| Project/Area Number |
05671517
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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 |
Morphological basic dentistry
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| Research Institution | TOKYO DENTAL COLLEGE |
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Principal Investigator |
YAMADA Marie Tokyo Dental College, Associate Professor, 歯学部, 助教授 (70115088)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Rat / Calvaria / Non-collagenous protein / Osteopontin / Immunocytochemistry / rat / calvaria / non-collagenous protein / osteopontin / immunocytochemistry / non‐collagenous proteins |
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| Research Abstract |
Osteopontin (OPN) is one of the major non-collagenous proteins in bone. Since the aminoacid sequence of OPN has been shown, the C-terminal 16 residues (Lys Phe Arg Ile Ser His Glu Leu Glu Ser Glu Val Asn)(synthetic peptide A) and the residues 147 to 163 from the N-terminal region (Glu Gln Tyr Pro Asp Ala Thr Asp Glu Asp Leu Thr Ser Arg Met Lys)(synthetic peptide B) of OPN were synthesized by a standard solid-phase method (J.G.Sutcliffe et al., 1980) and antibodies were raised to these two synthetic peptides.
Immunochemical preparations, these antibodies reacted to OPN obtained from newborn rat calvaria. For immunocytochemistry, newborn rat calvaria were fixed in 4% glutaraldehyde plus 1% paraformaldehyde solution and decalcified with 5% EDTA.All of them were embedded in GMA at -20ﾟC.We used post-embedding immunogold-silver staining method and transmission electron microscopy to detect ultrastructurally bound antigens in bone of newborn rats.
In newborn rat calvaria, an intense immunoreaction was observed over organic material (electron dense patch) at the initial calcification stage, after incubation with these two antibodies. And intense immunolabeling was also associated with a lamina limitans and cement (reverse) lines. Particularly anti-synthetic peptide A antibody labeling was associated with clear zone of osteroclasts. No immunolabeling was associated with collagen fibrils.
From these results, it was suggested that OPN may play an important role in initial mineralization process in bone.
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