Osteogenic and angiogenic activity of hypertrophic chondrocytes and its clinical application

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1.Establishment of the method to differentiate isolated mesenchymal stromal cells into chondrocytesWe isolated mesenchymal stromal cells from a transgenic mouse line which expressed green fluorescent protein (GFP) under the control of the osteoblast-specific type I collagen promoter. Using these cells as a detector, we screened combinations of various chondrogenic factors for the optimal signaling. As a result, we identified the combination of Sox5, Sox6, and Sox9 was the most efficient and cell-type independent signaling unit. Treatment with the combination induced mRNA expression of cartilage marker genes, such as type II collagen, type IX collagen, and type XI collagen, aggrecan, and cbondromodulin-1. The treatment also induced expression of cartilage-specific matrix detected by immunohistuchemistry for type II Collagen and toluidine Blue stainin2.Establishment of the method to induce hypertcophic differentiation of chondrocytesWe treated chondrocyte-differentiated mesenchymal stromal cells, a undifferentiated mesenchymal cell line C3H10T1/2, and primary rib chondrocytes with various signaling molecules to find out that the canonical Wnt signaling pathway and Runx2 signaling pathway promoted hypertrophic differentiation detected by real-time RT-PCRT for type X collagen. In contrast, the loss of the gene endoding a stimulatory G protein, a putative inhibitory factor for hypertrophy, resulted in accelerated hypertrophy of chondrocytes in vivo.3.Induction of angiogenesis and osteogenesis by mineralized hypertrophic chondrocytesWe isolated chondrocytes from rabbit ribs and treated them with the canonical Wnt signal in osteogenic medium to induce hypertrophic differentiation and calcification. We formed pellets from these cells and transplanted subcutaneously into the back of rabbits. Angiogenesis into the pellets was observed at 2 weeks after the surgery. At 4 weeks after the surgery, osteogenesis occurred with bone marrow space.