Subject(s)
Bone Substitutes , Neovascularization, Physiologic , Tissue Engineering/methods , Animals , Bone Regeneration , Computer-Aided Design , Humans , Lactic Acid/chemistry , Microsurgery , Polyesters/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Starch/chemistry , Stem Cells/cytologyABSTRACT
PURPOSE: To evaluate osteogenesis in canine bone marrow mesenchymal stem cells(BMSCs) transduced by point mutant HIF-1α. METHODS: Lenti-WT (wild type HIF-1α), Lenti-MT (mutant type HIF-1α), and Lenti-LacZ (the control group) were constructed with LR recombination system. And then Lenti-LacZ, Lenti-WT and Lenti-MT were used to transduce canine BMSCs. After transduction, total RNA and protein were extracted at 0 , 1 , 4 , 7, 14 d and 21 d, respectively. The mRNA and protein expression of osteogenic factors were detected by RT-PCR and Western blot in vitro under normoxic conditions.To further prove osteogenic differentiation of HIF-1α mediated BMSCs, the BMSCs were inoculated into 6-well plate (2×10(5)/well) and then the expression of calcium nodules was measured using Alizarin red staining(ARS) at 14 d and 21 d, respectively. RESULTS: At MOI=9, the transduced efficiency of BMSCs was up to 90%. After target gene being transduced to BMSCs, the mRNA and protein expression of osteogenic factors were significantly up-regulated at 4d, reached peak at 14-21 d and maintained a state of high expression (P<0.05). ARS results showed that the target gene can induce BMSCs to the osteogenic differentiation in normoxic conditions. CONCLUSIONS: Under normoxic conditions in vitro, mutant HIF-1α can stabilize the expression and maintain a high level of activity. Lenti-MT can significantly improve the osteogenic activity of canine BMSCs.
