[The study of osteogenic differentiation in canine bone marrow stromal cell induced by point mutant type of HIF-1α].

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.

Repairing critical-sized calvarial defects with BMSCs modified by a constitutively active form of hypoxia-inducible factor-1α and a phosphate cement scaffold.

Tissue engineering combined with gene therapy represents a promising approach for bone regeneration. The Hypoxia-inducible factor-1α (HIF-1α) gene is a pivotal regulator of vascular reactivity and angiogenesis. Our recent study has showed that HIF-1α could promote osteogenesis of bone mesenchymal stem cells (BMSCs) using a gene point mutant technique. To optimize the function of HIF-1α on inducing stem cells, another constitutively active form of HIF-1α (CA5) was constructed with truncation mutant method and its therapeutic potential on critical-sized bone defects was evaluated with calcium-magnesium phosphate cement (CMPC) scaffold in a rat model. BMSCs were treated with Lenti (lentivirus) -CA5, Lenti-WT (wild-type HIF-1α), and Lenti-LacZ. These genetically modified BMSCs were then combined with CMPC scaffolds to repair critical-sized calvarial defects in rats. The results showed that the overexpression of HIF-1α obviously enhanced the mRNA and protein expression of osteogenic markers in vitro and robust new bone formation with the higher local bone mineral density (BMD) was found in vivo in the CA5 and WT groups. Furthermore, CA5 showed significantly greater stability and osteogenic activity in BMSCs compared with WT. These data suggest that BMSCs transduced with truncation mutanted HIF-1α gene can promote the overexpression of osteogenic markers. CMPC could serve as a potential substrate for HIF-1α gene modified tissue engineered bone to repair critical sized bony defects.