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Abstract：
OBJECTIVE: Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro.
METHODS: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated.
RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression.
CONCLUSIONS: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.
METHODS: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated.
RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression.
CONCLUSIONS: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.
摘要：
目的：二膦酸盐用于治疗骨质疏松患者的骨吸收过度。然而,它的使用与潜在的不良反应有关，例如与药物相关的颌骨坏死，提示在牙槽骨手术前在患者中引入药物假期概念。此外，双膦酸盐停药已经在体内进行了研究，在人类中,和动物模型。然而,目前尚不清楚这种方法是否会在体外影响骨细胞。因此,这项研究的目的是研究双膦酸盐停药对体外成骨细胞前和成骨细胞活性的潜在影响。
方法：用浓度为1、5和10µM的双膦酸盐（阿仑膦酸盐）处理前成骨细胞（MC3T3）和成骨细胞。然后在不同的时间点撤回阿仑膦酸盐。阴性对照由未经处理的细胞（0μM）组成，而阳性对照由在整个实验中与阿仑膦酸盐孵育的细胞组成。细胞活力，细胞粘附，细胞骨架，矿化,和基因表达进行了调查。
结果：用5-10μM阿仑膦酸盐处理4天或更长时间后，前成骨细胞和成骨细胞显示细胞活力降低。与阳性对照相比,停用阿仑膦酸盐两天显著增加细胞活力。然而,这些水平没有达到阴性对照的水平.阿仑膦酸盐减少了骨结节的形成。停用阿仑膦酸钠可恢复骨结节形成。较长的停药期比较短的停药期在恢复结节形成方面更有效。添加阿仑膦酸盐导致死亡细胞百分比增加，which,反过来,停用阿仑膦酸盐后下降。阿仑膦酸盐通过分解肌动蛋白应力纤维影响细胞骨架。细胞粘附和细胞形态参数也受阿仑膦酸盐的影响。停用阿仑膦酸钠可恢复细胞粘附和这些参数。总的来说,阿仑膦酸钠停药后改善最大的是10µM.然而,阿仑膦酸钠治疗和停药不影响成骨细胞基因表达。
结论：停用阿仑膦酸钠有助于逆转药物对细胞活力的负面影响，细胞粘附，和矿化通过恢复细胞骨架。我们的数据表明药物假期和/或间歇性策略在细胞水平上给予阿仑膦酸盐的益处。
方法：用浓度为1、5和10µM的双膦酸盐（阿仑膦酸盐）处理前成骨细胞（MC3T3）和成骨细胞。然后在不同的时间点撤回阿仑膦酸盐。阴性对照由未经处理的细胞（0μM）组成，而阳性对照由在整个实验中与阿仑膦酸盐孵育的细胞组成。细胞活力，细胞粘附，细胞骨架，矿化,和基因表达进行了调查。
结果：用5-10μM阿仑膦酸盐处理4天或更长时间后，前成骨细胞和成骨细胞显示细胞活力降低。与阳性对照相比,停用阿仑膦酸盐两天显著增加细胞活力。然而,这些水平没有达到阴性对照的水平.阿仑膦酸盐减少了骨结节的形成。停用阿仑膦酸钠可恢复骨结节形成。较长的停药期比较短的停药期在恢复结节形成方面更有效。添加阿仑膦酸盐导致死亡细胞百分比增加，which,反过来,停用阿仑膦酸盐后下降。阿仑膦酸盐通过分解肌动蛋白应力纤维影响细胞骨架。细胞粘附和细胞形态参数也受阿仑膦酸盐的影响。停用阿仑膦酸钠可恢复细胞粘附和这些参数。总的来说,阿仑膦酸钠停药后改善最大的是10µM.然而,阿仑膦酸钠治疗和停药不影响成骨细胞基因表达。
结论：停用阿仑膦酸钠有助于逆转药物对细胞活力的负面影响，细胞粘附，和矿化通过恢复细胞骨架。我们的数据表明药物假期和/或间歇性策略在细胞水平上给予阿仑膦酸盐的益处。
