Background

Mesenchymal cells differentiate into skeletal elements by forming a cartilaginous nodule, which induces bone formation through endochondral ossification in the vertebral column and long bones (Karsenty et al., 2009). Endochondral ossification is required for proper skeletal development during embryogenesis and has been recently demonstrated to be involved in the bone regeneration and joint diseases postnatally (Kawaguchi, 2008). Mesenchymal cells could be used as a regenerative therapy for these diseases. To study the mechanisms regulating endochondral skeletal development, we have examined high-density micromass cultures of embryonic limb-bud mesenchymal cells (Gay and Kosher, 1984; Iezaki et al., 2018). The in vitro chondrogenic cell culture system is useful for the analysis cartilage nodule formation that results from the condensation of mesenchymal cells and differentiation into chondrocytes.

Several methods have been developed to generate cartilage from mesenchymal cells. Mesenchymal cells in cell pellet or micromass culture can be induced to form cartilage in chondrogenic medium containing cytokines such as transforming growth factor-β (TGF-β) (Sekiya et al., 2002). Mesenchymal cells have also been induced to differentiate into chondrocytes in extracellular matrices such as alginate or collagen gel (Xu et al., 2008). Although these methods are effective to analyze chondrogenesis, they can be time consuming and expensive because they require complex reagents and equipment.

For this reason, we have developed a culture method to form cartilage from mesenchymal cells using a very simple method. We induced the differentiation of cells harvested from mouse limb buds (embryonic day 12.5) in high-density micromass culture and induced the cells to form cartilage nodules in growth medium without chondrogenic cytokines. In comparison to other methods, the method described here is simple and does not require specialized equipment, expensive materials or complex reagents. This method could be really useful for reducing the cost and complexity of the procedure.