The Effect Of RhVEGF And RhBMP-7 On The Proliferation And Differentiation Of Rat Calvaria-derived Osteoblasts.

ObjectiveTo study the effect of recombinant human vascular endothelial growth factor (rhVEGF) and recombinant human bone morphogenetic protein-7 (rhBMP-7) on the proliferation and differentiation of rat calvaria-derived osteoblasts.Methods1. Isolation of osteoblasts. Osteoblasts were obtained from the calvaria of 24-h new born Sprague-Dawley rats by using primary culture.2. The cell cultured. The isolated cells were cultured in DMEM medium until cells were lined up in one layer. The cell were trypsinized and removed from the flask and collected for next step.3. Identification for osteoblasts.(1) Cell morphology on phase-contrast microscope(2) ALP staining (Gomori Ca-Co method)(3) Bone nodule staining4. To determine the effect ofrhVEGF (3.125, 6.250, 12.500, 25.000, 50.000ng/ml) and rhBMP-7 (0.500, 1.000, 5.000, 10.000, 50.000ng/ml) in different concentration on osteoblast activity and functions in the separateness or combination states.(1) To assay osteoblasts proliferation by MTT(2) To assay ALP activity of osteoblasts by PNPPResults1. The effect of rhVEGF (3.125, 6.250, 12.500, 25.000, 50.000ng/ml) on osteoblast activity and functions.(1) To assay osteoblasts proliferation by MTT Compared with control group, the osteoblasts proliferation was increased in treatment groups. The significantly higher levels of proliferation was observed in 3.125ng/ml, 6.250ng/ml, 12.500ng/ml groups (P＜0.05), and there was significant difference among groups (P＜0.05). The maximal effect on proliferation was at a concentration of 3.125 ng/ml.(2) To assay ALP activity of osteoblasts by PNPPCompared with control group, the alkaline phosphatase activity was increased in treatment groups. The significantly higher levels of proliferation was observed in6.250ng/ml, 12.500ng/ml, 25.000ng/ml groups (P＜0.05), but there was no significant difference among groups. The higher effect on the alkaline phosphatase activity was at a concentration of 12.500ng/ml.2. The effect of rhBMP-7 (0.500, 1.000, 5.000, 10.000, 50.000ng/ml) on osteoblast activity and functions.(1) To assay osteoblasts proliferation by MTTCompared with control group, the osteoblasts proliferation was increased in treatment groups.The significantly higher levels of proliferation was observed in 10.000ng/ml, 50.000ng/ml groups (P＜0.05), and there was significant difference among groups (P＜0.05). The maximal effect on proliferation was at a concentration of 50.000ng/ml.(2) To assay ALP activity of osteoblasts by PNPPCompared with control group, the alkaline phosphatase activity was increased in treatment groups.The significantly higher levels of proliferation was observed in 1.000ng/ml, 5.000ng/ml, 10.000ng/ml, 50.000ng/ml groups (P＜0.05), and there was significant difference among groups (P＜0.05). The maximal effect on the alkaline phosphatase activity was at a concentration of 50.000ng/ml.3. The effect on osteoblast proliferation with combination use of rhVEGF and rhBMP-7.(1) To assay osteoblasts proliferation by MTTCompared with control group, the osteoblasts proliferation were increased with combination use of recombinant human vascular endothelial growth factor and recombinant human bone morphogenetic protein-7 and there was significant difference among groups (P＜0.05). The maximal effect on proliferation was in the third day.(2) To assay ALP activity of osteoblasts by PNPP Compared with control group, the alkaline phosphatase activity was increased with combination use of rhVEGF and rhBMP-7. The maximal effect on the alkaline phosphatase activity was in the third day.ConclusionsIt showed that both rhVEGF and rhBMP-7could influence. The separateness or combination use of rhVEGF and rhBMP-7could significantly promote the proliferation and differentiation of osteoblasts. Therefore, the combination use of rhVEGF and rhBMP-7could increase bone formation.All above showed that these would be strong proof for clinic use both in bone tissue metabolism and in bone tissue engineering. The mechanism of combination use of rhVEGF and rhBMP-7 is worthy of further research.