Permalink : http://hdl.handle.net/2115/65481

KEYWORDS : osteoblast;compressive force;mechanical stress;hydroxyapatite;骨芽細胞;圧縮力;メカニカルストレス;ハイドロキシアパタイト

本研究の目的は，メカニカルストレスを想定したハイドロキシアパタイト（Hap）ブロックによる圧縮力が骨芽細胞様細胞（MC3T3-E1細胞）に与える影響を明らかにすることである．ポリスチレンディッシュで培養後に細胞上にHapを静置することにより圧縮力を負荷した群（UH群）とディッシュ上に静置したHap上で細胞を培養後にHapを反転し圧縮力を負荷した群（OH群）を実験群とし，ディッシュ上で通常培養したものをコントロール群（C群）とした．各群ともconfluence後に圧縮力を負荷し，１，２および３日間培養行い，総蛋白質量の測定と，Alkaline Phosphatase（ALP），Osteocalcin（Oc），RANKL，OPGのmRNA発現量をreal-time PCR法にて定量した．総蛋白質量は，UH群，OH群とも経時的に増加したが，C群と比較し有意に低い値を示したことから，細胞増殖の抑制が示された．UH群においては，OcとALPの遺伝子発現は経時的に増加したが，Ocは３日後，ALPはいずれの時点においても，C群に比較し有意に低い値を示した．RANKLは経時的に増加しC群に比較し有意に高い値を示したが，OPGは有意に低い値を示した．以上の結果から，骨芽細胞の分化は抑制され破骨細胞の分化が促進される可能性が示唆された．一方，OH群においては，OcとALPは経時的に増加し，Ocでは負荷３日後，ALPではいずれの計測時点においてもC群に比較し有意に高い値を示し，RANKLは経時的に減少しC群に比較し有意に低い値を示したことから，UH群と異なり，骨芽細胞の分化が促進され破骨細胞の分化は抑制される可能性が示唆された．以上より，MC3T3-E1細胞に対するHapによる圧縮力は，細胞増殖を抑制すること，また圧縮力の方向は骨形成に関する機能発現と破骨細胞の分化に影響を与えることが示唆された．
The impact of mechanical stress on the differentiation and functional expression of osteoblasts is not yet fully understood. The present study sought to clarify the effects of mechanical stress as represented by the compressive force of a hydroxyapatite (Hap) block on osteoblast-like cells (MC3T3-E1). The experimental groups consisted of two groups. The first was a UH group in which MC3T3-E1 cells were incubated in a polystyrene dish. A Hap block was fixed on the cells, and compressive force was applied (Group A). The second was an OH group in which MC3T3-E1 cells were incubated on a Hap block that was fixed on the dish and then inverted, and compressive force was applied (Group B). The control group used a standard culture of MC3T3-E1 cells grown on a dish (C group). In each experimental group, the compressive force was applied after cells reached confluence. On incubation days 1, 2, and 3, total protein levels were measured and real-time polymerase chain reaction assays were performed to determine mRNA expression of alkaline phosphatase (ALP), osteocalcin (Oc), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG). Total protein levels increased over time in both the UH and OH groups, but were significantly lower than in the C group. This suggests that compression inhibited cell growth. Oc and ALP levels increased over time in the UH group, but Oc after 3 days of compression and ALP at each time point were both significantly lower than in the C group. RANKL in the UH group also increased over time and was significantly higher than in the C group, but OPG was significantly lower. These findings suggest that the compressive force may have inhibited osteoblast differentiation and enhanced osteoclast differentiation. Meanwhile, Oc and ALP increased over time in the OH group, and Oc at Day 3 and ALP at each time point were both significantly higher than in the C group. Conversely, RANKL decreased over time and was significantly lower than in the C group. These findings suggest that, unlike in the UH group, compressive force may have enhanced osteoblast differentiation and inhibited osteoclast differentiation. Taken together, these findings suggest that compressive force applied to MC3T3-E1 cells using a Hap block may inhibit cell growth, and that the direction of force application may affect osteogenic functional expression and osteoclast differentiation.