|Budget Amount *help
¥6,200,000 (Direct Cost : ¥6,200,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥5,300,000 (Direct Cost : ¥5,300,000)
1.Long bones of 10-day-old rabbit were asepticly minced, agitated and supernatant unfractionated bone cells were cultured on collagen gel for 4h. Highly enriched mammalian osteoclasts were isolated by the sequential treatments of the collagen gel with pronase E/EDTA and collagenase.
2.Bone-like apatite can be formed on various substrates by the biomimetic method. Ca/P ratio and thickness of the bone-like apatite could be controlled by the concentration of simulated body fluid and the duration of soaking.
3.Bone-like apatite of the various thickness were made on the plastic disk, and bone cells including highly enriched osteoclasts were cultured on the surface of them. Using phase-contrast microscopy (LM) and scanning electron microscopy (SEM), osteoclasts and their resorption lacunae were examined, and bone-like apatite of 1.5mum thick was the best to observe both of them. The measurement of resorption area and volumn were also easy using an image analyzer.
4.Natural materials such as bone, dentine have various qualities, whose influence on osteoclastic resorbing activity is not negligible, whereas bone-like apatite made by the biomimetic method includes no organic materials. Resorbed area of bone-like apatite was about 70% of that of whale dentine, and the difference between the two groups was not significant. Using biomaterials for osteoclastic assay, the comparison between different groups can be easy because of their uniform and homogeneous nature.
5.Bone-like apatite-coated plastic disks have an advantage of translucency for the observation of osteoclasts. This is the first report of observation of actively resorbing osteoclasts during culture using LM.Osteoclasts isolated as described above maintained resorbing activity without contact ot stromal cells.
6.1-hydroxyethylidene-1,1-bisphosphonic acid (EHDP), used for treatment of osteoporosis, in culture medium at a concentration of 5 mug/ml decreased osteoclastic resorption partially.