Faculty of Dentistry, McGill University, Canada; Shriners Hospital for Children-Canada, Montreal, Canada.
Otto Schott Institute of Materials Research (OSIM), Friedrich Schiller University Jena, Löbdergraben 32, 07743 Jena, Germany.
Acta Biomater. 2015 Jul;20:140-146. doi: 10.1016/j.actbio.2015.03.026. Epub 2015 Mar 28.
Bone resorbing osteoclasts represent an important therapeutic target for diseases associated with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis. The quantification of osteoclast resorptive activity in vitro is widely used for screening new anti-resorptive medications. The aim of this paper was to develop a simplified semi-automated method for the quantification of osteoclastic resorption using fluorescently labeled biomimetic mineral layers which can replace time intensive, often subjective and clearly non-sustainable use of translucent slices of tusks from vulnerable or endangered species such as the elephant. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We confirmed that fluorescent labeling did not interfere with the biomimetic features of hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption. Correlation between our assay and traditional manual measurement techniques was found to be very strong (R(2)=0.99). In addition, we modified the technique to provide depth and volume data of the resorption pits by confocal imaging at defined depths. Thus, our method allows automatic quantification of total osteoclastic resorption as well as additional data not obtainable by the current tusk slice technique offering a better alternative for high throughput screening of potential antiresorptives.
破骨细胞是与骨骼和关节破坏相关疾病（如类风湿关节炎、牙周炎和骨质疏松症）的重要治疗靶点。体外破骨细胞吸收活性的定量分析广泛用于筛选新的抗吸收药物。本文旨在开发一种简化的半自动方法，使用荧光标记的仿生矿物层来定量检测破骨细胞的吸收，从而替代耗时、主观且明显不可持续地使用来自脆弱或濒危物种（如大象）的象牙半透明切片的方法。破骨细胞是从 RAW 264.7 小鼠单核细胞系使用核因子 kappa-B 配体（RANKL）的促吸收细胞因子受体激活剂形成的。我们证实荧光标记不会干扰羟基磷灰石的仿生特性，并开发了一种用于定量检测破骨细胞吸收的自动化方法。我们的测定法与传统的手动测量技术之间的相关性非常强（R²=0.99）。此外，我们修改了该技术，通过在定义的深度处进行共聚焦成像来提供吸收陷窝的深度和体积数据。因此，我们的方法不仅可以自动定量检测总破骨细胞吸收，还可以提供当前象牙切片技术无法获得的其他数据，为潜在抗吸收药物的高通量筛选提供了更好的替代方法。
