1991 Fiscal Year Final Research Report Summary
Establishment of the screening methods for bone-resorbing factors using in vitro osteoclast formation system.
| Project/Area Number |
01870078
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Showa University |
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Principal Investigator |
SUDA Tatsuo Showa Univ., School of Dentistry Professor, 歯学部, 教授 (90014034)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Takahisa Showa Univ., School of Dentistry Associate Professor, 歯学部, 助教授 (50129839)
YAMAGUCHI Akira Showa Univ., School of Dentistry Associate Professor, 歯学部, 助教授 (00142430)
TAKAHASHI Naoyuki Showa Univ., School of Dentistry Associate Professor, 歯学部, 助教授 (90119222)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Osteoclast / Bone resorbing factors / Osteoblast / Macrophage colony stimulating factor / M-CSF / 1alpha, 25(OH)_2D_3 / Bone resorption |
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| Research Abstract |
We have developed in vitro systems to examine the effects of osteotropic factors on the sequential process of osteoclastic bone resorption ; 1) proliferation of osteoclast progenitors, 2) differentiation of osteoclast precursors into multinucleated osteoclasts, and 3) pit formation by functionally active osteoclasts.
1. Evaluation of proliferation of osteoclast progenitors
We have developed a two-step culture system to determine the effect of osteotropic factors on proliferation of osteoclast progenitors. Bone marrow cells were first cultured in semisolid methylcellulose in the presence of various CSFs. Marrow cells were then isolated and further co-cultured with osteoblastic cells in the presence of 1alpha, 25(OH)_2D_3. After co-culture for 7 days, the number of osteoclasts formed were scored. On the basis of the number of marrow cells co-cultured with osteoblastic cells and that of osteoclasts formed, we were able to estimate the number of osteoclast progenitors present in marrow cell
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fractions. Using this method, we found that M-CSF was the most potent growth factor in inducing the growth of osteoclast progenitors.
2. Evaluation of differentiation of osteoclast precursors
We have reported that osteoclasts are formed in mouse marrow cultures and in co-cultures of mouse osteoblastic cells and spleen cells. Using these cultures, we have established a screening system for examining the effects of osteotropic factors on osteoclast differentiation. Bone-resorbing factors such as 1alpha, 25(OH)_2D_3, PTH, PGE_2 and IL-1 similarly stimulated differentiation of osteoclast precursors into functionally active osteoclasts.
3. Evaluation of bone-resorbing activity of osteoclasts
Osteoclasts formed on plastic dishes were hardly released from the dish surface. In contrast, when co-cultures were performed on collagen gel-coated dishes then treated with collagenase, most of the cells were easily released from the dishes. The osteoclast population was enriched by density gradient centrifugation. Using an osteoclast-enriched population and dentine slices, we have developed a simple bone resorption assay system. When isolated osteoclasts were cultured on dentine slices, they formed resorption pits within 24 hr. The area of resorption pits was quantitatively measured with an image analyzer. Using this system, we found that calcitonin and bafiromycin A_1 (an inhibitor of vacuolar H^+-ATPase) strongly inhibited pit formation by isolated osteoclasts.
These systems appear to be useful for examining the mechanism of action of osteotropic factors in osteoclastic bone resorption. Less
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