Division of Pediatrics, Department of Medicine, Faculty of Medicine, University of Oviedo, CP 33006, Oviedo, Asturias, Spain.
Fundacion para la Investigación Sanitaria del Principado de Asturias (FINBA), Oviedo, Spain.
Sci Rep. 2020 Apr 24;10(1):6935. doi: 10.1038/s41598-020-63978-6.
This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.
这篇手稿报告了一种通过共聚焦显微镜对生长板软骨细胞进行成像的新方法。该方法基于固定的未脱钙骨样本，采用伊红、环氧树脂包埋和研磨进行块内染色，以获得厚切片。它简单、廉价，并提供了整个软骨细胞在其天然腔中的三维图像。对生长板不同层的软骨细胞的体积、形状和细胞质密度进行定量分析，能够客观地将生长板软骨细胞分为七个不同的簇。然后，通过对一些明确的软骨细胞分化分子标志物的免疫组织化学分析，对这 7 类软骨细胞进行评估。此外，还对负责离子变化和水运输的蛋白质进行了免疫组织化学分析，这些蛋白质允许软骨细胞在肥大过程中肿胀。研究结果表明，在预备肥大区可以定义四个亚区，在肥大区可以定义三个亚区，这一事实表明，与根据主观细胞形态学标准定义不同区域时相比，生长板中的软骨细胞的同质性较差。本研究的结果提供了技术创新，并深入了解了生长板中软骨细胞分化过程的复杂性。
