Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation
Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation
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DOI：
10.1007/s00223-002-1057-y
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发表时间：
2003-05-01
影响因子：
4.2
通讯作者：
Boskey, A
中科院分区：
文献类型：
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作者：
Bonewald, LF;Harris, SE;Boskey, A
目前，许多技术被用于表征完整组织和细胞培养物中的生物矿化; von Kossa染色法、电子显微镜分析（EM）、X射线衍射和傅里叶变换红外光谱（FTIR）是最常见的。在这项研究中，我们利用这些方法中的三个培养的胎鼠颅骨细胞（FRC）和成骨细胞系2 T3和MC 3 T3-E1与大鼠颅骨的体内矿物质的矿化进行比较。在有或没有抗坏血酸（100 μ g/ml）和β-甘油磷酸盐（2.5、5或10 mM β GP）的情况下培养细胞，并在培养16至21天（FRC细胞和2 T3细胞）或30天（MC 3 T3-E1细胞）时收获。在FRC培养物中，在100 μ g/ml抗坏血酸存在下，用2.5和5 mM betaGP观察到最大von Kossa染色。当单独与β GP培养时，FRC细胞也显示出一些von Kossa染色。相比之下，用10 mM betaGP观察到MC 3 T3-E1细胞的最大von Kossa染色。只有同时接受抗坏血酸和betaGP的MC 3 T3-E1细胞培养物产生von Kossa阳性结构。2 T3培养物仅在用抗坏血酸和β GP处理后产生von Kossa阳性染色，骨形态发生蛋白-2（BMP-2）大大加速了这一过程。对FRC、MC 3 T3和2 T3培养物中产生的矿物质和基质进行FTIR，并将结果与来自16日龄大鼠颅骨的光谱进行比较。从大鼠颅骨的FTIR光谱计算的矿物基质比范围为2.97至7.44。FRC细胞生成骨样、结晶较差的磷灰石，并且随着betaGP的增加，矿物质-基质比呈统计学显著（P小于或等于0.02）剂量依赖性增加（2.5、5和10 mM betaGP分别为0.56 +/- 0.16、1.00 +/- 0.32和2.46 +/- 0.76）。FRC培养物的平均碳酸盐/磷酸盐比为0.015、0.012和0.008，以β GP浓度增加为序，而大鼠颅骨值为0.009-0.017。用BMP-2处理的2 T3细胞也产生骨样晶体，与FRC培养物中观察到的相似。相反。vonKossa阳性MC 3 T3-E1细胞的培养物没有显示出显著量的矿物质（最大矿物质-基质比为0.4）。因此，尽管FRC、2 T3和MC 3 T3-E1的von Kossa染色非常相似，但FTIR分析表明MC 3 T3培养物中不存在磷酸钙矿物质。通过EM，FRC细胞培养物和2 T3培养物中的矿物质通常与胶原蛋白相关，而MC 3 T3-E1培养物中罕见或稀疏的未知化学来源的营养不良性矿化是明显的。这些研究表明，单独使用von Kossa染色不适用于骨样矿物质的鉴定和定量，因此，应使用其他技术（如X射线衍射、EM或FTIR）来验证磷酸钙相的存在和质量。
Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 mug/ml) and beta-glycerophosphate (2.5, 5, or 10 mM betaGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM betaGP in the presence of 100 mug/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with betaGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM betaGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and betaGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining, only upon treatment with ascorbic acid and betaGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing betaGP, there was a statistically significant (P less than or equal to 0.02) dose-dependent increase in the mineral-to-matrix ratio (0.56 +/- 0.16, 1.00 +/- 0.32, and 2.46 +/- 0.76, for 2.5, 5, and 10 mM betaGP, respectively). The mean carbonate-to-phosphate ratios of the FRC cultures were 0.015, 0.012, and 0.008, in order of increasing betaGP concentration, compared with rat calvaria values of 0.009-0.017. The 2T3 cells treated with BMP-2 also made bonelike crystals, similar to those observed in FRC cultures. In contrast. the cultures of von Kossa positive MC3T3-E1 cells did not display a significant amount of mineral (maximum mineral-to-matrix ratio was 0.4). Thus, although the von Kossa stainings of FRC, 2T3, and MC3T3-E1 were very similar, FTIR analysis indicated that calcium phosphate mineral was not present in the MC3T3 cultures. By EM, the mineral in FRC cell cultures and 2T3 cultures was generally associated with collagen, whereas rare or sparse dystrophic mineralization of unknown chemical origin was evident in the MC3T3-E1 cultures. These studies demonstrate that von Kossa staining alone is not appropriate for the identification and quantitation of bonelike mineral and, hence, other techniques such as X-ray diffraction, EM, or FTIR should be utilized to verify the presence and quality of calcium phosphate phases.