Hard Tissue Research Group

■Object

The research of the group focuses on micromechanism of biomineralization, influence of mechanical stress on bone formation and physical properties and biological response of hydroxyapatite.

1) Elucidation of micromechanism of biomineralization in bone by biophysical and biochemical techniques, using cultured osteoblastic and osteoclastic cells.

2) Elucidation of influence of mechanical stress on bone formation, measuring signal transduction phenomena across plasma membrane of cultured cells.

3) Measurement of bending strength of single crystal hydroxyapatite and elucidation of hydroxyapatite crystal growth mechanism

■Results in Fiscal Year

1) For studying the roles of the bone proteins (collagen Type I and osteopontin) in the molecular level, we designed and synthesized the β-peptide(113-125) derived from a conserved sequence of theβsubunit of integrins. And the β-peptide proved to be a good tool for studying the adhesion properties of bone proteins to integrin by solid phase binding assay of the β-peptide to fibrinogen and fibronectin. Results from the solid phase binding assay suggest that the β-peptide could bind to the collagen Type I and osteopontin. Moreover the RBMO adhered more strongly to the osteopontin than collagen type I. It might suggest that pre-osteoblasts attach to the bone surface and differentiate into osteoblasts by recognizing osteopontins.

2) An increase in cytosolic free Ca concentration was observed by deforming plasma membrane of cultured cells such as osteoblast, endothelial cell with a micropipette set to a micromanipulator, suggesting the existence of unknown mechanisms which transduce mechanical stress to mobilization of second messengers such as Ca ions.

3) Biomineralization

Recently, we first succeeded in direct crystal growth observation of hydroxyapatite (Ca10(PO4)6(OH)2), a bone and tooth mineral. The result of direct observation suggested that the growth units of hydroxyapatite were left- and right-handed Ca9(PO4)6 clusters with size of 0.815-0.87 nm. The aim of the present study, therefore, is to show the evidence of the presence of Ca9(PO4)6 clusters in the simulated body fluid. A specially-improved dynamic light scattering technique was used for this aim. The size of particles in the fluid was found to be from 0.4 to 1.0 nm with a peak at 0.7 nm. However, a fluid excluding calcium and phosphate ions, showed only a peak at 0.5 m corresponding to the size of tris-aminomethane molecule used as a buffering reagent without having the fraction from 0.7 to 1.0 nm. The fraction from 0.7 to 1.0 nm also disappeared when the pH of the simulated body fluid was decreased to 3.0 by adding a HCl solution. Therefore, we concluded that the size fraction from 0.7 to 1.0 nm corresponded to Ca9(PO4)6 clusters.

Fig. 1 Mechanism of Remodeling of Bone

Fig. 2 Fluorescent Image of Bovine Endothelial Cells Stained with Fluo-3 and Transparent Image of Micropipette.

Fig. 3 Increase of Cytosolic Ca Ion Concentrations by Poking Bovine Endothelial Cell.

(upper: Ca transient in whole cell image)

(lower: Ca transient at fixed points)

Fig. 4 Three point bending strength of hydroxyapatite (HAP) and OH-car-bonated hydrox-yapatite (CHAP) single crystals as a function of crystal thickness.