Change of calpain and effect of mechanical stress on chondrocyte tube culture

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10671357
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: NAKAGAWA Yasuaki Kyoto University, Faculty of Medicine, assistantprof, 医学研究科, 助手 (90293860)
• Co-Investigator(Kenkyū-buntansha): NAKAMURA Takashi Kyoto University, Faculty of Medicine, professor, 医学研究科, 教授 (10201675)
• Project Period (FY): 1998 – 1999
• Keywords: tube culture / endochondral ossification / mechanical stress / centrifugation

Research Abstract

Rat chondrocyte cultures grown in centrifuge tubes with intermittent centrifugation differentiate into hypertrophic chondrocytes and form calcification. This culture system provides an extracellular matrix mineralization which is produced by chondrocytes per se, and we can assess this culture system as a model for the endochondral ossification in vivo. In our previous study, m-calpain was demonstrated immunohistichemically in epiphyseal chondrocytes, and immunoreactive m-calpain content in cells increased with terminal differentiation into hypertrophic cells. Using this culture system, we examined whether various degree of centrifugation changed the pattern of the endochondral ossification. Growth-plate chondrocytes were isolated from the rib cartilages of 5-week-old male Wister rats, and the cell suspension was transferred into a 15-ml plastic centrifuge tube at a density of 16X10ィイD14ィエD1 cells/ml/tube and centrifuged at 1, 500 rpm for 5 minutes. In the following, we divided three groups : group A was centrifuged at 3, 000 rpm for 30 minutes every day, group B was centrifuged at 1, 500 rpm for 5 minutes every other day, and group C was added no centrifugation. Group C also had the increase of alkaline phosphatase activity and terminal differentiation like group A and B. There were no significant differences in DNA content, proteoglycan content and alkaline phosphatase activity of all three groups. Calcium content of group A was significantly larger than that of group B at 3-week-culture, but at 4-week-culture, there was no significant difference between them.