Isolation, culture and differentiation of rabbit peripheral blood mesenchymal stem cells into osteoblasts in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND: A few mesenchymal stem cells (MSCs) can be found in peripheral blood.OBJECTIVE: To isolate rabbit peripheral blood MSCs and induce its differentiation into osteoblasts.DESIGN, TIME AND SE'I-I'ING The cytological in vitro study was performed at the Central Laboratory of Shangdong Provincial Hospital from June to December 2008.MATERIALS A total of 6 New Zealand rabbits were purchased from Shandong Academy of Agricultural Sciences. α-MEM and L-DiEM were bought from Hyclone.METHODS: Full-thickness skin (3 cm×3 cm) (dorsal muscular layer was left) was incised at various sites of rabbit back, every 3days. Incised skin was dressed following orthotopic transplantation. Each rabbit received four consecutive wounds. Peripheral blood was collected from femoral vein before injury and 1 week after injury. MSCs were harvested from peripheral blood by density gradient cantrifugation. MSCs were divided into 2 groups, which were respectively incubated in α-MEM supplemented with 10% fetal bovine serum and L-DiEM. Cells at passage 2 and 1 ×105/cm2 were incubated in a 12-well plate and induced with H-DiEM containing osteogenic inductor.MAIN OUTCOME MEASURES: The following parameters were measured cell morphology before and after injury; colony forming efficiency of MSCs; outcome of osteogenic induction.RESULTS: Following primary medium change and before injury, obtained cells were normal. Twenty-four hours following incubation and after injury, MSCs were spindle or polygonal, and adhered to the wall. 5-6 days later, cell colonies appeared.Compared with the L-DiEM group, the number of primary culture colony formation in α-MEM group was significantly greater (P < 0.05). Peripheral blood MSCs were spindle, tdangle or polygonal, with the presence of processes, 2-3 nuclei and cell division phase, and slowly proliferated following osteogenic induction. At day 7 after differentiation of MSCs into osteoblasts, positive rate of alkaline phosphates was above 80%. At day 21, calcium deposition was detected by Alizarin Red S (positive staining).CONCLUSION: MSCs could be harvested from peripheral blood of wounded rabbits, with characteristics of osteogenic differentiation, α -MEM was more suitable than L-DiEM for peripheral blood MSCs to growin vitro.