Objective: To investigate the proliferation and the secretion of vascular endothelial growth factor(VEGF), fibroblast growth factor-2(FGF-2) and insulin-like growth factor-1(IGF-1) of human adipose tissue-derived stromal cells(hADSCs) before and after osteogenic differentiation under the stimuli of recombinant human tumor necrosis factor alpha (rhTNF-alpha).
Methods: hADSCs were obtained from human lipoaspirates. All the cells used were at passage four. The proliferation of hADSCs was measured with MTT assays 48, 72, 96 hours after being treated with 0, 1, 5, 10, 50 or 100 microg/L rhTNF-alpha respectively. The secretion of VEGF, FGF-2 and IGF-1 of the undifferentiated hADSCs under stimuli of rhTNF-alpha with the above 5 concentration grades was observed and the secretion of these 3 growth factors of hADSCs at different stages of osteogenic differentiation under stimuli of 10 microg/L rhTNF-alpha was also observed. All the supernatants were harvested for measuring after 24 hours' incubation with rhTNF-alpha. The secretion of VEGF, FGF-2 and IGF-1 was measured with ELISA, and the values were normalized to the cell number of the corresponding wells.
Results: The effect of rhTNF-alpha on the proliferation of hADSCs varied with the concentration and time. Compared with the control(0 microg/L), 10 microg/L rhTNF-alpha showed no suppression or acceleration on proliferation of hADSCs at hour 48, but significantly promoted the proliferation at hour 96 (0.903+/-0.042 vs 0.810+/-0.011, P<0.01), 100 microg/L rhTNF-alpha seemed to suppress the proliferation at hour 48 (0.317+/-0.024 vs 0.458+/-0.046, P<0.01), but appeared to promote it (0.956+/-0.030 vs 0.810+/-0.011, P<0.01) at hour 96. rhTNF-alpha(1, 5, 10, 50 and 100 microg/L) significantly increased VEGF, FGF-2 and IGF-1 production of hADSCs versus the control (0 microg/L) (P<0.01). After osteogenic differention, the secretion of the three growth factors of hADSCs (without rhTNF-alpha treated) was elevated with the days increasing. Under the stimulus of 10 microg/L rhTNF-alpha, the hADSCs after 1 day of osteogenic differentiation significantly increased the secretion of VEGF (P<0.01) compared with the group without rhTNF-alpha treated; after 3 and 7 days of osteogenic differentiation, the hADSCs significantly increased the secretion of VEGF (P<0.01), FGF-2 (P<0.05)and IGF-1 (P<0.05). However, after 14 days of osteogenic differentiation, 10 microg/L rhTNF-alpha appeared to suppress the production of VEGF (P<0.01), FGF-2 (P<0.05) and IGF-1 (P<0.05) of the differentiated hADSCs.
Conclusion: Within certain concentration range, rhTNF-alpha can promote the proliferation of hADSCs and the production of VEGF, FGF-2 and IGF-1. The effect of 10 microg/L rhTNF-alpha on the production of VEGF, FGF-2 and IGF-1 of the differentiated hADSCs varied at different stages of osteogenic differentiation.