Combination Effect of Cyclic Tension Force and Glucosamine on Osteogenic Differentiation of Human Apical Papilla Cells

• Main Authors: Yi-Tzu Lin, 林怡孜
• Other Authors: Yi-Jane Chen
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/61690086860800669254

碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 101 === The three key ingredients for tissue engineering are cells, scaffolds, and growth-stimulation signals. Mesenchymal stem cells play an important role in bone tissue engineering. The source of mesenchymal stem cells could be embryonic or somatic origin. Previous studies reported that the progenitor cells obtained from apical papilla tissue of developing teeth exhibit the characteristic of mesenchymal stem cells. Apical papilla stem cells (APSCs) could commit to osteogenic differentiation in vitro as bone marrow mesenchymal stem cells. Our previous work revealed that low-level mechanical tensional force upregulated the osteogenic differentiation of human apical papilla cells. Moreover, the amino sugar glucosamine promoted the osteogenic differentiation of dental pulp stem cells through modulating the level of the TGF-β1 receptor. The purpose of the present study was to evaluate whether glucosamine could act synergistically with cyclic mechanical tensional force to induce osteogenic differentiation of human dental apical papilla cells. Human apical papilla cells (APCs) was obtained from apical tissues of developing third molars in young patient. Cells in passage 5 – 8 were used in the present study. After subjecting to cyclic tension force in the presence or absence of glucosamine, the mRNA expression of osteogenic genes in APCs were examined by using RT-PCR techanique, and the protein expression of Cbfa1 was analyzed by western blotting. The results revealed that the cytoskeleton in stretched APCs was aligned perpendicular to strain axis. The morphology of APCs exposed to glucosamine was not significantly different from the un-treated control cells. The treatment of cytochalasin D disrupted the organization of actin filaments, which became more obvious when cells subjecting to mechanical tensional force. Similarly, the treatment of jasplakinolide resulted in paradoxical stabilization of actin filament, which was not altered by mechanical tension force. Moreover, expression of Cbfa1/Runx2 (Core-binding factor alpha-1/Runt-related transcription factor-2) protein was inhibited by cytochalasin D and jasplakinolide. These findings confirmed that an intact, dynamic cytoskeleton is required for stretch-induced expression of Cbfa1/Runx2 in APCs. The expression of Cbfa1/Runx2 and ALP (alkaline phosphatase) genes were significantly upregulated in the APSCs subjected to cyclic mechanical tensional force and glucosamine treatment for 48 hours. The mRNA expression of DSPP (dentin sialophosphoprotein) gene was also upregulated. Moreover, the transcription factor Cbfa1/Runx2 was activated by either mechanical tensional force or glucosamine treatment, and the combination of both. The additive effect of the physical and chemical stimuli was noted for the phosphorylatiobn of Cbfa1, but not for the expression of Cbfa1 protein. To our knowledge, the present study was the first to report the combination effect of glucosamine and cyclic mechanical tensional force in upregulating the osteogenic differentiation in APSCs. Further study is required to further investigate the long-term effects, e.g. mineralized matrix deposition.