RUNX2 regulates the effects of TNFalpha on proliferation and apoptosis in SaOs-2 cells.

The runt-related transcriptional factor RUNX2 is an essential mediator of the osteoblast phenotype and plays a pivotal role in the process of osteoblast differentiation. The involvement of RUNX2 includes the regulation of genes that are important in committing cells to the osteoblast lineage. Increasing evidences are consistent with a requirement of RUNX2 for stringent control of osteoblast proliferation and recent data even suggested that RUNX2 might act as a proapoptotic factor. Among the cytokines described as modulators of osteoblast functions, TNFalpha affects both apoptosis and the differentiation rate from mesenchymal precursor cells of osteoblast. Thus we evaluated on the human osteosarcoma cell line SaOs-2 stably transfected with a RUNX2 dominant negative construct (DeltaRUNX2) the effects of serum and TNFalpha on proliferation and apoptosis. In this study we showed that SaOs-2 clones expressing high levels of DeltaRUNX2 presented a higher proliferation rate than clones transfected with an empty vector. This increase in cell growth was accompanied by a rise in cyclins A1, B1 and E1 expression and a decrease in the cyclin inhibitor p21. Moreover we observed that the expression of the RUNX2 transgene protected the SaOs-2 cells from the antiproliferative and the apoptotic effects induced by TNFalpha. This was accompanied by the inhibition of Bax and activation of Bcl2 expression. Experiments done on SaOs-2 cells transiently transfected with siRNA confirmed that RUNX2 represents a critical link between cell fate, proliferation and growth control. This study also suggested that RUNX2 might control osteoblastic growth depending on the differentiation stage of the cells by regulating expression of elements involved in hormones and cytokines sensitivity.

[Using perfusion bioreactor for mesenchymal stem cell proliferation in large tricalcium phosphate scaffold].

OBJECTIVE: To investigate the feasibility of using perfusion culture bioreactor for bone mesenchymal stem cell proliferation in large scale beta-tricalcium phosphate (beta-TCP) scaffold. METHODS: In the dynamic perfusion culture group, the porous beta-TCP cylindrical scaffolds combined with the sheep mesenchymal stem cells were continuously perfused with the complete alpha-MEM medium by a peristaltic pump for 1, 2 and 4 weeks. While in the static culture group, the hybrid constructs were immersed in the medium without perfusion for 2 and 4 weeks. The cell proliferation and distribution were examined by the daily glucose consumption, the cell viability and undecalcified histological study. RESULTS: The daily glucose consumption increased with time. The increase was much more evident in the first 2 weeks than in the last 2 weeks. The daily glucose consumption was higher in the dynamic culture group than in the static culture group. The cell viability also increased with time. It was higher in the dynamic culture group. In comparison to 2-week culture, the cell viability was significantly higher after 4-week culture in the dynamic culture group (P < 0.05), while it was not significantly different after 4-week culture in the static culture group (P > 0.05). Under dynamic perfusion culture, the mesenchymal stem cells survived and proliferated through the scaffolds. However, the mesenchymal stem cells survived and proliferated only in the peripheral pores of the scaffolds under static culture. Histomorphometrical study indicated that there were much more cells in dynamic culture group than in the static group. The cell/pore rate was not significantly different between 2-week static culture and 4-week static culture (P > 0.05). However, the cell/pore rate was significantly higher after 4-week dynamic culture than after 2-week dynamic culture (P < 0.05). CONCLUSION: Perfusion culture permitted the persistent nutrition supply and gas exchange into the centre of large scaffold. This perfusion bioreactor makes the mesenchymal stem cells survive and proliferate through a large three-dimensional scaffold.