A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes

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 thre...

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Bibliographic Details
Published in:Scientific reports 2020-04, Vol.10 (1), p.6935-6935, Article 6935
Main Authors: Fernández-Iglesias, Ángela, Fuente, Rocío, Gil-Peña, Helena, Alonso-Duran, Laura, García-Bengoa, María, Santos, Fernando, López, José M.
Format: Article
Language:English
Subjects:
13
13/51
14
14/19
631/136/818
631/80/2373
Animals
Cartilage - pathology
Cell Proliferation
Cell Shape
Chondrocytes
Chondrocytes - cytology
Chondrogenesis
Cluster Analysis
Confocal microscopy
Cytoplasm
Embedding
Epoxy resins
Female
Growth plate
Growth Plate - cytology
Humanities and Social Sciences
Hypertrophy
Immunohistochemistry
Microscopy
Microscopy, Confocal - methods
multidisciplinary
Proteins - metabolism
Rats, Sprague-Dawley
Science
Science (multidisciplinary)
Technological change
Tissue Fixation
Water transport
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-63978-6