Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.
Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.
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DOI：
10.1016/j.bone.2014.11.003
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发表时间：
2015-03
期刊：
影响因子：
4.1
通讯作者：
McKee MD
中科院分区：
文献类型：
--
作者：
Addison WN;Nelea V;Chicatun F;Chien YC;Tran-Khanh N;Buschmann MD;Nazhat SN;Kaartinen MT;Vali H;Tecklenburg MM;Franceschi RT;McKee MD
骨细胞培养系统是研究调节细胞外基质矿化的分子机制的重要工具。此类体外研究提供了有关矿化分子决定因素、矿化相关病理、组织工程和生物材料技术的深入信息。为了使细胞培养模型与体内生理发生的矿化过程相关，体外形成的矿物晶体必须具有正确的相、形状、大小、方向、位置和结晶度。 MC3T3-E1 细胞培养物是最常见的骨基质矿化体外模型。尽管它们被广泛使用，但尚未对这些培养物中产生的矿物质进行系统表征，并且细胞外基质矿化的机制仍不清楚。在本研究中，我们对细胞培养物生物矿物的形态、组成和超微结构进行了全面、多技术的表征，以确定 MC3T3-E1 培养物是否适合生物矿化研究，建立 MC3T3-E1 培养物矿化的明确参数，并深入了解体内和体外形成的培养矿物之间的重要相似点和差异。完整（非均质）树脂包埋培养物的 X 射线衍射 (XRD) 表明，与小鼠骨类似，培养物中的磷灰石晶体沿着 (100)、(101) 和 (111) 成核平面优先生长，这表明引导的生物生长，而不是营养不良的钙化。对分离的培养物晶体进行 XRD 分析表明，该矿物是一种结晶度较差的羟基磷灰石，具有 10-20 nm 尺寸的纳米微晶。与 XRD 观察结果一致，电子衍射图表明培养矿物具有生物磷灰石典型的低结晶度。正如预期的那样，培养矿物的能量色散 X 射线光谱显示矿物相中存在钙和磷，以及痕量的钠和镁。傅里叶变换红外光谱证实了磷灰石碳酸盐和磷酸盐的存在。通过使用所有技术，细胞培养矿物和颅骨非常相似，并且与合成羟基磷灰石不同，相反，合成羟基磷灰石表现出高结晶度和大晶体尺寸。使用扫描和透射电子显微镜进行的超微结构分析表明，培养物具有致密、组装的胶原基质，其中矿化随着出现小的 100 nm 胶原相关矿化焦点而进行，这些矿化焦点合并形成更大的矿物聚集体。此外，共焦成像和三维重建显示，一些细胞表现出树突状过程，并以类似骨细胞的方式嵌入矿物质中。总之，我们记录了 MC3T3-E1 细胞培养物中沉积的矿物相的特征，并确定这种矿物的结构和成分特性与小鼠颅骨的结构和成分特性相似。
Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. Such in vitro studies provide insightful information on molecular determinants of mineralization, mineralization-related pathologies, tissue engineering and biomaterial technologies. In order for cell culture models to have relevance to the mineralization process as it occurs physiologically in vivo, it is imperative that the mineral crystals formed in vitro be of a correct phase, shape, size, orientation, location and crystallinity. MC3T3-E1 cell cultures are the most prevalent in vitro model of bone matrix mineralization. Despite their widespread use, there is as yet no systematic characterization of the mineral produced in these cultures, and mechanisms of extracellular matrix mineralization remain unclear. In this study, we performed a comprehensive, multi-technique characterization of the morphology, composition and ultrastructure of cell culture biomineral to determine the suitability of MC3T3-E1 cultures for biomineralization studies, to establish definitive parameters of MC3T3-E1 culture mineralization, and to provide insight into important similarities and differences between culture mineral formed in vivo and in vitro. X-ray diffraction (XRD) on intact (nonhomogenized), resin-embedded cultures indicated that similar to mouse bone, apatite crystals in culture grow with preferential orientations along the (100), (101) and (111) nucleating planes indicative of a guided biogenic growth as opposed to dystrophic calcification. XRD analysis of isolated culture crystals revealed that the mineral was a poorly crystalline hydroxyapatite with 10–20 nm-sized nanocrystallites. Consistent with XRD observations, electron diffraction patterns indicated that culture mineral was of a low crystallinity typical of biological apatites. As expected, energy dispersive x-ray spectroscopy of the culture mineral showed the presence of calcium and phosphorus, and trace amounts of sodium and magnesium from the mineral phase. Fourier transform infrared spectroscopy confirmed the presence of apatitic carbonate and phosphate. With all the techniques utilized, cell culture mineral and calvarial bone were remarkably similar and were distinguished from synthetic hydroxyapatite which, in contrast, displayed high crystallinity and large crystal sizes. Ultrastructural analysis with scanning and transmission electron microscopy showed that cultures have a dense, assembled collagenous matrix where mineralization proceeds with the appearance of small 100 nm collagen-associated mineralization foci which coalesce to form larger mineral aggregates. Furthermore, confocal imaging and three-dimensional reconstructions revealed that some cells exhibited dendritic processes and become embedded within the mineral in an osteocyte-like manner. In summary, we have documented the characteristics of the mineral phase deposited in MC3T3-E1 cell cultures, and determined that the structural and compositional properties of this mineral are similar to that of mouse calvarial bone.
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影响因子：
6.9
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