In-situ visualization and quantification of mineralization of cultured osteogenetic cells
In-situ visualization and quantification of mineralization of cultured osteogenetic cells
复制标题
DOI：
10.1007/s00223-002-1052-3
复制
发表时间：
2003-12-01
影响因子：
4.2
通讯作者：
Ohgushi, H
中科院分区：
文献类型：
--
作者：
Uchimura, E;Machida, H;Ohgushi, H
成骨细胞系（HOS细胞）产生具有矿化的突出的类骨质基质。另一方面，成纤维细胞不表现出这种矿化。为了评估矿化程度，我们将钙黄绿素加入到培养基中，然后通过使用图像分析仪观察培养威尔斯孔。在HOS细胞中检测到钙黄绿素摄取到细胞/基质层中，但在成纤维细胞中未检测到钙黄绿素摄取。还通过使用图像分析仪原位定量钙黄绿素摄取，其显示HOS细胞中的高水平，其与矿化基质的钙含量良好相关。还将大鼠骨髓细胞培养在含有钙黄绿素、胎牛血清、P-甘油磷酸盐、L-抗坏血酸2-磷酸盐以及有或没有地塞米松的培养基中。使用地塞米松，细胞表现出成骨分化，导致约10天后矿化基质形成。基质形成与钙黄绿素摄取进入细胞/基质层的出现相一致，钙黄绿素摄取量随时间增加。相比之下，没有地塞米松的培养物没有表现出基质形成，并且钙黄绿素摄取可以忽略不计。在体外培养的HOS细胞和大鼠骨髓细胞的情况下，钙黄绿素不影响其碱性磷酸酶活性或骨钙素的产生的表达。此外，组织学观察显示，用钙黄绿素传代培养的大鼠骨髓细胞在体内植入后具有成骨能力。这些结果表明，目前检测培养物中钙黄绿素摄取的方法允许监测培养细胞的成骨能力，以及测量由成骨细胞产生的矿化量。鉴于成骨培养细胞/矿化基质用于骨重建手术，原位监测方法是非常宝贵的，因为它使我们能够评估体外结构的成骨能力。
An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, P-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.