Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth.