title: A newly established culture method highlights regulatory roles of retinoic acid on morphogenesis and calcification of mammalian limb cartilage.
abstract: 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.
authors.affiliation: Department of Bioscience, Kwansei Gakuin University, Sanda, Japan.
authors.forename: Eizo
authors.initials: E
authors.lastname: Masuda
authors.affiliation: Department of Bioscience, Kwansei Gakuin University, Sanda, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Shirai
authors.affiliation: Department of Bioscience, Kwansei Gakuin University, Sanda, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Maekubo
authors.affiliation: Department of Bioscience, Kwansei Gakuin University, Sanda, Japan.|Research Center for Intelligent Bio-Materials, Kwansei Gakuin University, Sanda, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Hirai
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002113:Calcification, Physiologic; D002356:Cartilage / Q000196:embryology* / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D005121:Extremities / Q000196:embryology* / Q000502:physiology; D005994:Glycerophosphates / Q000378:metabolism; D051379:Mice; D009024:Morphogenesis; D046509:Tissue Culture Techniques / Q000379:methods*; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D005994:Glycerophosphates; D014212:Tretinoin; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid
keywords: calcification; cartilage; chondrogenesis; limb; morphogenesis; retinoic acid; three dimensional culture