The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells
The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells
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DOI：
10.1371/journal.pone.0065943
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发表时间：
2013-06-18
期刊：
影响因子：
3.7
通讯作者：
Hoffmann, Andrea
中科院分区：
文献类型：
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作者：
Schaeck, Luisa M.;Noack, Sandra;Hoffmann, Andrea
对于骨髓间充质干细胞/间充质基质细胞通过二维细胞培养体外分化为成骨细胞，过去已经使用和评价了各种方案。特别是，用于诱导成矿的外部磷源在化学成分和浓度方面都有很大的不同。根据最近的发现，无机磷指导着对骨骼发育至关重要的基因的表达，在体外分化中显然需要一个标准化的磷酸盐来源。我们发现，化学成分(无机磷酸盐与有机磷酸盐来源)和磷酸盐补充浓度对通常用作成骨细胞形成标志的基因的基因表达结果以及所形成的矿物的组成有严重的影响。具体地说，基因表达的强度并不一定与高质量的矿化基质相关。我们的研究证明了用无机磷酸盐代替β-甘油磷酸盐的优点，并提出了钙离子和磷酸根离子的比色定量方法，作为X射线衍射和傅里叶变换红外光谱分析的经济和及时的替代方法，用于测定体外条件下形成的矿物基质的钙磷比和浓度。我们批判性地讨论了用于评估体外骨形成的不同方法的特异性，并提供了一种详细的体外实验方案，以帮助避免由于实验设计的差异而导致的相互矛盾的结果。
For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of beta-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost-and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.