Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures.
Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures.
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消除与水性溶剂的接触对于矿化 UMR106-01 成骨细胞培养物中钙和磷富集位点的早期检测和超微结构元素分析是必要的。
DOI：
10.1159/000324252
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Gorski,JeffreyP
中科院分区：
文献类型：
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作者：
Studer,Daniel;Hillmann-Marti,Therese;Huffman,NicholeT;Gorski,JeffreyP
骨矿化的机制已经得到了很好的研究，但仍然存在争议。矿化组织成像的固有困难和钙和磷酸盐的水溶性，结合形成骨矿物晶体的2个离子，限制了目前通过电子显微镜对不稳定的扩散，无定形和结晶中间体的分析。为了提高钙和磷的保留，我们开发了一种伪非水处理方法，并用它来表征生物矿化病灶，即成骨细胞培养中羟基磷灰石沉积的细胞外部位。由于UMR106-01成骨细胞的矿化是暂时同步的，并在镀后78小时开始，我们使用这些培养物来评估我们的方法在第一个矿物晶体形成之前应用于细胞时的有效性。我们的方法首次结合了三种成熟的方法和第四种方法，即干燥超薄切片。采用振荡金刚石刀干燥超薄切片，对高压冷冻和冷冻替代的矿化生物矿化灶进行电子能谱成像。为了比较，培养物也用常规处理和湿切片处理。结果表明，只有使用伪非水处理才能在76 h检测到早期钙和磷富集的细胞外位点，比检测生物矿化灶内的矿物晶体早几个小时。
The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, ie dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.