Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration.
Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration.
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DOI：
10.1016/j.bbrep.2017.02.001
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发表时间：
2017-07
影响因子：
2.7
通讯作者：
Gimsa J
中科院分区：
文献类型：
--
作者：
Galow AM;Rebl A;Koczan D;Bonk SM;Baumann W;Gimsa J
我们研究了碱性pH值对成骨细胞发育的影响。成骨细胞样细胞系 MC3T3-E1 的细胞最初在 pH 范围为 7.2 至 9.0 的 HEPES 缓冲培养基中培养六天。分析细胞计数、细胞 WST-1 代谢和 ATP 含量。三个参数显示最佳 pH 值为 8.4 左右，超出了 HEPES 在碱性侧面推荐的缓冲范围。因此，在更精细的日常研究中仅使用 pH 7.2、7.4、7.8 和 8.4 介质，以减少 24 小时 pH 控制间隔内 pH 变化的影响。所有参数都表现出相似的 pH 行为，大致显示在 pH 7.8 和 8.4 时增加到 130% 和 230%，而当使用 pH 7.4 数据作为参考时，在 pH 7.2 时减少到 70%。为了表征细胞分化和成骨细胞功能，将细胞在 pH 7.4 以及 pH 7.8 和 8.4 的碱性条件下培养 14 天。使用微阵列技术和茜素染色评估基因表达和矿化。在碱性条件下，ATF4（终末分化和功能的调节剂）以及 DMP1（成骨细胞转变为骨细胞的潜在标志物）显着上调，暗示分化过程加速。 21 天后，仅在碱性 pH 值下才检测到显着的矿化作用。我们的结论是，升高的 pH 值有利于骨细胞的培养，也可能在骨再生疗法中提供治疗价值。在严重碱性 pH 条件下，细胞计数、WST 代谢和 ATP 含量都会增加。碱性pH值可促进成骨细胞增殖。终末分化/功能标记 (ATF4/DMP1) 在 pH 7.8 时上调。碱性pH值加速终末分化和钙化。 pH值升高有利于骨细胞培养。
We investigated the effects of alkaline pH on developing osteoblasts. Cells of the osteoblast-like cell line MC3T3-E1 were initially cultured for six days in HEPES-buffered media with pH ranging from 7.2 to 9.0. Cell count, cellular WST-1 metabolism, and ATP content were analyzed. The three parameters showed a pH optimum around pH 8.4, exceeding the recommended buffer range of HEPES at the alkaline flank. Therefore, only pH 7.2, 7.4, 7.8, and 8.4 media were used in more elaborate, daily investigations to reduce the effects of pH change within the pH control intervals of 24 h. All parameters exhibited similar pH behaviors, roughly showing increases to 130% and 230% at pH 7.8 and 8.4, as well as decreases to 70% at pH 7.2 when using the pH 7.4 data for reference. To characterize cell differentiation and osteoblastic cell function, cells were cultured at pH 7.4 and under alkaline conditions at pH 7.8 and 8.4 for 14 days. Gene expression and mineralization were evaluated using microarray technology and Alizarin staining. Under alkaline conditions, ATF4, a regulator for terminal differentiation and function as well as DMP1, a potential marker for the transition of osteoblasts into osteocytes, were significantly upregulated, hinting at an accelerated differentiation process. After 21 days, significant mineralization was only detected at alkaline pH. We conclude that elevated pH is beneficial for the cultivation of bone cells and may also provide therapeutic value in bone regeneration therapies. Cell count, WST-metabolism and ATP-content are increased at severely alkaline pH. Alkaline pH enhances osteoblast proliferation. Markers for terminal differentiation/function (ATF4/DMP1) are upregulated at pH 7.8. Alkaline pH accelerates terminal differentiation and calcification. Elevated pH is beneficial for bone cell cultivation.