Age-related distribution of non-collagenous proteins in bone

Research Project

Project/Area Number	07671990
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Morphological basic dentistry
Research Institution	TOKYO DENTAL COLLEGE
Principal Investigator	YAMADA Marie Tokyo Dental College, Department of Dentistry, Associate Professor, 歯学部, 助教授 (70115088)
Project Period (FY)	1995 – 1996
Project Status	Completed (Fiscal Year 1996)
Budget Amount *help	¥2,500,000 (Direct Cost: ¥2,500,000) Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000) Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywords	Non-collagenous proteins / Bone / Immunocytochemistry / Osteopontin / Rat / Aging
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 (synthetic peptide A) and the residues 147 to 163 from the N-terminal region (synthetic peptide B) of OPN were synthesized by a standard solid-phase method 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% paraformaldehyde plus 1% glutaraldehyde solution and decalcified with 5% EDTA.All of them were embedded in GMA or L.R.White. We used post-embedding immunogold-silver staining method and transmission electron microscopy to detect ultrastructurally bound antigens in bone (calvaria, tibia, mandible) of newborn, 13,54 and 107 week-old 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 osteoclasts. No immunolabeling was associated with collagen fibrils. In adult rat bone, anti-synthetic peptide A antibody labeling was generally faint, and no gold particles were concentrated over patches of organic material among the collagen fibrils of the mineralized bone matrix and lamina limitans. While anti-synthetic peptide B antibody labeling was clearly observed along a thick, lamina limitans and bone canaliculi. From these results, it was suggested that OPN may play an important role in initial mineralization process in bone and that OPN may have heterogeneity in the amino acid composition.