The decrease in bone density occurs by the increase in bone resorption, due to the breakdown in the balance of resorption of bone by osteoclasts dissolving and bone formation by osteoblasts. Osteoporosis is a representative disease caused by the decrease in the bone density. Therefore, osteoclasts are one of the important target cells for the treatment of osteoporosis. Recently, a real-time cellular analysis system (RTCA) was developed to analyze the quantity of cytomorphology and the cell adhesion in vitro by measuring the electronic impedance (Cell Index; CI) of the electrode in a plate. To date, there was no report on the use of RTCA for osteoclast differentiation until our report. Therefore, we thought that RTCA is useful for the screening of therapeutic drugs for osteoporosis. We reported inhibition of osteoclast differentiation by epigallocatechin gallate (EGCG). In this study, we attempted to quantify the inhibitory effect of EGCG for osteoclast differentiation using a new method. We have taken bone marrow cells from femur and tibia bone of male mice (5-8 weeks of age). We cultured bone marrow cells of 1.0 × 10
5 cells/well. We added EGCG to culture media of bone marrow cells in 1µM, 10µM and 100µM with CO
2 incubator and induced osteoclasts. We cultured osteoclasts for 188 hours in total, and exchanged the culture media and added EGCG after 24 and 72 hours. We counted the cells for osteoclasts which appeared as tartrate-resistant acid phosphatase (TRAP)-positive and had three or more nuclei. The multinuclear osteoclast number by TRAP staining and the CI level were inhibited concentration dependent by EGCG. Furthermore, the CI level and the multinuclear osteoclast number had a strong correlation. In addition, we found that 1µM EGCG of low-concentration inhibited the osteoclast differentiation. There is a report that the highest plasma concentration of EGCG becomes 2µM by drinking one cup of green tea (400mg of catechin components). Therefore, it is strongly suggested that EGCG has an osteoporosis prophylaxis effect. RTCA does not require the staining and the measurement is possible sequentially. RTCA is relatively simple and easy, and the screening of drugs for osteoclasts is enabled. It is suggested that this RTCA system might be useful to determine the appropriate level and the site of action of osteoporotic therapeutic drugs.
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