Atsuko Nemoto

Mechanical Strain Effect on Bone-Resorbing Activity and Messenger RNA Expressions of Marker Enzymes in Isolated Osteoclast Culture

Adaptive modeling and remodeling are controlled by the activities of osteoblasts and osteoclasts, which are capable of sensing their mechanical environments and regulating deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts have been scarcely examined because it has proven to be difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. Recently, we developed an apparatus for applying mechanical stretching to the ivory slice/plastic plate component on which cells could be cultured. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Using this apparatus, we examined the role of mechanical stretching on the bone‐resorbing activity of the osteoclasts. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component while being subjected to intermittent tensile strain. The stretched osteoclasts showed enhanced messenger RNA (mRNA) expression levels of osteoclast marker enzymes, tartrate‐resistant acid phosphatase (TRAP), and cathepsin K and increases of resorbed‐pit formation, suggesting that the mechanical stretching up‐regulated the bone‐resorbing activity of the osteoclasts. A stretch‐activated cation (SA‐cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching. This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving a SA‐cat channel in the absence of mesenchymal cells and, as a result, up‐regulated their bone‐resorbing activity.

Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells.

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106‐6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.

Osteopontin involvement in bone remodeling and its effects on in vivo osteogenic potential of bone marrow-derived osteoblasts/porous hydroxyapatite constructs

Abstract This review article will present our work regarding osteopontin (OPN) function in bone remodeling and its application to tissue engineering in bone. OPN, one of the major non-collagenous bone proteins, has an important role in bone remodeling. Our study suggested that OPN acts not only as a trigger for osteoblast early differentiation but activates osteoclast resorption. On this point of view, we proposed a model of bone remodeling via integrin α V β 3 –OPN binding, in which OPN could act as a signal molecule between osteoblasts and osteoclasts in the process of remodeling. Moreover, OPN was shown to induce the in vivo osteogenesis of a bone marrow-derived osteoblast/porous hydroxyapatite (HA) constructs. Our application of adenovirus-mediated gene transfer of osteopontin to primary osteoblasts is also presented.

The role of sialoproteins in recognition of bone surface by osteoblasts via integrin

Abstract To investigate the roles of collagenous and non-collagenous bone proteins in the early mineralization process by osteoblasts, we have examined the expression of several typical bone proteins (collagen Type I, fibronectin, osteopontin, BSP, osteonectin and osteocalcin) on rat bone marrow derived osteoblastic primary cells (RBMO), using SEM, AFM and laser confocal microscopes. The results suggest that the collagen Type I, osteopontin and BSP play important roles in early biomineralization. To study the roles of these bone proteins at the molecular level, we designed and synthesized the β -peptide (113–125) derived from a conserved sequence of the β subunit of integrins. 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. The solid phase binding assay suggested that the β -peptide could bind to the collagen Type I and osteopontin. Moreover, the RBMO adhered more strongly to sialoproteins such as osteopontin than to collagen Type I. This suggests that pre-osteoblasts attach to the bone surface and differentiate into osteoblasts by recognizing sialoproteins. This paper reviews our recent results, relating the role of sialoproteins in the recognition of bone surface via integrin by osteoblasts.