OBJECTIVE: To investigate the effect of recombinant human tumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced human adipose-derived stromal cells (hASCs) in vitro. METHODS: hASCs at passage 4 were divided into four groups according to culturing conditions: basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 μg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + β-glycerophosphate) and OM with 10 μg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . RESULTS: rhTNF-α promoted ALP activities of induced hASCs on day 14 (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells. CONCLUSION: The findings indicate that 10 μg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.
OBJECTIVE: To investigate the effect of recombinant humantumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced human adipose-derived stromal cells (hASCs) in vitro. METHODS: hASCs at passage 4 were divided into four groups according to culturing conditions: basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 μg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + β-glycerophosphate) and OM with 10 μg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . RESULTS: rhTNF-α promoted ALP activities of induced hASCs on day 14 (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells. CONCLUSION: The findings indicate that 10 μg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.