Ultrastructural and Cytochemical Studies of Bone Formation by Cultured Osteoblasts.

Research Project

Project/Area Number	60480399
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	Morphological basic dentistry
Research Institution	Tokyo Dental College
Principal Investigator	YAMADA Marie Tokyo Dental College,School of Dentistry, Associate Professor, 歯学部, 助教授 (70115088)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥5,500,000 (Direct Cost: ¥5,500,000) Fiscal Year 1986: ¥1,300,000 (Direct Cost: ¥1,300,000) Fiscal Year 1985: ¥4,200,000 (Direct Cost: ¥4,200,000)
Keywords	Osteoblasts / Cultured cells / Ultrastructure / ALPase / Ca-ATPase / Location of Calcium / Non-collagenous protein / ラット頭蓋骨
Research Abstract	It is well known that osteoblasts must be involved in bone matrix formation and in bone calcification. Osteoblasts are also important in Ca homeostasis. In order to ascertain the function of osteoblasts, bone cells were cultured in vitro. In the present report, studies have been made of the ultrastructural details and cytochemical characteristics of osteoblasts in the process of bone formation. Bone cells isolated from rat pup calvaria by repetitive collagenase digestions were incubated in modified BGJ medium. After 5-days culture, the confluent cells showed round or ovoid shapes. In some areas, they formed multiple cell layers. These cells contained a well-developed Golgi apparatus, r-ER, and mitochondria. There were some cells containing numerous glycogen granules, presumably preosteoblasts. The location of ALPase and Ca-ATPase activities in the cultured cells were examined. Intense reaction products were mainly seen on the plasma membrane and caveolae, and also in the Golgi structures … More and intracellular vesicular structures. K-pyroantimonate fixation method was utilized to locate Ca in the cultured cells in normal medium and cells which were exposed to Ca-free medium before the termination of the cultivation. Following the fixation, precipitates were located in mainly mitochondria, vesicular structures and on the plasma membrane when cells were in media of normal Ca concentration. When cells were deprived of extracellular Ca a decrease occurred at these intracellular Ca sites. This method may represent the experimental system in attempt to better understand role of osteoblasts in Ca-transport mechanism. Non-collagenous protein, which may play the important role in bone mineralization, was prepared from rat pup calvaria according to the method by Termine et al(1981). The proteins were induced in BALB/c mice by repeated injection of antigen. After the last booster injection, the presence of antibody in the immune serum was checked by ELISA. So far, however, the antibody against the protein fraction has not been obtained. Less