Video-based calcification assay: A novel method for kinetic analysis of osteogenesis in live cultures.

The method is less error-prone than absorption-based assays since it takes longitudinal measurements from the same cultures It is cost effective as it foregoes the use of calcium-sensitive dyes It is automatable and amenable to high-throughput and thus allows the concurrent quantification of multiple parameters of differentiation Traditional methods of quantifying osteoblast calcification in culture require the use of calcium sensitive dyes, such as Arsenazo III or Alizarin Red S, which have been successfully used for decades to assess osteogenesis. Because these dyes elicit a colorimetric change when reacted with a cell lysate and are cytotoxic to live cells, they forfeit the ability to trace calcification longitudinally over time. Here, we demonstrate that image analysis and quantification of calcification can be performed from a series of time-lapse images acquired from videos. This method capitalizes on the unique facet of the mineralized extracellular matrix to appear black when viewed with phase contrast optics. This appearance of calcified areas had been previously documented to be characteristic to the formation of bone nodules in vitro. Due to this distinguishable appearance, extracting the information corresponding to calcification through segmentation allowed us to threshold only the pixels that comprise the mineralized areas in the image. Ultimately, this method can be used to quantify calcification yield, rates and kinetics facilitating the analyses of bone-supportive properties of growth factors and morphogens as well as of adverse effects elicited by toxicants. It may also be used on images that were acquired manually.

该方法比基于吸收的检测方法更不易出错，因为它对相同的培养物进行纵向测量。 它具有成本效益，因为它无需使用钙敏感染料。 它可自动化，适用于高通量，因此能够同时对分化的多个参数进行定量。 传统的培养物中定量成骨细胞钙化的方法需要使用钙敏感染料，如偶氮胂III或茜素红S，几十年来它们已成功用于评估成骨作用。由于这些染料在与细胞裂解物反应时会引起比色变化，并且对活细胞具有细胞毒性，它们丧失了随时间纵向追踪钙化的能力。在此，我们证明可以从视频获取的一系列延时图像中进行钙化的图像分析和定量。这种方法利用了矿化细胞外基质的独特特征，即当用相差光学器件观察时呈现黑色。钙化区域的这种外观先前已被记录为体外骨结节形成的特征。由于这种独特的外观，通过分割提取与钙化对应的信息使我们能够仅对图像中构成矿化区域的像素进行阈值处理。最终，这种方法可用于量化钙化产量、速率和动力学，有助于分析生长因子和形态发生素的骨支持特性以及毒物引起的不良影响。它也可用于手动获取的图像。