Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure
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Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.
KeywordsChondrogenesis Hydrostatic pressure Micromass culture Embryonic cells Hypertrophy
AS was funded by Trinity College and Government of Ireland studentships. RR was funded by a Trinity Collage Innovation Bursary. All animal work was subject to ethical approval within Trinity College and carried out under licence (to AS) by the Health Products Regulatory Authority (formerly the Irish Medicines Board). Thanks to Prof Greg Lunstrum, Research Centre Shriners Hospital for Children, Portland USA, for providing the collagen X antibody. Thanks to Peter O’Reilly, Mechanical engineering, Peter Stafford and Alison Boyce, Zoology, for technical support.
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