Addison W N, Nelea V, Chicatun F, Chien Y-C, Tran-Khanh N, Buschmann M D, Nazhat S N, Kaartinen M T, Vali H, Tecklenburg M M, Franceschi R T, McKee M D
Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
Department of Mining and Materials, McGill University, Montreal, Quebec, Canada.
Bone. 2015 Feb;71:244-56. doi: 10.1016/j.bone.2014.11.003. Epub 2014 Nov 13.
Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.
骨细胞培养系统是研究调节细胞外基质矿化分子机制的重要工具。MC3T3-E1成骨细胞培养是骨基质矿化最常用的体外模型。尽管该细胞系在生物矿化研究中广泛应用，但尚未对这些培养物中产生的矿相进行系统表征。在此，我们对这种细胞培养矿物质和细胞外基质进行了全面的多技术生物物理表征，并将其与小鼠骨骼和合成磷灰石矿物质标准进行比较，以确定MC3T3-E1培养物用于生物矿化研究的适用性。能量色散X射线光谱（EDS）进行的元素组成分析表明，两种生物样品中均含有钙、磷以及微量的钠和镁。对树脂包埋的完整培养物进行的X射线衍射（XRD）表明，与1月龄小鼠骨骼相似，磷灰石晶体沿（100）、（101）和（111）矿面优先取向生长，这表明是有导向的生物成因生长，而非营养不良性钙化。从培养物中分离出的晶体的XRD显示，矿相为结晶度较差的羟基磷灰石，纳米微晶尺寸为10至20nm。与XRD观察结果一致，电子衍射图谱表明培养矿物质具有生物磷灰石典型的低结晶度。傅里叶变换红外光谱（FTIR）证实了生物样品中存在磷灰石碳酸盐和磷酸盐。使用所有技术，细胞培养矿物质和小鼠骨骼矿物质非常相似。扫描电子显微镜（SEM）和透射电子显微镜（TEM）显示，培养物具有致密的纤维状胶原基质，有小的、100nm大小的与胶原纤维相关的矿化灶，这些矿化灶融合形成更大的矿物质聚集体，矿化部位显示出矿质结合蛋白骨桥蛋白的积累。光学显微镜、共聚焦显微镜和三维重建显示，一些细胞具有树突状突起，并以骨细胞样方式嵌入矿物质中。总之，我们记录了MC3T3-E1成骨细胞培养物的矿物质和基质相特征，并确定该矿物质的结构和组成特性与小鼠骨骼高度相似。
