 0.15% collagenase, and isolated OCLs were exposed to PEMFs with different stimulation times. There were four groups: Control, PEMF-1 (exposure for 1 hr ), PEMF-8 (exposure for 8 hr), and PEMF-16 (exposure for 16 hr). The PEMF parameters were: single pulse, 7.5 Hz of frequency, and 2 mV/cm of induced electric field intensity.
Apoptosis rates of OCLs were assayed by PI-stain method individually at 0, 24, 48, and 72 hr after PEMF exposure, where the time point after isolation was defined as 0 hr.
For qualitative analysis of OCLs there were two processes. In one, Tartrate-resistant acid phosphatase (TRAP) was used as a marker of acid phosphatase of OCLs, and TRAP-positive multinucleated (more than 3 nuclei) cells (TRAP-PMCs) were identified as OCLs. In the other, bone resorption pits on the surface of porcine cortical bone slices formed by OCLs were examined by scanning electron microscope (SEM).
The results showed that: (1) Numerous OCLs (TRAP-PMCs) were observed in an optimal coculture system established by OB (1x106 cells/6-cm dish) of new-born mice and BMCs (2x107 cells/6-cm dish) of adult female Wistar rats. However, the other five coculture models failed to develop TRAP-PMCs. Considering the uniformity of PEMFs, a 3.5-cm dish was substituted for the 6-cm dish and the optimal cell densities of OB and BMCs subsequently change into 4x105 and 2x107 respectively, and numerous TRAP-PMCs still appeared in this coculture system. (2) The results of apoptosis assay revealed that the rate of apoptotic OCLs increased when the apoptosis assay time increased, reaching the highest rate of 98%. Nuclear fragments of apoptotic OCLs appeared clearly after a longer time period after isolation. Compared with PEMF-1 and Control groups, the rates of apoptotic OCLs between 0~72 hr were similar. Compared with Control, the rate of apoptotic OCLs in PEMF-8 and PEMF-16 were both higher at 48 hr of apoptosis assay time points, and others were similar at 0, 24, and 72 hr of apoptosis assay time points.
It suggested that both PEMF stimulation time and apoptosis assay time points are important roles in inducing OCL apoptosis by PEMFs with specific parameters. The rate of apoptotic OCLs increased at 48 hr after exposure to PEMFs for 8 and 16 hr.
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