Mechanical Force Induces Osteogenic Differentiation of Human Ligamentum Flavum Cells

• Main Authors: Shih-Yung Huang, 黃詩詠
• Other Authors: Chun-Min Lo
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/80767775292001487591

碩士 === 國立陽明大學 === 醫學工程研究所 === 100 === Calcification of the ligamentum flavum was frequently observed in patients with lumbar spinal canal stenosis and those with cauda equina symptoms. It is reported to appear more often in combination with ligamentum flavum hypertrophy of the spine. The study aims to elucidate the pathomechani- sms of calcification of the ligamentum flavum in vitro and to further investigate the effect of mechanical force and BMP-2 on the osteogenic differentiation of human ligamentum flavum cells. Human ligamenturn flavum cells were obtained from 12 patients undergoing lumbar spine surgery. 9 patients were female, and 3 were male. The mean age of the patients at the time of surgery was 62.17 years (range, 39 to 79 years). Mechanical force was applied by subjecting human ligamentum flavum cells in monolayer culture to centrifugal force. Mechanical force, BMP-2 and mechanical force in combination with BMP-2 were applied to cultured ligamentum flavum cells. Osteogenic phenotypes of human LF cells, such as alkaline phosphatase, Alizarin red-S stain, and gene expression of osteogenic makers including alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OP), Runx2/Cbfa1(Runx2) were further evaluated. Osteoinductive factors, BMP-2 and TG2, were evaluated by real-time PCR and ELISA. It was found that mechanical force in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical force significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OP and Runx2 gene expression on day 7. No significant effect was found on BMP-2 gene expression following mechanical stimulation. Mechanical force significantly induced tissue transglutaminase (TG2) gene expression and enzyme activity in human ligamentum flavum cells. To further identify the role of TG2 in osteogenic induction of LF cells, exogenous TG2 was delivered into culture medium. The result showed that exogenous TG2 increased ALP and OC gene expression. In summary, mechanical force-induced TG2 expression may be involved in the osteogenic differentiation of human ligamentum flavum cells and the progression of the calcification of ligamentum flavum.