A newly established culture method highlights regulatory roles of retinoic acid on morphogenesis and calcification of mammalian limb cartilage

During mammalian embryogenesis, sclerotome-derived chondrocytes in the limb bud are arranged into a complicated bone shape with specific areas undergoing hypertrophy and calcification, creating a region-specific mineralized pattern in the cartilage. To follow chondrogenesis progression in vitro, we...

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Bibliographic Details
Published in:	BioTechniques Vol. 58; no. 6; pp. 318 - 324
Main Authors:	Masuda, Eizo, Shirai, Kota, Maekubo, Kenji, Hirai, Yohei
Format:	Journal Article
Language:	English
Published:	England Taylor & Francis Group 01-06-2015
Subjects:	Animals Ascorbic Acid - metabolism calcification Calcification, Physiologic cartilage Cartilage - embryology Cartilage - physiology Cell Differentiation Cell Line chondrogenesis Extremities - embryology Extremities - physiology Glycerophosphates - metabolism limb Mice Morphogenesis retinoic acid three dimensional culture Tissue Culture Techniques - methods Tretinoin - metabolism limb retinoic acid morphogenesis three dimensional culture cartilage calcification chondrogenesis
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Summary:	During mammalian embryogenesis, sclerotome-derived chondrocytes in the limb bud are arranged into a complicated bone shape with specific areas undergoing hypertrophy and calcification, creating a region-specific mineralized pattern in the cartilage. To follow chondrogenesis progression in vitro, we isolated limb cartilage from mice on embryonic day 13 (E13) and cultured it at the air-liquid interface after microsurgical removal of the ectoderm/epidermis. Explants underwent proper morphogenesis, giving rise to complete templates for limb bones in vitro. We found that region-specific calcification patterns resembling limbs of prepartum mature embryos could be induced in explants using culture medium containing high concentrations of CaCl2 (Ca), ascorbic acid (AA), and β-glycerophosphoric acid (BGP). In this culture system, excess amounts of all-trans retinoic acid (RA) severely disrupted morphogenesis and calcification patterns in limb cartilage. These effects were more pronounced in forearms than in phalanges. Although dissociated, the nascent chondrocytes in culture did not give rise to cartilage units even though augmented calcification was induced in these cell aggregates in the presence of RA. Taken together, our newly established culture system revealed that RA independently regulates three-dimensional morphogenesis and calcification.
ISSN:	0736-6205 1940-9818
DOI:	10.2144/000114300