柚皮苷為天然黃酮類，是一種HMG-CoA之抑制劑。過去的文獻指出柚皮苷能促進骨生成，但高濃度的柚皮苷會造成細胞毒性。為了控制柚皮苷的濃度及釋放時間，我們將柚皮苷固定於幾丁聚醣表面。考慮到傳統交聯系統可能的毒性或低接枝效率，在本研究中，我們利用臭氧改質激發幾丁聚醣表面建立自由基，接著利用熱誘導法固定柚皮苷於表面。通過紫外光譜定量材料表面的柚皮苷，並藉由掃描式電子顯微鏡（SEM）及紅外線光譜（FTIR）分析改質後幾丁聚醣基材的特性。結果表明幾丁聚醣成功且有效地接枝於幾丁聚醣上，同時也發現被固定柚皮苷被緩慢地釋放，經由這釋放程序，可以良好地控制培養液中柚皮苷濃度。
細胞實驗結果指出，表面固定柚皮苷之幾丁聚醣基材無細胞毒性且能促進細胞的活性與貼附。更重要的是，柚皮苷大幅促進了骨細胞的分化，早期骨分化指標的鹼性磷酸酶（alkaline phosphatase, ALP）表現在經過臭氧與柚皮苷改質的基材上被促進了一倍。通過骨鈣素(OCN, osteocalcin)與骨涎蛋白(BSP, bone sialoprotein)的表現得知：固定化柚皮苷也明顯地促進骨細胞中後期的分化。
為了進一步研究固定化柚皮苷的效果，我們利用SEM觀察骨母細胞的細胞骨架。結果指出在具有固定化柚皮苷的基材表面上，細胞層狀偽足(lamellipodia)的形成明顯地被促進了，除了證明細胞有較佳的貼附表現外，也顯示細胞內部之細胞骨架在此一表面上處於一高度活化的狀況，這項發現與本實驗中免疫螢光染色的結果吻合，證實固定化柚皮苷能高度激發細胞骨架中之肌動蛋白。利用生物原子力顯微鏡(bio-AFM, bio-atomic force microscope)測定骨母細胞機械性質的結果顯示固定化柚皮苷能大幅增加細胞與基材的交互作用力。而Runx2的結果亦指出：基材表面的柚皮苷誘發了細胞骨架相關的骨分化訊息傳遞鏈。因此，我們推測固定化柚皮苷提高了細胞與基材間的交互作用力，此一強作用力活化細胞骨架，進而協調貼附依賴性的信號傳導而提升骨細胞分化。

Naringin, a flavonoid available commonly in citrus fruits, is a HMG-CoA reductase inhibitor in osteoblastic cells. The promotion of bone formation by naringin has been proved in the previous studies, but naringin with high concentration would be cytotoxic. To control the concentration and duration of naringin, naringin was immobilized onto chitosan, a biocompatible material However, convectional crosslinkers, such as GA, are not biocompatible, resulting in difficulties in clinical applications. In this research, ozone treatment was thus applied to activate chitosan surfaces with radicals, followed by the immobilization of naringin after thermal induction. The amount of immobilized naringin was analyzed by UV spectra. The chitosan substrate with immobilized naringin was characterized by SEM and FTIR. Besides, the in vitro release of naringin from chitosan substrates in PBS for two weeks was also performed. The results indicated that naringin was successfully and efficiently grafted onto chitosan. It was also found that the immobilized naringin was slowly released, where the concentration of naringin in the medium was well controlled by this delivery process. The optimization of ozone treatment and naringin concentration was then carried out in this study.
The results of cell culture showed the cell activity, attachment and proliferation were promoted with the immobilized naringin on chitosan substrates. With sutiable naringin densities, the chitosan substrates showed no cytotoxicity. The marker of early osteogenic differentiation, ALPase expression, also increased. Besides, the osteogenic differentiation in middle and late stages were alos promoted by immobilized naringin, revealed by the increase in the expression of OCN and BSP. To further investigate the effects of immobilized naringin, the cytoskeleton projection of osteoblasts was observed under SEM. The results indicated that immobilized naringin enhanced cell spreading, and the lamellipodia was clearly formed on the chitosan substrates due to the naringin modification. The results from florescence staining also support that the actin filament in cytoskeleton was highly activated due to the immobilized naringin.The mechanical properties of osteoblasts measured with bioAFM indicates that the cell-substrate interforce greatly increased by naringin immobilization. The expression of Runx2 also proved the positive regulation of signal transduction induced by cytoskeleton activation. Thus, we interfere that the strong interforce caused by immobilized naringin activated cytoskeleton, which coordinated the anchorage-dependent signal transduction to enhance the osteogenic differentiation. . It would explain the mechanism that immobilized naringin was effective on the differentiation of osteoblasts.

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