1994 Fiscal Year Final Research Report Summary
Establishment of a osteoblastic cell line derived from calvarial of rat embryo.
| Project/Area Number |
04671117
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
HISADA Yoh Hokkaido Univ., School of Dentistry., Ass.Prof., 歯学部, 助教授 (20001018)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIMURA Yoshitaka Univ., School of Dentistry., Assistant., 歯学部, 助手 (30230816)
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| Project Period (FY) |
1992 – 1994
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| Keywords | Rat Osteoblastic cell / Cell line / Calcification / ALP ase / Establichment |
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| Research Abstract |
An osteoblastic cell line that retains the capacity to mineralize in vitro would be a very useful model system for the study of the mechanism of calcification.However a few lines have established including the origin of cancer cells.The present study aimed to establish the cell line from calvariae of 18-21-old rat embryo.
Isolation of osteoblastic cells : (1) Single-cell suspensions from enzymatic digestions of the calvariae.(2) Outgrowth from the flagments of calvariae.
Culture medium : alpha-MEM with 10% FBS AND 60mug/ml Kanamycine sulfate.The changes of media at every 2 days.Subculture at 7-10 days with confluence state, in the culture.
Passage culture : Cells obtained by the populations to form opaque regions representing mineralized areas within the nodules.
ALPase activity : Assay by the method with pNPP.
Electoum Microscopy : Scanniry and transmission electron microscope observations.
[Results]About 18 cell lines induced from both of isolated methods have established showing the nodul
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es with mineralization.These lines were stored in liguid nitrogen at-80ﾟC.Some cell lines continued to more 80th passage culture.Especially, these cell lines were studied to characterize the processes of differentiation of cells and calcification in vitro.ALP activity in each cell line showed very low at the beginning of culture, then increased after the confluence state of cells.These results strongly suggested that the cells were an osteogenic cell line.
In the cells of 52-55 passage culture, the formation of mineralized tissue were observed from the 2-days to 28 weeks in the culture period as follows : Monolayr of osteoblast-like cells at 2-days.Two or more layrs of the cells at 7-days.Appearance of nodules and collagenouse matrix at 2-weeks.The nodules comprised a collagenouse matrix containing ovoid osteocyte-like cells and needle-shape crystals containing Ca^<2+> and Pi within 28 weeks.
From these results, these cell lines are an established clonal osteoblastic cell line, and it should be a useful model system for study of the mechanism of biological calcification. Less
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